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1
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Lemieux G, Pérez-Vargas J, Désilets A, Hassanzadeh M, Thompson CAH, Gravel-Trudeau A, Joushomme A, Ennis S, Villanueva I, Marouseau É, Fraser BJ, Champagne W, Lepage M, Niikura M, Arrowsmith CH, Jean F, Leduc R, Boudreault PL. From N-0385 to N-0920: Unveiling a Host-Directed Protease Inhibitor with Picomolar Antiviral Efficacy against Prevalent SARS-CoV-2 Variants. J Med Chem 2025; 68:7119-7136. [PMID: 40163818 PMCID: PMC11998928 DOI: 10.1021/acs.jmedchem.4c02468] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2024] [Revised: 01/30/2025] [Accepted: 02/28/2025] [Indexed: 04/02/2025]
Abstract
The worldwide spread of new SARS-CoV-2 variants emphasizes the need to diversify existing therapeutic strategies. TMPRSS2, a host protease crucial for SARS-CoV-2 entry, has garnered significant research attention as a potential target for therapeutic intervention. Here, we optimized N-0385, a previously reported TMPRSS2 ketobenzothiazole-based peptidomimetic inhibitor, by screening 135 derivatives for target affinity and antiviral potency. Among the top candidates, N-0695 exhibited low nanomolar Ki values against three TTSPs associated with respiratory virus entry: TMPRSS2, matriptase, and TMPRSS13. Notably, N-0920 demonstrated exceptional potency in reducing SARS-CoV-2 variants EG.5.1 and JN.1 entry in Calu-3 cells, representing the first in cellulo picomolar inhibitor with EC50 values of 300 and 90 pM, respectively. Additionally, molecular modeling provided insights into the binding interactions between the compounds and their targets. This study underscores the effectiveness of our screening approach in refining an existing peptidomimetic scaffold to enhance selectivity and antiviral activity.
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Affiliation(s)
- Gabriel Lemieux
- Department
of Pharmacology-Physiology, Institut de Pharmacologie de Sherbrooke,
Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke J1H5N4, Quebec, Canada
| | - Jimena Pérez-Vargas
- Department
of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver V6T 1Z3, British Columbia, Canada
| | - Antoine Désilets
- Department
of Pharmacology-Physiology, Institut de Pharmacologie de Sherbrooke,
Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke J1H5N4, Quebec, Canada
| | - Malihe Hassanzadeh
- Department
of Pharmacology-Physiology, Institut de Pharmacologie de Sherbrooke,
Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke J1H5N4, Quebec, Canada
| | - Connor A. H. Thompson
- Department
of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver V6T 1Z3, British Columbia, Canada
| | - Alice Gravel-Trudeau
- Department
of Pharmacology-Physiology, Institut de Pharmacologie de Sherbrooke,
Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke J1H5N4, Quebec, Canada
| | - Alexandre Joushomme
- Department
of Pharmacology-Physiology, Institut de Pharmacologie de Sherbrooke,
Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke J1H5N4, Quebec, Canada
| | - Siobhan Ennis
- Faculty
of Health Sciences, Simon Fraser University, Burnaby J1H5N4, British Columbia, Canada
| | - Ivan Villanueva
- Department
of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver V6T 1Z3, British Columbia, Canada
| | - Étienne Marouseau
- Department
of Pharmacology-Physiology, Institut de Pharmacologie de Sherbrooke,
Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke J1H5N4, Quebec, Canada
| | - Bryan J. Fraser
- Department
of Medical Biophysics, University of Toronto, Toronto M5S 1A1, Ontario, Canada
- Structural
Genomics Consortium, University of Toronto, Toronto M5S 1A1, Ontario, Canada
| | - William Champagne
- Department
of Pharmacology-Physiology, Institut de Pharmacologie de Sherbrooke,
Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke J1H5N4, Quebec, Canada
| | - Matthieu Lepage
- Department
of Pharmacology-Physiology, Institut de Pharmacologie de Sherbrooke,
Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke J1H5N4, Quebec, Canada
| | - Masahiro Niikura
- Faculty
of Health Sciences, Simon Fraser University, Burnaby J1H5N4, British Columbia, Canada
| | - Cheryl H. Arrowsmith
- Department
of Medical Biophysics, University of Toronto, Toronto M5S 1A1, Ontario, Canada
- Structural
Genomics Consortium, University of Toronto, Toronto M5S 1A1, Ontario, Canada
- Princess
Margaret Cancer Centre, Toronto M5S 1A1, Ontario, Canada
| | - François Jean
- Department
of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver V6T 1Z3, British Columbia, Canada
| | - Richard Leduc
- Department
of Pharmacology-Physiology, Institut de Pharmacologie de Sherbrooke,
Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke J1H5N4, Quebec, Canada
| | - Pierre-Luc Boudreault
- Department
of Pharmacology-Physiology, Institut de Pharmacologie de Sherbrooke,
Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke J1H5N4, Quebec, Canada
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2
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Lundgren JG, Flynn MG, List K. GPI-anchored serine proteases: essential roles in development, homeostasis, and disease. Biol Chem 2025; 406:1-28. [PMID: 40094301 DOI: 10.1515/hsz-2024-0135] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2024] [Accepted: 02/23/2025] [Indexed: 03/19/2025]
Abstract
The glycosylphosphatidylinositol (GPI)-anchored serine proteases, prostasin and testisin, have essential roles in diverse physiological functions including development, reproduction, homeostasis and barrier function of epithelia, angiogenesis, coagulation, and fibrinolysis. Important functions in pathological conditions such as cancer, kidney disease and cardiovascular disease have also been reported. In this review, we summarize current knowledge of the cellular and in vivo roles of prostasin and testisin in physiology and pathophysiology and explore the underlying molecular mechanisms. We discuss how new insights of their role in cancer and cardiovascular disease may facilitate translation into clinical settings in the future.
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Affiliation(s)
- Joseph G Lundgren
- Department of Pharmacology, Wayne State University, Detroit, MI 48201, USA
- Department of Oncology, Wayne State University, Detroit, MI 48201, USA
| | - Michael G Flynn
- Department of Pharmacology, Wayne State University, Detroit, MI 48201, USA
| | - Karin List
- Department of Pharmacology, Wayne State University, Detroit, MI 48201, USA
- Department of Oncology, Wayne State University, Detroit, MI 48201, USA
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3
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Jin Z, Zhang Y, Chen W, Li H, Shi L, Wang D, Zhu R, Zhang C. Intracellular autoactivation and surface location of hepsin, TMPRSS2, and TMPRSS13. Life Sci 2025; 361:123299. [PMID: 39643034 DOI: 10.1016/j.lfs.2024.123299] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2024] [Revised: 12/03/2024] [Accepted: 12/03/2024] [Indexed: 12/09/2024]
Abstract
AIMS Hepsin (HPN), a Type II transmembrane serine protease (TTSP), is involved in hepatocyte metabolism and various diseases. It undergoes autoactivation on the surface of human hepatoma cells, a mechanism not observed in other cell types. This study aims to explore HPN activation and surface expression in endometrial epithelial cells. MATERIALS AND METHODS We studied HPN zymogen activation and cell surface expression in human embryonic kidney 293 and endometrial epithelial AN3CA and Ishikawa cells using site-directed mutagenesis, Western blotting, flow cytometry, and immunostaining. Treatments with brefeldin A (BFA) and monensin, along with co-transfection assays, were employed to assess HPN activation and expression before reaching the cell surface. We also analyzed the activation and expression of TMPRSS2 and TMPRSS13 and examined the effect of the serine protease inhibitor HAI-1 on these proteases. KEY FINDINGS HPN zymogen autoactivates in the endoplasmic reticulum (ER) and Golgi apparatus. Its active form reduces cell surface expression through trans-autodegradation, a mechanism also applicable to in TMPRSS2 and TMPRSS13. Additionally, HAI-1 interacts with these TTSPs in different ways: it inhibits HPN activation and stabilizes its cell-surface expression; it inhibits TMPRSS2 activation without affecting its cell-surface expression; and it facilitates TMPRSS13 activation, protecting it from degradation and stabilizing its cell surface expression. SIGNIFICANCE These results revealed an intracellular autoactivation and expression mechanism of HPN, TMPRSS2, and TMPRSS13, differing from the extracellular activated TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation, potentially aiding in treating TTSP-related endometrial diseases.
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Affiliation(s)
- Zili Jin
- State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjin, China
| | - Yue Zhang
- Medical Science and Technology Innovation Center, Central Laboratory, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, China
| | - Wenjun Chen
- The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, China
| | - Hui Li
- Medical Science and Technology Innovation Center, Central Laboratory, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, China
| | - Lingyun Shi
- The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, China
| | - Di Wang
- Center for Human Reproduction and Genetics, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, China
| | - Rui Zhu
- Center for Human Reproduction and Genetics, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, China.
| | - Ce Zhang
- Center for Human Reproduction and Genetics, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, China.
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Peng T, Wen J, Huang G, Zhao H, Liu J. First-generation high-affinity ST14 radiopharmaceutical: Design, synthesis, and preclinical PET imaging evaluation for pancreatic cancer detection. Bioorg Chem 2025; 154:108085. [PMID: 39721147 DOI: 10.1016/j.bioorg.2024.108085] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2024] [Revised: 12/11/2024] [Accepted: 12/18/2024] [Indexed: 12/28/2024]
Abstract
The non-specificity of 18F-FDG, coupled with high false-positive rates in pancreatitis, underscores an unmet clinical need for using specific positron emission tomography (PET) radiopharmaceuticals in noninvasive pancreatic cancer detection. ST14, a trypsin-like protease and a member of the type II transmembrane serine protease family, is overexpressed in various solid malignancies, including pancreatic cancer. This study aimed to develop a 68Ga-labeled PET radiopharmaceutical targeting ST14 for pancreatic cancer detection. A precursor ST14-06 was designed, and molecular docking was employed to preliminarily predict the binding mode. ST14-06 emerged as the preferred precursor with experimental inhibition assays confirming its high affinity for ST14 (IC50 = 1.06 ± 0.08 nM). 68Ga-ST14-06 was successfully synthesized with high radiochemical purity (RCP, >95 %) and molar activity (25-30 GBq/μmol) and was stable in saline and serum. In vitro studies demonstrated specific binding of the tracer to ST14-positive AsPC-1 cells compared to the blocking group (11.45 ± 0.12 % vs. 2.48 ± 0.34 %, P < 0.01). PET/CT imaging in AsPC-1 tumor-bearing mice confirmed ST14-specific uptake, which was reduced by co-administration of an excess blocking agent. Biodistribution studies revealed higher distribution in AsPC-1 tumors (0.99 ± 0.08 %ID/g) than in PANC-1 tumors (0.32 ± 0.02 %ID/g) and the blocking group (0.32 ± 0.04 %ID/g) at 1 h post-injection. Immunohistochemistry results showed that ST14 was highly positive in AsPC-1 tumors, but was negative in PANC-1 tumors. These preliminary findings suggest that 68Ga-ST14-06 has potential as a first-generation PET radiopharmaceutical for ST14-specific imaging, offering a promising tool for pancreatic cancer detection.
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Affiliation(s)
- Tukang Peng
- Department of Nuclear Medicine, Institute of Clinical Nuclear Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 210000, China
| | - Jun Wen
- Department of Nuclear Medicine, Institute of Clinical Nuclear Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 210000, China
| | - Gang Huang
- Department of Nuclear Medicine, Institute of Clinical Nuclear Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 210000, China; Shanghai Key Laboratory of Molecular Imaging, Shanghai University of Medicine and Health Sciences, Shanghai 210000, China.
| | - Haitao Zhao
- Department of Nuclear Medicine, Institute of Clinical Nuclear Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 210000, China.
| | - Jianjun Liu
- Department of Nuclear Medicine, Institute of Clinical Nuclear Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 210000, China.
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5
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Desgagné M, Désilets A, Ferková S, Lepage M, Perreault O, Joushomme A, Lemieux G, Guerrab W, Froehlich U, Comeau C, Sarret P, Leduc R, Boudreault PL. Rational In Silico Design of Selective TMPRSS6 Peptidomimetic Inhibitors via Exploitation of the S2 Subpocket. J Med Chem 2024; 67:12969-12983. [PMID: 39028865 PMCID: PMC11321340 DOI: 10.1021/acs.jmedchem.4c00922] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2024] [Revised: 06/17/2024] [Accepted: 06/21/2024] [Indexed: 07/21/2024]
Abstract
TMPRSS6 is a potential therapeutic target for the treatment of iron overload due to its role in regulating levels of hepcidin. Although potent TMPRSS6 inhibitors have been previously developed, their lack of specificity requires optimization to avoid potential side effects before pursuing preclinical development with in vivo models. Here, using computer-aided drug design based on a TMPRSS6 homology model, we reveal that the S2 position of TMPRSS6 offers a potential avenue to achieve selectivity against other members of the TTSP family. Accordingly, we synthesized novel peptidomimetic molecules containing lipophilic amino acids at the P2 position to exploit this unexplored pocket. This enabled us to identify TMPRSS6-selective small molecules with low nanomolar affinity. Finally, pharmacokinetic parameters were determined, and a compound was found to be potent in cellulo toward its primary target while retaining TTSP-subtype selectivity and showing no signs of alteration in in vitro TEER experiments.
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Affiliation(s)
- Michael Desgagné
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
| | - Antoine Désilets
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
| | - Sára Ferková
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
| | - Matthieu Lepage
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
| | - Olivier Perreault
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
| | - Alexandre Joushomme
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
| | - Gabriel Lemieux
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
| | - Walid Guerrab
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
| | - Ulrike Froehlich
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
| | - Christian Comeau
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
| | - Philippe Sarret
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
| | - Richard Leduc
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
| | - Pierre-Luc Boudreault
- Department of Pharmacology-Physiology,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC J1H 5N4, Canada
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6
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Koelink PJ, Gómez-Mellado VE, Duijst S, van Roest M, Meisner S, Ho-Mok KS, Frank S, Appelman BS, Bloemendaal LT, Vogel GF, van de Graaf SFJ, Bosma PJ, Oude Elferink RPJ, Wildenberg ME, Paulusma CC. The Phospholipid Flippase ATP8B1 is Involved in the Pathogenesis of Ulcerative Colitis via Establishment of Intestinal Barrier Function. J Crohns Colitis 2024; 18:1134-1146. [PMID: 38366839 PMCID: PMC11302967 DOI: 10.1093/ecco-jcc/jjae024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/07/2023] [Revised: 01/30/2024] [Accepted: 02/15/2024] [Indexed: 02/18/2024]
Abstract
AIMS Patients with mutations in ATP8B1 develop progressive familial intrahepatic cholestasis type 1 [PFIC1], a severe liver disease that requires life-saving liver transplantation. PFIC1 patients also present with gastrointestinal problems, including intestinal inflammation and diarrhoea, which are aggravated after liver transplantation. Here we investigate the intestinal function of ATP8B1 in relation to inflammatory bowel diseases. METHODS ATP8B1 expression was investigated in intestinal samples of patients with Crohn's disease [CD] or ulcerative colitis [UC] as well as in murine models of intestinal inflammation. Colitis was induced in ATP8B1-deficient mice with dextran sodium sulphate [DSS] and intestinal permeability was investigated. Epithelial barrier function was assessed in ATP8B1 knockdown Caco2-BBE cells. Co-immunoprecipitation experiments were performed in Caco2-BBE cells overexpressing ATP8B1-eGFP. Expression and localization of ATP8B1 and tight junction proteins were investigated in cells and in biopsies of UC and PFIC1 patients. RESULTS ATP8B1 expression was decreased in UC and DSS-treated mice, and was associated with a decreased tight junctional pathway transcriptional programme. ATP8B1-deficient mice were extremely sensitive to DSS-induced colitis, as evidenced by increased intestinal barrier leakage. ATP8B1 knockdown cells showed delayed barrier establishment that affected Claudin-4 [CLDN4] levels and localization. CLDN4 immunohistochemistry showed a tight junctional staining in control tissue, whereas in UC and intestinal PFIC1 samples, CLDN4 was not properly localized. CONCLUSION ATP8B1 is important in the establishment of the intestinal barrier. Downregulation of ATP8B1 levels in UC, and subsequent altered localization of tight junctional proteins, including CLDN4, might therefore be an important mechanism in UC pathophysiology.
