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He X, Wang Z, Cheng L, Wang H, Sun Y. An antagonistic role of clock genes and lima1 in kidney regeneration. Commun Biol 2025; 8:29. [PMID: 39789202 PMCID: PMC11718004 DOI: 10.1038/s42003-025-07455-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2024] [Accepted: 01/02/2025] [Indexed: 01/12/2025] Open
Abstract
The circadian clock genes are known important for kidney development, maturation and physiological functions. However, whether and how they play a role in renal regeneration remain elusive. Here, by using the single cell RNA-sequencing (scRNA-seq) technology, we investigated the dynamic gene expression profiles and cell states after acute kidney injury (AKI) by gentamicin treatment in zebrafish. The core clock genes such as per1/2 and nr1d1, which encode transcriptional repressors of the circadian system, are strongly induced in the proximal tubule epithelial cells (PTECs). By generating mutant zebrafish lines, we show that per1a and nr1d1 are required for proper renal regeneration, by facilitating the expression of renal progenitor cell (RPC) genes. In per1a and nr1d1 mutants, the expression of RPC genes and the number of RPCs were decreased, resulting in a marked delay in nephron regeneration. lima1a, which encodes a cytoskeleton binding protein that functions to negatively regulate epithelial to mesenchymal transition (EMT), is identified as the direct target of the clock proteins. Down-regulation of lima1a is associated with enhanced EMT, increased expression of cell migration- and RPC markers, and accelerated nephron regeneration. We propose that per1a and nr1d1 are important for the formation of nephrongenic RPCs by repressing lima1a. Our findings using zebrafish provide important insights into the roles of the clock genes in kidney repair.
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Affiliation(s)
- Xian He
- Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Institute of Hydrobiology, Chinese Academy of Sciences, 430072, Wuhan, China
- The Innovation of Seed Design, Chinese Academy of Sciences, 430072, Wuhan, China
- University of Chinese Academy of Sciences, 100049, Beijing, PR China
| | - Ziming Wang
- Center for Circadian Clocks, and School of Basic Medical Sciences, Suzhou Medical College, Soochow University, 215123, Suzhou, China
| | - Linxi Cheng
- Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Institute of Hydrobiology, Chinese Academy of Sciences, 430072, Wuhan, China
- The Innovation of Seed Design, Chinese Academy of Sciences, 430072, Wuhan, China
- University of Chinese Academy of Sciences, 100049, Beijing, PR China
| | - Han Wang
- Center for Circadian Clocks, and School of Basic Medical Sciences, Suzhou Medical College, Soochow University, 215123, Suzhou, China
| | - Yuhua Sun
- Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Institute of Hydrobiology, Chinese Academy of Sciences, 430072, Wuhan, China.
- The Innovation of Seed Design, Chinese Academy of Sciences, 430072, Wuhan, China.
- University of Chinese Academy of Sciences, 100049, Beijing, PR China.
- Hubei Hongshan Laboratory, 430070, Wuhan, China.
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2
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Zeng J, Wang C, Ruge F, Ji EK, Martin TA, Sanders AJ, Jia S, Hao C, Jiang WG. EPLIN, a prospective oncogenic molecule with contribution to growth, migration and drug resistance in pancreatic cancer. Sci Rep 2024; 14:30850. [PMID: 39730634 DOI: 10.1038/s41598-024-81485-w] [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: 03/26/2024] [Accepted: 11/26/2024] [Indexed: 12/29/2024] Open
Abstract
Most pancreatic cancer patients are diagnosed at advanced stages, with poor survival rates and drug resistance making pancreatic cancer one of the highest causes of cancer death in the UK. Understanding the underlying mechanism behind its carcinogenesis, metastasis and drug resistance has become an essential task for researchers. We have discovered that a well-established tumour suppressor, EPLIN, has an oncogenic rather than suppressive role in pancreatic cancer. Notably, upregulation of EPLIN was observed in pancreatic cancer samples compared to normal samples at RNA and protein levels. Moreover, the presence of EPLIN resulted in poor clinical outcomes in patients. We also report that inhibition of EPLIN led to reduced cellular growth and migration in pancreatic cancer cells. EPLIN regulates expression and phosphorylation levels of several key players in MAPK and PIK3CA-AKT signalling pathways, as well as key contributors of EMT. Furthermore, EPLIN mediates the inhibitory ability PIK3 kinases, MEK and ERK inhibitors have on cell migration. EPLIN was also found to have an impact on pancreatic cancer cells response to chemotherapeutic and EGFR/HER2 targeted therapeutic agents, namely gemcitabine, fluorouracil (5FU) and neratinib (Nerlynx).
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Affiliation(s)
- Jianyuan Zeng
- School of Medicine, Cardiff University, Henry Wellcome Building, Cardiff, CF14 4XN, UK
| | - Cai Wang
- School of Medicine, Cardiff University, Henry Wellcome Building, Cardiff, CF14 4XN, UK
| | - Fiona Ruge
- School of Medicine, Cardiff University, Henry Wellcome Building, Cardiff, CF14 4XN, UK
| | - Edison Ke Ji
- Gastrointestinal Cancer Centre, Peking University Cancer Hospital, Peking University, Fucheng Road, Haidian District, Beijing, China
| | - Tracey A Martin
- School of Medicine, Cardiff University, Henry Wellcome Building, Cardiff, CF14 4XN, UK
| | - Andrew J Sanders
- School of Education and Science, University of Gloucestershire, Francis Close Hall, Swindon Road, Cheltenham, GL50 4AZ, UK
| | - Shuqin Jia
- Gastrointestinal Cancer Centre, Peking University Cancer Hospital, Peking University, Fucheng Road, Haidian District, Beijing, China
| | - Chunyi Hao
- Gastrointestinal Cancer Centre, Peking University Cancer Hospital, Peking University, Fucheng Road, Haidian District, Beijing, China
| | - Wen G Jiang
- School of Medicine, Cardiff University, Henry Wellcome Building, Cardiff, CF14 4XN, UK.
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Hecht M, Alber N, Marhoffer P, Johnsson N, Gronemeyer T. The concerted action of SEPT9 and EPLIN modulates the adhesion and migration of human fibroblasts. Life Sci Alliance 2024; 7:e202201686. [PMID: 38719752 PMCID: PMC11077590 DOI: 10.26508/lsa.202201686] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Revised: 04/29/2024] [Accepted: 04/29/2024] [Indexed: 05/12/2024] Open
Abstract
Septins are cytoskeletal proteins that participate in cell adhesion, migration, and polarity establishment. The septin subunit SEPT9 directly interacts with the single LIM domain of epithelial protein lost in neoplasm (EPLIN), an actin-bundling protein. Using a human SEPT9 KO fibroblast cell line, we show that cell adhesion and migration are regulated by the interplay between both proteins. The low motility of SEPT9-depleted cells could be partly rescued by increased levels of EPLIN. The normal organization of actin-related filopodia and stress fibers was directly dependent on the expression level of SEPT9 and EPLIN. Increased levels of SEPT9 and EPLIN enhanced the size of focal adhesions in cell protrusions, correlating with stabilization of actin bundles. Conversely, decreased levels had the opposite effect. Our work thus establishes the interaction between SEPT9 and EPLIN as an important link between the septin and the actin cytoskeleton, influencing cell adhesion, motility, and migration.
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Affiliation(s)
- Matthias Hecht
- Institute of Molecular Genetics and Cell Biology, James Franck Ring N27, Ulm University, Ulm, Germany
| | - Nane Alber
- Institute of Molecular Genetics and Cell Biology, James Franck Ring N27, Ulm University, Ulm, Germany
| | - Pia Marhoffer
- Institute of Molecular Genetics and Cell Biology, James Franck Ring N27, Ulm University, Ulm, Germany
| | - Nils Johnsson
- Institute of Molecular Genetics and Cell Biology, James Franck Ring N27, Ulm University, Ulm, Germany
| | - Thomas Gronemeyer
- Institute of Molecular Genetics and Cell Biology, James Franck Ring N27, Ulm University, Ulm, Germany
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4
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Lindell E, Zhang X. Exploring the Enigma: The Role of the Epithelial Protein Lost in Neoplasm in Normal Physiology and Cancer Pathogenesis. Int J Mol Sci 2024; 25:4970. [PMID: 38732188 PMCID: PMC11084159 DOI: 10.3390/ijms25094970] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2024] [Revised: 04/28/2024] [Accepted: 04/29/2024] [Indexed: 05/13/2024] Open
Abstract
The cytoskeleton plays a pivotal role in maintaining the epithelial phenotype and is vital to several hallmark processes of cancer. Over the past decades, researchers have identified the epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) as a key regulator of cytoskeletal dynamics, cytoskeletal organization, motility, as well as cell growth and metabolism. Dysregulation of EPLIN is implicated in various aspects of cancer progression, such as tumor growth, invasion, metastasis, and therapeutic resistance. Its altered expression levels or activity can disrupt cytoskeletal dynamics, leading to aberrant cell motility and invasiveness characteristic of malignant cells. Moreover, the involvement of EPLIN in cell growth and metabolism underscores its significance in orchestrating key processes essential for cancer cell survival and proliferation. This review provides a comprehensive exploration of the intricate roles of EPLIN across diverse cellular processes in both normal physiology and cancer pathogenesis. Additionally, this review discusses the possibility of EPLIN as a potential target for anticancer therapy in future studies.
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Affiliation(s)
| | - Xiaonan Zhang
- Department of Immunology, Genetics and Pathology, Uppsala University, SE-751 85 Uppsala, Sweden;
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5
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Ruperti F, Becher I, Stokkermans A, Wang L, Marschlich N, Potel C, Maus E, Stein F, Drotleff B, Schippers KJ, Nickel M, Prevedel R, Musser JM, Savitski MM, Arendt D. Molecular profiling of sponge deflation reveals an ancient relaxant-inflammatory response. Curr Biol 2024; 34:361-375.e9. [PMID: 38181793 DOI: 10.1016/j.cub.2023.12.021] [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: 08/17/2023] [Revised: 11/03/2023] [Accepted: 12/07/2023] [Indexed: 01/07/2024]
Abstract
A hallmark of animals is the coordination of whole-body movement. Neurons and muscles are central to this, yet coordinated movements also exist in sponges that lack these cell types. Sponges are sessile animals with a complex canal system for filter-feeding. They undergo whole-body movements resembling "contractions" that lead to canal closure and water expulsion. Here, we combine live 3D optical coherence microscopy, pharmacology, and functional proteomics to elucidate the sequence and detail of shape changes, the tissues and molecular physiology involved, and the control of these movements. Morphometric analysis and targeted perturbation suggest that the movement is driven by the relaxation of actomyosin stress fibers in epithelial canal cells, which leads to whole-body deflation via collapse of the incurrent and expansion of the excurrent canal system. Thermal proteome profiling and quantitative phosphoproteomics confirm the control of cellular relaxation by an Akt/NO/PKG/PKA pathway. Agitation-induced deflation leads to differential phosphorylation of proteins forming epithelial cell junctions, implying their mechanosensitive role. Unexpectedly, untargeted metabolomics detect a concomitant decrease in antioxidant molecules during deflation, reflecting an increase in reactive oxygen species. Together with the secretion of proteinases, cytokines, and granulin, this indicates an inflammation-like state of the deflating sponge reminiscent of vascular endothelial cells experiencing oscillatory shear stress. These results suggest the conservation of an ancient relaxant-inflammatory response of perturbed fluid-carrying systems in animals and offer a possible mechanism for whole-body coordination through diffusible paracrine signals and mechanotransduction.
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Affiliation(s)
- Fabian Ruperti
- Developmental Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; Collaboration for joint Ph.D. degree between EMBL and Heidelberg University, Faculty of Biosciences 69117 Heidelberg, Germany
| | - Isabelle Becher
- Genome Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | | | - Ling Wang
- Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
| | - Nick Marschlich
- Developmental Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; Centre for Organismal Studies (COS), University of Heidelberg, 69120 Heidelberg, Germany
| | - Clement Potel
- Genome Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Emanuel Maus
- Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Frank Stein
- Proteomics Core Facility, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Bernhard Drotleff
- Metabolomics Core Facility, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Klaske J Schippers
- Developmental Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Michael Nickel
- Bionic consulting Dr. Michael Nickel, 71686 Remseck am Neckar, Germany
| | - Robert Prevedel
- Developmental Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Jacob M Musser
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.
| | - Mikhail M Savitski
- Genome Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; Proteomics Core Facility, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
| | - Detlev Arendt
- Developmental Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; Centre for Organismal Studies (COS), University of Heidelberg, 69120 Heidelberg, Germany.
