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Wang J, Hao Z, Hu L, Qiao L, Luo Y, Hu J, Liu X, Li S, Zhao F, Shen J, Li M, Zhao Z. MicroRNA-199a-3p regulates proliferation and milk fat synthesis of ovine mammary epithelial cells by targeting VLDLR. Front Vet Sci 2022; 9:948873. [PMID: 35990270 PMCID: PMC9391033 DOI: 10.3389/fvets.2022.948873] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2022] [Accepted: 07/11/2022] [Indexed: 11/18/2022] Open
Abstract
In our previous study, microRNA (miR)-199a-3p was found to be the most upregulated miRNA in mammary gland tissue during the non-lactation period compared with the peak-lactation period. However, there have been no reports describing the function of miR-199a-3p in ovine mammary epithelial cells (OMECs) and the biological mechanisms by which the miRNA affects cell proliferation and milk fat synthesis in sheep. In this study, the effect of miR-199a-3p on viability, proliferation, and milk fat synthesis of OMECs was investigated, and the target relationship of the miRNA with very low-density lipoprotein receptor (VLDLR) was also verified. Transfection with a miR-199a-3p mimic increased the viability of OMECs and the number of Edu-labeled positive OMECs. In contrast, a miR-199-3p inhibitor had the opposite effect with the miR-199a-3p mimic. The expression levels of three marker genes were also regulated by both the miR-199a-3p mimic and miR-199-3p inhibitor in OMECs. Together, these results suggest that miR-199a-3p promotes the viability and proliferation of OMECs. A dual luciferase assay confirmed that miR-199a-3p can target VLDLR by binding to the 3′-untranslated regions (3'UTR) of the gene. Further studies found a negative correlation in the expression of miR-199a-3p with VLDLR. The miR-199a-3p mimic decreased the content of triglycerides, as well as the expression levels of six milk fat synthesis marker genes in OMECs, namely, lipoprotein lipase gene (LPL), acetyl-CoA carboxylase alpha gene (ACACA), fatty acid binding protein 3 gene (FABP3), CD36, stearoyl-CoA desaturase gene (SCD), and fatty acid synthase gene (FASN). The inhibition of miR-199a-3p increased the level of triglycerides and the expression of LPL, ACACA, FABP3, SCD, and FASN in OMECs. These findings suggest that miR-199a-3p inhibited milk fat synthesis of OMECs. This is the first study to reveal the molecular mechanisms by which miR-199a-3p regulates the proliferation and milk fat synthesis of OMECs in sheep.
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Shang L, Xue G, Gong L, Zhang Y, Peng S, Yuan C, Huang M. A novel ELISA for the detection of active form of plasminogen activator inhibitor-1 based on a highly specific trapping agent. Anal Chim Acta 2019; 1053:98-104. [DOI: 10.1016/j.aca.2018.12.005] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2018] [Revised: 11/26/2018] [Accepted: 12/08/2018] [Indexed: 10/27/2022]
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Dlugosz P, Nimpf J. The Reelin Receptors Apolipoprotein E receptor 2 (ApoER2) and VLDL Receptor. Int J Mol Sci 2018; 19:E3090. [PMID: 30304853 PMCID: PMC6213145 DOI: 10.3390/ijms19103090] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2018] [Revised: 10/03/2018] [Accepted: 10/03/2018] [Indexed: 01/28/2023] Open
Abstract
Apolipoprotein E receptor 2 (ApoER2) and VLDL receptor belong to the low density lipoprotein receptor family and bind apolipoprotein E. These receptors interact with the clathrin machinery to mediate endocytosis of macromolecules but also interact with other adapter proteins to perform as signal transduction receptors. The best characterized signaling pathway in which ApoER2 and VLDL receptor (VLDLR) are involved is the Reelin pathway. This pathway plays a pivotal role in the development of laminated structures of the brain and in synaptic plasticity of the adult brain. Since Reelin and apolipoprotein E, are ligands of ApoER2 and VLDLR, these receptors are of interest with respect to Alzheimer's disease. We will focus this review on the complex structure of ApoER2 and VLDLR and a recently characterized ligand, namely clusterin.
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Affiliation(s)
- Paula Dlugosz
- Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University Vienna, 1030 Vienna, Austria.
| | - Johannes Nimpf
- Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University Vienna, 1030 Vienna, Austria.