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Affiliation(s)
- Pim J Koelink
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
- Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands
| | - Valentina E Gómez-Mellado
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
- Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands
| | - Suzanne Duijst
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
- Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands
| | - Manon van Roest
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
- Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands
| | - Sander Meisner
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
- Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands
| | - Kam S Ho-Mok
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
- Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands
| | - Sabrina Frank
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
| | - Babette S Appelman
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
| | - Lysbeth ten Bloemendaal
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
- Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands
| | - Georg F Vogel
- Department of Paediatrics I, Medical University of Innsbruck, 6020 Innsbruck, Austria
- Institute of Cell Biology, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria
| | - Stan F J van de Graaf
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
- Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands
- Department of Gastroenterology and Hepatology, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands
| | - Piter J Bosma
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
- Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands
- Department of Gastroenterology and Hepatology, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands
| | - Ronald P J Oude Elferink
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
- Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands
- Department of Gastroenterology and Hepatology, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands
| | - Manon E Wildenberg
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
- Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands
- Department of Gastroenterology and Hepatology, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands
| | - Coen C Paulusma
- Amsterdam University Medical Centers, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands
- Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam, The Netherlands
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7
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Higashi T, Saito AC, Chiba H. Damage control of epithelial barrier function in dynamic environments. Eur J Cell Biol 2024; 103:151410. [PMID: 38579602 DOI: 10.1016/j.ejcb.2024.151410] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2023] [Revised: 03/27/2024] [Accepted: 03/30/2024] [Indexed: 04/07/2024] Open
Abstract
Epithelial tissues cover the surfaces and lumens of the internal organs of multicellular animals and crucially contribute to internal environment homeostasis by delineating distinct compartments within the body. This vital role is known as epithelial barrier function. Epithelial cells are arranged like cobblestones and intricately bind together to form an epithelial sheet that upholds this barrier function. Central to the restriction of solute and fluid diffusion through intercellular spaces are occluding junctions, tight junctions in vertebrates and septate junctions in invertebrates. As part of epithelial tissues, cells undergo constant renewal, with older cells being replaced by new ones. Simultaneously, the epithelial tissue undergoes relative rearrangement, elongating, and shifting directionally as a whole. The movement or shape changes within the epithelial sheet necessitate significant deformation and reconnection of occluding junctions. Recent advancements have shed light on the intricate mechanisms through which epithelial cells sustain their barrier function in dynamic environments. This review aims to introduce these noteworthy findings and discuss some of the questions that remain unanswered.
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Affiliation(s)
- Tomohito Higashi
- Department of Basic Pathology, Fukushima Medical University, Fukushima 960-1295, Japan.
| | - Akira C Saito
- Department of Basic Pathology, Fukushima Medical University, Fukushima 960-1295, Japan
| | - Hideki Chiba
- Department of Basic Pathology, Fukushima Medical University, Fukushima 960-1295, Japan
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8
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Zeng N, Wu F, Lu J, Li X, Lin S, Zhou L, Wang Z, Wu G, Huang Q, Zheng D, Gao J, Wu S, Chen X, Chen M, Meng F, Shang H, He Y, Chen P, Wei H, Li Z, Zhou H. High-fat diet impairs gut barrier through intestinal microbiota-derived reactive oxygen species. SCIENCE CHINA. LIFE SCIENCES 2024; 67:879-891. [PMID: 37202543 DOI: 10.1007/s11427-022-2283-4] [Citation(s) in RCA: 19] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/25/2022] [Accepted: 02/19/2023] [Indexed: 05/20/2023]
Abstract
Gut barrier disruption is a key event in bridging gut microbiota dysbiosis and high-fat diet (HFD)-associated metabolic disorders. However, the underlying mechanism remains elusive. In the present study, by comparing HFD- and normal diet (ND)-treated mice, we found that the HFD instantly altered the composition of the gut microbiota and subsequently damaged the integrity of the gut barrier. Metagenomic sequencing revealed that the HFD upregulates gut microbial functions related to redox reactions, as confirmed by the increased reactive oxygen species (ROS) levels in fecal microbiota incubation in vitro and in the lumen, which were detected using in vivo fluorescence imaging. This microbial ROS-producing capability induced by HFD can be transferred through fecal microbiota transplantation (FMT) into germ-free (GF) mice, downregulating the gut barrier tight junctions. Similarly, mono-colonizing GF mice with an Enterococcus strain excelled in ROS production, damaged the gut barrier, induced mitochondrial malfunction and apoptosis of the intestinal epithelial cells, and exacerbated fatty liver, compared with other low-ROS-producing Enterococcus strains. Oral administration of recombinant high-stability-superoxide dismutase (SOD) significantly reduced intestinal ROS, protected the gut barrier, and improved fatty liver against the HFD. In conclusion, our study suggests that extracellular ROS derived from gut microbiota play a pivotal role in HFD-induced gut barrier disruption and is a potential therapeutic target for HFD-associated metabolic diseases.
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Affiliation(s)
- Nianyi Zeng
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Fan Wu
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Junqi Lu
- Department of Environmental Health, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, 510515, China
| | - Xiang Li
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Shaomei Lin
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Lang Zhou
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Zhongwei Wang
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Guangyan Wu
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Qingfa Huang
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Daowen Zheng
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Jie Gao
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Shan Wu
- Department of Environmental Health, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, 510515, China
| | - Xiaojiao Chen
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Muxuan Chen
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Fanguo Meng
- Redox Medical Center for Public Health, Soochow University, Suzhou, 215301, China
| | - Haitao Shang
- Precision Medicine Institute, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China
| | - Yan He
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China
| | - Peng Chen
- Department of Pathophysiology, Guangdong Provincial Key Laboratory of Proteomics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China
| | - Hong Wei
- Precision Medicine Institute, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China.
| | - Zhuang Li
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China.
| | - Hongwei Zhou
- Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, China.
- Department of Environmental Health, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, 510515, China.
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9
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Ye F, Yuan Z, Tang Y, Li J, Liu X, Sun X, Chen S, Ye X, Zeng Z, Zhang XK, Zhou H. Endocytic activation and exosomal secretion of matriptase stimulate the second wave of EGF signaling to promote skin and breast cancer invasion. Cell Rep 2024; 43:114002. [PMID: 38547126 DOI: 10.1016/j.celrep.2024.114002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2023] [Revised: 10/26/2023] [Accepted: 03/11/2024] [Indexed: 04/28/2024] Open
Abstract
The dysfunction of matriptase, a membrane-anchored protease, is highly related to the progression of skin and breast cancers. Epidermal growth factor (EGF)-induced matriptase activation and cancer invasion are known but with obscure mechanisms. Here, we demonstrate a vesicular-trafficking-mediated interplay between matriptase and EGF signaling in cancer promotion. We found that EGF induces matriptase to undergo endocytosis together with the EGF receptor, followed by acid-induced activation in endosomes. Activated matriptase is then secreted extracellularly on exosomes to catalyze hepatocyte growth factor precursor (pro-HGF) cleavage, resulting in autocrine HGF/c-Met signaling. Matriptase-induced HGF/c-Met signaling represents the second signal wave of EGF, which promotes cancer cell scattering, migration, and invasion. These findings demonstrate a role of vesicular trafficking in efficient activation and secretion of membrane matriptase and a reciprocal regulation of matriptase and EGF signaling in cancer promotion, providing insights into the physiological functions of vesicular trafficking and the molecular pathological mechanisms of skin and breast cancers.
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Affiliation(s)
- Fang Ye
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian 361102, China
| | - Zhikang Yuan
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian 361102, China
| | - Ying Tang
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian 361102, China
| | - Jiamei Li
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian 361102, China
| | - Xingxing Liu
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian 361102, China
| | - Xuedi Sun
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian 361102, China
| | - Shuang Chen
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian 361102, China
| | - Xiaohong Ye
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian 361102, China; High Throughput Drug Screening Platform, Xiamen University, Xiamen, Fujian 361102, China
| | - Zhiping Zeng
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian 361102, China; High Throughput Drug Screening Platform, Xiamen University, Xiamen, Fujian 361102, China
| | - Xiao-Kun Zhang
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian 361102, China; High Throughput Drug Screening Platform, Xiamen University, Xiamen, Fujian 361102, China
| | - Hu Zhou
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian 361102, China; High Throughput Drug Screening Platform, Xiamen University, Xiamen, Fujian 361102, China.
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10
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Rickman OJ, Guignard E, Chabanon T, Bertoldi G, Auberson M, Hummler E. Tmprss2 maintains epithelial barrier integrity and transepithelial sodium transport. Life Sci Alliance 2024; 7:e202302304. [PMID: 38171596 PMCID: PMC10765116 DOI: 10.26508/lsa.202302304] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2023] [Revised: 12/21/2023] [Accepted: 12/22/2023] [Indexed: 01/05/2024] Open
Abstract
The mouse cortical collecting duct cell line presents a tight epithelium with regulated ion and water transport. The epithelial sodium channel (ENaC) is localized in the apical membrane and constitutes the rate-limiting step for sodium entry, thereby enabling transepithelial transport of sodium ions. The membrane-bound serine protease Tmprss2 is co-expressed with the alpha subunit of ENaC. αENaC gene expression followed the Tmprss2 expression, and the absence of Tmprss2 resulted not only in down-regulation of αENaC gene and protein expression but also in abolished transepithelial sodium transport. In addition, RNA-sequencing analyses unveiled drastic down-regulation of the membrane-bound protease CAP3/St14, the epithelial adhesion molecule EpCAM, and the tight junction proteins claudin-7 and claudin-3 as also confirmed by immunohistochemistry. In summary, our data clearly demonstrate a dual role of Tmprss2 in maintaining not only ENaC-mediated transepithelial but also EpCAM/claudin-7-mediated paracellular barrier; the tight epithelium of the mouse renal mCCD cells becomes leaky. Our working model proposes that Tmprss2 acts via CAP3/St14 on EpCAM/claudin-7 tight junction complexes and through regulating transcription of αENaC on ENaC-mediated sodium transport.
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Affiliation(s)
- Olivia J Rickman
- Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland
| | - Emma Guignard
- Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland
| | - Thomas Chabanon
- Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland
| | - Giovanni Bertoldi
- Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland
| | - Muriel Auberson
- Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland
| | - Edith Hummler
- Department of Biomedical Sciences, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland
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11
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Wen W, Xu Y, Qian W, Huang L, Gong J, Li Y, Zhu W, Guo Z. PUFAs add fuel to Crohn's disease-associated AIEC-induced enteritis by exacerbating intestinal epithelial lipid peroxidation. Gut Microbes 2023; 15:2265578. [PMID: 37800577 PMCID: PMC10561586 DOI: 10.1080/19490976.2023.2265578] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/06/2023] [Accepted: 09/27/2023] [Indexed: 10/07/2023] Open
Abstract
Polyunsaturated fatty acids (PUFAs) have been shown to exacerbate Crohn's disease (CD) by promoting lipid peroxidation (LPO) of intestinal epithelial cells (IECs). Dysbiosis of the gut microbiota may play a crucial role in this process. CD patients often exhibit an increased abundance of Escherichia coli (E. coli) in the gut, and the colonization of adherent-invasive E. coli (AIEC) is implicated in the initiation of intestinal inflammation in CD. However, the impact of AIEC on LPO remains unclear. In this study, we observed that AIEC colonization in the terminal ileum of CD patients was associated with decreased levels of glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH) in the intestinal epithelium, along with elevated levels of 4-Hydroxynonenal (4-HNE). In vitro experiments demonstrated that AIEC infection reduced the levels of GPX4 and FTH, increased LPO, and induced ferroptosis in IECs. Furthermore, arachidonic acid (AA) and docosahexaenoic acid (DHA) supplementation in AIEC-infected IECs significantly aggravated LPO and ferroptosis. However, overexpression of GPX4 rescued AIEC-induced LPO and ferroptosis in IECs. Our results further confirmed that AIEC with AA supplementation, associated with excessive LPO and cell death in IECs, worsened colitis in the DSS mouse model and induced enteritis in the antibiotic cocktail pre-treatment mouse model in vivo. Moreover, treatment with ferrostatin-1, a ferroptosis inhibitor, alleviated AIEC with AA supplementation-induced enteritis in mice, accompanied by reduced LPO and cell death in IECs. Our findings suggest that AIEC, in combination with PUFA supplementation, can induce and exacerbate intestinal inflammation, primarily through increased LPO and ferroptosis in IECs.
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Affiliation(s)
- Weiwei Wen
- Department of General Surgery, Jinling Hospital, Medical School of Southeast University, Nanjing, China
- Department of General Surgery, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
| | - Yihan Xu
- Department of General Surgery, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
| | - Wenwei Qian
- Department of General Surgery, Jinling Hospital, Medical School of Southeast University, Nanjing, China
- Department of General Surgery, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
| | - Liangyu Huang
- Department of Colorectal Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Jianfeng Gong
- Department of General Surgery, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
| | - Yi Li
- Department of General Surgery, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
| | - Weiming Zhu
- Department of General Surgery, Jinling Hospital, Medical School of Southeast University, Nanjing, China
- Department of General Surgery, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
| | - Zhen Guo
- Department of General Surgery, Jinling Hospital, Medical School of Southeast University, Nanjing, China
- Department of General Surgery, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China
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12
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Kim HW, Lee SY, Hur SJ, Kil DY, Kim JH. Effects of functional nutrients on chicken intestinal epithelial cells induced with oxidative stress. JOURNAL OF ANIMAL SCIENCE AND TECHNOLOGY 2023; 65:1040-1052. [PMID: 37969347 PMCID: PMC10640939 DOI: 10.5187/jast.2023.e22] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/18/2023] [Revised: 02/23/2023] [Accepted: 02/24/2023] [Indexed: 11/17/2023]
Abstract
The objective of this study was to investigate the protective effects of functional nutrients including various functional amino acids, vitamins, and minerals on chicken intestinal epithelial cells (cIECs) treated with oxidative stress. The cIECs were isolated from specific pathogen free eggs. Cells were exposed to 0 mM supplement (control), 20 mM threonine (Thr), 0.4 mM tryptophan (Trp), 1 mM glycine (Gly), 10 μM vitamin C (VC), 40 μM vitamin E (VE), 5 μM vitamin A (VA), 34 μM chromium (Cr), 0.42 μM selenium (Se), and 50 μM zinc (Zn) for 24 h with 6 replicates for each treatment. After 24 h, cells were further incubated with fresh culture medium (positive control, PC) or 1 mM H2O2 with different supplements (negative control, NC and each treatment). Oxidative stress was measured by cell proliferation, whereas tight junction barrier function was analyzed by fluorescein isothiocyanate (FITC)-dextran permeability and transepithelial electrical resistance (TEER). Results indicated that cell viability and TEER values were less (p < 0.05) in NC treatments with oxidative stress than in PC treatments. In addition, FITC-dextran values were greater (p < 0.05) in NC treatments with oxidative stress than in PC treatments. The supplementations of Thr, Trp, Gly, VC, and VE in cells treated with H2O2 showed greater (p < 0.05) cell viability than the supplementation of VA, Cr, Se, and Zn. The supplementations of Trp, Gly, VC, and Se in cells treated with H2O2 showed the least (p < 0.05) cellular permeability. In addition, the supplementation of Thr, VE, VA, Cr, and Zn in cells treated with H2O2 decreased (p < 0.05) cellular permeability. At 48 h, the supplementations of Thr, Trp, and Gly in cells treated with H2O2 showed the greatest (p < 0.05) TEER values among all treatments, and the supplementations of VC and VE in cells treated with H2O2 showed greater (p < 0.05) TEER values than the supplementations of VA, Cr, Se, and Zn in cells treated with H2O2. In conclusion, Thr, Trp, Gly, and VC supplements were effective in improving cell viability and intestinal barrier function of cIECs exposed to oxidative stress.