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Ng GYQ, Loh ZWL, Fann DY, Mallilankaraman K, Arumugam TV, Hande MP. Role of Mitogen-Activated Protein (MAP) Kinase Pathways in Metabolic Diseases. Genome Integr 2024; 15:e20230003. [PMID: 38770527 PMCID: PMC11102075 DOI: 10.14293/genint.14.1.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/22/2024] Open
Abstract
Physiological processes that govern the normal functioning of mammalian cells are regulated by a myriad of signalling pathways. Mammalian mitogen-activated protein (MAP) kinases constitute one of the major signalling arms and have been broadly classified into four groups that include extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38, and ERK5. Each signalling cascade is governed by a wide array of external and cellular stimuli, which play a critical part in mammalian cells in the regulation of various key responses, such as mitogenic growth, differentiation, stress responses, as well as inflammation. This evolutionarily conserved MAP kinase signalling arm is also important for metabolic maintenance, which is tightly coordinated via complicated mechanisms that include the intricate interaction of scaffold proteins, recognition through cognate motifs, action of phosphatases, distinct subcellular localisation, and even post-translational modifications. Aberration in the signalling pathway itself or their regulation has been implicated in the disruption of metabolic homeostasis, which provides a pathophysiological foundation in the development of metabolic syndrome. Metabolic syndrome is an umbrella term that usually includes a group of closely associated metabolic diseases such as hyperglycaemia, hyperlipidaemia, and hypertension. These risk factors exacerbate the development of obesity, diabetes, atherosclerosis, cardiovascular diseases, and hepatic diseases, which have accounted for an increase in the worldwide morbidity and mortality rate. This review aims to summarise recent findings that have implicated MAP kinase signalling in the development of metabolic diseases, highlighting the potential therapeutic targets of this pathway to be investigated further for the attenuation of these diseases.
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Affiliation(s)
- Gavin Yong Quan Ng
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Zachary Wai-Loon Loh
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - David Y. Fann
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Healthy Longevity Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Karthik Mallilankaraman
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Healthy Longevity Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Thiruma V. Arumugam
- School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea
- Department of Physiology, Anatomy & Microbiology, School of Life Sciences, La Trobe University, Bundoora, Victoria, Australia
| | - M. Prakash Hande
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
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7
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Ruperti F, Becher I, Stokkermans A, Wang L, Marschlich N, Potel C, Maus E, Stein F, Drotleff B, Schippers K, Nickel M, Prevedel R, Musser JM, Savitski MM, Arendt D. Molecular profiling of sponge deflation reveals an ancient relaxant-inflammatory response. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.02.551666. [PMID: 37577507 PMCID: PMC10418225 DOI: 10.1101/2023.08.02.551666] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/15/2023]
Abstract
A hallmark of animals is the coordination of whole-body movement. Neurons and muscles are central to this, yet coordinated movements also exist in sponges that lack these cell types. Sponges are sessile animals with a complex canal system for filter-feeding. They undergo whole-body movements resembling "contractions" that lead to canal closure and water expulsion. Here, we combine 3D optical coherence microscopy, pharmacology, and functional proteomics to elucidate anatomy, molecular physiology, and control of these movements. We find them driven by the relaxation of actomyosin stress fibers in epithelial canal cells, which leads to whole-body deflation via collapse of the incurrent and expansion of the excurrent system, controlled by an Akt/NO/PKG/A pathway. A concomitant increase in reactive oxygen species and secretion of proteinases and cytokines indicate an inflammation-like state reminiscent of vascular endothelial cells experiencing oscillatory shear stress. This suggests an ancient relaxant-inflammatory response of perturbed fluid-carrying systems in animals.
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Affiliation(s)
- Fabian Ruperti
- Developmental Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
- Collaboration for joint Ph.D. degree between EMBL and Heidelberg University, Faculty of Biosciences 69117 Heidelberg, Germany
| | - Isabelle Becher
- Genome Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | | | - Ling Wang
- Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Nick Marschlich
- Developmental Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
- Centre for Organismal Studies (COS), University of Heidelberg, 69120 Heidelberg, Germany
| | - Clement Potel
- Genome Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Emanuel Maus
- Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Frank Stein
- Proteomics Core Facility, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Bernhard Drotleff
- Metabolomics Core Facility, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Klaske Schippers
- Developmental Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Michael Nickel
- Bionic Consulting Dr. Michael Nickel, 71686 Remseck am Neckar, Germany
| | - Robert Prevedel
- Developmental Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
- Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Jacob M Musser
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA
| | - Mikhail M Savitski
- Genome Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
- Proteomics Core Facility, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
| | - Detlev Arendt
- Developmental Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
- Centre for Organismal Studies (COS), University of Heidelberg, 69120 Heidelberg, Germany
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8
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Wang X, Zhang C, Song H, Yuan J, Zhang X, Yuan Y, Zhang L, He J. Characterization of LIMA1 and its emerging roles and potential therapeutic prospects in cancers. Front Oncol 2023; 13:1115943. [PMID: 37274282 PMCID: PMC10235525 DOI: 10.3389/fonc.2023.1115943] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2022] [Accepted: 05/02/2023] [Indexed: 06/06/2023] Open
Abstract
Actin is the most abundant and highly conserved cytoskeletal protein present in all eukaryotic cells. Remodeling of the actin cytoskeleton is controlled by a variety of actin-binding proteins that are extensively involved in biological processes such as cell motility and maintenance of cell shape. LIM domain and actin-binding protein 1 (LIMA1), as an important actin cytoskeletal regulator, was initially thought to be a tumor suppressor frequently downregulated in epithelial tumors. Importantly, the deficiency of LIMA1 may be responsible for dysregulated cytoskeletal dynamics, altered cell motility and disrupted cell-cell adhesion, which promote tumor proliferation, invasion and migration. As research progresses, the roles of LIMA1 extend from cytoskeletal dynamics and cell motility to cell division, gene regulation, apical extrusion, angiogenesis, cellular metabolism and lipid metabolism. However, the expression of LIMA1 in malignant tumors and its mechanism of action have not yet been elucidated, and many problems and challenges remain to be addressed. Therefore, this review systematically describes the structure and biological functions of LIMA1 and explores its expression and regulatory mechanism in malignant tumors, and further discusses its clinical value and therapeutic prospects.
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Affiliation(s)
- Xiaoxiao Wang
- Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Taiyuan, China
| | - Chao Zhang
- Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Taiyuan, China
| | - Huangqin Song
- Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Taiyuan, China
| | - Junlong Yuan
- Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Taiyuan, China
| | - Xiaomin Zhang
- Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Taiyuan, China
| | - Yiran Yuan
- Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Taiyuan, China
| | - Lei Zhang
- Department of Hepatobiliary Surgery, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan, China
- Hepatic Surgery Center, Institute of Hepato-Pancreato-Biliary Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Jiefeng He
- Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Taiyuan, China
- Department of Hepatobiliary Surgery, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan, China
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9
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Voigt AL, Dardari R, Lara NLM, He T, Steele H, Dufour A, Orwig KE, Dobrinski I. Multiomics approach to profiling Sertoli cell maturation during development of the spermatogonial stem cell niche. Mol Hum Reprod 2023; 29:gaad004. [PMID: 36688722 PMCID: PMC9976880 DOI: 10.1093/molehr/gaad004] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Revised: 11/10/2022] [Indexed: 01/24/2023] Open
Abstract
Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, a complex process supported by a specialized microenvironment, called the SSC niche. Postnatal development of SSCs is characterized by distinct metabolic transitions from prepubertal to adult stages. An understanding of the niche factors that regulate these maturational events is critical for the clinical application of SSCs in fertility preservation. To investigate the niche maturation events that take place during SSC maturation, we combined different '-omics' technologies. Serial single cell RNA sequencing analysis revealed changes in the transcriptomes indicative of niche maturation that was initiated at 11 years of age in humans and at 8 weeks of age in pigs, as evident by Monocle analysis of Sertoli cells and peritubular myoid cell (PMC) development in humans and Sertoli cell analysis in pigs. Morphological niche maturation was associated with lipid droplet accumulation, a characteristic that was conserved between species. Lipidomic profiling revealed an increase in triglycerides and a decrease in sphingolipids with Sertoli cell maturation in the pig model. Quantitative (phospho-) proteomics analysis detected the activation of distinct pathways with porcine Sertoli cell maturation. We show here that the main aspects of niche maturation coincide with the morphological maturation of SSCs, which is followed by their metabolic maturation. The main aspects are also conserved between the species and can be predicted by changes in the niche lipidome. Overall, this knowledge is pivotal to establishing cell/tissue-based biomarkers that could gauge stem cell maturation to facilitate laboratory techniques that allow for SSC transplantation for restoration of fertility.
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Affiliation(s)
- A L Voigt
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Canada
- Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | - R Dardari
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Canada
- Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | - N L M Lara
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Canada
- Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | - T He
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Canada
- Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | - H Steele
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Canada
- Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | - A Dufour
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Canada
- Department of Physiology and Pharmacology, University of Calgary, Calgary, Canada
| | - K E Orwig
- Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Women’s Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - I Dobrinski
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Canada
- Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
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10
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Samimi H, Tavakoli R, Fallah P, Naderi Sohi A, Amini Shirkouhi M, Naderi M, Haghpanah V. BI-847325, a selective dual MEK and Aurora kinases inhibitor, reduces aggressive behavior of anaplastic thyroid carcinoma on an in vitro three-dimensional culture. Cancer Cell Int 2022; 22:388. [PMID: 36482411 PMCID: PMC9730667 DOI: 10.1186/s12935-022-02813-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2022] [Accepted: 11/28/2022] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Anaplastic thyroid carcinoma (ATC) is the most aggressive subtype of thyroid cancer. In this study, we used a three-dimensional in vitro system to evaluate the effect of a dual MEK/Aurora kinase inhibitor, BI-847325 anticancer drug, on several cellular and molecular processes involved in cancer progression. METHODS Human ATC cell lines, C643 and SW1736, were grown in alginate hydrogel and treated with IC50 values of BI-847325. The effect of BI-847325 on inhibition of kinases function of MEK1/2 and Aurora kinase B (AURKB) was evaluated via Western blot analysis of phospho-ERK1/2 and phospho-Histone H3 levels. Sodium/iodide symporter (NIS) and thyroglobulin (Tg), as two thyroid-specific differentiation markers, were measured by qRT-PCR as well as flow cytometry and immunoradiometric assay. Apoptosis was assessed by Annexin V/PI flow cytometry and BIM, NFκB1, and NFκB2 expressions. Cell cycle distribution and proliferation were determined via P16, AURKA, and AURKB expressions as well as PI and CFSE flow cytometry assays. Multidrug resistance was evaluated by examining the expression of MDR1 and MRP1. Angiogenesis and invasion were investigated by VEGF expression and F-actin labeling with Alexa Fluor 549 Phalloidin. RESULTS Western blot results showed that BI-847325 inhibits MEK1/2 and AURKB functions by decreasing phospho-ERK1/2 and phospho-Histone H3 levels. BI-847325 induced thyroid differentiation markers and apoptosis in ATC cell lines. Inversely, BI-847325 intervention decreased multidrug resistance, cell cycle progression, proliferation, angiogenesis, and invasion at the molecular and/or cellular levels. CONCLUSION The results of the present study suggest that BI-857,325 might be an effective multi-targeted anticancer drug for ATC treatment.
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Affiliation(s)
- Hilda Samimi
- grid.411705.60000 0001 0166 0922Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran ,grid.411463.50000 0001 0706 2472Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
| | - Rezvan Tavakoli
- grid.420169.80000 0000 9562 2611Hepatitis and HIV Department, Pasteur Institute of Iran, Tehran, Iran
| | - Parviz Fallah
- grid.411705.60000 0001 0166 0922Department of Laboratory Science, Faculty of Allied Medicine, Alborz University of Medical Sciences, Karaj, Iran
| | - Alireza Naderi Sohi
- grid.411705.60000 0001 0166 0922Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
| | - Maryam Amini Shirkouhi
- grid.411705.60000 0001 0166 0922Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
| | - Mahmood Naderi
- grid.411705.60000 0001 0166 0922Digestive Diseases Research Center, Digestive Diseases Research Institute, Tehran University of Medical Sciences, Tehran, Iran
| | - Vahid Haghpanah
- grid.411705.60000 0001 0166 0922Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran ,grid.411705.60000 0001 0166 0922Personalized Medicine Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
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11
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Bruno C, Paparo L, Pisapia L, Romano A, Cortese M, Punzo E, Berni Canani R. Protective effects of the postbiotic deriving from cow's milk fermentation with L. paracasei CBA L74 against Rotavirus infection in human enterocytes. Sci Rep 2022; 12:6268. [PMID: 35428750 PMCID: PMC9012738 DOI: 10.1038/s41598-022-10083-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2021] [Accepted: 03/11/2022] [Indexed: 01/17/2023] Open
Abstract
Rotavirus (RV) is the leading cause of acute gastroenteritis-associated mortality in early childhood. Emerging clinical evidence suggest the efficacy of the postbiotic approach based on cow's milk fermentation with the probiotic Lacticaseibacillus paracasei CBAL74 (FM-CBAL74) in preventing pediatric acute gastroenteritis, but the mechanisms of action are still poorly characterized. We evaluated the protective action of FM-CBAL74 in an in vitro model of RV infection in human enterocytes. The number of infected cells together with the relevant aspects of RV infection were assessed: epithelial barrier damage (tight-junction proteins and transepithelial electrical resistance evaluation), and inflammation (reactive oxygen species, pro-inflammatory cytokines IL-6, IL-8 and TNF-α, and mitogen-activated protein kinase pathway activation). Pre-incubation with FM-CBA L74 resulted in an inhibition of epithelial barrier damage and inflammation mediated by mitogen-activated protein kinase pathway activation induced by RV infection. Modulating several protective mechanisms, the postbiotic FM-CBAL74 exerted a preventive action against RV infection. This approach could be a disrupting nutritional strategy against one of the most common killers for the pediatric age.