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Banerjee K, Yakovlev S, Gruschus JM, Medved L, Tjandra N. Nuclear Magnetic Resonance Solution Structure of the Recombinant Fragment Containing Three Fibrin-Binding Cysteine-Rich Domains of the Very Low Density Lipoprotein Receptor. Biochemistry 2018; 57:4395-4403. [PMID: 29965730 PMCID: PMC6657517 DOI: 10.1021/acs.biochem.8b00349] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Our previous studies revealed that interaction of fibrin with the very low density lipoprotein (VLDL) receptor plays a prominent role in transendothelial migration of leukocytes and thereby inflammation. The major goal of our subsequent studies is to establish the structural basis for this interaction. As the first step toward this goal, we localized the fibrin-binding sites within cysteine-rich (CR) domains 2-4 of the VLDL receptor. In this study, we have made a next step toward this goal by establishing the nuclear magnetic resonance solution structure of the recombinant VLDLR(2-4) fragment containing all three fibrin-binding CR domains of this receptor. The structure revealed that all three CR domains have a similar general fold. Each domain contains a calcium-binding loop, and the loop in the CR3 domain has a unique conformation relative to the other two. Domains CR2 and CR3 interact with each other, while CR4 is flexible relative to the other two domains. In addition, analysis of the electrostatic potential surface of VLDLR(2-4) revealed extended negatively charged regions in each of its CR domains. The presence of these regions suggests that they may interact with three positively charged clusters of the fibrin βN domain whose involvement in interaction with the VLDL receptor was demonstrated earlier. Altogether, these findings provide a solid background for our next step toward establishing the structural basis for fibrin-VLDL receptor interaction.
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Affiliation(s)
- Koyeli Banerjee
- Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, United States
| | - Sergiy Yakovlev
- Center for Vascular and Inflammatory Diseases and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, United States
| | - James M. Gruschus
- Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, United States
| | - Leonid Medved
- Center for Vascular and Inflammatory Diseases and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, United States
| | - Nico Tjandra
- Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, United States
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Yakovlev S, Medved L. Interaction of Fibrin with the Very Low-Density Lipoprotein (VLDL) Receptor: Further Characterization and Localization of the VLDL Receptor-Binding Site in Fibrin βN-Domains. Biochemistry 2017; 56:2518-2528. [PMID: 28437098 DOI: 10.1021/acs.biochem.7b00087] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
Our recent study revealed that fibrin and the very low-density lipoprotein receptor (VLDLR) interact with each other through a pair of fibrin βN-domains and CR domains of the receptor and this interaction promotes transendothelial migration of leukocytes and thereby inflammation. The major objectives of this study were to further clarify the molecular mechanism of fibrin-VLDLR interaction and to identify amino acid residues in the βN-domains involved in this interaction. Our binding experiments with the (β15-66)2 fragment, which corresponds to a pair of fibrin βN-domains, and the VLDLR(1-8) fragment, consisting of eight CR domains of VLDLR, revealed that interaction between them strongly depends on ionic strength and chemical modification of all Lys or Arg residues in (β15-66)2 results in abrogation of this interaction. To identify which of these residues are involved in the interaction, we mutated all Lys or Arg residues in each of the three positively charged Lys/Arg clusters of the (β15-66)2 fragment, as well as single Arg17 and Arg30, and tested the affinity of the mutants obtained for VLDLR(1-8) by an enzyme-linked immunosorbent assay and surface plasmon resonance. The experiments revealed that the second and third Lys/Arg clusters make the major contribution to this interaction while the contribution of the first cluster is moderate. The results obtained suggest that interaction between fibrin and the VLDL receptor employs the "double-Lys/Arg" recognition mode previously proposed for the interaction of the LDL receptor family members with their ligands. They also provide valuable information for the development of highly specific peptide-based inhibitors of fibrin-VLDLR interaction.