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Affiliation(s)
- Hyun Woo Kim
- Department of Animal Science and
Technology, Chung-Ang University, Anseong 17546, Korea
| | - Seung Yun Lee
- Department of Animal Science (BK21 Four),
Institute of Agriculture Life Science, Gyeongsang National
University, Jinju 52725, Korea
| | - Sun Jin Hur
- Department of Animal Science and
Technology, Chung-Ang University, Anseong 17546, Korea
| | - Dong Yong Kil
- Department of Animal Science and
Technology, Chung-Ang University, Anseong 17546, Korea
| | - Jong Hyuk Kim
- Department of Animal Science, Chungbuk
National University, Cheongju 28644, Korea
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13
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Wu Q, Li S, Zhang X, Dong N. Type II Transmembrane Serine Proteases as Modulators in Adipose Tissue Phenotype and Function. Biomedicines 2023; 11:1794. [PMID: 37509434 PMCID: PMC10376093 DOI: 10.3390/biomedicines11071794] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2023] [Revised: 06/21/2023] [Accepted: 06/22/2023] [Indexed: 07/30/2023] Open
Abstract
Adipose tissue is a crucial organ in energy metabolism and thermoregulation. Adipose tissue phenotype is controlled by various signaling mechanisms under pathophysiological conditions. Type II transmembrane serine proteases (TTSPs) are a group of trypsin-like enzymes anchoring on the cell surface. These proteases act in diverse tissues to regulate physiological processes, such as food digestion, salt-water balance, iron metabolism, epithelial integrity, and auditory nerve development. More recently, several members of the TTSP family, namely, hepsin, matriptase-2, and corin, have been shown to play a role in regulating lipid metabolism, adipose tissue phenotype, and thermogenesis, via direct growth factor activation or indirect hormonal mechanisms. In mice, hepsin deficiency increases adipose browning and protects from high-fat diet-induced hyperglycemia, hyperlipidemia, and obesity. Similarly, matriptase-2 deficiency increases fat lipolysis and reduces obesity and hepatic steatosis in high-fat diet-fed mice. In contrast, corin deficiency increases white adipose weights and cell sizes, suppresses adipocyte browning and thermogenic responses, and causes cold intolerance in mice. These findings highlight an important role of TTSPs in modifying cellular phenotype and function in adipose tissue. In this review, we provide a brief description about TTSPs and discuss recent findings regarding the role of hepsin, matriptase-2, and corin in regulating adipose tissue phenotype, energy metabolism, and thermogenic responses.
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Affiliation(s)
- Qingyu Wu
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Prevention, Soochow University, Suzhou 215123, China
| | - Shuo Li
- Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
| | - Xianrui Zhang
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Prevention, Soochow University, Suzhou 215123, China
| | - Ningzheng Dong
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Prevention, Soochow University, Suzhou 215123, China
- NHC Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, Soochow University, Suzhou 215006, China
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14
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Haroun E, Kumar PA, Saba L, Kassab J, Ghimire K, Dutta D, Lim SH. Intestinal barrier functions in hematologic and oncologic diseases. J Transl Med 2023; 21:233. [PMID: 37004099 PMCID: PMC10064590 DOI: 10.1186/s12967-023-04091-w] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2023] [Accepted: 03/26/2023] [Indexed: 04/03/2023] Open
Abstract
The intestinal barrier is a complex structure that not only regulates the influx of luminal contents into the systemic circulation but is also involved in immune, microbial, and metabolic homeostasis. Evidence implicating disruption in intestinal barrier functions in the development of many systemic diseases, ranging from non-alcoholic steatohepatitis to autism, or systemic complications of intestinal disorders has increased rapidly in recent years, raising the possibility of the intestinal barrier as a potential target for therapeutic intervention to alter the course and mitigate the complications associated with these diseases. In addition to the disease process being associated with a breach in the intestinal barrier functions, patients with hematologic and oncologic diseases are particularly at high risks for the development of increased intestinal permeability, due to the frequent use of broad-spectrum antibiotics and chemoradiation. They also face a distinct challenge of being intermittently severely neutropenic due to treatment of the underlying conditions. In this review, we will discuss how hematologic and oncologic diseases are associated with disruption in the intestinal barrier and highlight the complications associated with an increase in the intestinal permeability. We will explore methods to modulate the complication. To provide a background for our discussion, we will first examine the structure and appraise the methods of evaluation of the intestinal barrier.
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Affiliation(s)
- Elio Haroun
- Division of Hematology and Oncology, State University of New York Upstate Medical University, SUNY Upstate Medical University, 750 E Adams, Syracuse, NY, 13210, USA
| | - Prashanth Ashok Kumar
- Division of Hematology and Oncology, State University of New York Upstate Medical University, SUNY Upstate Medical University, 750 E Adams, Syracuse, NY, 13210, USA
| | - Ludovic Saba
- Department of Medicine, Saint-Joseph University of Beirut, Beirut, Lebanon
| | - Joseph Kassab
- Department of Medicine, Saint-Joseph University of Beirut, Beirut, Lebanon
| | - Krishna Ghimire
- Division of Hematology and Oncology, State University of New York Upstate Medical University, SUNY Upstate Medical University, 750 E Adams, Syracuse, NY, 13210, USA
| | - Dibyendu Dutta
- Division of Hematology and Oncology, State University of New York Upstate Medical University, SUNY Upstate Medical University, 750 E Adams, Syracuse, NY, 13210, USA.
| | - Seah H Lim
- Division of Hematology and Oncology, State University of New York Upstate Medical University, SUNY Upstate Medical University, 750 E Adams, Syracuse, NY, 13210, USA.
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15
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Salari A, Zhou K, Nikolovska K, Seidler U, Amiri M. Human Colonoid-Myofibroblast Coculture for Study of Apical Na +/H + Exchangers of the Lower Cryptal Neck Region. Int J Mol Sci 2023; 24:ijms24054266. [PMID: 36901695 PMCID: PMC10001859 DOI: 10.3390/ijms24054266] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Revised: 02/17/2023] [Accepted: 02/18/2023] [Indexed: 02/25/2023] Open
Abstract
Cation and anion transport in the colonocyte apical membrane is highly spatially organized along the cryptal axis. Because of lack of experimental accessibility, information about the functionality of ion transporters in the colonocyte apical membrane in the lower part of the crypt is scarce. The aim of this study was to establish an in vitro model of the colonic lower crypt compartment, which expresses the transit amplifying/progenitor (TA/PE) cells, with accessibility of the apical membrane for functional study of lower crypt-expressed Na+/H+ exchangers (NHEs). Colonic crypts and myofibroblasts were isolated from human transverse colonic biopsies, expanded as three-dimensional (3D) colonoids and myofibroblast monolayers, and characterized. Filter-grown colonic myofibroblast-colonic epithelial cell (CM-CE) cocultures (myofibroblasts on the bottom of the transwell and colonocytes on the filter) were established. The expression pattern for ion transport/junctional/stem cell markers of the CM-CE monolayers was compared with that of nondifferentiated (EM) and differentiated (DM) colonoid monolayers. Fluorometric pHi measurements were performed to characterize apical NHEs. CM-CE cocultures displayed a rapid increase in transepithelial electrical resistance (TEER), paralleled by downregulation of claudin-2. They maintained proliferative activity and an expression pattern resembling TA/PE cells. The CM-CE monolayers displayed high apical Na+/H+ exchange activity, mediated to >80% by NHE2. Human colonoid-myofibroblast cocultures allow the study of ion transporters that are expressed in the apical membrane of the nondifferentiated colonocytes of the cryptal neck region. The NHE2 isoform is the predominant apical Na+/H+ exchanger in this epithelial compartment.
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Affiliation(s)
- Azam Salari
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30625 Hannover, Germany
| | - Kunyan Zhou
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30625 Hannover, Germany
- Department of Thyroid Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310027, China
| | - Katerina Nikolovska
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30625 Hannover, Germany
| | - Ursula Seidler
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30625 Hannover, Germany
- Correspondence: (U.S.); (M.A.); Tel.: +49-511-532-9427 (U.S.); Fax: +49-511-532-8428 (U.S.)
| | - Mahdi Amiri
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30625 Hannover, Germany
- Correspondence: (U.S.); (M.A.); Tel.: +49-511-532-9427 (U.S.); Fax: +49-511-532-8428 (U.S.)
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16
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Xu Q, Zhao J, Jian H, Ye J, Gong M, Zou X, Dong X. Linoleic acid ameliorates intestinal mucosal barrier injury in early weaned pigeon squabs (Columba livia). J Anim Sci 2023; 101:skad125. [PMID: 37186172 PMCID: PMC10195202 DOI: 10.1093/jas/skad125] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Accepted: 04/22/2023] [Indexed: 05/17/2023] Open
Abstract
The study aimed to investigate whether linoleic acid could improve the intestinal barrier function of squabs under weaning stress conditions. Totally 320 7-d-old weaned squabs were randomly divided into four treatment groups, including control group (CON), 0.7% linoleic acid addition group (LA007), 1.4% linoleic acid addition group (LA014) and 2.1% linoleic acid addition group (LA021). At 21 d, eight squabs were randomly selected from each treatment group for sampling and determination. The results showed that adding linoleic acid could improve (P < 0.05) the body weight of weaned squabs, and LA014 had the best effect. With the increase of linoleic acid dosage, villi height and villi area increased linearly or quadratically (P < 0.05), and reached the maximum in LA021 or LA014, respectively. The linoleic acid supplementation could improve the intestinal tight junction of weaned squabs, and the LA014 was the most significant (P < 0.05). With the linoleic acid increasing, the levels of intestinal IL-6 and TNF-α decreased linearly (P < 0.05), while intestinal IL-10 increased quadratically (P < 0.05) and reached the maximum in LA014. Serum endotoxin and diamine oxidase levels decreased linearly (P < 0.05) and reached the lowest level in LA014. The ultrastructure of villi revealed that the length of ileal microvilli in LA014 was significantly increased (P < 0.05) and the microvilli became dense, and the mitochondria in epithelial cells returned to normal state. Further exploring the mechanism of linoleic acid alleviating intestinal injury caused by weaning stress in squabs, it was found that linoleic acid down-regulated (P < 0.05) the relative protein expression of TLR4, MyD88, phosphorylated JNK, and phosphorylated p38, reducing secretion of pro-inflammatory factors IL-6 and TNF-α. This study indicated that linoleic acid could alleviate intestinal barrier injury of early weaned squabs by down-regulating TLR4-MyD88-JNK/p38-IL6/TNF-α pathway.
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Affiliation(s)
- Qianqian Xu
- Key Laboratory of Characteristic Agricultural Product Quality and Hazardous Substance Control Technology of Zhejiang Province, Institute of Food Nutrition and Quality Safety, College of Life Science, China Jiliang University, Hangzhou 310018, China
| | - Jin Zhao
- Key Laboratory of Characteristic Agricultural Product Quality and Hazardous Substance Control Technology of Zhejiang Province, Institute of Food Nutrition and Quality Safety, College of Life Science, China Jiliang University, Hangzhou 310018, China
| | - Huafeng Jian
- Key laboratory for Molecular Animal Nutrition of Ministry of Education, Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Feed Science Institute, College of Animal Science, Zhejiang University (Zijingang Campus), Hangzhou 310058, China
| | - Jiangcheng Ye
- Key Laboratory of Characteristic Agricultural Product Quality and Hazardous Substance Control Technology of Zhejiang Province, Institute of Food Nutrition and Quality Safety, College of Life Science, China Jiliang University, Hangzhou 310018, China
| | - Mingxiu Gong
- Key Laboratory of Characteristic Agricultural Product Quality and Hazardous Substance Control Technology of Zhejiang Province, Institute of Food Nutrition and Quality Safety, College of Life Science, China Jiliang University, Hangzhou 310018, China
| | - Xiaoting Zou
- Key laboratory for Molecular Animal Nutrition of Ministry of Education, Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Feed Science Institute, College of Animal Science, Zhejiang University (Zijingang Campus), Hangzhou 310058, China
| | - Xinyang Dong
- Key laboratory for Molecular Animal Nutrition of Ministry of Education, Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Feed Science Institute, College of Animal Science, Zhejiang University (Zijingang Campus), Hangzhou 310058, China
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17
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Rodrat M, Wongdee K, Teerapornpuntakit J, Thongbunchoo J, Tanramluk D, Aeimlapa R, Thammayon N, Thonapan N, Wattano P, Charoenphandhu N. Vasoactive intestinal peptide and cystic fibrosis transmembrane conductance regulator contribute to the transepithelial calcium transport across intestinal epithelium-like Caco-2 monolayer. PLoS One 2022; 17:e0277096. [PMID: 36399482 PMCID: PMC9674163 DOI: 10.1371/journal.pone.0277096] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2022] [Accepted: 10/20/2022] [Indexed: 11/19/2022] Open
Abstract
Vasoactive intestinal peptide (VIP) as a neurocrine factor released by enteric neurons has been postulated to participate in the regulation of transcellular active calcium transport across intestinal epithelium, but the preceding evidence is scant and inconclusive. Herein, transepithelial calcium flux and epithelial electrical parameters were determined by Ussing chamber technique with radioactive tracer in the intestinal epithelium-like Caco-2 monolayer grown on Snapwell. After 3-day culture, Caco-2 cells expressed mRNA of calcium transporters, i.e., TRPV6, calbindin-D9k, PMCA1b and NCX1, and exhibited transepithelial resistance of ~200 Ω cm2, a characteristic of leaky epithelium similar to the small intestine. VIP receptor agonist was able to enhance transcellular calcium flux, whereas VIP receptor antagonist totally abolished calcium fluxes induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Since the intestinal cystic fibrosis transmembrane conductance regulator (CFTR) could be activated by VIP and calciotropic hormones, particularly parathyroid hormone, we sought to determine whether CFTR also contributed to the 1,25(OH)2D3-induced calcium transport. A selective CFTR inhibitor (20-200 μM CFTRinh-172) appeared to diminish calcium fluxes as well as transepithelial potential difference and short-circuit current, both of which indicated a decrease in electrogenic ion transport. On the other hand, 50 μM genistein-a molecule that could rapidly activate CFTR-was found to increase calcium transport. Our in silico molecular docking analysis confirmed direct binding of CFTRinh-172 and genistein to CFTR channels. In conclusion, VIP and CFTR apparently contributed to the intestinal calcium transport, especially in the presence of 1,25(OH)2D3, thereby supporting the existence of the neurocrine control of intestinal calcium absorption.