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Affiliation(s)
- Cristina Bruno
- Department of Translational Medical Science, University of Naples Federico II, Naples, Italy.,ImmunoNutritionLab at CEINGE Advanced Biotechnologies, University of Naples Federico II, Naples, Italy
| | - Lorella Paparo
- Department of Translational Medical Science, University of Naples Federico II, Naples, Italy.,ImmunoNutritionLab at CEINGE Advanced Biotechnologies, University of Naples Federico II, Naples, Italy
| | - Laura Pisapia
- Institute of Genetics and Biophysics, CNR, Naples, Italy
| | - Alessia Romano
- ImmunoNutritionLab at CEINGE Advanced Biotechnologies, University of Naples Federico II, Naples, Italy
| | - Maddalena Cortese
- Department of Translational Medical Science, University of Naples Federico II, Naples, Italy.,ImmunoNutritionLab at CEINGE Advanced Biotechnologies, University of Naples Federico II, Naples, Italy
| | - Erika Punzo
- Department of Translational Medical Science, University of Naples Federico II, Naples, Italy.,ImmunoNutritionLab at CEINGE Advanced Biotechnologies, University of Naples Federico II, Naples, Italy
| | - Roberto Berni Canani
- Department of Translational Medical Science, University of Naples Federico II, Naples, Italy. .,ImmunoNutritionLab at CEINGE Advanced Biotechnologies, University of Naples Federico II, Naples, Italy. .,European Laboratory for the Investigation of Food-Induced Diseases, University of Naples Federico II, Naples, Italy. .,Task Force for Microbiome Studies, University of Naples Federico II, Naples, Italy. .,Task Force for Nutraceuticals and Functional Foods, University of Naples Federico II, Naples, Italy.
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12
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Identification and validation of new ERK substrates by phosphoproteomic technologies including Phos-tag SDS-PAGE. J Proteomics 2022; 258:104543. [DOI: 10.1016/j.jprot.2022.104543] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2021] [Revised: 02/15/2022] [Accepted: 02/15/2022] [Indexed: 11/17/2022]
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13
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Duethorn B, Groll F, Rieger B, Drexler HCA, Brinkmann H, Kremer L, Stehling M, Borowski MT, Mildner K, Zeuschner D, Zernicka-Goetz M, Stemmler MP, Busch KB, Vaquerizas JM, Bedzhov I. Lima1 mediates the pluripotency control of membrane dynamics and cellular metabolism. Nat Commun 2022; 13:610. [PMID: 35105859 PMCID: PMC8807836 DOI: 10.1038/s41467-022-28139-5] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2021] [Accepted: 01/10/2022] [Indexed: 12/13/2022] Open
Abstract
Lima1 is an extensively studied prognostic marker of malignancy and is also considered to be a tumour suppressor, but its role in a developmental context of non-transformed cells is poorly understood. Here, we characterise the expression pattern and examined the function of Lima1 in mouse embryos and pluripotent stem cell lines. We identify that Lima1 expression is controlled by the naïve pluripotency circuit and is required for the suppression of membrane blebbing, as well as for proper mitochondrial energetics in embryonic stem cells. Moreover, forcing Lima1 expression enables primed mouse and human pluripotent stem cells to be incorporated into murine pre-implantation embryos. Thus, Lima1 is a key effector molecule that mediates the pluripotency control of membrane dynamics and cellular metabolism.
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Affiliation(s)
- Binyamin Duethorn
- Embryonic Self-Organization research group, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149, Münster, Germany
| | - Fabian Groll
- Regulatory Genomics group, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149, Münster, Germany
| | - Bettina Rieger
- Institut für Integrative Zellbiologie und Physiologie, University of Münster, Schlossplatz 5, 48149, Münster, Germany
| | - Hannes C A Drexler
- Mass Spectrometry Unit, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149, Münster, Germany
| | - Heike Brinkmann
- Embryonic Self-Organization research group, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149, Münster, Germany
| | - Ludmila Kremer
- Transgenic Facility, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149, Münster, Germany
| | - Martin Stehling
- Flow Cytometry Unit, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149, Münster, Germany
| | - Marie-Theres Borowski
- Institut für Integrative Zellbiologie und Physiologie, University of Münster, Schlossplatz 5, 48149, Münster, Germany
| | - Karina Mildner
- Electron Microscopy Facility, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149, Münster, Germany
| | - Dagmar Zeuschner
- Electron Microscopy Facility, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149, Münster, Germany
| | - Magdalena Zernicka-Goetz
- Mammalian Embryo and Stem Cell Group, Department of Physiology, Development, and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK.,Plasticity and Self-Organization Group, Division of Biology and Biological Engineering, California Institute of Technology (Caltech), Pasadena, CA, 91125, USA
| | - Marc P Stemmler
- Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, FAU University Erlangen-Nürnberg, Erlangen, Germany
| | - Karin B Busch
- Institut für Integrative Zellbiologie und Physiologie, University of Münster, Schlossplatz 5, 48149, Münster, Germany
| | - Juan M Vaquerizas
- Regulatory Genomics group, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149, Münster, Germany.,MRC London Institute of Medical Sciences, Du Cane Road, W12 0NN, London, UK.,Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK
| | - Ivan Bedzhov
- Embryonic Self-Organization research group, Max Planck Institute for Molecular Biomedicine, Röntgenstraße 20, 48149, Münster, Germany.
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14
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Zeng J, Jiang WG, Sanders AJ. Epithelial Protein Lost in Neoplasm, EPLIN, the Cellular and Molecular Prospects in Cancers. Biomolecules 2021; 11:biom11071038. [PMID: 34356662 PMCID: PMC8301816 DOI: 10.3390/biom11071038] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2021] [Revised: 07/11/2021] [Accepted: 07/13/2021] [Indexed: 11/16/2022] Open
Abstract
Epithelial Protein Lost In Neoplasm (EPLIN), also known as LIMA1 (LIM Domain And Actin Binding 1), was first discovered as a protein differentially expressed in normal and cancerous cell lines. It is now known to be key to the progression and metastasis of certain solid tumours. Despite a slow pace in understanding the biological role in cells and body systems, as well as its clinical implications in the early years since its discovery, recent years have witnessed a rapid progress in understanding the mechanisms of this protein in cells, diseases and indeed the body. EPLIN has drawn more attention over the past few years with its roles expanding from cell migration and cytoskeletal dynamics, to cell cycle, gene regulation, angiogenesis/lymphangiogenesis and lipid metabolism. This concise review summarises and discusses the recent progress in understanding EPLIN in biological processes and its implications in cancer.
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15
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Kong J, Shen S, Zhang Z, Wang W. Identification of hub genes and pathways in cholangiocarcinoma by coexpression analysis. Cancer Biomark 2020; 27:505-517. [PMID: 32116234 DOI: 10.3233/cbm-190038] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
BACKGROUND Cholangiocarcinoma (CCA) is the most common biliary malignancy worldwide. However, the molecular mechanisms of its tumorigenesis and progression are still largely unclear. OBJECTIVE This study aimed to explore the hub genes and pathways associated with CCA prognosis by coexpression analysis. METHODS A coexpression network complex was constructed using the top 20% most variant genes in the GSE89748 dataset to find modules associated with prognosis related clinical trait-histology. The hub genes in the clinically significant modules were defined as candidates if they were common in both the coexpression network and protein-protein interaction (PPI) network. Afterwards, survival analysis, expression level analysis and a series of bioinformatic analysis were used to validate the hub genes. RESULTS Twenty-five modules were obtained, and the cyan, light cyan and red modules regarded as closely associated with histology were selected. Subsequently, combining the PPI network complexes and coexpression networks, we screened 20 candidates. After expression and survival analysis, 10 real hub genes (LIMA1, HDAC1, ITGA3, ACTR3, GSK3B, ITGA2, THOC2, PTGES3, HEATR1 and ILF2) were finally identified. Additionally, functional enrichment analysis revealed that the hub genes were mainly enriched in cell cycle-related pathways. CONCLUSIONS Overall, this study identified 10 hub genes and cell cycle-related pathways were closely related to CCA development, progression and prognosis, which may contribute to CCA diagnosis and treatment.
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16
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Taha M, Aldirawi M, März S, Seebach J, Odenthal-Schnittler M, Bondareva O, Bojovic V, Schmandra T, Wirth B, Mietkowska M, Rottner K, Schnittler H. EPLIN-α and -β Isoforms Modulate Endothelial Cell Dynamics through a Spatiotemporally Differentiated Interaction with Actin. Cell Rep 2020; 29:1010-1026.e6. [PMID: 31644899 DOI: 10.1016/j.celrep.2019.09.043] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Revised: 08/08/2019] [Accepted: 09/13/2019] [Indexed: 12/14/2022] Open
Abstract
Actin-binding proteins are essential for linear and branched actin filament dynamics that control shape change, cell migration, and cell junction remodeling in vascular endothelium (endothelial cells [ECs]). The epithelial protein lost in neoplasm (EPLIN) is an actin-binding protein, expressed as EPLIN-α and EPLIN-β by alternative promoters; however, the isoform-specific functions are not yet understood. Aortic compared to cava vein ECs and shear stress-exposed cultured ECs express increased EPLIN-β levels that stabilize stress fibers. In contrast, EPLIN-α expression is increased in growing and migrating ECs, is targeted to membrane protrusions, and terminates their growth via interaction with the Arp2/3 complex. The data indicate that EPLIN-α controls protrusion dynamics while EPLIN-β has an actin filament stabilizing role, which is consistent with FRAP analyses demonstrating a lower EPLIN-β turnover rate compared to EPLIN-α. Together, EPLIN isoforms differentially control actin dynamics in ECs, essential in shear stress responses, cell migration, and barrier function.
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Affiliation(s)
- Muna Taha
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms University of Münster, 48149 Münster, Germany; Cells-in-Motion Cluster of Excellence (EXC 1003 CiM), Westfälische Wilhelms University of Münster, 48149 Münster, Germany
| | - Mohammed Aldirawi
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms University of Münster, 48149 Münster, Germany; Cells-in-Motion Cluster of Excellence (EXC 1003 CiM), Westfälische Wilhelms University of Münster, 48149 Münster, Germany
| | - Sigrid März
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms University of Münster, 48149 Münster, Germany; Cells-in-Motion Cluster of Excellence (EXC 1003 CiM), Westfälische Wilhelms University of Münster, 48149 Münster, Germany
| | - Jochen Seebach
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms University of Münster, 48149 Münster, Germany; Cells-in-Motion Cluster of Excellence (EXC 1003 CiM), Westfälische Wilhelms University of Münster, 48149 Münster, Germany
| | - Maria Odenthal-Schnittler
- Cells-in-Motion Cluster of Excellence (EXC 1003 CiM), Westfälische Wilhelms University of Münster, 48149 Münster, Germany; Department of Ophthalmology, Westfälische Wilhelms University of Münster, Medical Center, 48149 Münster, Germany
| | - Olga Bondareva
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms University of Münster, 48149 Münster, Germany; Cells-in-Motion Cluster of Excellence (EXC 1003 CiM), Westfälische Wilhelms University of Münster, 48149 Münster, Germany
| | - Vesna Bojovic
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms University of Münster, 48149 Münster, Germany
| | - Thomas Schmandra
- Heart and Vascular Clinic Bad Neustadt, Bad Neustadt a.d. Saale, Germany
| | - Benedikt Wirth
- Cells-in-Motion Cluster of Excellence (EXC 1003 CiM), Westfälische Wilhelms University of Münster, 48149 Münster, Germany; Institute for Analysis and Numerics, Westfälische Wilhelms University of Münster, 48149 Münster Germany
| | - Magdalena Mietkowska
- Division of Molecular Cell Biology, Zoological Institute, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany; Molecular Cell Biology Group, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany
| | - Klemens Rottner
- Division of Molecular Cell Biology, Zoological Institute, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany; Molecular Cell Biology Group, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany
| | - Hans Schnittler
- Institute of Anatomy and Vascular Biology, Westfälische Wilhelms University of Münster, 48149 Münster, Germany; Cells-in-Motion Cluster of Excellence (EXC 1003 CiM), Westfälische Wilhelms University of Münster, 48149 Münster, Germany.