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Affiliation(s)
- Sergiy Yakovlev
- Center for Vascular and Inflammatory Diseases and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine , Baltimore, Maryland 21201, United States
| | - Leonid Medved
- Center for Vascular and Inflammatory Diseases and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine , Baltimore, Maryland 21201, United States
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Li X, Zhao D, Guo Z, Li T, Qili M, Xu B, Qian M, Liang H, E X, Chege Gitau S, Wang L, Huangfu L, Wu Q, Xu C, Shan H. Overexpression of SerpinE2/protease nexin-1 Contribute to Pathological Cardiac Fibrosis via increasing Collagen Deposition. Sci Rep 2016; 6:37635. [PMID: 27876880 PMCID: PMC5120308 DOI: 10.1038/srep37635] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2016] [Accepted: 11/01/2016] [Indexed: 01/01/2023] Open
Abstract
Although increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis, the molecular mechanisms of pressure overload or AngII -induced cardiac interstitial fibrosis remain elusive. In this study, serpinE2/protease nexin-1 was over-expressed in a cardiac fibrosis model induced by pressure-overloaded via transverse aortic constriction (TAC) in mouse. Knockdown of serpinE2 attenuates cardiac fibrosis in a mouse model of TAC. At meantime, the results showed that serpinE2 significantly were increased with collagen accumulations induced by AngII or TGF-β stimulation in vitro. Intriguingly, extracellular collagen in myocardial fibroblast was reduced by knockdown of serpinE2 compared with the control in vitro. In stark contrast, the addition of exogenous PN-1 up-regulated the content of collagen in myocardial fibroblast. The MEK1/2- ERK1/2 signaling probably promoted the expression of serpinE2 via transcription factors Elk1 in myocardial fibroblast. In conclusion, stress-induced the ERK1/2 signaling pathway activation up-regulated serpinE2 expression, consequently led accumulation of collagen protein, and contributed to cardiac fibrosis.
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Affiliation(s)
- Xuelian Li
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China
| | - Dandan Zhao
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China
| | - Zhenfeng Guo
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China.,The second Clinical Medical School of Inner Mongolia University for Nationalities, Inner Mongolia Forestry General Hospital, Inner Mongolia, China
| | - Tianshi Li
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China
| | - Muge Qili
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China
| | - Bozhi Xu
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China
| | - Ming Qian
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China
| | - Haihai Liang
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China
| | - Xiaoqiang E
- Department of Orthopaedics, the First Affiliated Hospital, Harbin Medical University, Harbin, China
| | - Samuel Chege Gitau
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China.,Department of Pharmacy and Complementary Medicine, School of Health Sciences, Kenyatta University, Nairobi, Kenya
| | - Lu Wang
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China
| | - Longtao Huangfu
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China
| | - Qiuxia Wu
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China
| | - Chaoqian Xu
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China
| | - Hongli Shan
- Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, China
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Fredriksson K, Mishra A, Lam JK, Mushaben EM, Cuento RA, Meyer KS, Yao X, Keeran KJ, Nugent GZ, Qu X, Yu ZX, Yang Y, Raghavachari N, Dagur PK, McCoy JP, Levine SJ. The very low density lipoprotein receptor attenuates house dust mite-induced airway inflammation by suppressing dendritic cell-mediated adaptive immune responses. THE JOURNAL OF IMMUNOLOGY 2014; 192:4497-509. [PMID: 24733846 DOI: 10.4049/jimmunol.1301234] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The very low density lipoprotein receptor (VLDLR) is a member of the low-density lipoprotein receptor family that binds multiple ligands and plays a key role in brain development. Although the VLDLR mediates pleiotropic biological processes, only a limited amount of information is available regarding its role in adaptive immunity. In this study, we identify an important role for the VLDLR in attenuating house dust mite (HDM)-induced airway inflammation in experimental murine asthma. We show that HDM-challenged Vldlr(-/-) mice have augmented eosinophilic and lymphocytic airway inflammation with increases in Th2 cytokines, C-C chemokines, IgE production, and mucous cell metaplasia. A genome-wide analysis of the lung transcriptome identified that mRNA levels of CD209e (DC-SIGNR4), a murine homolog of DC-SIGN, were increased in the lungs of HDM-challenged Vldlr(-/-) mice, which suggested that the VLDLR might modify dendritic cell (DC) function. Consistent with this, VLDLR expression by human monocyte-derived DCs was increased by HDM stimulation. In addition, 55% of peripheral blood CD11c(+) DCs from individuals with allergy expressed VLDLR under basal conditions. Lastly, the adoptive transfer of HDM-pulsed, CD11c(+) bone marrow-derived DCs (BMDCs) from Vldlr(-/-) mice to the airways of wild type recipient mice induced augmented eosinophilic and lymphocytic airway inflammation upon HDM challenge with increases in Th2 cytokines, C-C chemokines, IgE production, and mucous cell metaplasia, as compared with the adoptive transfer of HDM-pulsed, CD11c(+) BMDCs from wild type mice. Collectively, these results identify a novel role for the VLDLR as a negative regulator of DC-mediated adaptive immune responses in HDM-induced allergic airway inflammation.