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Affiliation(s)
- Mayuree Rodrat
- Faculty of Science, Center of Calcium and Bone Research (COCAB), Mahidol University, Bangkok, Thailand
- Faculty of Science, Department of Physiology, Mahidol University, Bangkok, Thailand
- Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand
- Center of Research and Development for Biomedical Instrumentation, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand
| | - Kannikar Wongdee
- Faculty of Science, Center of Calcium and Bone Research (COCAB), Mahidol University, Bangkok, Thailand
- Faculty of Allied Health Sciences, Burapha University, Chonburi, Thailand
| | - Jarinthorn Teerapornpuntakit
- Faculty of Science, Center of Calcium and Bone Research (COCAB), Mahidol University, Bangkok, Thailand
- Department of Physiology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand
| | - Jirawan Thongbunchoo
- Faculty of Science, Center of Calcium and Bone Research (COCAB), Mahidol University, Bangkok, Thailand
- Faculty of Science, Department of Physiology, Mahidol University, Bangkok, Thailand
| | - Duangrudee Tanramluk
- Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand
- Integrative Computational BioScience (ICBS) Center, Mahidol University, Nakhon Pathom, Thailand
| | - Ratchaneevan Aeimlapa
- Faculty of Science, Center of Calcium and Bone Research (COCAB), Mahidol University, Bangkok, Thailand
- Faculty of Science, Department of Physiology, Mahidol University, Bangkok, Thailand
| | - Nithipak Thammayon
- Faculty of Science, Center of Calcium and Bone Research (COCAB), Mahidol University, Bangkok, Thailand
- Faculty of Science, Graduate Program in Molecular Medicine, Mahidol University, Bangkok, Thailand
| | - Natchayaporn Thonapan
- Faculty of Science, Center of Calcium and Bone Research (COCAB), Mahidol University, Bangkok, Thailand
- Faculty of Science, Graduate Program in Molecular Medicine, Mahidol University, Bangkok, Thailand
| | - Pathnaree Wattano
- Faculty of Science, Center of Calcium and Bone Research (COCAB), Mahidol University, Bangkok, Thailand
| | - Narattaphol Charoenphandhu
- Faculty of Science, Center of Calcium and Bone Research (COCAB), Mahidol University, Bangkok, Thailand
- Faculty of Science, Department of Physiology, Mahidol University, Bangkok, Thailand
- Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand
- The Academy of Science, The Royal Society of Thailand, Bangkok, Thailand
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18
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Higashi T, Saito AC, Fukazawa Y, Furuse M, Higashi AY, Ono M, Chiba H. EpCAM proteolysis and release of complexed claudin-7 repair and maintain the tight junction barrier. J Cell Biol 2022; 222:213688. [PMID: 36378161 PMCID: PMC9671161 DOI: 10.1083/jcb.202204079] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2022] [Revised: 09/09/2022] [Accepted: 09/28/2022] [Indexed: 11/16/2022] Open
Abstract
TJs maintain the epithelial barrier by regulating paracellular permeability. Since TJs are under dynamically fluctuating intercellular tension, cells must continuously survey and repair any damage. However, the underlying mechanisms allowing cells to sense TJ damage and repair the barrier are not yet fully understood. Here, we showed that proteinases play an important role in the maintenance of the epithelial barrier. At TJ break sites, EpCAM-claudin-7 complexes on the basolateral membrane become accessible to apical membrane-anchored serine proteinases (MASPs) and the MASPs cleave EpCAM. Biochemical data and imaging analysis suggest that claudin-7 released from EpCAM contributes to the rapid repair of damaged TJs. Knockout (KO) of MASPs drastically reduced barrier function and live-imaging of TJ permeability showed that MASPs-KO cells exhibited increased size, duration, and frequency of leaks. Together, our results reveal a novel mechanism of TJ maintenance through the localized proteolysis of EpCAM at TJ leaks, and provide a better understanding of the dynamic regulation of epithelial permeability.
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Affiliation(s)
- Tomohito Higashi
- Department of Basic Pathology, Fukushima Medical University, Fukushima, Japan,Correspondence to Tomohito Higashi:
| | - Akira C. Saito
- Department of Basic Pathology, Fukushima Medical University, Fukushima, Japan
| | - Yugo Fukazawa
- Division of Brain Structure and Function, Faculty of Medical Science, Life Science Innovation Center, University of Fukui, Fukui, Japan
| | - Mikio Furuse
- Division of Cell Structure, National Institute for Physiological Sciences, Okazaki, Aichi, Japan,Department of Physiological Sciences, School of Life Science, SOKENDAI (Graduate University for Advanced Studies), Okazaki, Aichi, Japan
| | - Atsuko Y. Higashi
- Department of Basic Pathology, Fukushima Medical University, Fukushima, Japan
| | - Masahiro Ono
- Department of Basic Pathology, Fukushima Medical University, Fukushima, Japan
| | - Hideki Chiba
- Department of Basic Pathology, Fukushima Medical University, Fukushima, Japan
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19
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Anand D, Hummler E, Rickman OJ. ENaC activation by proteases. Acta Physiol (Oxf) 2022; 235:e13811. [PMID: 35276025 PMCID: PMC9540061 DOI: 10.1111/apha.13811] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2021] [Revised: 03/04/2022] [Accepted: 03/08/2022] [Indexed: 12/13/2022]
Abstract
Proteases are fundamental for a plethora of biological processes, including signalling and tissue remodelling, and dysregulated proteolytic activity can result in pathogenesis. In this review, we focus on a subclass of membrane‐bound and soluble proteases that are defined as channel‐activating proteases (CAPs), since they induce Na+ ion transport through an autocrine mechanism when co‐expressed with the highly amiloride‐sensitive epithelial sodium channel (ENaC) in Xenopus oocytes. These experiments first identified CAP1 (channel‐activating protease 1, prostasin) followed by CAP2 (channel‐activating protease 2, TMPRSS4) and CAP3 (channel‐activating protease 3, matriptase) as in vitro mediators of ENaC current. Since then, more serine‐, cysteine‐ and metalloproteases were confirmed as in vitro CAPs that potentially cleave and regulate ENaC, and thus this nomenclature was not further followed, but is accepted as functional term or alias. The precise mechanism of ENaC modulation by proteases has not been fully elucidated. Studies in organ‐specific protease knockout models revealed evidence for their role in increasing ENaC activity, although the proteases responsible for ENaC activation are yet to be identified. We summarize recent findings in animal models of these CAPs with respect to their implication in ENaC activation. We discuss the consequences of dysregulated CAPs underlying epithelial phenotypes in pathophysiological conditions, and the role of selected protease inhibitors. We believe that these proteases may present interesting therapeutic targets for diseases with aberrant sodium homoeostasis.
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Affiliation(s)
- Deepika Anand
- Department of Biomedical Sciences University of Lausanne Lausanne Switzerland
- National Center of Competence in Research, Kidney.CH Lausanne Switzerland
| | - Edith Hummler
- Department of Biomedical Sciences University of Lausanne Lausanne Switzerland
- National Center of Competence in Research, Kidney.CH Lausanne Switzerland
| | - Olivia J. Rickman
- Department of Biomedical Sciences University of Lausanne Lausanne Switzerland
- National Center of Competence in Research, Kidney.CH Lausanne Switzerland
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20
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Yuan M, Chen X, Su T, Zhou Y, Sun X. Supplementation of Kiwifruit Polyphenol Extract Attenuates High Fat Diet Induced Intestinal Barrier Damage and Inflammation via Reshaping Gut Microbiome. Front Nutr 2021; 8:702157. [PMID: 34527688 PMCID: PMC8435571 DOI: 10.3389/fnut.2021.702157] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Accepted: 08/05/2021] [Indexed: 01/06/2023] Open
Abstract
Background: Impaired intestinal integrity and barrier function is associated with various diseases, including inflammatory bowel disease and metabolic syndrome. In recent years, plant-derived polyphenols have attracted much attention on regulating intestinal barrier function. Kiwifruit was recorded as a traditional Chinese medicine which can treat gastrointestinal diseases, but the mechanism was still unclear. In this study we investigated the effects of kiwifruit polyphenol extracts (KPE) on high fat diet induced intestinal permeability and its possible mechanism. Results: Dietary supplementation of KPE with 50 or 100 mg/kg bw could inhibit the increase of intestinal permeability caused by HFD and promote the expression of tight junction protein (Claudin-1, Occludin and ZO-1). From microbial diversity and RT-PCR, KPE administration reshaping gut microbiome, the relative abundance of Lactobacillus and Bifidobacterium were increased, and the relative abundance of Clostridium and Desulfovibrionaceae were decreased. The changes in microbe may influence intestinal inflammatory status. Then the expression of TLRs and cytokines were detected. KPE supplementation showed anti-inflammatory effect, the expression of IL-10 was increased and the expression of TLR-2, TLR-4, TNF-α and IL-1β were decreased. Correlation analysis indicated that the expression of tight junction protein was negative correlation with TLR-2, TLR-4, TNF-α and IL-1β expression, but positively correlated with Bacteroidete, Bifidobacterium and IL-10 expression; the expression of Bacteroidete, Lactobacillusand and Bifidobacterium were negative correlation with TLR4, TNF-α, and IL-1β expression. Conclusion: KPE treatment relieve the intestinal damage caused by HFD, which was related to the regulation of Bacteroidete, Lactobacillusand, and Bifidobacterium expression and inhibit intestinal inflammation. KPE could be a functional component for preventing gut damage and its related disease.
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Affiliation(s)
- Minlan Yuan
- School of Public Health, The Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang, China
| | - Xiao Chen
- School of Public Health, The Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang, China
| | - Tianxia Su
- School of Public Health, The Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang, China
| | - Yan Zhou
- School of Public Health, The Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang, China
| | - Xiaohong Sun
- School of Public Health, The Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang, China
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21
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Marchelletta RR, Krishnan M, Spalinger MR, Placone TW, Alvarez R, Sayoc-Becerra A, Canale V, Shawki A, Park YS, Bernts LH, Myers S, Tremblay ML, Barrett KE, Krystofiak E, Kachar B, McGovern DP, Weber CR, Hanson EM, Eckmann L, McCole DF. T cell protein tyrosine phosphatase protects intestinal barrier function by restricting epithelial tight junction remodeling. J Clin Invest 2021; 131:138230. [PMID: 34623320 DOI: 10.1172/jci138230] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Accepted: 07/22/2021] [Indexed: 12/12/2022] Open
Abstract
Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.
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Affiliation(s)
- Ronald R Marchelletta
- Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Moorthy Krishnan
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, Riverside, California, USA
| | - Marianne R Spalinger
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, Riverside, California, USA
| | - Taylaur W Placone
- Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Rocio Alvarez
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, Riverside, California, USA
| | - Anica Sayoc-Becerra
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, Riverside, California, USA
| | - Vinicius Canale
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, Riverside, California, USA
| | - Ali Shawki
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, Riverside, California, USA
| | - Young Su Park
- Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Lucas Hp Bernts
- Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Stephen Myers
- Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Michel L Tremblay
- Department of Biochemistry and Goodman Cancer Research Centre, Faculty of Medicine and Health Sciences, McGill University, Montréal, Québec, Canada
| | - Kim E Barrett
- Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Evan Krystofiak
- National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland, USA
| | - Bechara Kachar
- National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland, USA
| | - Dermot Pb McGovern
- F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA
| | | | - Elaine M Hanson
- Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Lars Eckmann
- Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Declan F McCole
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, Riverside, California, USA
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22
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Lei H, Crawford MS, McCole DF. JAK-STAT Pathway Regulation of Intestinal Permeability: Pathogenic Roles and Therapeutic Opportunities in Inflammatory Bowel Disease. Pharmaceuticals (Basel) 2021; 14:840. [PMID: 34577540 PMCID: PMC8466350 DOI: 10.3390/ph14090840] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2021] [Revised: 08/17/2021] [Accepted: 08/19/2021] [Indexed: 12/15/2022] Open
Abstract
The epithelial barrier forms the interface between luminal microbes and the host immune system and is the first site of exposure to many of the environmental factors that trigger disease activity in chronic inflammatory bowel disease (IBD). Disruption of the epithelial barrier, in the form of increased intestinal permeability, is a feature of IBD and other inflammatory diseases, including celiac disease and type 1 diabetes. Variants in genes that regulate or belong to the JAK-STAT signaling pathway are associated with IBD risk. Inhibitors of the JAK-STAT pathway are now effective therapeutic options in IBD. This review will discuss emerging evidence that JAK inhibitors can be used to improve defects in intestinal permeability and how this plays a key role in resolving intestinal inflammation.
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Affiliation(s)
| | | | - Declan F. McCole
- Division of Biomedical Sciences, School of Medicine, University of California, Riverside, CA 92521, USA; (H.L.); (M.S.C.)
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23
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Hosic S, Bindas AJ, Puzan ML, Lake W, Soucy JR, Zhou F, Koppes RA, Breault DT, Murthy SK, Koppes AN. Rapid Prototyping of Multilayer Microphysiological Systems. ACS Biomater Sci Eng 2021; 7:2949-2963. [PMID: 34275297 PMCID: PMC8290094 DOI: 10.1021/acsbiomaterials.0c00190] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Microfluidic organs-on-chips aim to realize more biorelevant in vitro experiments compared to traditional two-dimensional (2D) static cell culture. Often such devices are fabricated via poly(dimethylsiloxane) (PDMS) soft lithography, which offers benefits (e.g., high feature resolution) along with drawbacks (e.g., prototyping time/costs). Here, we report benchtop fabrication of multilayer, PDMS-free, thermoplastic organs-on-chips via laser cut and assembly with double-sided adhesives that overcome some limitations of traditional PDMS lithography. Cut and assembled chips are economical to prototype ($2 per chip), can be fabricated in parallel within hours, and are Luer compatible. Biocompatibility was demonstrated with epithelial line Caco-2 cells and primary human small intestinal organoids. Comparable to control static Transwell cultures, Caco-2 and organoids cultured on chips formed confluent monolayers expressing tight junctions with low permeability. Caco-2 cells-on-chip differentiated ∼4 times faster, including increased mucus, compared to controls. To demonstrate the robustness of cut and assemble, we fabricated a dual membrane, trilayer chip integrating 2D and 3D compartments with accessible apical and basolateral flow chambers. As proof of concept, we cocultured a human, differentiated monolayer and intact 3D organoids within multilayered contacting compartments. The epithelium exhibited 3D tissue structure and organoids expanded close to the adjacent monolayer, retaining proliferative stem cells over 10 days. Taken together, cut and assemble offers the capability to rapidly and economically manufacture microfluidic devices, thereby presenting a compelling fabrication technique for developing organs-on-chips of various geometries to study multicellular tissues.
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Affiliation(s)
- Sanjin Hosic
- Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, Massachusetts 02115, United States
| | - Adam J Bindas
- Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, Massachusetts 02115, United States
| | - Marissa L Puzan
- Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, Massachusetts 02115, United States
| | - Will Lake
- Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, Massachusetts 02115, United States
| | - Jonathan R Soucy
- Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, Massachusetts 02115, United States
| | - Fanny Zhou
- Division of Endocrinology, Boston Children's Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115, United States
| | - Ryan A Koppes
- Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, Massachusetts 02115, United States
| | - David T Breault
- Division of Endocrinology, Boston Children's Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115, United States
- Department of Pediatrics, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115, United States
- Principal Faculty, Harvard Stem Cell Institute, 7 Divinity Ave, Cambridge, Massachusetts 02138, United States
| | - Shashi K Murthy
- Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, Massachusetts 02115, United States
| | - Abigail N Koppes
- Department of Chemical Engineering, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, Massachusetts 02115, United States
- Department of Biology, Northeastern University, 360 Huntington Ave., 313 Snell Engineering, Boston, Massachusetts 02115, United States
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24
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Van Spaendonk H, Ceuleers H, Smet A, Berg M, Joossens J, Van der Veken P, Francque SM, Lambeir AM, De Man JG, De Meester I, Augustyns K, De Winter BY. The Effect of a Novel Serine Protease Inhibitor on Inflammation and Intestinal Permeability in a Murine Colitis Transfer Model. Front Pharmacol 2021; 12:682065. [PMID: 34248633 PMCID: PMC8264366 DOI: 10.3389/fphar.2021.682065] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Accepted: 06/14/2021] [Indexed: 12/17/2022] Open
Abstract
Background: A protease/antiprotease disbalance is observed in inflammatory bowel diseases (IBD). We therefore studied the effect of the novel serine protease inhibitor UAMC-00050 on intestinal inflammation and permeability in a chronic colitis T cell transfer mouse model to get further insight into the regulation of T cell-mediated immunopathology. Methods: Colitis was induced in severe combined immunodeficient (SCID) mice, by the adoptive transfer of CD4+CD25-CD62L+ T cells. Animals were treated intraperitoneally (i.p.) 2x/day with vehicle or UAMC-00050 (5 mg/kg) from week 2 onwards. Colonic inflammation was assessed by clinical parameters, colonoscopy, macroscopy, microscopy, myeloperoxidase activity and cytokine expression levels. At week 4, 4 kDa FITC-dextran intestinal permeability was evaluated and T helper transcription factors, protease-activated receptors and junctional proteins were quantified by RT-qPCR. Results: Adoptive transfer of CD4+CD25-CD62L+ T cells resulted in colonic inflammation and an altered intestinal permeability. The serine protease inhibitor UAMC-00050 ameliorated both the inflammatory parameters and the intestinal barrier function. Furthermore, a decrease in colonic mRNA expression of Tbet and PAR4 was observed in colitis mice after UAMC-00050 treatment. Conclusion: The beneficial effect of UAMC-00050 on inflammation was apparent via a reduction of Tbet, IFN-γ, TNF-α, IL-1β and IL-6. Based on these results, we hypothesize a pivotal effect of serine protease inhibition on the Th1 inflammatory profile potentially mediated via PAR4.