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17
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Bhattacharya A, Ghosh P, Prasad R, Ghosh A, Das K, Roy A, Mallik S, Sinha DK, Sen P. MAP Kinase driven actomyosin rearrangement is a crucial regulator of monocyte to macrophage differentiation. Cell Signal 2020; 73:109691. [PMID: 32531262 DOI: 10.1016/j.cellsig.2020.109691] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2019] [Revised: 06/05/2020] [Accepted: 06/06/2020] [Indexed: 12/31/2022]
Abstract
Rearrangement of actin cytoskeleton correlates significantly with the immune responses as the perturbation of cytoskeletal dynamics leads to many immune deficiencies. Mechanistic insights into this correlation remain unknown. Cellular spreading, the most characteristic phenotype associated with monocyte to macrophage differentiation, led us to investigate the contribution of actomyosin dynamics in monocyte differentiation. Our observation revealed that actomyosin reorganization intrinsically governs the process of monocyte to macrophage differentiation. Further, we established that the MAPK-driven signaling pathways regulate the cellular actomyosin dynamics that direct monocyte to macrophage differentiation. We also identified P42/44 Mitogen-Activated Protein Kinase (P42/44 MAPK), P38 Mitogen-Activated Protein Kinase (P38 MAPK), MAP Kinase Activated Protein Kinase 2 (MK-2), Heat Shock Protein 27 (Hsp-27), Lim Kinase (Lim K), non-muscle cofilin (n-cofilin), Myosin Light Chain Kinase (MLCK) and Myosin Light Chain (MLC) as critical components of the signaling network. Moreover, we have shown the involvement of the same signaling cascade in 3D gel-like microenvironment induced spontaneous monocyte to macrophage differentiation and in human blood-derived PBMC differentiation. Our study reveals new mechanistic insights into the process of monocyte to macrophage differentiation.
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Affiliation(s)
- Anindita Bhattacharya
- Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India
| | - Purnam Ghosh
- Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India
| | - Ramesh Prasad
- Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India
| | - Arnab Ghosh
- Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India
| | - Kaushik Das
- Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India
| | - Abhishek Roy
- Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India
| | - Suman Mallik
- Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India
| | - Deepak Kumar Sinha
- Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India
| | - Prosenjit Sen
- Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India.
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18
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Early Events in Actin Cytoskeleton Dynamics and E-Cadherin-Mediated Cell-Cell Adhesion during Epithelial-Mesenchymal Transition. Cells 2020; 9:cells9030578. [PMID: 32121325 PMCID: PMC7140442 DOI: 10.3390/cells9030578] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2020] [Accepted: 02/28/2020] [Indexed: 12/21/2022] Open
Abstract
Epithelial-mesenchymal transition (EMT) plays an important role in development and also in initiation of metastasis during cancer. Disruption of cell-cell contacts during EMT allowing cells to detach from and migrate away from their neighbors remains poorly understood. Using immunofluorescent staining and live-cell imaging, we analyzed early events during EMT induced by epidermal growth factor (EGF) in IAR-20 normal epithelial cells. Control cells demonstrated stable adherens junctions (AJs) and robust contact paralysis, whereas addition of EGF caused rapid dynamic changes at the cell-cell boundaries: fragmentation of the circumferential actin bundle, assembly of actin network in lamellipodia, and retrograde flow. Simultaneously, an actin-binding protein EPLIN was phosphorylated, which may have decreased the stability of the circumferential actin bundle. Addition of EGF caused gradual replacement of linear E-cadherin–based AJs with dynamic and unstable punctate AJs, which, unlike linear AJs, colocalized with the mechanosensitive protein zyxin, confirming generation of centripetal force at the sites of cell-cell contacts during EMT. Our data show that early EMT promotes heightened dynamics at the cell-cell boundaries—replacement of stable AJs and actin structures with dynamic ones—which results in overall weakening of cell-cell adhesion, thus priming the cells for front-rear polarization and eventual migration.
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19
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Farmer LK, Rollason R, Whitcomb DJ, Ni L, Goodliff A, Lay AC, Birnbaumer L, Heesom KJ, Xu SZ, Saleem MA, Welsh GI. TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility. J Am Soc Nephrol 2019; 30:1910-1924. [PMID: 31416818 DOI: 10.1681/asn.2018070729] [Citation(s) in RCA: 55] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Accepted: 06/17/2019] [Indexed: 11/03/2022] Open
Abstract
BACKGROUND Mutations in the transient receptor potential channel 6 (TRPC6) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with TRPC6 mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified role within the target cell for FSGS, the kidney podocyte. METHODS We generated a stable TRPC6 knockout podocyte cell line from TRPC6 knockout mice. These cells were engineered to express wild-type TRPC6, a dominant negative TRPC6 mutation, or either of two disease-causing mutations of TRPC6, G109S or K874*. We extensively characterized these cells using motility, detachment, and calpain activity assays; immunofluorescence; confocal or total internal reflection fluorescence microscopy; and western blotting. RESULTS Compared with wild-type cells, TRPC6-/- podocytes are less motile and more adhesive, with an altered actin cytoskeleton. We found that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon (a calmodulin- and actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, and motility via cleavage of paxillin, focal adhesion kinase, and talin). Knockdown or expression of the truncated K874* mutation (but not expression of the gain-of-function G019S mutation or dominant negative mutant of TRPC6) results in the mislocalization of calpain 1 and 2 and significant downregulation of calpain activity; this leads to altered podocyte cytoskeleton, motility, and adhesion-characteristics of TRPC6 -/- podocytes. CONCLUSIONS Our data demonstrate that independent of TRPC6 channel activity, the physical interaction between TRPC6 and calpain in the podocyte is important for cell motility and detachment and demonstrates a scaffolding role of the TRPC6 protein in disease.
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Affiliation(s)
| | | | - Daniel J Whitcomb
- Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, Bristol Medical School, and
| | - Lan Ni
- Bristol Renal, Bristol Medical School
| | | | | | - Lutz Birnbaumer
- Neurobiology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina.,Faculty of Medical Sciences, Institute of Biomedical Research, Catholic University of Argentina, Buenos Aires, Argentina; and
| | - Kate J Heesom
- Proteomics Facility, University of Bristol, Bristol, United Kingdom
| | - Shang-Zhong Xu
- Centre for Cardiovascular and Metabolic Research, Hull York Medical School, University of Hull, Hull, United Kingdom
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20
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Collins RJ, Morgan LD, Owen S, Ruge F, Jiang WG, Sanders AJ. Mechanistic insights of epithelial protein lost in neoplasm in prostate cancer metastasis. Int J Cancer 2018; 143:2537-2550. [PMID: 30098000 DOI: 10.1002/ijc.31786] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2018] [Revised: 07/12/2018] [Accepted: 07/24/2018] [Indexed: 01/21/2023]
Abstract
EPLIN is frequently downregulated or lost in various cancers. The purpose of this study was to evaluate the importance of EPLIN in prostate cancer progression, with particular focus on the mechanistic implications to elucidate EPLIN's tumor suppressive function in cancer. EPLIN expression was evaluated in prostate cancer cell lines and tissues. PC-3 and LNCaP EPLINα overexpression models were generated through transfection with EPLINα sequence and EPLIN knockdown was achieved using shRNA in CA-HPV-10 cells. Functional assays were performed to evaluate cellular characteristics and potential mechanisms were evaluated using a protein microarray, and validated using western blot analysis. EPLIN expression was reduced in clinical prostate cancer sections, including hyperplasia (p ≤ 0.001) and adenocarcinoma (p = 0.005), when compared to normal prostate tissue. EPLINα overexpression reduced cell growth, migration and invasion, and influenced transcript, protein and phosphoprotein expression of paxillin, FAK and Src. EPLIN knockdown increased the invasive and migratory nature of CA-HPV-10 cells and also induced changes to FAK and Src total and/or phospho expression. Functional characterization of cellular migration and invasion in addition to FAK and Src inhibition demonstrated differential effects between control and EPLINα overexpression and EPLIN knockdown cell lines. This study highlights that EPLIN expression in prostate cancer is able to influence several aspects of cancer cell characteristics, including cell growth, migration and invasion. The mechanism of the tumor suppressive action of EPLIN remains to be fully elucidated; and this study proposes a role for EPLIN's ability to regulate the aggressive characteristics of prostate cancer cells partially through regulating FAK/Src signaling.
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Affiliation(s)
- Ross J Collins
- Cardiff China Medical Research Collaborative (CCMRC), Cardiff University School of Medicine, Cardiff, United Kingdom
| | - Liam D Morgan
- Cardiff China Medical Research Collaborative (CCMRC), Cardiff University School of Medicine, Cardiff, United Kingdom
| | - Sioned Owen
- Cardiff China Medical Research Collaborative (CCMRC), Cardiff University School of Medicine, Cardiff, United Kingdom
| | - Fiona Ruge
- Cardiff China Medical Research Collaborative (CCMRC), Cardiff University School of Medicine, Cardiff, United Kingdom
| | - Wen G Jiang
- Cardiff China Medical Research Collaborative (CCMRC), Cardiff University School of Medicine, Cardiff, United Kingdom
| | - Andrew J Sanders
- Cardiff China Medical Research Collaborative (CCMRC), Cardiff University School of Medicine, Cardiff, United Kingdom
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21
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Miao Y, Tipakornsaowapak T, Zheng L, Mu Y, Lewellyn E. Phospho-regulation of intrinsically disordered proteins for actin assembly and endocytosis. FEBS J 2018; 285:2762-2784. [PMID: 29722136 DOI: 10.1111/febs.14493] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2017] [Revised: 04/04/2018] [Accepted: 04/26/2018] [Indexed: 12/13/2022]
Abstract
Actin filament assembly contributes to the endocytic pathway pleiotropically, with active roles in clathrin-dependent and clathrin-independent endocytosis as well as subsequent endosomal trafficking. Endocytosis comprises a series of dynamic events, including the initiation of membrane curvature, bud invagination, vesicle abscission and subsequent vesicular transport. The ultimate success of endocytosis requires the coordinated activities of proteins that trigger actin polymerization, recruit actin-binding proteins (ABPs) and organize endocytic proteins (EPs) that promote membrane curvature through molecular crowding or scaffolding mechanisms. A particularly interesting phenomenon is that multiple EPs and ABPs contain a substantial percentage of intrinsically disordered regions (IDRs), which can contribute to protein coacervation and phase separation. In addition, intrinsically disordered proteins (IDPs) frequently contain sites for post-translational modifications (PTMs) such as phosphorylation, and these modifications exhibit a high preference for IDR residues [Groban ES et al. (2006) PLoS Comput Biol 2, e32]. PTMs are implicated in regulating protein function by modulating the protein conformation, protein-protein interactions and the transition between order and disorder states of IDPs. The molecular mechanisms by which IDRs of ABPs and EPs fine-tune actin assembly and endocytosis remain mostly unexplored and elusive. In this review, we analyze protein sequences of budding yeast EPs and ABPs, and discuss the potential underlying mechanisms for regulating endocytosis and actin assembly through the emerging concept of IDR-mediated protein multivalency, coacervation, and phase transition, with an emphasis on the phospho-regulation of IDRs. Finally, we summarize the current understanding of how these mechanisms coordinate actin cytoskeleton assembly and membrane curvature formation during endocytosis in budding yeast.
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Affiliation(s)
- Yansong Miao
- School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.,School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore
| | | | - Liangzhen Zheng
- School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
| | - Yuguang Mu
- School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
| | - Eric Lewellyn
- Department of Biology, Division of Natural Sciences, St Norbert College, De Pere, WI, USA
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22
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Pal R, Bondar VV, Adamski CJ, Rodney GG, Sardiello M. Inhibition of ERK1/2 Restores GSK3β Activity and Protein Synthesis Levels in a Model of Tuberous Sclerosis. Sci Rep 2017. [PMID: 28646232 PMCID: PMC5482840 DOI: 10.1038/s41598-017-04528-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Tuberous sclerosis (TS) is a multi-organ autosomal dominant disorder that is best characterized by neurodevelopmental deficits and the presence of benign tumors. TS pathology is caused by mutations in tuberous sclerosis complex (TSC) genes and is associated with insulin resistance, decreased glycogen synthase kinase 3β (GSK3β) activity, activation of the mammalian target of rapamycin complex 1 (mTORC1), and subsequent increase in protein synthesis. Here, we show that extracellular signal–regulated kinases (ERK1/2) respond to insulin stimulation and integrate insulin signaling to phosphorylate and thus inactivate GSK3β, resulting in increased protein synthesis that is independent of Akt/mTORC1 activity. Inhibition of ERK1/2 in Tsc2−/− cells—a model of TS—rescues GSK3β activity and protein synthesis levels, thus highlighting ERK1/2 as a potential therapeutic target for the treatment of TS.
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Affiliation(s)
- Rituraj Pal
- Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA.,Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA
| | - Vitaliy V Bondar
- Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA
| | - Carolyn J Adamski
- Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA
| | - George G Rodney
- Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA.,Cardiovascular Research Institute, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA.,Center for Space Medicine, Baylor College of Medicine Bioscience Research Collaborative, Houston, TX, 77030, USA
| | - Marco Sardiello
- Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA. .,Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX, 77030, USA.