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Affiliation(s)
- Karin Fredriksson
- Laboratory of Asthma and Lung Inflammation, Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892
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8
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Simone TM, Higgins CE, Czekay RP, Law BK, Higgins SP, Archambeault J, Kutz SM, Higgins PJ. SERPINE1: A Molecular Switch in the Proliferation-Migration Dichotomy in Wound-"Activated" Keratinocytes. Adv Wound Care (New Rochelle) 2014; 3:281-290. [PMID: 24669362 DOI: 10.1089/wound.2013.0512] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2013] [Accepted: 01/13/2014] [Indexed: 11/13/2022] Open
Abstract
Significance: A highly interactive serine protease/plasmin/matrix metalloproteinase axis regulates stromal remodeling in the wound microenvironment. Current findings highlight the importance of stringent controls on protease expression and their topographic activities in cell proliferation, migration, and tissue homeostasis. Targeting elements in this cascading network may lead to novel therapeutic approaches for fibrotic diseases and chronic wounds. Recent Advances: Matrix-active proteases and their inhibitors orchestrate wound site tissue remodeling, cell migration, and proliferation. Indeed, the serine proteases urokinase plasminogen activator and tissue-type plasminogen activator (uPA/tPA) and their major phsyiological inhibitor, plasminogen activator inhibitor-1 (PAI-1; serine protease inhibitor clade E member 1 [SERPINE1]), are upregulated in several cell types during injury repair. Coordinate expression of proteolytic enzymes and their inhibitors in the wound bed provides a mechanism for fine control of focal proteolysis to facilitate matrix restructuring and cell motility in complex environments. Critical Issues: Cosmetic and tissue functional consequences of wound repair anomalies affect the quality of life of millions of patients in the United States alone. The development of novel therapeutics to manage individuals most affected by healing anomalies will likely derive from the identification of critical, translationally accessible, control elements in the wound site microenvironment. Future Directions: Activation of the PAI-1 gene early after wounding, its prominence in the repair transcriptome and varied functions suggest a key role in the global cutaneous injury response program. Targeting PAI-1 gene expression and/or PAI-1 function with molecular genetic constructs, neutralizing antibodies or small molecule inhibitors may provide a novel, therapeutically relevant approach, to manage the pathophysiology of wound healing disorders associated with deficient or excessive PAI-1 levels.
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Affiliation(s)
- Tessa M. Simone
- Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York
| | - Craig E. Higgins
- Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York
| | - Ralf-Peter Czekay
- Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York
| | - Brian K. Law
- Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida
| | - Stephen P. Higgins
- Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York
| | - Jaclyn Archambeault
- Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York
| | - Stacie M. Kutz
- Department of Biology, Sage College of Albany, Albany, New York
| | - Paul J. Higgins
- Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York
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Cartier-Michaud A, Malo M, Charrière-Bertrand C, Gadea G, Anguille C, Supiramaniam A, Lesne A, Delaplace F, Hutzler G, Roux P, Lawrence DA, Barlovatz-Meimon G. Matrix-bound PAI-1 supports cell blebbing via RhoA/ROCK1 signaling. PLoS One 2012; 7:e32204. [PMID: 22363817 PMCID: PMC3283740 DOI: 10.1371/journal.pone.0032204] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2011] [Accepted: 01/24/2012] [Indexed: 11/19/2022] Open
Abstract
The microenvironment of a tumor can influence both the morphology and the behavior of cancer cells which, in turn, can rapidly adapt to environmental changes. Increasing evidence points to the involvement of amoeboid cell migration and thus of cell blebbing in the metastatic process; however, the cues that promote amoeboid cell behavior in physiological and pathological conditions have not yet been clearly identified. Plasminogen Activator Inhibitor type-1 (PAI-1) is found in high amount in the microenvironment of aggressive tumors and is considered as an independent marker of bad prognosis. Here we show by immunoblotting, activity assay and immunofluorescence that, in SW620 human colorectal cancer cells, matrix-associated PAI-1 plays a role in the cell behavior needed for amoeboid migration by maintaining cell blebbing, localizing PDK1 and ROCK1 at the cell membrane and maintaining the RhoA/ROCK1/MLC-P pathway activation. The results obtained by modeling PAI-1 deposition around tumors indicate that matrix-bound PAI-1 is heterogeneously distributed at the tumor periphery and that, at certain spots, the elevated concentrations of matrix-bound PAI-1 needed for cancer cells to undergo the mesenchymal-amoeboid transition can be observed. Matrix-bound PAI-1, as a matricellular protein, could thus represent one of the physiopathological requirements to support metastatic formation.