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Affiliation(s)
- Hanne Van Spaendonk
- Laboratory of Experimental Medicine and Pediatrics, University of Antwerp, Antwerp, Belgium
- Infla-Med, Centre of Excellence, University of Antwerp, Antwerp, Belgium
| | - Hannah Ceuleers
- Laboratory of Experimental Medicine and Pediatrics, University of Antwerp, Antwerp, Belgium
- Infla-Med, Centre of Excellence, University of Antwerp, Antwerp, Belgium
| | - Annemieke Smet
- Laboratory of Experimental Medicine and Pediatrics, University of Antwerp, Antwerp, Belgium
- Infla-Med, Centre of Excellence, University of Antwerp, Antwerp, Belgium
| | - Maya Berg
- Infla-Med, Centre of Excellence, University of Antwerp, Antwerp, Belgium
| | - Jurgen Joossens
- Laboratory of Medicinal Chemistry, University of Antwerp, Antwerp, Belgium
| | - Pieter Van der Veken
- Infla-Med, Centre of Excellence, University of Antwerp, Antwerp, Belgium
- Laboratory of Medicinal Chemistry, University of Antwerp, Antwerp, Belgium
| | - Sven M. Francque
- Laboratory of Experimental Medicine and Pediatrics, University of Antwerp, Antwerp, Belgium
- Infla-Med, Centre of Excellence, University of Antwerp, Antwerp, Belgium
- Division of Gastroenterology and Hepatology, Antwerp University Hospital, Antwerp, Belgium
| | - Anne-Marie Lambeir
- Infla-Med, Centre of Excellence, University of Antwerp, Antwerp, Belgium
- Laboratory of Medical Biochemistry, University of Antwerp, Antwerp, Belgium
| | - Joris G. De Man
- Laboratory of Experimental Medicine and Pediatrics, University of Antwerp, Antwerp, Belgium
- Infla-Med, Centre of Excellence, University of Antwerp, Antwerp, Belgium
| | - Ingrid De Meester
- Infla-Med, Centre of Excellence, University of Antwerp, Antwerp, Belgium
- Laboratory of Medical Biochemistry, University of Antwerp, Antwerp, Belgium
| | - Koen Augustyns
- Infla-Med, Centre of Excellence, University of Antwerp, Antwerp, Belgium
- Laboratory of Medicinal Chemistry, University of Antwerp, Antwerp, Belgium
| | - Benedicte Y. De Winter
- Laboratory of Experimental Medicine and Pediatrics, University of Antwerp, Antwerp, Belgium
- Infla-Med, Centre of Excellence, University of Antwerp, Antwerp, Belgium
- Division of Gastroenterology and Hepatology, Antwerp University Hospital, Antwerp, Belgium
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25
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Pászti-Gere E, Pomothy J, Jerzsele Á, Pilgram O, Steinmetzer T. Exposure of human intestinal epithelial cells and primary human hepatocytes to trypsin-like serine protease inhibitors with potential antiviral effect. J Enzyme Inhib Med Chem 2021; 36:659-668. [PMID: 33641565 PMCID: PMC7928042 DOI: 10.1080/14756366.2021.1886093] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 μM in HIEC-6. These inhibitors did not cause elevations in extracellular H2O2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 μM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 μM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.
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Affiliation(s)
- Erzsébet Pászti-Gere
- Department of Pharmacology and Toxicology, University of Veterinary Medicine, Budapest, Hungary
| | - Judit Pomothy
- Department of Pharmacology and Toxicology, University of Veterinary Medicine, Budapest, Hungary
| | - Ákos Jerzsele
- Department of Pharmacology and Toxicology, University of Veterinary Medicine, Budapest, Hungary
| | - Oliver Pilgram
- Faculty of Pharmacy, Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marburg, Germany
| | - Torsten Steinmetzer
- Faculty of Pharmacy, Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marburg, Germany
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Talyuli OAC, Bottino-Rojas V, Polycarpo CR, Oliveira PL, Paiva-Silva GO. Non-immune Traits Triggered by Blood Intake Impact Vectorial Competence. Front Physiol 2021; 12:638033. [PMID: 33737885 PMCID: PMC7960658 DOI: 10.3389/fphys.2021.638033] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2020] [Accepted: 02/08/2021] [Indexed: 11/13/2022] Open
Abstract
Blood-feeding arthropods are considered an enormous public health threat. They are vectors of a plethora of infectious agents that cause potentially fatal diseases like Malaria, Dengue fever, Leishmaniasis, and Lyme disease. These vectors shine due to their own physiological idiosyncrasies, but one biological aspect brings them all together: the requirement of blood intake for development and reproduction. It is through blood-feeding that they acquire pathogens and during blood digestion that they summon a collection of multisystemic events critical for vector competence. The literature is focused on how classical immune pathways (Toll, IMD, and JAK/Stat) are elicited throughout the course of vector infection. Still, they are not the sole determinants of host permissiveness. The dramatic changes that are the hallmark of the insect physiology after a blood meal intake are the landscape where a successful infection takes place. Dominant processes that occur in response to a blood meal are not canonical immunological traits yet are critical in establishing vector competence. These include hormonal circuitries and reproductive physiology, midgut permeability barriers, midgut homeostasis, energy metabolism, and proteolytic activity. On the other hand, the parasites themselves have a role in the outcome of these blood triggered physiological events, consistently using them in their favor. Here, to enlighten the knowledge on vector-pathogen interaction beyond the immune pathways, we will explore different aspects of the vector physiology, discussing how they give support to these long-dated host-parasite relationships.
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Affiliation(s)
- Octavio A C Talyuli
- Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
| | - Vanessa Bottino-Rojas
- Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
| | - Carla R Polycarpo
- Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.,Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil
| | - Pedro L Oliveira
- Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.,Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil
| | - Gabriela O Paiva-Silva
- Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.,Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil
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Li S, Wang L, Sun S, Wu Q. Hepsin: a multifunctional transmembrane serine protease in pathobiology. FEBS J 2020; 288:5252-5264. [PMID: 33300264 DOI: 10.1111/febs.15663] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2020] [Revised: 11/30/2020] [Accepted: 12/07/2020] [Indexed: 12/14/2022]
Abstract
Cell membrane-bound serine proteases are important in the maintenance of physiological homeostasis. Hepsin is a type II transmembrane serine protease highly expressed in the liver. Recent studies indicate that hepsin activates prohepatocyte growth factor in the liver to enhance Met signaling, thereby regulating glucose, lipid, and protein metabolism. In addition, hepsin functions in nonhepatic tissues, including the adipose tissue, kidney, and inner ear, to regulate adipocyte differentiation, urinary protein processing, and auditory function, respectively. In mouse models, hepsin deficiency lowers blood glucose, lipid, and protein levels, impairs uromodulin assembly in renal epithelial cells, and causes hearing loss. Elevated hepsin expression has also been found in many cancers. As a type II transmembrane protease, cell surface expression and zymogen activation are essential for hepsin activity. In this review, we discuss the current knowledge regarding hepsin biosynthesis, activation, and functions in pathobiology.
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Affiliation(s)
- Shuo Li
- Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, OH, USA
| | - Lina Wang
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Prevention, Soochow University, Suzhou, China
| | - Shijin Sun
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Prevention, Soochow University, Suzhou, China
| | - Qingyu Wu
- Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, OH, USA.,Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Prevention, Soochow University, Suzhou, China
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Maternal dietary linoleic acid altered intestinal barrier function in domestic pigeons ( Columba livia). Br J Nutr 2020; 126:1003-1016. [PMID: 33298208 DOI: 10.1017/s0007114520004973] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Linoleic acid (LA) is predominantly essential for poultry. Poultry lacking LA show retarded growth and reduced disease resistance. Intestinal barrier function plays an important role in pigeon squab growth, whereas research on the effects of LA on intestinal health in altrices is scant. Considering that squabs are fed by their parents, the study aimed to explore the effects of maternal dietary LA on intestinal morphology, tight junction proteins, immune cytokines and microbial flora in squabs. A completely randomised design with a control group, 1 % LA supplementation group, 2 % LA supplementation group and 4 % LA supplementation group was used. Six squabs from each treatment were randomly sampled at 21 d post-hatching. The results indicated that LA supplementation improved intestinal morphology, as reflected by increased villus height, villus area and the ratio of villi to crypts. Also, 1 % LA supplementation elevated the density of goblet cells in the intestine and strengthened tight junctions by up-regulating claudin-3 and occludin gene expression but down-regulating claudin-2 gene expression. Moreover, 1 % LA supplementation reduced the secretion of proinflammatory cytokines and partly increased anti-inflammatory cytokines. The intestinal microbial diversity in the 1 % LA supplementation group was higher than that in the other groups. As beneficial bacteria, Butyrivibrio was the biomarker of 1 % LA supplementation. However, excessive (4 %) LA supplementation led to adverse impacts on intestinal immunity and microbiota. In conclusion, maternal dietary LA might alter intestinal barrier function in pigeon squabs in a dose-dependent manner. Supplementation with 1 % LA was suggested in parental pigeons.
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Nojkov B, Zhou SY, Dolan RD, Davis EM, Appelman HD, Guo X, Jackson K, Sturm MB, Wang TD, Owyang C, Liu JJ, Chey WD. Evidence of Duodenal Epithelial Barrier Impairment and Increased Pyroptosis in Patients With Functional Dyspepsia on Confocal Laser Endomicroscopy and "Ex Vivo" Mucosa Analysis. Am J Gastroenterol 2020; 115:1891-1901. [PMID: 33156108 PMCID: PMC8409129 DOI: 10.14309/ajg.0000000000000827] [Citation(s) in RCA: 46] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
INTRODUCTION Duodenal epithelial barrier impairment and immune activation may play a role in the pathogenesis of functional dyspepsia (FD). This study was aimed to evaluate the duodenal epithelium of patients with FD and healthy individuals for detectable microscopic structural abnormalities. METHODS This is a prospective study using esophagogastroduodenoscopy enhanced with duodenal confocal laser endomicroscopy (CLE) and mucosal biopsies in patients with FD (n = 16) and healthy controls (n = 18). Blinded CLE images analysis evaluated the density of epithelial gaps (cell extrusion zones), a validated endoscopic measure of the intestinal barrier status. Analyses of the biopsied duodenal mucosa included standard histology, quantification of mucosal immune cells/cytokines, and immunohistochemistry for inflammatory epithelial cell death called pyroptosis. Transepithelial electrical resistance (TEER) was measured using Ussing chambers. Epithelial cell-to-cell adhesion proteins expression was assessed by real-time polymerase chain reaction. RESULTS Patients with FD had significantly higher epithelial gap density on CLE in the distal duodenum than that of controls (P = 0.002). These mucosal abnormalities corresponded to significant changes in the duodenal biopsy samples of patients with FD, compared with controls, including impaired mucosal integrity by TEER (P = 0.009) and increased number of epithelial cells undergoing pyroptosis (P = 0.04). Reduced TEER inversely correlated with the severity of certain dyspeptic symptoms. Furthermore, patients with FD demonstrated altered duodenal expression of claudin-1 and interleukin-6. No differences in standard histology were found between the groups. DISCUSSION This is the first report of duodenal CLE abnormalities in patients with FD, corroborated by biopsy findings of epithelial barrier impairment and increased cell death, implicating that duodenal barrier disruption is a pathogenesis factor in FD and introducing CLE a potential diagnostic biomarker in FD.
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Affiliation(s)
- Borko Nojkov
- Division of Gastroenterology and Hepatology, Michigan Medicine, Ann Arbor, Michigan, USA
| | - Shi-Yi Zhou
- Division of Gastroenterology and Hepatology, Michigan Medicine, Ann Arbor, Michigan, USA
| | - Russell D. Dolan
- Department of Internal Medicine, Michigan Medicine, Ann Arbor, Michigan, USA
| | - Elisabeth M. Davis
- Division of Gastroenterology and Hepatology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - Henry D. Appelman
- Department of Pathology, Michigan Medicine, Ann Arbor, Michigan, USA
| | - Xueyan Guo
- Division of Gastroenterology and Hepatology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - Kenya Jackson
- Division of Gastroenterology and Hepatology, Michigan Medicine, Ann Arbor, Michigan, USA
| | - Matthew B. Sturm
- Division of Gastroenterology and Hepatology, Michigan Medicine, Ann Arbor, Michigan, USA
| | - Thomas D. Wang
- Division of Gastroenterology and Hepatology, Michigan Medicine, Ann Arbor, Michigan, USA
| | - Chung Owyang
- Division of Gastroenterology and Hepatology, Michigan Medicine, Ann Arbor, Michigan, USA
| | - Julia J. Liu
- Division of Gastroenterology and Hepatology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - William D. Chey
- Division of Gastroenterology and Hepatology, Michigan Medicine, Ann Arbor, Michigan, USA
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30
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Zhang F, Liu L, Zhang H, Liu ZL. Effect of Platelet-Activating Factor on Barrier Function of ARPE-19 Cells. DRUG DESIGN DEVELOPMENT AND THERAPY 2020; 14:4205-4214. [PMID: 33116408 PMCID: PMC7567541 DOI: 10.2147/dddt.s251941] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/29/2020] [Accepted: 09/25/2020] [Indexed: 12/17/2022]
Abstract
Aim To examine the effects of platelet-activating factor (PAF) on the barrier functions of cultured retinal pigment epithelial (RPE) cells. Methods A human RPE cell line (ARPE-19) was cultured on microporous filter supports and treated with PAF and WEB 2086, a specific PAF-receptor (PAF-R) antagonist. The permeability of the RPE monolayer was measured using transepithelial electrical resistance (TER) and sodium fluorescein flux. The expression of the tight junction protein zonula occludens (ZO)-1 and the adherens junction protein N-cadherin was assessed using immunohistochemistry and Western blotting. We also measured the vascular endothelial growth factor (VEGF) concentrations in PAF-treated cultures and re-measured RPE monolayer permeability in the presence of VEGF-neutralizing antibodies. Results PAF significantly decreased the TER and enhanced the sodium fluorescein flux of the RPE monolayer and downregulated the expression of ZO-1 and N-cadherin. These effects were abolished by WEB 2086-mediated blockage of the PAF-R. PAF stimulation increased VEGF expression in RPE cells, and the antibody-mediated neutralization of VEGF caused a partial recovery of the barrier properties. Conclusion The barrier functions of ARPE-19 cells were altered by PAF, and these effects were partly mediated by an upregulation of VEGF expression in these cells. Our results contribute to the growing body of evidence supporting the role of PAF in choroidal neovascularization. Our findings suggest that PAF is a novel target in the development of therapies for increased permeability of the RPE monolayer.