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23
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Human phosphatase CDC14A regulates actin organization through dephosphorylation of epithelial protein lost in neoplasm. Proc Natl Acad Sci U S A 2017; 114:5201-5206. [PMID: 28465438 DOI: 10.1073/pnas.1619356114] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
CDC14 is an essential dual-specificity phosphatase that counteracts CDK1 activity during anaphase to promote mitotic exit in Saccharomyces cerevisiae Surprisingly, human CDC14A is not essential for cell cycle progression. Instead, it regulates cell migration and cell adhesion. Little is known about the substrates of hCDC14A and the counteracting kinases. Here, we combine phospho-proteome profiling and proximity-dependent biotin identification to identify hCDC14A substrates. Among these targets were actin regulators, including the tumor suppressor eplin. hCDC14A counteracts EGF-induced rearrangements of actin cytoskeleton by dephosphorylating eplin at two known extracellular signal-regulated kinase sites, serine 362 and 604. hCDC14APD and eplin knockout cell lines exhibited down-regulation of E-cadherin and a reduction in α/β-catenin at cell-cell adhesions. Reduction in the levels of hCDC14A and eplin mRNA is frequently associated with colorectal carcinoma and is correlated with poor prognosis. We therefore propose that eplin dephosphorylation by hCDC14A reduces actin dynamics to restrict tumor malignancy.
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24
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Wu D. Epithelial protein lost in neoplasm (EPLIN): Beyond a tumor suppressor. Genes Dis 2017; 4:100-107. [PMID: 30258911 PMCID: PMC6136588 DOI: 10.1016/j.gendis.2017.03.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2016] [Accepted: 03/25/2017] [Indexed: 12/25/2022] Open
Abstract
The majority of cancer-related deaths are caused by tumor recurrence, metastasis and therapeutic resistance. During the late stages of tumor progression, multiple factors are involved, including the downregulation and/or loss of function of metastasis suppressors. Epithelial protein lost in neoplasm (EPLIN), an actin-binding protein, was initially identified as a putative tumor suppressor that is frequently downregulated in epithelial tumors. Recent evidence indicates that EPLIN may negatively regulate epithelia-to-mesenchymal transition (EMT), a crucial process by which cancer cells acquire invasive capabilities and therapeutic resistance. Importantly, downregulation of EPLIN is associated with clinical metastasis in a variety of solid tumors, suggesting that EPLIN could be a suppressor of metastasis. In this review, I will discuss the regulation and function of EPLIN in human cancer cells and explore the clinical significance of EPLIN in metastatic disease.
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Affiliation(s)
- Daqing Wu
- Georgia Cancer Center and Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, Augusta, GA, USA,MetCure Therapeutics LLC, Atlanta, GA, USA,Corresponding author. Georgia Cancer Center and Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, Augusta, GA, USA.
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25
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Schroeder CC, Scariot JSVR, Ribeiro JCZGM, Deliberador TM, Giovanini AMMAF. Platelet Rich Plasma (PRP) Produces an Atherofibrotic Histophenotype During Craniofacial Bone Repair Due to Changes of Immunohistochemical Expression of Erk1/2, p38α/β, Adiponectin and Elevated Presence of Cells Exhibiting B-scavenger Receptor (CD36+). Braz Dent J 2017; 27:243-54. [PMID: 27224555 DOI: 10.1590/0103-6440201602450] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2015] [Accepted: 04/18/2016] [Indexed: 11/22/2022] Open
Abstract
The platelet-extracellular matrix interaction in platelet rich plasma (PRP) through thrombospondin receptor-CD36 induces the secretion of growth factors responsible for cellular proliferation and differentiation during the repair process. Since CD36 also acts as a class B-scavenger-receptor for development of foam-like cells and mitogen-activated kinases, such as Erk1/2 and p38α/β, are important proteins activated by platelet growth factor, the aim of this study was to evaluate the immunohistochemical presence of CD36, Erk1/2, p38α/β during the bone repair treated and non-treated with PRP and to compare these results with the histomorphometry of repair. Simultaneously, the immunopresence of adiponectin was analyzed, which may contribute to osteogenesis at the same time it inhibits fibrosis and impairs adipogenesis and foam cell formation in the medullary area. An artificial bone defect measuring 5×1 mm was produced in the calvaria of 56 Wistar rats. The defects were randomly treated with autograft, autograft+PRP, PRP alone and sham. The animals were euthanized at 2 and 6 weeks post-surgery. Data were analyzed by ANOVA followed by non-parametric test Student Newman-Keuls (p<0.05) for histomorphometric and immunohistochemical interpretation. The results revealed that in specimens that received PRP the immunopositivity for Erk1/2, p38α/β and CD36 proteins increased significantly while the immunohistochemical expression of adiponectin decreased simultaneously. There was also an accentuated reduction of bone matrix deposition and increase of the medullary area represented by fibrosis and/or presence of foam-like cells, which exhibited immunophenotype CD36+adiponectin. The findings of this study suggest that PRP acted as an inhibitor of osteogenesis during the craniofacial bone repair and induced a pathological condition that mimics an atherofibrotic condition.
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26
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Kosako H, Motani K. Global Identification of ERK Substrates by Phosphoproteomics Based on IMAC and 2D-DIGE. Methods Mol Biol 2017; 1487:137-149. [PMID: 27924564 DOI: 10.1007/978-1-4939-6424-6_10] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Extracellular signal-regulated kinase (ERK) regulates various cellular functions through phosphorylation of numerous downstream substrates, which have not yet been fully characterized. To date, several phosphoproteomic approaches have been employed to identify novel substrates for ERK. In this chapter, we describe a method to globally identify ERK substrates by combining immobilized metal affinity chromatography (IMAC) and two-dimensional difference gel electrophoresis (2D-DIGE) followed by mass spectrometry. Phosphoprotein enrichment by IMAC enables the subsequent detection of many protein spots with different fluorescence intensities between ERK-inhibited and -activated cells in 2D-DIGE analysis. Furthermore, the advanced sensitivity and resolution of liquid chromatography coupled with tandem mass spectrometry allow for a direct identification of proteins obtained from silver-stained 2D-DIGE gels. Validation experiments such as Phos-tag Western blotting are important steps to further elucidate the functional roles of ERK-mediated phosphorylation of these newly identified substrates.
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Affiliation(s)
- Hidetaka Kosako
- Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan.
| | - Kou Motani
- Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan
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27
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Tochiki KK, Maiarú M, Norris C, Hunt SP, Géranton SM. The mitogen and stress-activated protein kinase 1 regulates the rapid epigenetic tagging of dorsal horn neurons and nocifensive behaviour. Pain 2016; 157:2594-2604. [PMID: 27482631 PMCID: PMC5065054 DOI: 10.1097/j.pain.0000000000000679] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2016] [Revised: 07/15/2016] [Accepted: 07/20/2016] [Indexed: 12/15/2022]
Abstract
Phosphorylation of histone H3 at serine 10 (p-H3S10) is a marker of active gene transcription. Using cognitive models of neural plasticity, p-H3S10 was shown to be downstream of extracellular signal-regulated kinase (ERK) signalling in the hippocampus. In this study, we show that nociceptive signalling after peripheral formalin injection increased p-H3S10 expression in the ipsilateral dorsal horn. This increase was maximal 30 minutes after formalin injection and occurred mainly within p-ERK-positive neurons. Spinal p-H3S10-enhanced expression was also observed in neurokinin 1 receptor (NK1R), c-Fos, and Zif268 positive neurons and was inhibited by ablation of serotonergic descending controls. The mitogen and stress-activated protein kinase 1 (MSK1) is downstream of ERK and can induce p-H3S10. We found that, after formalin injection, most phospho-MSK1 (p-MSK1)-positive cells (87% ± 3%) expressed p-ERK and the majority of p-H3S10-positive cells (85% ± 5%) expressed p-MSK1. Inhibition of ERK activity with the MEK inhibitor SL327 reduced formalin-induced p-ERK, p-MSK1, and p-H3S10, demonstrating that spinal p-MSK1 and p-H3S10 were at least partly downstream of ERK signalling. Crucially, pharmacological blockade of spinal MSK1 activity with the novel MSK1 inhibitor SB727651A inhibited formalin-induced spinal p-H3S10 and nocifensive behaviour. These findings are the first to establish the involvement of p-H3S10 and its main kinase, MSK1, in ERK regulation of nociception. Given the general importance of ERK signalling in pain processing, our results suggest that p-H3S10 could play a role in the response to injury.
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Affiliation(s)
- Keri K. Tochiki
- Department of Cell and Developmental Biology, University College London, London, United Kingdom
| | - Maria Maiarú
- Department of Cell and Developmental Biology, University College London, London, United Kingdom
| | - Caspar Norris
- Department of Cell and Developmental Biology, University College London, London, United Kingdom
| | - Stephen P. Hunt
- Department of Cell and Developmental Biology, University College London, London, United Kingdom
| | - Sandrine M. Géranton
- Department of Cell and Developmental Biology, University College London, London, United Kingdom
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28
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Abstract
Treatment of malignant disease is of paramount importance in modern medicine. In 2012, it was estimated that 162,000 people died from cancer in the UK which illustrates a fundamental problem. Traditional treatments for cancer have various drawbacks, and this creates a considerable need for specific, molecular targets to overcome cancer spread. Epithelial protein lost in neoplasm (EPLIN) is an actin-associated molecule which has been implicated in the development and progression of various cancers including breast, prostate, oesophageal and lung where EPLIN expression is frequently lost as the cancer progresses. EPLIN is important in the regulation of actin dynamics and has multiple associations at epithelial cells junctions. Thus, EPLIN loss in cancer may have significant effects on cancer cell migration and invasion, increasing metastatic potential. Overexpression of EPLIN has proved to be an effective tool for manipulating cancerous traits such as reducing cell growth and cell motility and rendering cells less invasive illustrating the therapeutic potential of EPLIN. Here, we review the current state of knowledge of EPLIN, highlighting EPLIN involvement in regulating cytoskeletal dynamics, signalling pathways and implications in cancer and metastasis.
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Affiliation(s)
- Ross J Collins
- Cardiff China Medical Research Collaborative (CCMRC), Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff, CF14 4XN, UK.
| | - Wen G Jiang
- Cardiff China Medical Research Collaborative (CCMRC), Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff, CF14 4XN, UK
| | - Rachel Hargest
- Cardiff China Medical Research Collaborative (CCMRC), Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff, CF14 4XN, UK
| | - Malcolm D Mason
- Department of Clinical Oncology, Cardiff University School of Medicine, Cardiff, UK
| | - Andrew J Sanders
- Cardiff China Medical Research Collaborative (CCMRC), Cardiff University School of Medicine, Henry Wellcome Building, Heath Park, Cardiff, CF14 4XN, UK
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29
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Liao W, Sharma S. Modulation of B-cell receptor and microenvironment signaling by a guanine exchange factor in B-cell malignancies. Cancer Biol Med 2016; 13:277-85. [PMID: 27458535 PMCID: PMC4944547 DOI: 10.20892/j.issn.2095-3941.2016.0026] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
OBJECTIVE Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cells over-express a guanine exchange factor (GEF), Rasgrf-1. This GEF increases active Ras as it catalyzes the removal of GDP from Ras so that GTP can bind and activate Ras. This study aims to study the mechanism of action of Rasgrf-1 in B-cell malignancies. METHODS N-terminus truncated Rasgrf-1 variants have a higher GEF activity as compared to the full-length transcript therefore a MCL cell line with stable over-expression of truncated Rasgrf-1 was established. The B-cell receptor (BCR) and chemokine signaling pathways were compared in the Rasgrf-1 over-expressing and a control transfected cell line. RESULTS Cells over-expressing truncated form of Rasgrf-1 have a higher proliferative rate as compared to control transfected cells. BCR was activated by lower concentrations of anti-IgM antibody in Rasgrf-1 over-expressing cells as compared to control cells indicating that these cells are more sensitive to BCR signaling. BCR signaling also phosphorylates Rasgrf-1 that further increases its GEF function and amplifies BCR signaling. This activation of Rasgrf-1 in over-expressing cells resulted in a higher expression of phospho-ERK, AKT, BTK and PKC-alpha as compared to control cells. Besides BCR, Rasgrf-1 over-expressing cells were also more sensitive to microenvironment stimuli as determined by resistance to apoptosis, chemotaxis and ERK pathway activation. CONCLUSIONS This GEF protein sensitizes B-cells to BCR and chemokine mediated signaling and also upregulates a number of other signaling pathways which promotes growth and survival of these cells.
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Affiliation(s)
- Wei Liao
- Division of Hematology-Oncology, Greater Los Angeles VA Healthcare Center, UCLA School of Medicine, Los Angeles, CA 90073, USA
| | - Sanjai Sharma
- Division of Hematology-Oncology, Greater Los Angeles VA Healthcare Center, UCLA School of Medicine, Los Angeles, CA 90073, USA
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30
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Morandi EM, Verstappen R, Zwierzina ME, Geley S, Pierer G, Ploner C. ITGAV and ITGA5 diversely regulate proliferation and adipogenic differentiation of human adipose derived stem cells. Sci Rep 2016; 6:28889. [PMID: 27363302 PMCID: PMC4929468 DOI: 10.1038/srep28889] [Citation(s) in RCA: 86] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2016] [Accepted: 06/09/2016] [Indexed: 12/19/2022] Open
Abstract
The fate of human adipose tissue stem cells (ASCs) is largely determined by biochemical and mechanical cues from the extracellular matrix (ECM), which are sensed and transmitted by integrins. It is well known that specific ECM constituents influence ASC proliferation and differentiation. Nevertheless, knowledge on how individual integrins regulate distinct processes is still limited. We performed gene profiling of 18 alpha integrins in sorted ASCs and adipocytes, identifying downregulations of RGD-motif binding integrins integrin-alpha-V (ITGAV) and integrin-alpha-5 (ITGA5), upregulation of laminin binding and leukocyte-specific integrins and individual regulations of collagen and LDV-receptors in differentiated adipocytes in-vivo. Gene function analyses in in-vitro cultured ASCs unraveled differential functions of ITGA5 and ITGAV. Knockdown of ITGAV, but not ITGA5 reduced proliferation, caused p21Cip1 induction, repression of survivin and specific regulation of Hippo pathway mediator TAZ. Gene knockdown of both integrins promoted adipogenic differentiation, while transgenic expression impaired adipogenesis. Inhibition of ITGAV using cilengitide resulted in a similar phenotype, mimicking loss of pan-ITGAV expression using RNAi. Herein we show ASC specific integrin expression patterns and demonstrate distinct regulating roles of both integrins in human ASCs and adipocyte physiology suggesting a negative impact of RDG-motif signaling on adipogenic differentiation of ASCs via ITGA5 and ITGAV.