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Affiliation(s)
| | - Michel Malo
- IBISC EA 4526, Evry Val d'Essonne University, Evry, France
| | - Cécile Charrière-Bertrand
- IBISC EA 4526, Evry Val d'Essonne University, Evry, France
- University Paris-Est Créteil, Créteil, France
| | - Gilles Gadea
- CRBM UMR 5237 CNRS, Montpellier University, Montpellier, France
| | | | | | - Annick Lesne
- Institut des Hautes Etudes Scientifiques, Bures-sur-Yvette, France
- LPTMC UMR 7600 CNRS, Paris, France
| | | | | | - Pierre Roux
- CRBM UMR 5237 CNRS, Montpellier University, Montpellier, France
| | - Daniel A. Lawrence
- Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan, United States of America
| | - Georgia Barlovatz-Meimon
- IBISC EA 4526, Evry Val d'Essonne University, Evry, France
- University Paris-Est Créteil, Créteil, France
- * E-mail:
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Complex Regulation of the Pericellular Proteolytic Microenvironment during Tumor Progression and Wound Repair: Functional Interactions between the Serine Protease and Matrix Metalloproteinase Cascades. Biochem Res Int 2012; 2012:454368. [PMID: 22454771 PMCID: PMC3290807 DOI: 10.1155/2012/454368] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2011] [Accepted: 11/21/2011] [Indexed: 01/08/2023] Open
Abstract
Spatial and temporal regulation of the pericellular proteolytic environment by local growth factors, such as EGF and TGF-β, initiates a wide repertoire of cellular responses coupled to a plasmin/matrix metalloproteinase (MMP) dependent stromal-remodeling axis. Cell motility and invasion, tumor metastasis, wound healing, and organ fibrosis, for example, represent diverse events controlled by expression of a subset of genes that encode various classes of tissue remodeling proteins. These include members of the serine protease and MMP families that functionally constitute a complex system of interacting protease cascades and titrated by their respective inhibitors. Several structural components of the extracellular matrix are upregulated by TGF-β as are matrix-active proteases (e.g., urokinase (uPA), plasmin, MMP-1, -3, -9, -10, -11, -13, -14). Stringent controls on serine protease/MMP expression and their topographic activity are essential for maintaining tissue homeostasis. Targeting individual elements in this highly interactive network may lead to novel therapeutic approaches for the treatment of cancer, fibrotic diseases, and chronic wounds.
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PAI-1: An Integrator of Cell Signaling and Migration. Int J Cell Biol 2011; 2011:562481. [PMID: 21837240 PMCID: PMC3151495 DOI: 10.1155/2011/562481] [Citation(s) in RCA: 136] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2011] [Revised: 05/09/2011] [Accepted: 05/17/2011] [Indexed: 12/23/2022] Open
Abstract
Cellular migration, over simple surfaces or through complex stromal barriers, requires coordination between detachment/re-adhesion cycles, involving structural components of the extracellular matrix and their surface-binding elements (integrins), and the precise regulation of the pericellular proteolytic microenvironment. It is now apparent that several proteases and protease inhibitors, most notably urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1), also interact with several cell surface receptors transducing intracellular signals that significantly affect both motile and proliferative programs. These events appear distinct from the original function of uPA/PAI-1 as modulators of the plasmin-based proteolytic cascade. The multifaceted interactions of PAI-1 with specific matrix components (i.e., vitronectin), the low-density lipoprotein receptor-related protein-1 (LRP1), and the uPA/uPA receptor complex have dramatic consequences on the migratory phenotype and may underlie the pathophysiologic sequalae of PAI-1 deficiency and overexpression. This paper focuses on the increasingly intricate role of PAI-1 as a major mechanistic determinant of the cellular migratory phenotype.