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Affiliation(s)
- Fan Zhang
- Department of Ophthalmology, The Fourth Affiliated Hospital of China Medical University, Eye Hospital of China Medical University, Key Lens Research Laboratory of Liaoning Province, Shenyang, Liaoning, People's Republic of China
| | - Lei Liu
- Department of Ophthalmology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, People's Republic of China
| | - Han Zhang
- Department of Ophthalmology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, People's Republic of China
| | - Zhe-Li Liu
- Department of Ophthalmology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, People's Republic of China
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31
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Solà-Tapias N, Vergnolle N, Denadai-Souza A, Barreau F. The Interplay Between Genetic Risk Factors and Proteolytic Dysregulation in the Pathophysiology of Inflammatory Bowel Disease. J Crohns Colitis 2020; 14:1149-1161. [PMID: 32090263 DOI: 10.1093/ecco-jcc/jjaa033] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Crohn's disease [CD] and ulcerative colitis [UC] are the two main forms of inflammatory bowel disease [IBD]. Previous studies reported increased levels of proteolytic activity in stool and tissue samples from IBD patients, whereas the re-establishment of the proteolytic balance abrogates the development of experimental colitis. Furthermore, recent data suggest that IBD occurs in genetically predisposed individuals who develop an abnormal immune response to intestinal microbes once exposed to environmental triggers. In this review, we highlight the role of proteases in IBD pathophysiology, and we showcase how the main cellular pathways associated with IBD influence proteolytic unbalance and how functional proteomics are allowing the unambiguous identification of dysregulated proteases in IBD, paving the way to the development of new protease inhibitors as a new potential treatment.
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Affiliation(s)
- Núria Solà-Tapias
- IRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, Toulouse, France
| | - Nathalie Vergnolle
- IRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, Toulouse, France.,Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada
| | - Alexandre Denadai-Souza
- Department of Chronic Diseases, Metabolism and Ageing, University of Leuven, Leuven, Belgium
| | - Frédérick Barreau
- IRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, Toulouse, France
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32
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Membrane-anchored serine proteases as regulators of epithelial function. Biochem Soc Trans 2020; 48:517-528. [PMID: 32196551 PMCID: PMC9869603 DOI: 10.1042/bst20190675] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Revised: 02/28/2020] [Accepted: 03/04/2020] [Indexed: 02/07/2023]
Abstract
Cleavage of proteins in the extracellular milieu, including hormones, growth factors and their receptors, ion channels, and various cell adhesion and extracellular matrix molecules, plays a key role in the regulation of cell behavior. Among more than 500 proteolytic enzymes encoded by mammalian genomes, membrane-anchored serine proteases (MASPs), which are expressed on the surface of epithelial cells of all major organs, are excellently suited to mediate signal transduction across the epithelia and are increasingly being recognized as important regulators of epithelial development, function, and disease [ 1-3]. In this minireview, we summarize current knowledge of the in vivo roles of MASPs in acquisition and maintenance of some of the defining functions of epithelial tissues, such as barrier formation, ion transport, and sensory perception.
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33
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Wu CJ, Lu M, Feng X, Nakato G, Udey MC. Matriptase Cleaves EpCAM and TROP2 in Keratinocytes, Destabilizing Both Proteins and Associated Claudins. Cells 2020; 9:cells9041027. [PMID: 32326212 PMCID: PMC7226414 DOI: 10.3390/cells9041027] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2020] [Revised: 04/17/2020] [Accepted: 04/19/2020] [Indexed: 02/07/2023] Open
Abstract
The homologs EpCAM and TROP2, which both interact with claudin-1 and claudin-7, are frequently coexpressed in epithelia including skin. Intestine uniquely expresses high levels of EpCAM but not TROP2. We previously identified EpCAM as a substrate of the membrane-anchored protease matriptase and linked HAI-2, matriptase, EpCAM and claudin-7 in a pathway that is pivotal for intestinal epithelial cells (IEC) homeostasis. Herein, we reveal that TROP2 is also a matriptase substrate. Matriptase cleaved TROP2 when purified recombinant proteins were mixed in vitro. TROP2, like EpCAM, was also cleaved after co-transfection of matriptase in 293T cells. Neither EpCAM nor TROP2 cleavage was promoted by protease-disabled matriptase or matriptase that harbored the ichthyosis-associated G827R mutation. We confirmed that EpCAM and TROP2 are both expressed in skin and detected cleavage of these proteins in human keratinocytes (HaCaT cells) after the physiologic inhibition of matriptase by HAI proteins was relieved by siRNA knockdown. Knockdown of EpCAM or TROP2 individually had only small effects on claudin-1 and claudin-7 levels, whereas elimination of both markedly diminished claudin levels. HAI-1 knockdown promoted EpCAM and TROP2 cleavage accompanied by reductions in claudins, whereas HAI-2 knockdown had little impact. Double knockdown of HAI-1 and HAI-2 induced nearly complete cleavage of EpCAM and TROP2 and drastic reductions of claudins. These effects were eliminated by concurrent matriptase knockdown. Decreases in claudin levels were also diminished by the lysosomal inhibitor chloroquine and cleaved EpCAM/TROP2 fragments accumulated preferentially. We demonstrate that TROP2 and EpCAM exhibit redundancies with regard to regulation of claudin metabolism and that an HAI, matriptase, EpCAM and claudin pathway analogous to what we described in IECs exists in keratinocytes. This study may offer insights into the mechanistic basis for matriptase dysregulation-induced ichthyosis.
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Affiliation(s)
- Chuan-Jin Wu
- Laboratory of Immune Cell Biology, National Cancer Institute, Bethesda, MD 20892, USA
- Correspondence: (C.-J.W.); (M.C.U.); Tel.: +1-301-760-7452 (C.-J.W.); +1-314-454-8547 (M.C.U.)
| | - Michael Lu
- Experimental Immunology Branch, National Cancer Institute, Bethesda, MD 20892, USA;
| | - Xu Feng
- Retired from National Cancer Institute, Bethesda, MD 20892, USA;
| | - Gaku Nakato
- Kanagawa Institute of Industrial Science and Technology, Tonomachi, Kawasaki-ku, Kawasaki-shi, Kanagawa 210-0821, Japan;
| | - Mark C. Udey
- Dermatology Division, Department of Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA
- Correspondence: (C.-J.W.); (M.C.U.); Tel.: +1-301-760-7452 (C.-J.W.); +1-314-454-8547 (M.C.U.)
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34
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Kriaa A, Jablaoui A, Mkaouar H, Akermi N, Maguin E, Rhimi M. Serine proteases at the cutting edge of IBD: Focus on gastrointestinal inflammation. FASEB J 2020; 34:7270-7282. [PMID: 32307770 DOI: 10.1096/fj.202000031rr] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Revised: 03/27/2020] [Accepted: 03/28/2020] [Indexed: 12/15/2022]
Abstract
Serine proteases have been long recognized to coordinate many physiological processes and play key roles in regulating the inflammatory response. Accordingly, their dysregulation has been regularly associated with several inflammatory disorders and suggested as a central mechanism in the pathophysiology of digestive inflammation. So far, studies addressing the proteolytic homeostasis in the gut have mainly focused on host serine proteases as candidates of interest, while largely ignoring the potential contribution of their bacterial counterparts. The human gut microbiota comprises a complex ecosystem that contributes to host health and disease. Yet, our understanding of microbially produced serine proteases and investigation of whether they are causally linked to IBD is still in its infancy. In this review, we highlight recent advances in the emerging roles of host and bacterial serine proteases in digestive inflammation. We also discuss the application of available tools in the gut to monitor disease-related serine proteases. An exhaustive representation and understanding of such functional potential would help in closing existing gaps in mechanistic knowledge.
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Affiliation(s)
- Aicha Kriaa
- Microbiota Interaction with Human and Animal Team (MIHA), Micalis Institute, AgroParisTech, Université Paris-Saclay, INRAE, Jouy-en-Josas, France
| | - Amin Jablaoui
- Microbiota Interaction with Human and Animal Team (MIHA), Micalis Institute, AgroParisTech, Université Paris-Saclay, INRAE, Jouy-en-Josas, France
| | - Héla Mkaouar
- Microbiota Interaction with Human and Animal Team (MIHA), Micalis Institute, AgroParisTech, Université Paris-Saclay, INRAE, Jouy-en-Josas, France
| | - Nizar Akermi
- Microbiota Interaction with Human and Animal Team (MIHA), Micalis Institute, AgroParisTech, Université Paris-Saclay, INRAE, Jouy-en-Josas, France
| | - Emmanuelle Maguin
- Microbiota Interaction with Human and Animal Team (MIHA), Micalis Institute, AgroParisTech, Université Paris-Saclay, INRAE, Jouy-en-Josas, France
| | - Moez Rhimi
- Microbiota Interaction with Human and Animal Team (MIHA), Micalis Institute, AgroParisTech, Université Paris-Saclay, INRAE, Jouy-en-Josas, France
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Yoon J, Cho Y, Kim KY, Yoon MJ, Lee HS, Jeon SD, Cho Y, Kim C, Kim MG. A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CβII. J Biol Chem 2020; 295:7168-7177. [PMID: 32241917 PMCID: PMC7242708 DOI: 10.1074/jbc.ra119.011206] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2019] [Revised: 03/04/2020] [Indexed: 12/28/2022] Open
Abstract
Serine protease 14 (Prss14)/epithin is a transmembrane serine protease that plays essential roles in tumor progression and metastasis and therefore is a promising target for managing cancer. Prss14/epithin shedding may underlie its activity in cancer and worsen outcomes; accordingly, a detailed understanding of the molecular mechanisms in Prss14/epithin shedding may inform the design of future cancer therapies. On the basis of our previous observation that an activator of PKC, phorbol 12-myristate 13-acetate (PMA), induces Prss14/epithin shedding, here we further investigated the intracellular signaling pathway involved in this process. While using mitogen-activated protein kinase inhibitors to investigate possible effectors of downstream PKC signaling, we unexpectedly found that an inhibitor of c-Jun N-terminal kinase (JNK), SP600125, induces Prss14/epithin shedding even in the absence of PMA. SP600125-induced shedding, like that stimulated by PMA, was mediated by tumor necrosis factor-α–converting enzyme. In contrast, a JNK activator, anisomycin, partially abolished the effects of SP600125 on Prss14/epithin shedding. Moreover, the results from loss-of-function experiments with specific inhibitors, short hairpin RNA–mediated knockdown, and overexpression of dominant-negative PKCβII variants indicated that PKCβII is a major player in JNK inhibition– and PMA-mediated Prss14/epithin shedding. SP600125 increased phosphorylation of PKCβII and tumor necrosis factor-α–converting enzyme and induced their translocation into the plasma membrane. Finally, in vitro cell invasion experiments and bioinformatics analysis of data in The Cancer Genome Atlas breast cancer database revealed that JNK and PKCβII are important for Prss14/epithin-mediated cancer progression. These results provide important information regarding strategies against tumor metastasis.
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Affiliation(s)
- Joobyoung Yoon
- School of Biological Sciences, Seoul National University, Seoul 08826, Korea
| | - Youngkyung Cho
- School of Biological Sciences, Seoul National University, Seoul 08826, Korea.,Department of Life Sciences, Korea University, Seoul 02841, Korea
| | - Ki Yeon Kim
- Department of Biological Sciences, Inha University, Incheon 22212, Korea
| | - Min Ji Yoon
- Department of Life Sciences, Korea University, Seoul 02841, Korea
| | - Hyo Seon Lee
- School of Biological Sciences, Seoul National University, Seoul 08826, Korea
| | - Sangjun Davie Jeon
- Department of Biological Sciences, Inha University, Incheon 22212, Korea
| | - Yongcheol Cho
- Department of Life Sciences, Korea University, Seoul 02841, Korea
| | - Chungho Kim
- Department of Life Sciences, Korea University, Seoul 02841, Korea
| | - Moon Gyo Kim
- Department of Biological Sciences, Inha University, Incheon 22212, Korea
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36
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Holt-Danborg L, Vodopiutz J, Nonboe AW, De Laffolie J, Skovbjerg S, Wolters VM, Müller T, Hetzer B, Querfurt A, Zimmer KP, Jensen JK, Entenmann A, Heinz-Erian P, Vogel LK, Janecke AR. SPINT2 (HAI-2) missense variants identified in congenital sodium diarrhea/tufting enteropathy affect the ability of HAI-2 to inhibit prostasin but not matriptase. Hum Mol Genet 2020; 28:828-841. [PMID: 30445423 DOI: 10.1093/hmg/ddy394] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2018] [Revised: 11/07/2018] [Accepted: 11/08/2018] [Indexed: 11/13/2022] Open
Abstract
The syndromic form of congenital sodium diarrhea (SCSD) is caused by bi-allelic mutations in SPINT2, which encodes a Kunitz-type serine protease inhibitor (HAI-2). We report three novel SCSD patients, two novel SPINT2 mutations and review published cases. The most common findings in SCSD patients were choanal atresia (20/34) and keratitis of infantile onset (26/34). Characteristic epithelial tufts on intestinal histology were reported in 13/34 patients. Of 13 different SPINT2 variants identified in SCSD, 4 are missense variants and localize to the second Kunitz domain (KD2) of HAI-2. HAI-2 has been implicated in the regulation of the activities of several serine proteases including prostasin and matriptase, which are both important for epithelial barrier formation. No patient with bi-allelic stop mutations was identified, suggesting that at least one SPINT2 allele encoding a protein with residual HAI-2 function is necessary for survival. We show that the SCSD-associated HAI-2 variants p.Phe161Val, p.Tyr163Cys and p.Gly168Ser all display decreased ability to inhibit prostasin-catalyzed cleavage. However, the SCSD-associated HAI-2 variants inhibited matriptase as efficiently as the wild-type HAI-2. Homology modeling indicated limited solvent exposure of the mutated amino acids, suggesting that they induce misfolding of KD2. This suggests that prostasin needs to engage with an exosite motif located on KD2 in addition to the binding loop (Cys47/Arg48) located on the first Kunitz domain in order to inhibit prostasin. In conclusion our data suggests that SCSD is caused by lack of inhibition of prostasin or a similar protease in the secretory pathway or on the plasma membrane.