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Affiliation(s)
- E M Morandi
- Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria
| | - R Verstappen
- Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria
| | - M E Zwierzina
- Department of Anatomy, Histology and Embryology, Medical University of Innsbruck, Müllerstrasse 59, 6020 Innsbruck, Austria
| | - S Geley
- Division of Molecular Pathophysiology, Medical University of Innsbruck, Innrain 80, 6020 Innsbruck, Austria
| | - G Pierer
- Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria
| | - C Ploner
- Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria
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31
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Abstract
The ROS1 gene belongs to the sevenless subfamily of tyrosine kinase insulin receptor genes. A literature review identified a ROS1 fusion in 2.54% of the patients with lung adenocarcinoma and even higher frequencies in spitzoid neoplasms and inflammatory myofibroblastic tumors. At present, 26 genes were found to fuse with ROS1, some of them already known to fuse with RET and ALK. All the fusion proteins retain the ROS1 kinase domain, but rarely its transmembrane domain. Most of the partners have dimerization domains that are retained in the fusion, presumably leading to constitutive ROS1 tyrosine kinase activation. Some partners have transmembrane domains that are retained or not in the chimeric proteins. Therefore, different ROS1 fusions have distinct subcellular localization, suggesting that they may activate different substrates in vivo.
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Affiliation(s)
- Arnaud Uguen
- Faculté de Médecine et des Sciences de la Santé, Université de Brest, Brest, France.,Institut National de la Santé et de la Recherche Médicale (INSERM), U1078, Brest, France.,Service d'Anatomie et Cytologie Pathologiques, Hôpital Morvan, CHRU Brest, Brest, France
| | - Marc De Braekeleer
- Faculté de Médecine et des Sciences de la Santé, Université de Brest, Brest, France.,Institut National de la Santé et de la Recherche Médicale (INSERM), U1078, Brest, France.,Service de Cytogénétique et Biologie de la Reproduction, Hôpital Morvan, CHRU Brest, Brest, France
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32
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Liu R, Martin TA, Jordan NJ, Ruge F, Ye L, Jiang WG. Epithelial protein lost in neoplasm-α (EPLIN-α) is a potential prognostic marker for the progression of epithelial ovarian cancer. Int J Oncol 2016; 48:2488-96. [PMID: 27035883 DOI: 10.3892/ijo.2016.3462] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2015] [Accepted: 01/13/2016] [Indexed: 11/05/2022] Open
Abstract
Epithelial protein lost in neoplasm-α (EPLIN-α) is a cytoskeletal protein whose expression is often lost or is aberrant in cancerous cells and tissues and whose loss is believed to be involved in aggressive phenotypes. This study examined this molecule in human epithelial ovarian tissues and investigated the cellular impact of EPLIN-α on ovarian cancer cells (EOC), SKOV3 and COV504. The expression of EPLIN-α in human ovarian tissues and EOC was assessed at both the mRNA and protein levels using reverse transcription-PCR (RT-PCR) and immunohistochemistry, respectively. In vitro assays for cellular matrix adhesion and migration (confirmed by an electrical cell substrate impedance sensing (ECIS) based method), invasion and cell growth were employed in order to assess the biological influence of EPLIN-α expression on EOC cells. Immunohistochemical analysis of ovarian cancer samples demonstrated that only a small number expressed EPLIN-α protein. Downregulation of EPLIN-α protein in EOC cell lines increased the growth, invasion, adhesion and migration in vitro. This EPLIN-α downregulation may have a prognostic value. From these data, we conclude that downregulation of EPLIN-α may be associated with poorer patient prognosis, and that this molecule appears to play a tumour suppressor role by inhibition of EOC growth and migration.
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Affiliation(s)
- Rong Liu
- Cardiff China Medical Research Collaborative, Cardiff University School of Medicine, Cardiff, CF14 4XN, UK
| | - Tracey A Martin
- Cardiff China Medical Research Collaborative, Cardiff University School of Medicine, Cardiff, CF14 4XN, UK
| | - Nicola J Jordan
- Cardiff China Medical Research Collaborative, Cardiff University School of Medicine, Cardiff, CF14 4XN, UK
| | - Fiona Ruge
- Cardiff China Medical Research Collaborative, Cardiff University School of Medicine, Cardiff, CF14 4XN, UK
| | - Lin Ye
- Cardiff China Medical Research Collaborative, Cardiff University School of Medicine, Cardiff, CF14 4XN, UK
| | - Wen G Jiang
- Cardiff China Medical Research Collaborative, Cardiff University School of Medicine, Cardiff, CF14 4XN, UK
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33
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Jungke P, Hans S, Gupta M, Machate A, Zöller D, Brand M. Generation of a conditionallima1aallele in zebrafish using the FLEx switch technology. Genesis 2015; 54:19-28. [DOI: 10.1002/dvg.22909] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2015] [Revised: 10/30/2015] [Accepted: 11/12/2015] [Indexed: 01/18/2023]
Affiliation(s)
- Peggy Jungke
- Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology; Tatzberg Dresden 47-49, 01307 Germany
| | - Stefan Hans
- Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology; Tatzberg Dresden 47-49, 01307 Germany
| | - Mansi Gupta
- Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology; Tatzberg Dresden 47-49, 01307 Germany
| | - Anja Machate
- Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology; Tatzberg Dresden 47-49, 01307 Germany
| | - Daniela Zöller
- Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology; Tatzberg Dresden 47-49, 01307 Germany
| | - Michael Brand
- Biotechnology Center and Center for Regenerative Therapies Dresden, Dresden University of Technology; Tatzberg Dresden 47-49, 01307 Germany
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34
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Chen YL, Song JJ, Chen XC, Xu W, Zhi Q, Liu YP, Xu HZ, Pan JS, Ren JL, Guleng B. Mechanisms of pyruvate kinase M2 isoform inhibits cell motility in hepatocellular carcinoma cells. World J Gastroenterol 2015; 21:9093-9102. [PMID: 26290635 PMCID: PMC4533040 DOI: 10.3748/wjg.v21.i30.9093] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/26/2014] [Revised: 01/08/2015] [Accepted: 06/09/2015] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate biological mechanisms underlying pyruvate kinase M2 isoform (PKM2) regulation of cell migration and invasion in hepatocellular carcinoma cells. METHODS HepG2 and Huh-7 hepatocellular carcinoma cell lines were stably transfected and cultured in DMEM (HyClone, Logan, UT, United States). To investigate the effects of PKM2 on cellular proliferation, hepatocellular carcinoma cells were subjected to the Cell Counting Kit-8 (Dojindo, Kamimashiki-gun, Kumamoto, Japan). And investigate the effects of PKM2 on cell signal pathway related with migration and invasion, Western immunoblotting were used to find out the differential proteins. All the antibody used was purchaseed from Cell Signal Technology. In order to explore cell motility used Transwell invasion and wound healing assays. The transwell plate with 0.5 mg/mL collagen type I (BD Bioscience, San Jose, CA)-coated filters. The wound-healing assay was performed in 6-well plates. Total RNA was extracted using TRIzol reagent (Invitrogen, CA, United States) and then reverse transcription was conducted. Quantitative reverse transcription-polymerase chain reaction (PCR) analysis was performed with the ABI 7500 real-time PCR system (Applied Biosystems). We further use digital gene expression tag profiling and identification of differentially expressed genes. RESULTS The cells seeded in four 96-well plates were measured OD450 by conducted Cell Counting Kit-8. From this conduction we observed that both HepG2 and Huh-7 hepatocellular carcinoma cells with silenced PKM2 turn on a proliferate inhibition; however, cell migration and invasion were enhanced compared with the control upon stimulation with epidermal growth factor (EGF). Our results indicate that the knockdown of PKM2 decreased the expression of E-cadherin and enhanced the activity of the EGF/EGFR signaling pathway, furthermore up-regulate the subsequent signal molecular the PLCγ1 and extracellular signal-regulated kinase 1/2 expression in the hepatocellular carcinoma cell lines HepG2 and Huh-7, which regulates cell motility. These variations we observed were due to the activation of the transforming growth factor beta (TGFβ) signaling pathway after PKM2 knockdown. We also found that the expression of TGFBRI was increased and the phosphorylation of Smad2 was enhanced. Taken together, our findings demonstrate that PKM2 can regulate cell motility through the EGF/EGFR and TGFβ/TGFR signaling pathways in hepatocellular carcinoma cells. CONCLUSION PKM2 play different roles in modulating the proliferation and metastasis of hepatocellular carcinoma cells, and this finding could help to guide the future targeted therapies.
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Chapnick DA, Bunker E, Liu X. A biosensor for the activity of the "sheddase" TACE (ADAM17) reveals novel and cell type-specific mechanisms of TACE activation. Sci Signal 2015; 8:rs1. [PMID: 25714465 DOI: 10.1126/scisignal.2005680] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Diverse environmental conditions stimulate protein "shedding" from the cell surface through proteolytic cleavage. The protease TACE [tumor necrosis factor-α (TNFα)--converting enzyme, encoded by ADAM17] mediates protein shedding, thereby regulating the maturation and release of various extracellular substrates, such as growth factors and cytokines, that induce diverse cellular responses. We developed a FRET (fluorescence resonance energy transfer)-based biosensor called TSen that quantitatively reports the kinetics of TACE activity in live cells. In combination with chemical biology approaches, we used TSen to probe the dependence of TACE activation on the induction of the kinases p38 and ERK (extracellular signal-regulated kinase) in various epithelial cell lines. Using TSen, we found that disruption of the actin cytoskeleton in keratinocytes induced rapid and robust TSen cleavage and the accumulation of TACE at the plasma membrane. Cytoskeletal disruption also increased the cleavage of endogenous TACE substrates, including transforming growth factor-α. Thus, TSen is a useful tool for unraveling the mechanisms underlying the spatiotemporal activation of TACE in live cells.
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Affiliation(s)
- Douglas A Chapnick
- Department of Chemistry and Biochemistry, 596 UCB, University of Colorado, Jennie Smoly Caruthers Biotechnology Building (JSCBB), 3415 Colorado Avenue, Boulder, CO 80303, USA
| | - Eric Bunker
- Department of Chemistry and Biochemistry, 596 UCB, University of Colorado, Jennie Smoly Caruthers Biotechnology Building (JSCBB), 3415 Colorado Avenue, Boulder, CO 80303, USA
| | - Xuedong Liu
- Department of Chemistry and Biochemistry, 596 UCB, University of Colorado, Jennie Smoly Caruthers Biotechnology Building (JSCBB), 3415 Colorado Avenue, Boulder, CO 80303, USA.
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Karaköse E, Geiger T, Flynn K, Lorenz-Baath K, Zent R, Mann M, Fässler R. The focal adhesion protein PINCH-1 associates with EPLIN at integrin adhesion sites. J Cell Sci 2015; 128:1023-33. [PMID: 25609703 DOI: 10.1242/jcs.162545] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
PINCH-1 is a LIM-only domain protein that forms a ternary complex with integrin-linked kinase (ILK) and parvin (to form the IPP complex) downstream of integrins. Here, we demonstrate that PINCH-1 (also known as Lims1) gene ablation in the epidermis of mice caused epidermal detachment from the basement membrane, epidermal hyperthickening and progressive hair loss. PINCH-1-deficient keratinocytes also displayed profound adhesion, spreading and migration defects in vitro that were substantially more severe than those of ILK-deficient keratinocytes indicating that PINCH-1 also exerts functions in an ILK-independent manner. By isolating the PINCH-1 interactome, the LIM-domain-containing and actin-binding protein epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) was identified as a new PINCH-1-associated protein. EPLIN localized, in a PINCH-1-dependent manner, to integrin adhesion sites of keratinocytes in vivo and in vitro and its depletion severely attenuated keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions. Given that the low PINCH-1 levels in ILK-deficient keratinocytes were sufficient to recruit EPLIN to integrin adhesions, our findings suggest that PINCH-1 regulates integrin-mediated adhesion of keratinocytes through the interactions with ILK as well as EPLIN.