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Affiliation(s)
- Lasse Holt-Danborg
- Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, Denmark
| | - Julia Vodopiutz
- Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna
| | - Annika W Nonboe
- Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, Denmark
| | - Jan De Laffolie
- Abteilung Allgemeine Pädiatrie und Neonatologie, Zentrum für Kinderheilkunde und Jugendmedizin, Justus-Liebig-Universität, Gießen, Germany
| | - Signe Skovbjerg
- Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, Denmark
| | - Victorien M Wolters
- Department of Pediatric Gastroenterology, WKZ/ UMC Utrecht, Utrecht, The Netherlands
| | - Thomas Müller
- Department of Pediatrics I, Medical University of Innsbruck, Innsbruck, Austria
| | - Benjamin Hetzer
- Department of Pediatrics I, Medical University of Innsbruck, Innsbruck, Austria
| | - Alexander Querfurt
- Gesundheit Nord gGmbH, Klinikverbund Bremen, Klinik für Kinder und Jugendmedizin, Professor-Hess-Kinderklinik, Klinikum Bremen-Mitte, Bremen, Germany
| | - Klaus-Peter Zimmer
- Abteilung Allgemeine Pädiatrie und Neonatologie, Zentrum für Kinderheilkunde und Jugendmedizin, Justus-Liebig-Universität, Gießen, Germany
| | - Jan K Jensen
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
| | - Andreas Entenmann
- Department of Pediatrics I, Medical University of Innsbruck, Innsbruck, Austria
| | - Peter Heinz-Erian
- Department of Pediatrics I, Medical University of Innsbruck, Innsbruck, Austria
| | - Lotte K Vogel
- Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, Denmark
| | - Andreas R Janecke
- Department of Pediatrics I, Medical University of Innsbruck, Innsbruck, Austria.,Division of Human Genetics, Medical University of Innsbruck, Innsbruck, Austria
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37
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Barna RF, Pomothy JM, Paréj Z, Pásztiné Gere E. Investigation of sphingosin-1-phosphate-triggered matriptase activation using a rat primary hepatocyte model. Acta Vet Hung 2019; 67:578-587. [PMID: 31842605 DOI: 10.1556/004.2019.057] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Sphingosine-1-phosphate (S1P) has been reported as a matriptase activator. The aim of this study was to reveal if S1P can influence hepcidin production. Furthermore, we investigated how S1P can affect the viability and the redox status of primary hepatocytes. Rat primary hepatocytes were cultivated for 72 h and were treated with 50, 200, 1000 ng/ml S1P. Cell-free supernatants were collected every 24 h. Cell viability was tested by a colorimetric method using tetrazolium compound (MTS). The hepcidin levels in the cell-free supernatants were examined with hepcidin sandwich ELISA to determine the effect of S1P on the hepcidin-modulating ability of matriptase. In order to estimate the extent of S1P-generated oxidative stress, extracellular H2O2 measurements were performed by the use of fluorescent dye. Based on the findings, S1P treatment did not cause cell death for 72 h at concentrations up to 1000 ng/ml. S1P did not influence the extracellular H2O2 production for 72 h. The hepcidin levels were significantly suppressed in hepatocytes exposed to S1P treatment. Further studies would be needed to explore the exact mechanism of action of S1P.
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Affiliation(s)
- Réka Fanni Barna
- Department of Pharmacology and Toxicology, University of Veterinary Medicine Budapest, István u. 2, H-1078 Budapest, Hungary
| | - Judit Mercédesz Pomothy
- Department of Pharmacology and Toxicology, University of Veterinary Medicine Budapest, István u. 2, H-1078 Budapest, Hungary
| | - Zsuzsanna Paréj
- Department of Pharmacology and Toxicology, University of Veterinary Medicine Budapest, István u. 2, H-1078 Budapest, Hungary
| | - Erzsébet Pásztiné Gere
- Department of Pharmacology and Toxicology, University of Veterinary Medicine Budapest, István u. 2, H-1078 Budapest, Hungary
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38
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Béliveau F, Tarkar A, Dion SP, Désilets A, Ghinet MG, Boudreault PL, St-Georges C, Marsault É, Paone D, Collins J, Macphee CH, Campobasso N, Groy A, Cottom J, Ouellette M, Pope AJ, Leduc R. Discovery and Development of TMPRSS6 Inhibitors Modulating Hepcidin Levels in Human Hepatocytes. Cell Chem Biol 2019; 26:1559-1572.e9. [DOI: 10.1016/j.chembiol.2019.09.004] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2018] [Revised: 06/06/2019] [Accepted: 09/03/2019] [Indexed: 02/06/2023]
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39
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Konishi S, Yano T, Tanaka H, Mizuno T, Kanoh H, Tsukita K, Namba T, Tamura A, Yonemura S, Gotoh S, Matsumoto H, Hirai T, Tsukita S. Vinculin is critical for the robustness of the epithelial cell sheet paracellular barrier for ions. Life Sci Alliance 2019; 2:2/4/e201900414. [PMID: 31399484 PMCID: PMC6689668 DOI: 10.26508/lsa.201900414] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2019] [Revised: 07/23/2019] [Accepted: 07/24/2019] [Indexed: 12/12/2022] Open
Abstract
Vinculin in the apical junctional complex maintains the paracellular barrier function specifically for ions, but not for large solutes, by buffering mechanical fluctuations. The paracellular barrier function of tight junctions (TJs) in epithelial cell sheets is robustly maintained against mechanical fluctuations, by molecular mechanisms that are poorly understood. Vinculin is an adaptor of a mechanosensory complex at the adherens junction. Here, we generated vinculin KO Eph4 epithelial cells and analyzed their confluent cell-sheet properties. We found that vinculin is dispensable for the basic TJ structural integrity and the paracellular barrier function for larger solutes. However, vinculin is indispensable for the paracellular barrier function for ions. In addition, TJs stochastically showed dynamically distorted patterns in vinculin KO cell sheets. These KO phenotypes were rescued by transfecting full-length vinculin and by relaxing the actomyosin tension with blebbistatin, a myosin II ATPase activity inhibitor. Our findings indicate that vinculin resists mechanical fluctuations to maintain the TJ paracellular barrier function for ions in epithelial cell sheets.
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Affiliation(s)
- Satoshi Konishi
- Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka, Japan.,Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Tomoki Yano
- Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka, Japan
| | - Hiroo Tanaka
- Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka, Japan.,Department of Pharmacology, Teikyo University, Tokyo, Japan.,Strategic Innovation and Research Center, Teikyo University, Tokyo, Japan
| | - Tomoaki Mizuno
- Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka, Japan
| | - Hatsuho Kanoh
- Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka, Japan.,Graduate School of Biostudies, Kyoto University, Kyoto, Japan
| | - Kazuto Tsukita
- Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka, Japan.,Department of Neurology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Toshinori Namba
- Graduate School of Arts and Sciences, Tokyo University, Tokyo, Japan
| | - Atsushi Tamura
- Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka, Japan.,Department of Pharmacology, Teikyo University, Tokyo, Japan.,Strategic Innovation and Research Center, Teikyo University, Tokyo, Japan
| | - Shigenobu Yonemura
- Department of Cell Biology, Tokushima University Graduate School of Medical Science, Tokushima, Japan.,Laboratory for Ultrastructural Research, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan
| | - Shimpei Gotoh
- Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.,Department of Drug Discovery for Lung Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Hisako Matsumoto
- Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Toyohiro Hirai
- Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Sachiko Tsukita
- Laboratory of Biological Science, Graduate School of Frontier Biosciences and Graduate School of Medicine, Osaka University, Osaka, Japan .,Strategic Innovation and Research Center, Teikyo University, Tokyo, Japan
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40
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Zhai Q, Gong X, Wang C, Zhao J, Zhang H, Tian F, Chen W. Food-borne patulin toxicity is related to gut barrier disruption and can be prevented by docosahexaenoic acid and probiotic supplementation. Food Funct 2019; 10:1330-1339. [PMID: 30741300 DOI: 10.1039/c8fo02292e] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Patulin (PAT) is a mycotoxin widely found in fruits and vegetables. Several reviews and studies have hypothesized that in vivo PAT toxicity is related to gut barrier dysfunction, but evidence for this is not substantial. The goal of the present study was to further demonstrate the role of the gut barrier in food-borne PAT toxicity. In vitro assays showed that PAT exposure induced significant cell death, inhibited the mRNA expressions of tight junction proteins and increased gut permeability in Caco-2 cell monolayers. An acute PAT exposure animal trial reported for the first time an association between PAT-induced disruption of the gut barrier and endotoxemia in mice. Sub-chronic PAT exposure also inhibited the expression of ZO-1 in the gut and induced both intestinal and systematic inflammation in mice. Dietary supplements with previously reported protective effects on the gut barrier, such as docosahexaenoic acid and Lactobacillus plantarum CCFM8610, were able to recover the PAT-induced gut barrier dysfunction and significantly alleviate PAT toxicity in vivo. Another L. plantarum strain, CCFM11, with poor gut barrier modulation ability, failed to exhibit identical protective effects against PAT toxicity to L. plantarum CCFM8610. Our results indicated that PAT-induced disruption of the gut barrier and bacterial translocation may be another toxic mechanism of PAT besides its inherent cytotoxicity. Gut barrier protection may be considered an important target for the prevention of PAT toxicity.
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Affiliation(s)
- Qixiao Zhai
- State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China.
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41
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Pawar NR, Buzza MS, Antalis TM. Membrane-Anchored Serine Proteases and Protease-Activated Receptor-2-Mediated Signaling: Co-Conspirators in Cancer Progression. Cancer Res 2019; 79:301-310. [PMID: 30610085 DOI: 10.1158/0008-5472.can-18-1745] [Citation(s) in RCA: 54] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2018] [Revised: 10/12/2018] [Accepted: 11/07/2018] [Indexed: 12/18/2022]
Abstract
Pericellular proteolysis provides a significant advantage to developing tumors through the ability to remodel the extracellular matrix, promote cell invasion and migration, and facilitate angiogenesis. Recent advances demonstrate that pericellular proteases can also communicate directly to cells by activation of a unique group of transmembrane G-protein-coupled receptors (GPCR) known as protease-activated receptors (PAR). In this review, we discuss the specific roles of one of four mammalian PARs, namely PAR-2, which is overexpressed in advanced stage tumors and is activated by trypsin-like serine proteases that are highly expressed or otherwise dysregulated in many cancers. We highlight recent insights into the ability of different protease agonists to bias PAR-2 signaling and the newly emerging evidence for an interplay between PAR-2 and membrane-anchored serine proteases, which may co-conspire to promote tumor progression and metastasis. Interfering with these pathways might provide unique opportunities for the development of new mechanism-based strategies for the treatment of advanced and metastatic cancers.
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Affiliation(s)
- Nisha R Pawar
- Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland.,Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland
| | - Marguerite S Buzza
- Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland.,Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland.,University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland
| | - Toni M Antalis
- Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland. .,Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland.,University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland
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42
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Drees L, Königsmann T, Jaspers MHJ, Pflanz R, Riedel D, Schuh R. Conserved function of the matriptase-prostasin proteolytic cascade during epithelial morphogenesis. PLoS Genet 2019; 15:e1007882. [PMID: 30601807 PMCID: PMC6331135 DOI: 10.1371/journal.pgen.1007882] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2018] [Revised: 01/14/2019] [Accepted: 12/06/2018] [Indexed: 01/08/2023] Open
Abstract
Extracellular matrix (ECM) assembly and remodelling is critical during development and organ morphogenesis. Dysregulation of ECM is implicated in many pathogenic conditions, including cancer. The type II transmembrane serine protease matriptase and the serine protease prostasin are key factors in a proteolytic cascade that regulates epithelial ECM differentiation during development in vertebrates. Here, we show by rescue experiments that the Drosophila proteases Notopleural (Np) and Tracheal-prostasin (Tpr) are functional homologues of matriptase and prostasin, respectively. Np mediates morphogenesis and remodelling of apical ECM during tracheal system development and is essential for maintenance of the transepithelial barrier function. Both Np and Tpr degrade the zona pellucida-domain (ZP-domain) protein Dumpy, a component of the transient tracheal apical ECM. Furthermore, we demonstrate that Tpr zymogen and the ZP domain of the ECM protein Piopio are cleaved by Np and matriptase in vitro. Our data indicate that the evolutionarily conserved ZP domain, present in many ECM proteins of vertebrates and invertebrates, is a novel target of the conserved matriptase-prostasin proteolytic cascade. Epithelial tissue covers the outside of the animal body and lines internal organs. Its disorganization is the source of approximately 90% of all human cancers. Elaboration of the basic epithelial characteristics has led to an understanding of how complex structures such as the branched tubular networks of vertebrate lung or invertebrate tracheal system are organized. Aside from obvious morphological differences, specific compositions of the epithelial extracellular matrix (ECM) have been noted. For example, while the flexible ECM of the vertebrate skin mainly consists of collagen and elastic fibers, the rigid ECM of invertebrates is chitin-based to serve as an inflexible exoskeleton. We show that a central regulator of ECM differentiation and epithelial development in vertebrates, the matriptase-prostasin proteolytic cascade (MPPC), is conserved and essential for both Drosophila ECM morphogenesis and physiology. The functionally conserved components of the MPPC mediate cleavage of zona pellucida-domain (ZP-domain) proteins, which play crucial roles in organizing apical structures of the ECM in both vertebrates and invertebrates. Our data indicate that ZP-proteins are molecular targets of the conserved MPPC and that cleavage within the ZP-domains is a conserved mechanism of ECM development and differentiation.
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Affiliation(s)
- Leonard Drees
- Research Group Molecular Organogenesis, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany
| | - Tatiana Königsmann
- Research Group Molecular Organogenesis, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany
| | - Martin H. J. Jaspers
- Research Group Molecular Organogenesis, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany
| | - Ralf Pflanz
- Research Group Mass Spectrometry, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany
| | - Dietmar Riedel
- Electron Microscopy Group, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany
| | - Reinhard Schuh
- Research Group Molecular Organogenesis, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany
- * E-mail:
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43
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Sakurai N, Nishio S, Akiyama Y, Miyata S, Oshima K, Nadano D, Matsuda T. Apical-to-basolateral transepithelial transport of cow's milk caseins by intestinal Caco-2 cell monolayers: MS-based quantitation of cellularly degraded α- and β-casein fragments. J Biochem 2018; 164:113-125. [PMID: 29490044 DOI: 10.1093/jb/mvy034] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2018] [Accepted: 02/21/2018] [Indexed: 11/12/2022] Open
Abstract
Casein (CN) is the major milk protein to nourish infants but, in certain population, it causes cow's milk allergy, indicating the uptake of antigenic CN and their peptides through the intestinal epithelium. Using human intestinal Caco-2 cell monolayers, the apical-to-basal transepithelial transport of CN was investigated. Confocal microscopy using component-specific antibodies showed that αs1-CN antigens became detectable as punctate signals at the apical-side cytoplasm and reached to the cytoplasm at a tight-junction level within a few hours. Such intracellular CN signals were more remarkable than those of the other antigens, β-lactoglobulin and ovalbumin, colocalized in part with an early endosome marker protein (EEA1) and decreased in the presence of cytochalasin D or sodium azide and also at lowered temperature at 4°C. Liquid chromatography coupled with mass spectroscopy analysis of the protein fraction in the basal-side medium identified the αs1-CB fragment including the N-terminal region and the αs2-CN fragment containing the central part of polypeptide at 100-1,000 fmol per well levels. Moreover, β-CN C-terminal overlapping peptides were identified in the peptide fraction below 10 kDa of the basal medium. These results suggest that CNs are partially degraded by cellular proteases and/or peptidases and immunologically active CN fragments are transported to basal side of the cell monolayers.
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Affiliation(s)
- Nao Sakurai
- Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
| | - Shunsuke Nishio
- Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
| | - Yuka Akiyama
- Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
| | - Shinji Miyata
- Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
| | - Kenzi Oshima
- Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
| | - Daita Nadano
- Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
| | - Tsukasa Matsuda
- Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
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44
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Danielsen ET, Olsen AK, Coskun M, Nonboe AW, Larsen S, Dahlgaard K, Bennett EP, Mitchelmore C, Vogel LK, Troelsen JT. Intestinal regulation of suppression of tumorigenicity 14 (ST14) and serine peptidase inhibitor, Kunitz type -1 (SPINT1) by transcription factor CDX2. Sci Rep 2018; 8:11813. [PMID: 30087389 PMCID: PMC6081401 DOI: 10.1038/s41598-018-30216-z] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2018] [Accepted: 07/23/2018] [Indexed: 12/14/2022] Open
Abstract
The type II membrane-anchored serine protease, matriptase, encoded by suppression of tumorgenicity-14 (ST14) regulates the integrity of the intestinal epithelial barrier in concert with its inhibitor, HAI-1 encoded by serine peptidase inhibitor, Kunitz type -1 (SPINT1). The balance of the protease/inhibitor gene expression ratio is vital in preventing the oncogenic potential of matriptase. The intestinal cell lineage is regulated by a transcriptional regulatory network where the tumor suppressor, Caudal homeobox 2 (CDX2) is considered to be an intestinal master transcription factor. In this study, we show that CDX2 has a dual function in regulating both ST14 and SPINT1, gene expression in intestinal cells. We find that CDX2 is not required for the basal ST14 and SPINT1 gene expression; however changes in CDX2 expression affects the ST14/SPINT1 mRNA ratio. Exploring CDX2 ChIP-seq data from intestinal cell lines, we identified genomic CDX2-enriched enhancer elements for both ST14 and SPINT1, which regulate their corresponding gene promoter activity. We show that CDX2 displays both repressive and enhancing regulatory abilities in a cell specific manner. Together, these data reveal new insight into transcriptional mechanisms controlling the intestinal matriptase/inhibitor balance.