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Affiliation(s)
- Esra Karaköse
- Department of Molecular Medicine, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
| | - Tamar Geiger
- Department of Proteomics and Signal Transductions, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
| | - Kevin Flynn
- Department of Molecular Medicine, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
| | - Katrin Lorenz-Baath
- Department of Molecular Medicine, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
| | - Roy Zent
- Division of Nephrology, Department of Medicine, Vanderbilt Medical Center, Nashville, TN, 37232, USA Department of Medicine, Nashville Veterans Affairs Medical Center, Nashville, TN, 37232, USA
| | - Matthias Mann
- Department of Proteomics and Signal Transductions, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
| | - Reinhard Fässler
- Department of Molecular Medicine, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
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Gulino-Debrac D. Mechanotransduction at the basis of endothelial barrier function. Tissue Barriers 2014; 1:e24180. [PMID: 24665386 PMCID: PMC3879236 DOI: 10.4161/tisb.24180] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2012] [Revised: 03/01/2013] [Accepted: 03/02/2013] [Indexed: 01/03/2023] Open
Abstract
Destabilization of cell-cell contacts involved in the maintenance of endothelial barrier function can lead to increased endothelial permeability. This increase in endothelial permeability results in an anarchical movement of fluid, solutes and cells outside the vasculature and into the surrounding tissues, thereby contributing to various diseases such as stroke or pulmonary edema. Thus, a better understanding of the molecular mechanisms regulating endothelial cell junction integrity is required for developing new therapies for these diseases. In this review, we describe the mechanotransduction mechanism at the basis of adherens junction strengthening at endothelial cell-cell contacts. More particularly, we report on the emerging role of α-catenin and EPLIN that act as a mechanotransmitter of myosin-IIgenerated traction forces. The interplay between α-catenin, EPLIN and the myosin-II machinery initiates the junctional recruitment of vinculin and α-actinin leading to a drastic remodeling of the actin cytoskeleton and to cortical actin ring reshaping. The pathways initiated by tyrosine phosphorylation of VE-cadherin at the basis of endothelial cell-cell junction remodeling is also reported, as it may be interrelated to α-catenin/ EPLIN-mediated mechanotransduction mechanisms. We also describe the junctional mechanosensory complex composed of PECAM-1, VE-cadherin and VEGFR2 that is able to transmit signaling pathway under the onset of shear stress. This mechanosensing mechanism, involved in the earliest events promoting atherogenesis, is required for endothelial cell alignment along flow direction.
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Affiliation(s)
- Danielle Gulino-Debrac
- Biology of Cancer and Infection Laboratory; U INSERM 1036, iRTSV; Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA); Université Joseph Fourier; Grenoble, France
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Kabekkodu SP, Bhat S, Radhakrishnan R, Aithal A, Mascarenhas R, Pandey D, Rai L, Kushtagi P, Mundyat GP, Satyamoorthy K. DNA promoter methylation-dependent transcription of the double C2-like domain β (DOC2B) gene regulates tumor growth in human cervical cancer. J Biol Chem 2014; 289:10637-10649. [PMID: 24570007 PMCID: PMC4036182 DOI: 10.1074/jbc.m113.491506] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2013] [Revised: 02/18/2014] [Indexed: 11/06/2022] Open
Abstract
Double C2-like domain β (DOC2B) gene encodes for a calcium-binding protein, which is involved in neurotransmitter release, sorting, and exocytosis. We have identified the promoter region of the DOC2B gene as hypermethylated in pre-malignant, malignant cervical tissues, and cervical cancer cell lines by methylation-sensitive dimethyl sulfoxide-polymerase chain reaction and bisulfite genome sequencing; whereas, it was unmethylated in normal cervical tissues (p < 0.05). The promoter hypermethylation was inversely associated with mRNA expression in SiHa, CaSki, and HeLa cells and treatment with demethylating agent 5-aza-2-deoxycytidine restored DOC2B expression. The region -630 to +25 bp of the DOC2B gene showed robust promoter activity by a luciferase reporter assay and was inhibited by in vitro artificial methylation with Sss1 methylase prior to transient transfections. Overexpression of the DOC2B gene in SiHa cells when compared with controls showed significantly reduced colony formation, cell proliferation, induced cell cycle arrest, and repressed cell migration and invasion (p < 0.05). Ectopic expression of DOC2B resulted in anoikis-mediated cell death and repressed tumor growth in a nude mice xenograft model (p < 0.05). DOC2B expressing cells showed a significant increase in intracellular calcium level (p < 0.05), impaired AKT1 and ERK1/2 signaling, and induced actin cytoskeleton remodeling. Our results show that promoter hypermethylation and silencing of the DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth.
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Affiliation(s)
- Shama Prasada Kabekkodu
- Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India
| | - Samatha Bhat
- Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India
| | - Raghu Radhakrishnan
- Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India
| | - Abhijit Aithal
- Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India
| | - Roshan Mascarenhas
- Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India
| | - Deeksha Pandey
- Department of OBGYN, Kasturba Medical College, Manipal University, Manipal 576104, India
| | - Lavanya Rai
- Department of OBGYN, Kasturba Medical College, Manipal University, Manipal 576104, India
| | - Pralhad Kushtagi
- Department of OBGYN, Kasturba Medical College, Manipal University, Mangalore 575001, India
| | - Gopinath Puthiya Mundyat
- Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India
| | - Kapaettu Satyamoorthy
- Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India.
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Tsurumi H, Harita Y, Kurihara H, Kosako H, Hayashi K, Matsunaga A, Kajiho Y, Kanda S, Miura K, Sekine T, Oka A, Ishizuka K, Horita S, Hattori M, Hattori S, Igarashi T. Epithelial protein lost in neoplasm modulates platelet-derived growth factor-mediated adhesion and motility of mesangial cells. Kidney Int 2014; 86:548-57. [PMID: 24694988 DOI: 10.1038/ki.2014.85] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2013] [Revised: 02/03/2014] [Accepted: 02/06/2014] [Indexed: 01/16/2023]
Abstract
Mesangial cell migration, regulated by several growth factors, is crucial after glomerulopathy and during glomerular development. Directional migration requires the establishment of a polarized cytoskeletal arrangement, a process regulated by coordinated actin dynamics and focal adhesion turnover at the peripheral ruffles in migrating cells. Here we found high expression of the actin cross-linking protein EPLIN (epithelial protein lost in neoplasm) in mesangial cells. EPLIN was localized in mesangial angles, which consist of actin-containing microfilaments extending underneath the capillary endothelium, where they attach to the glomerular basement membrane. In cultured mesangial cells, EPLIN was localized in peripheral actin bundles at focal adhesions and formed a protein complex with paxillin. The MEK-ERK (extracellular signal-regulated kinase) cascade regulated EPLIN-paxillin interaction and induced translocalization of EPLIN from focal adhesion sites to peripheral ruffles. Knockdown of EPLIN in mesangial cells enhanced platelet-derived growth factor-induced focal adhesion disassembly and cell migration. Furthermore, EPLIN expression was decreased in mesangial proliferative nephritis in rodents and humans in vivo. These results shed light on the coordinated actin remodeling in mesangial cells during restorative remodeling. Thus, changes in expression and localization of cytoskeletal regulators underlie phenotypic changes in mesangial cells in glomerulonephritis.
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Affiliation(s)
- Haruko Tsurumi
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Yutaka Harita
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Hidetake Kurihara
- Department of Anatomy, Juntendo University School of Medicine, Tokyo, Japan
| | - Hidetaka Kosako
- Division of Cell Signaling, Fujii Memorial Institute of Medical Sciences, The University of Tokushima, Tokushima, Japan
| | - Kenji Hayashi
- Department of Molecular Biology, Yokohama City University School of Medicine, Kanagawa, Japan
| | - Atsuko Matsunaga
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Yuko Kajiho
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Shoichiro Kanda
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Kenichiro Miura
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Takashi Sekine
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Akira Oka
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Kiyonobu Ishizuka
- Department of Pediatric Nephrology, Tokyo Women's Medical University, School of Medicine, Tokyo, Japan
| | - Shigeru Horita
- Department of Pediatric Nephrology, Tokyo Women's Medical University, School of Medicine, Tokyo, Japan
| | - Motoshi Hattori
- Department of Pediatric Nephrology, Tokyo Women's Medical University, School of Medicine, Tokyo, Japan
| | - Seisuke Hattori
- Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan
| | - Takashi Igarashi
- 1] Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan [2] National Center for Child Health and Development, Tokyo, Japan
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Smith TC, Fridy PC, Li Y, Basil S, Arjun S, Friesen RM, Leszyk J, Chait BT, Rout MP, Luna EJ. Supervillin binding to myosin II and synergism with anillin are required for cytokinesis. Mol Biol Cell 2013; 24:3603-19. [PMID: 24088567 PMCID: PMC3842989 DOI: 10.1091/mbc.e12-10-0714] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II-binding proteins anillin and supervillin, act earlier. Anillin's role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831-1281, which bind central spindle proteins, and residues 1-170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis.
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Affiliation(s)
- Tara C Smith
- Program in Cell and Developmental Dynamics, Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655 Laboratory of Cellular and Structural Biology, Rockefeller University, New York, NY 10065 Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, New York, NY 10065 Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Shrewsbury, MA 01545
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Grossman GH, Ebke LA, Beight CD, Jang GF, Crabb JW, Hagstrom SA. Protein partners of dynamin-1 in the retina. Vis Neurosci 2013; 30:129-39. [PMID: 23746204 PMCID: PMC3936680 DOI: 10.1017/s0952523813000138] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Dynamin proteins are involved in vesicle generation, providing mechanical force to excise newly formed vesicles from membranes of cellular compartments. In the brain, dynamin-1, dynamin-2, and dynamin-3 have been well studied; however, their function in the retina remains elusive. A retina-specific splice variant of dynamin-1 interacts with the photoreceptor-specific protein Tubby-like protein 1 (Tulp1), which when mutated causes an early onset form of autosomal recessive retinitis pigmentosa. Here, we investigated the role of the dynamins in the retina, using immunohistochemistry to localize dynamin-1, dynamin-2, and dynamin-3 and immunoprecipitation followed by mass spectrometry to explore dynamin-1 interacting proteins in mouse retina. Dynamin-2 is primarily confined to the inner segment compartment of photoreceptors, suggesting a role in outer segment protein transport. Dynamin-3 is present in the terminals of photoreceptors and dendrites of second-order neurons but is most pronounced in the inner plexiform layer where second-order neurons relay signals from photoreceptors. Dynamin-1 appears to be the dominant isoform in the retina and is present throughout the retina and in multiple compartments of the photoreceptor cell. This suggests that it may function in multiple cellular pathways. Surprisingly, dynamin-1 expression and localization did not appear to be disrupted in tulp1−/− mice. Immunoprecipitation experiments reveal that dynamin-1 associates primarily with proteins involved in cytoskeletal-based membrane dynamics. This finding is confirmed by western blot analysis. Results further implicate dynamin-1 in vesicular protein transport processes relevant to synaptic and post-Golgi pathways and indicate a possible role in photoreceptor stability.
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Affiliation(s)
- Gregory H Grossman
- Department of Ophthalmic Research, Cleveland Clinic Cole Eye Institute, Cleveland, Ohio
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Gaji RY, Huynh MH, Carruthers VB. A novel high throughput invasion screen identifies host actin regulators required for efficient cell entry by Toxoplasma gondii. PLoS One 2013; 8:e64693. [PMID: 23741372 PMCID: PMC3669402 DOI: 10.1371/journal.pone.0064693] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2013] [Accepted: 04/17/2013] [Indexed: 01/08/2023] Open
Abstract
Toxoplasma gondii critically relies on cell invasion as a survival strategy to evade immune clearance during infection. Although it was widely thought that Toxoplasma entry is parasite directed and that the host cell is largely a passive victim, recent studies have suggested that host components such as microfilaments and microtubules indeed contribute to entry. Hence to identify additional host factors, we performed a high-throughput siRNA screen of a human siRNA library targeting druggable proteins using a novel inducible luciferase based invasion assay. The top 100 hits from the primary screen that showed the strongest decreases in invasion were subjected to confirmation by secondary screening, revealing 24 proteins that are potentially involved in Toxoplasma entry into host cells. Interestingly, 6 of the hits appear to affect parasite invasion by modifying host cell actin dynamics, resulting in increased deposition of F-actin at the periphery of the cell. These findings support the emerging notion that host actin dynamics are important for Toxoplasma invasion along with identifying several novel host factors that potentially participate in parasite entry.