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Affiliation(s)
- E Thomas Danielsen
- Department of Science and Environment, Roskilde University, Roskilde, Denmark.,Institute of Cellular and Molecular Medicine, the Panum Institute, University of Copenhagen, Copenhagen, Denmark
| | - Anders Krüger Olsen
- Institute of Cellular and Molecular Medicine, the Panum Institute, University of Copenhagen, Copenhagen, Denmark
| | - Mehmet Coskun
- Department of Gastroenterology, University of Copenhagen, DK-2730, Herlev, Denmark
| | - Annika W Nonboe
- Institute of Cellular and Molecular Medicine, the Panum Institute, University of Copenhagen, Copenhagen, Denmark
| | - Sylvester Larsen
- Department of Science and Environment, Roskilde University, Roskilde, Denmark.,Department of Clinical Immunology, Naestved Hospital, Naestved, Region Zealand, Denmark
| | - Katja Dahlgaard
- Department of Science and Environment, Roskilde University, Roskilde, Denmark
| | - Eric Paul Bennett
- Copenhagen Center for Glycomics, Department of Odontology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Cathy Mitchelmore
- Department of Science and Environment, Roskilde University, Roskilde, Denmark
| | - Lotte Katrine Vogel
- Institute of Cellular and Molecular Medicine, the Panum Institute, University of Copenhagen, Copenhagen, Denmark
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45
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Sabirzhanov B, Faden AI, Aubrecht T, Henry R, Glaser E, Stoica BA. MicroRNA-711-Induced Downregulation of Angiopoietin-1 Mediates Neuronal Cell Death. J Neurotrauma 2018; 35:2462-2481. [PMID: 29774773 DOI: 10.1089/neu.2017.5572] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Angiopoietin-1 (Ang-1) is a well-known endothelial growth factor, but its effects on neurons have yet to be elucidated. We show that Ang-1 is rapidly downregulated in the injured brain after controlled cortical impact (CCI), a mouse experimental traumatic brain injury (TBI) model and in etoposide-induced neuronal apoptosis in vitro. Ang-1 treatment inhibits etoposide-induced upregulation of proapoptotic B-cell lymphoma 2 (Bcl-2) family members Noxa, p53 upregulated modulator of apoptosis (Puma), Bcl-2 interacting mediator of cell death (Bim), and Bcl-2-associated X protein (Bax); reduces markers of caspase-dependent (cytochrome c release/caspase activation) and caspase-independent (apoptosis-inducing factor release) pathways; and limits neuronal cell death. Ang-1 treatment phosphorylates receptors Tunica interna endothelial cell kinase 2 (Tie2), and β1-integrin and limits the etoposide-induced decrease in protein kinase B (Akt) activity. Blocking Tie2 and β1-integrin signaling reduces Ang-1 neuroprotective effects. After both TBI and etoposide treatment microRNA (miR)-711 are upregulated, consistent with its putative role as a negative regulator of Ang-1. We show that miR-711 directly targets the Ang-1 messenger RNA (mRNA), decreasing Ang-1 expression. Increased levels of miR-711 and Ang-1 mRNA are found in the RNA-induced silencing complex complex site of miR-mediated degradation of target mRNAs after etoposide treatment and the miR-711mimic downregulates Ang-1. Administration of miR-711 inhibitor elevates Ang-1 after TBI whereas Ang-1 administration increases Akt activation; reduces Puma, Noxa, Bim, and Bax levels; and attenuates caspase-dependent and -independent neuronal apoptosis 24 h after TBI. Ang-1 also attenuates neuronal degeneration, increases gene expression of molecules that maintain blood-brain barrier integrity, and reduces post-traumatic lesion volume/edema 24 h after TBI. Although we only observed short-term neuroprotective effects after Ang-1 administration, miR-711-dependent downregulation of Ang-1, followed by Akt pathway inhibition, may play a role in neuronal cell death after neuronal injury in vitro and after experimental TBI.
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Affiliation(s)
- Boris Sabirzhanov
- Department of Anesthesiology and Center for Shock, Trauma, and Anesthesiology Research (STAR), University of Maryland , School of Medicine, Baltimore, Maryland
| | - Alan I Faden
- Department of Anesthesiology and Center for Shock, Trauma, and Anesthesiology Research (STAR), University of Maryland , School of Medicine, Baltimore, Maryland
| | - Taryn Aubrecht
- Department of Anesthesiology and Center for Shock, Trauma, and Anesthesiology Research (STAR), University of Maryland , School of Medicine, Baltimore, Maryland
| | - Rebecca Henry
- Department of Anesthesiology and Center for Shock, Trauma, and Anesthesiology Research (STAR), University of Maryland , School of Medicine, Baltimore, Maryland
| | - Ethan Glaser
- Department of Anesthesiology and Center for Shock, Trauma, and Anesthesiology Research (STAR), University of Maryland , School of Medicine, Baltimore, Maryland
| | - Bogdan A Stoica
- Department of Anesthesiology and Center for Shock, Trauma, and Anesthesiology Research (STAR), University of Maryland , School of Medicine, Baltimore, Maryland
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46
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Activated matriptase as a target to treat breast cancer with a drug conjugate. Oncotarget 2018; 9:25983-25992. [PMID: 29899836 PMCID: PMC5995259 DOI: 10.18632/oncotarget.25414] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2018] [Accepted: 03/21/2018] [Indexed: 01/01/2023] Open
Abstract
The antitumor effects of a novel antibody drug conjugate (ADC) was tested against human solid tumor cell lines and against human triple negative breast cancer (TNBC) xenografts in immunosuppressed mice. The ADC targeting activated matriptase of tumor cells was synthesized by using the potent anti-tubulin toxin, monomethyl auristatin-E linked to the activated matriptase-specific monoclonal antibody (M69) via a lysosomal protease-cleavable dipeptide linker. This ADC was found to be cytotoxic against multiple activated matriptase-positive epithelial carcinoma cell lines in vitro and markedly inhibited growth of triple negative breast cancer xenografts and a primary human TNBC (PDX) in vivo. Overexpression of activated matriptase may be a biomarker for response to this ADC. The ADC had potent anti-tumor activity, while the unconjugated M69 antibody was ineffective in a mouse model study using MDA-MB-231 xenografts in mice. Treatment of a human TNBC (MDA-MB-231) showed potent anti-tumor effects in combination with cisplatin in mice. This ADC alone or in combination with cisplatin has the potential to improve the treatment outcomes of patients with TNBC as well as other tumors overexpressing activated matriptase.
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47
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Szabo R, Bugge TH. Loss of HAI-2 in mice with decreased prostasin activity leads to an early-onset intestinal failure resembling congenital tufting enteropathy. PLoS One 2018; 13:e0194660. [PMID: 29617460 PMCID: PMC5884512 DOI: 10.1371/journal.pone.0194660] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2017] [Accepted: 03/07/2018] [Indexed: 01/15/2023] Open
Abstract
Prostasin (CAP1/PRSS8) is a glycosylphosphatidylinositol (GPI)-anchored serine protease that is essential for epithelial development and overall survival in mice. Prostasin is regulated primarily by the transmembrane serine protease inhibitor, hepatocyte growth factor activator inhibitor (HAI)-2, and loss of HAI-2 function leads to early embryonic lethality in mice due to an unregulated prostasin activity. We have recently reported that critical in vivo functions of prostasin can be performed by proteolytically-inactive or zymogen-locked variants of the protease. Here we show that the zymogen form of prostasin does not bind to HAI-2 and, as a result, loss of HAI-2 does not affect prenatal development and survival of mice expressing only zymogen-locked variant of prostasin (Prss8 R44Q). Indeed, HAI-2-deficient mice homozygous for R44Q mutation (Spint2-/-;Prss8R44Q/R44Q) are born in the expected numbers and do not exhibit any obvious developmental abnormality at birth. However, postnatal growth in these mice is severely impaired and they all die within 4 to 7 days after birth due to a critical failure in the development of small and large intestines, characterized by a widespread villous atrophy, tufted villi, near-complete loss of mucin-producing goblet cells, loss of colonic crypt structure, and bleeding into the intestinal lumen. Intestines of Spint2-/-;Prss8R44Q/R44Q mice showed altered expression of epithelial junctional proteins, including reduced levels of EpCAM, E-cadherin, occludin, claudin-1 and -7, as well as an increased level of claudin-4, indicating that the loss of HAI-2 compromises intestinal epithelial barrier function. Our data indicate that the loss of HAI-2 in Prss8R44Q/R44Q mice leads to development of progressive intestinal failure that at both histological and molecular level bears a striking resemblance to human congenital tufting enteropathy, and may provide important clues for understanding and treating this debilitating human disease.
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Affiliation(s)
- Roman Szabo
- Proteases and Tissue Remodeling Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, United States of America
- * E-mail: (RS); (THB)
| | - Thomas H. Bugge
- Proteases and Tissue Remodeling Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, United States of America
- * E-mail: (RS); (THB)
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Schepis A, Barker A, Srinivasan Y, Balouch E, Zheng Y, Lam I, Clay H, Hsiao CD, Coughlin SR. Protease signaling regulates apical cell extrusion, cell contacts, and proliferation in epithelia. J Cell Biol 2018; 217:1097-1112. [PMID: 29301867 PMCID: PMC5839797 DOI: 10.1083/jcb.201709118] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2017] [Revised: 11/27/2017] [Accepted: 12/07/2017] [Indexed: 11/22/2022] Open
Abstract
Mechanisms that sense and regulate epithelial morphogenesis, integrity, and homeostasis are incompletely understood. Protease-activated receptor 2 (Par2), the Par2-activating membrane-tethered protease matriptase, and its inhibitor, hepatocyte activator inhibitor 1 (Hai1), are coexpressed in most epithelia and may make up a local signaling system that regulates epithelial behavior. We explored the role of Par2b in matriptase-dependent skin abnormalities in Hai1a-deficient zebrafish embryos. We show an unexpected role for Par2b in regulation of epithelial apical cell extrusion, roles in regulating proliferation that were opposite in distinct but adjacent epithelial monolayers, and roles in regulating cell-cell junctions, mobility, survival, and expression of genes involved in tissue remodeling and inflammation. The epidermal growth factor receptor Erbb2 and matrix metalloproteinases, the latter induced by Par2b, may contribute to some matriptase- and Par2b-dependent phenotypes and be permissive for others. Our results suggest that local protease-activated receptor signaling can coordinate cell behaviors known to contribute to epithelial morphogenesis and homeostasis.
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Affiliation(s)
- Antonino Schepis
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA
| | - Adrian Barker
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA
| | - Yoga Srinivasan
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA
| | - Eaman Balouch
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA
| | - Yaowu Zheng
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA
| | - Ian Lam
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA
| | - Hilary Clay
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA
| | - Chung-Der Hsiao
- Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li, Taiwan
| | - Shaun R Coughlin
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA
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Böttcher-Friebertshäuser E, Garten W, Klenk HD. Membrane-Anchored Serine Proteases: Host Cell Factors in Proteolytic Activation of Viral Glycoproteins. ACTIVATION OF VIRUSES BY HOST PROTEASES 2018. [PMCID: PMC7122464 DOI: 10.1007/978-3-319-75474-1_8] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Over one third of all known proteolytic enzymes are serine proteases. Among these, the trypsin-like serine proteases comprise one of the best characterized subfamilies due to their essential roles in blood coagulation, food digestion, fibrinolysis, or immunity. Trypsin-like serine proteases possess primary substrate specificity for basic amino acids. Most of the well-characterized trypsin-like proteases such as trypsin, plasmin, or urokinase are soluble proteases that are secreted into the extracellular environment. At the turn of the millennium, a number of novel trypsin-like serine proteases have been identified that are anchored in the cell membrane, either by a transmembrane domain at the N- or C-terminus or via a glycosylphosphatidylinositol (GPI) linkage. Meanwhile more than 20 membrane-anchored serine proteases (MASPs) have been identified in human and mouse, and some of them have emerged as key regulators of mammalian development and homeostasis. Thus, the MASP corin and TMPRSS6/matriptase-2 have been demonstrated to be the activators of the atrial natriuretic peptide (ANP) and key regulator of hepcidin expression, respectively. Furthermore, MASPs have been recognized as host cell factors activating respiratory viruses including influenza virus as well as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses. In particular, transmembrane protease serine S1 member 2 (TMPRSS2) has been shown to be essential for proteolytic activation and consequently spread and pathogenesis of a number of influenza A viruses in mice and as a factor associated with severe influenza virus infection in humans. This review gives an overview on the physiological functions of the fascinating and rapidly evolving group of MASPs and a summary of the current knowledge on their role in proteolytic activation of viral fusion proteins.
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Affiliation(s)
| | - Wolfgang Garten
- 0000 0004 1936 9756grid.10253.35Institut für Virologie, Philipps Universität, Marburg, Germany
| | - Hans Dieter Klenk
- 0000 0004 1936 9756grid.10253.35Institut für Virologie, Philipps-Universität, Marburg, Germany
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Sun X, Du M, Navarre DA, Zhu MJ. Purple Potato Extract Promotes Intestinal Epithelial Differentiation and Barrier Function by Activating AMP-Activated Protein Kinase. Mol Nutr Food Res 2018; 62:10.1002/mnfr.201700536. [PMID: 29193691 PMCID: PMC7192330 DOI: 10.1002/mnfr.201700536] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2017] [Revised: 09/01/2017] [Indexed: 12/20/2022]
Abstract
SCOPE Perturbation of gut epithelial barrier function induces inflammation and other health problems that originate from the gut. Purple potato contains a high content of beneficial polyphenolic compounds. The objective of this study is to evaluate the effect of purple potato extract (PPE) on intestinal differentiation and barrier function, and explore its underlying mechanism using Caco-2 cells and ex vivo cultured gut tissues. METHODS AND RESULTS PPE increases transepithelial electrical resistance and decreases FITC-dextran paracellular flux in Caco-2 cells, which are associated with strengthened intestinal epithelial differentiation in both Caco-2 cells and ex vivo guts. Furthermore, PPE treatment enhances AMP-activated protein kinase (AMPK) activity, concomitant with the increased expression of CDX2, a key transcriptional factor regulating intestinal epithelial differentiation. Knocking out AMPK using CRISPR/Cas9 system abolishes the positive effects of PPE on intestinal epithelial differentiation and barrier function, in junction with the reduced expression of CDX2. CONCLUSION PPE improves gut epithelial differentiation and barrier function via activating AMPK, indicating that PPE, as well as associated purple potato consumption, could be used as a supportive dietary therapeutic strategy for improving gut epithelial health.
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Affiliation(s)
- Xiaofei Sun
- School of Food Science, Washington State University, Pullman, WA 99164
| | - Min Du
- Department of Animal Science, Washington State University, Pullman, WA 99164, USA
| | - Duroy A. Navarre
- Yakima Agricultural Research Laboratory, USDA-ARS, Prosser, WA 99350, USA
| | - Mei-Jun Zhu
- School of Food Science, Washington State University, Pullman, WA 99164
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