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Affiliation(s)
- Rajshekhar Y. Gaji
- Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America
| | - My-Hang Huynh
- Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America
| | - Vern B. Carruthers
- Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America
- * E-mail:
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Zhang S, Wang X, Iqbal S, Wang Y, Osunkoya AO, Chen Z, Chen Z, Shin DM, Yuan H, Wang YA, Zhau HE, Chung LWK, Ritenour C, Kucuk O, Wu D. Epidermal growth factor promotes protein degradation of epithelial protein lost in neoplasm (EPLIN), a putative metastasis suppressor, during epithelial-mesenchymal transition. J Biol Chem 2013; 288:1469-79. [PMID: 23188829 PMCID: PMC3548460 DOI: 10.1074/jbc.m112.438341] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Aberrant expression of EGF receptors has been associated with hormone-refractory and metastatic prostate cancer (PCa). However, the molecular mechanism for EGF signaling in promoting PCa metastasis remains elusive. Using experimental models of PCa metastasis, we demonstrated that EGF could induce robust epithelial-mesenchymal transition (EMT) and increase invasiveness. Interestingly, EGF was found to be capable of promoting protein turnover of epithelial protein lost in neoplasm (EPLIN), a putative suppressor of EMT and tumor metastasis. Mechanistic study revealed that EGF could activate the phosphorylation, ubiquitination, and degradation of EPLIN through an extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent signaling cascade. Pharmacological inhibition of the ERK1/2 pathway effectively antagonized EGF-induced EPLIN degradation. Two serine residues, i.e. serine 362 and serine 604, were identified as putative ERK1/2 phosphorylation sites in human EPLIN, whose point mutation rendered resistance to EGF-induced protein turnover. This study elucidated a novel molecular mechanism for EGF regulation of EMT and invasiveness in PCa cells, indicating that blockade of EGF signaling could be beneficial in preventing and retarding PCa metastasis at early stages.
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Affiliation(s)
- Shumin Zhang
- From the Department of Urology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322
| | - Xu Wang
- the Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322
| | - Shareen Iqbal
- From the Department of Urology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322
| | - Yanru Wang
- From the Department of Urology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322
| | - Adeboye O. Osunkoya
- From the Department of Urology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322, ,the Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322
| | - Zhengjia Chen
- the Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University, Atlanta, Georgia 30322
| | - Zhuo Chen
- the Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322
| | - Dong M. Shin
- the Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322
| | - Hongwei Yuan
- the Department of Pathology, The Affiliated Hospital Inner Mongolia Medical College, Hohhot, Inner Mongolia Autonomous Region 10050, China
| | | | - Haiyen E. Zhau
- the Uro-Oncology Research Program, Cedars-Sinai Medical Center, Los Angeles, California 90048
| | - Leland W. K. Chung
- the Uro-Oncology Research Program, Cedars-Sinai Medical Center, Los Angeles, California 90048
| | - Chad Ritenour
- From the Department of Urology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322
| | - Omer Kucuk
- From the Department of Urology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322
| | - Daqing Wu
- From the Department of Urology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322, , To whom correspondence should be addressed: Dept. of Urology, Emory University School of Medicine, 1365 Clifton Rd., NE., Clinic B, B5107, Atlanta, GA 30322. Tel.: 404-778-4845; E-mail:
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Regulation of adherens junction dynamics by phosphorylation switches. JOURNAL OF SIGNAL TRANSDUCTION 2012; 2012:125295. [PMID: 22848810 PMCID: PMC3403498 DOI: 10.1155/2012/125295] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/05/2012] [Revised: 05/21/2012] [Accepted: 05/22/2012] [Indexed: 12/15/2022]
Abstract
Adherens junctions connect the actin cytoskeleton of neighboring cells through transmembrane cadherin receptors and a network of adaptor proteins. The interactions between these adaptors and cadherin as well as the activity of actin regulators localized to adherens junctions are tightly controlled to facilitate cell junction assembly or disassembly in response to changes in external or internal forces and/or signaling. Phosphorylation of tyrosine, serine, or threonine residues acts as a switch on the majority of adherens junction proteins, turning "on" or "off" their interactions with other proteins and/or their enzymatic activity. Here, we provide an overview of the kinases and phosphatases regulating phosphorylation of adherens junction proteins and bring examples of phosphorylation events leading to the assembly or disassembly of adherens junctions, highlighting the important role of phosphorylation switches in regulating their dynamics.
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Faure E, Garrouste F, Parat F, Monferran S, Leloup L, Pommier G, Kovacic H, Lehmann M. P2Y2 receptor inhibits EGF-induced MAPK pathway to stabilise keratinocyte hemidesmosomes. J Cell Sci 2012; 125:4264-77. [PMID: 22718344 DOI: 10.1242/jcs.097600] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
α6β4 integrin is the main component of hemidesmosomes (HD) that stably anchor the epithelium to the underlying basement membrane. Epithelial cell migration requires HD remodelling, which can be promoted by epidermal growth factor (EGF). We previously showed that extracellular nucleotides inhibit growth factor-induced keratinocyte migration. Here, we investigate the effect of extracellular nucleotides on α6β4 integrin localisation in HD during EGF-induced cell migration. Using a combination of pharmacological inhibition and gene silencing approaches, we found that UTP activates the P2Y2 purinergic receptor and Gαq protein to inhibit EGF/ERK1/2-induced cell migration in keratinocytes. Using a keratinocyte cell line expressing an inducible form of the Raf kinase, we show that UTP inhibits the EGF-induced ERK1/2 pathway activation downstream of Raf. Moreover, we established that ERK1/2 activation by EGF leads to the mobilisation of α6β4 integrin from HD. Importantly, activation of P2Y2R and Gαq by UTP promotes HD formation and protects these structures from EGF-triggered dissolution as revealed by confocal analysis of the distribution of α6β4 integrin, plectin, BPAG1, BPAG2 and CD151 in keratinocytes. Finally, we demonstrated that the activation of p90RSK, downstream of ERK1/2, is sufficient to promote EGF-mediated HD dismantling and that UTP does not stabilise HD in cells expressing an activated form of p90RSK. Our data underline an unexpected role of P2Y2R and Gαq in the inhibition of the ERK1/2 signalling pathway and in the modulation of hemidesmosome dynamics and keratinocyte migration.
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Affiliation(s)
- Emilie Faure
- Aix-Marseille Université, INSERM UMR 911, Centre de Recherche en Oncologie Biologique et en Oncopharmacologie, Marseille 13005, France
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Liu Y, Sanders AJ, Zhang L, Jiang WG. Expression Profile of Epithelial Protein Lost in Neoplasm-Alpha (EPLIN-α) in Human Pulmonary Cancer and Its Impact on SKMES-1 Cells <i>in vitro</i>. ACTA ACUST UNITED AC 2012. [DOI: 10.4236/jct.2012.324058] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Asano E, Maeda M, Hasegawa H, Ito S, Hyodo T, Yuan H, Takahashi M, Hamaguchi M, Senga T. Role of palladin phosphorylation by extracellular signal-regulated kinase in cell migration. PLoS One 2011; 6:e29338. [PMID: 22216253 PMCID: PMC3247243 DOI: 10.1371/journal.pone.0029338] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2011] [Accepted: 11/25/2011] [Indexed: 12/30/2022] Open
Abstract
Phosphorylation of actin-binding proteins plays a pivotal role in the remodeling of the actin cytoskeleton to regulate cell migration. Palladin is an actin-binding protein that is phosphorylated by growth factor stimulation; however, the identity of the involved protein kinases remains elusive. In this study, we report that palladin is a novel substrate of extracellular signal-regulated kinase (ERK). Suppression of ERK activation by a chemical inhibitor reduced palladin phosphorylation, and expression of active MEK alone was sufficient for phosphorylation. In addition, an in vitro kinase assay demonstrated direct palladin phosphorylation by ERK. We found that Ser77 and Ser197 are essential residues for phosphorylation. Although the phosphorylation of these residues was not required for actin cytoskeletal organization, we found that expression of non-phosphorylated palladin enhanced cell migration. Finally, we show that phosphorylation inhibits the palladin association with Abl tyrosine kinase. Taken together, our results indicate that palladin phosphorylation by ERK has an anti-migratory function, possibly by modulating interactions with molecules that regulate cell migration.
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Affiliation(s)
- Eri Asano
- Division of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Masao Maeda
- Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Hitoki Hasegawa
- Division of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Satoko Ito
- Division of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Toshinori Hyodo
- Division of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Hong Yuan
- Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Masahide Takahashi
- Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Michinari Hamaguchi
- Division of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Takeshi Senga
- Division of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
- * E-mail:
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Chervin-Pétinot A, Courçon M, Almagro S, Nicolas A, Grichine A, Grunwald D, Prandini MH, Huber P, Gulino-Debrac D. Epithelial protein lost in neoplasm (EPLIN) interacts with α-catenin and actin filaments in endothelial cells and stabilizes vascular capillary network in vitro. J Biol Chem 2011; 287:7556-72. [PMID: 22194609 DOI: 10.1074/jbc.m111.328682] [Citation(s) in RCA: 57] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Adherens junctions are required for vascular endothelium integrity. These structures are formed by the clustering of the homophilic adhesive protein VE-cadherin, which recruits intracellular partners, such as β- and α-catenins, vinculin, and actin filaments. The dogma according to which α-catenin bridges cadherin·β-catenin complexes to the actin cytoskeleton has been challenged during the past few years, and the link between the VE-cadherin·catenin complex and the actin cytoskeleton remains unclear. Recently, epithelial protein lost in neoplasm (EPLIN) has been proposed as a possible bond between the E-cadherin·catenin complex and actin in epithelial cells. Herein, we show that EPLIN is expressed at similar levels in endothelial and epithelial cells and is located at interendothelial junctions in confluent cells. Co-immunoprecipitation and GST pulldown experiments provided evidence that EPLIN interacts directly with α-catenin and tethers the VE-cadherin·catenin complex to the actin cytoskeleton. In the absence of EPLIN, vinculin was delocalized from the junctions. Furthermore, suppression of actomyosin tension using blebbistatin triggered a similar vinculin delocalization from the junctions. In a Matrigel assay, EPLIN-depleted endothelial cells exhibited a reduced capacity to form pseudocapillary networks because of numerous breakage events. In conclusion, we propose a model in which EPLIN establishes a link between the cadherin·catenin complex and actin that is independent of actomyosin tension. This link acts as a mechanotransmitter, allowing vinculin binding to α-catenin and formation of a secondary molecular bond between the adherens complex and the cytoskeleton through vinculin. In addition, we provide evidence that the EPLIN clutch is necessary for stabilization of capillary structures in an angiogenesis model.
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The vaccinia virus O1 protein is required for sustained activation of extracellular signal-regulated kinase 1/2 and promotes viral virulence. J Virol 2011; 86:2323-36. [PMID: 22171261 DOI: 10.1128/jvi.06166-11] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Sustained activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway in infected cells has been shown to be crucial for full replication efficiency of orthopoxviruses in cell culture. In infected cells, this pathway is mainly activated by the vaccinia virus growth factor (VGF), an epidermal growth factor (EGF)-like protein. We show here that chorioallantois vaccinia virus Ankara (CVA), but not modified vaccinia virus Ankara (MVA), induced sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in infected human 293 cells, although both viruses direct secretion of functional VGF. A CVA mutant lacking the O1L gene (CVA-ΔO1L) demonstrated that the O1 protein was required for sustained upregulation of the ERK1/2 pathway in 293 cells as well as in other mammalian cell lines. The highly conserved orthopoxvirus O1L gene encodes a predicted 78-kDa protein with a hitherto-unknown function. CVA-ΔO1L showed reduced plaque size and an attenuated cytopathic effect (CPE) in infected cell cultures and reduced virulence and spread from lungs to ovaries in intranasally infected BALB/c mice. Reinsertion of an intact O1L gene into MVA, which in its original form harbors a fragmented O1L open reading frame (ORF), restored ERK1/2 activation in 293 cells but did not increase replication and spread of MVA in human or other mammalian cell lines. Thus, the O1 protein was crucial for sustained ERK1/2 activation in CVA- and MVA-infected human cells, complementing the autocrine function of VGF, and enhanced virulence in vivo.
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Min J, Defea K. β-arrestin-dependent actin reorganization: bringing the right players together at the leading edge. Mol Pharmacol 2011; 80:760-8. [PMID: 21836019 DOI: 10.1124/mol.111.072470] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
First identified as mediators of G-protein-coupled receptor desensitization and internalization and later as signaling platforms, β-arrestins play a requisite role in chemotaxis and reorganization of the actin cytoskeleton, downstream of multiple receptors. However, the precise molecular mechanisms underlying their involvement have remained elusive. Initial interest in β-arrestins as facilitators of cell migration and actin reorganization stemmed from the known interplay between receptor endocytosis and actin filament formation, because disruption of the actin cytoskeleton inhibits these β-arrestin-dependent events. With growing interest in the mechanisms by which cells can sense a gradient of agonist during cell migration, investigators began to hypothesize that β-arrestins may contribute to directed migration by controlling chemotactic receptor turnover at the plasma membrane. Finally, increasing evidence emerged that β-arrestins are more than just clathrin adaptor proteins involved in turning off receptor signals; they are actually capable of generating their own signals by scaffolding signaling molecules and controlling the activity of multiple cellular enzymes. This new role of β-arrestins as signaling scaffolds has led to the hypothesis that they can facilitate cell migration by sequestering actin assembly activities and upstream regulators of actin assembly at the leading edge. This Minireview discusses recent advances in our understanding of how β-arrestin scaffolds contribute to cell migration, focusing on recently identified β-arrestin interacting proteins and phosphorylation targets that have known roles in actin reorganization.
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Affiliation(s)
- Jungah Min
- Division of Biomedical Sciences and Graduate Program in Cell, Molecular and Developmental Biology, University of California, Riverside, CA, USA
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