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He L, Lu Y, Wang P, Zhang J, Yin C, Qu S. Up-regulated expression of type II very low density lipoprotein receptor correlates with cancer metastasis and has a potential link to β-catenin in different cancers. BMC Cancer 2010; 10:601. [PMID: 21047397 PMCID: PMC2988033 DOI: 10.1186/1471-2407-10-601] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2010] [Accepted: 11/03/2010] [Indexed: 11/24/2022] Open
Abstract
Background Very low density lipoprotein receptor (VLDLR) has been considered as a multiple function receptor due to binding numerous ligands, causing endocytosis and regulating cellular signaling. Our group previously reported that enhanced activity of type II VLDLR (VLDLR II), one subtype of VLDLR, promotes adenocarcinoma SGC7901 cells proliferation and migration. The aim of this study is to explore the expression levels of VLDLR II in human gastric, breast and lung cancer tissues, and to investigate its relationship with clinical characteristics and β-catenin expression status. Methods VLDLR II expression was examined using immunohistochemistry (IHC) and Western blot in tumor tissues from 213 gastric, breast and lung cancer patients, tumor adjacent noncancerous tissues by same methods. Correlations between VLDLR II and clinical features, as well as β-catenin expression status were evaluated by statistical analysis. Results The immunohistochemical staining of VLDLR II showed statistical difference between tumor tissues and tumor adjacent noncancerous tissues in gastric, breast and lung cancers (P = 0.034, 0.018 and 0.043, respectively). Moreover, using Western, we found higher VLDLR II expression levels were associated with lymph node and distant metastasis in gastric and breast cancer (P < 0.05). Furthermore, highly significant positive correlations were found between VLDLR II and β-catenin in gastric cancer (r = 0.689; P < 0.001)breast cancer (r = 0.594; P < 0.001). Conclusions According to the results of the current study, high VLDLR II expression is correlated with lymph node and distant metastasis in gastric and breast cancer patients, the data suggest that VLDLR II may be a clinical marker in cancers, and has a potential link with β-catenin signaling pathway. This is the first to reveal the closer relationship of VLDLR II with clinical information.
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Affiliation(s)
- Lei He
- Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
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13
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Madsen JB, Dupont DM, Andersen TB, Nielsen AF, Sang L, Brix DM, Jensen JK, Broos T, Hendrickx MLV, Christensen A, Kjems J, Andreasen PA. RNA aptamers as conformational probes and regulatory agents for plasminogen activator inhibitor-1. Biochemistry 2010; 49:4103-15. [PMID: 20387790 DOI: 10.1021/bi100066j] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The hallmark of serpins is the ability to undergo the so-called "stressed-to-relaxed" switch during which the surface-exposed reactive center loop (RCL) becomes incorporated as strand 4 in central beta-sheet A. RCL insertion drives not only the inhibitory reaction of serpins with their target serine proteases but also the conversion to the inactive latent state. RCL insertion is coupled to conformational changes in the flexible joint region flanking beta-sheet A. One interesting serpin is plasminogen activator inhibitor-1 (PAI-1), a fast and specific inhibitor of the serine proteases tissue-type and urokinase-type plasminogen activator. Via its flexible joints' region, native PAI-1 binds vitronectin and relaxed, protease-complexed PAI-1 certain endocytosis receptors. From a library of 35-nucleotides long 2'-fluoropyrimidine-containing RNA oligonucleotides, we have isolated two aptamers binding PAI-1 by the flexible joint region with low nanomolar K(D) values. One of the aptamers exhibited measurable binding to native PAI-1 only, while the other also bound relaxed PAI-1. While none of the aptamers inhibited the antiproteolytic effect of PAI-1, both aptamers inhibited vitronectin binding and the relaxed PAI-1-binding aptamer also endocytosis receptor binding. The aptamer binding exclusively to native PAI-1 increased the half-life for the latency transition to more than 6 h, manyfold more than vitronectin. Contact with Lys124 in the flexible joint region was critical for strong inhibition of the latency transition and the lack of binding to relaxed PAI-1. We conclude that aptamers yield important information about the serpin conformational switch and, because they can compete with high-affinity protein-protein interactions, may provide leads for pharmacological intervention.
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Affiliation(s)
- Jeppe B Madsen
- Danish-Chinese Center for Proteases and Cancer, Aarhus University,10C Gustav Wieds Vej, 8000 Aarhus C, Denmark
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14
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Czekay RP, Loskutoff DJ. Plasminogen activator inhibitors regulate cell adhesion through a uPAR-dependent mechanism. J Cell Physiol 2009; 220:655-63. [PMID: 19472211 DOI: 10.1002/jcp.21806] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Binding of type-1 plasminogen activator inhibitor (PAI-1) to cell surface urokinase (uPA) promotes inactivation and internalization of adhesion receptors (e.g., urokinase receptor (uPAR), integrins) and leads to cell detachment from a variety of extracellular matrices. In this report, we begin to examine the mechanism of this process. We show that neither specific antibodies to uPA, nor active site inhibitors of uPA, can detach the cells. Thus, cell detachment is not simply the result of the binding of macromolecules to uPA and/or of the inactivation of uPA. We further demonstrate that another uPA inhibitor, protease nexin-1 (PN-1), also stimulates cell detachment in a uPA/uPAR-dependent manner. The binding of both inhibitors to uPA leads to the specific inactivation of the matrix-engaged integrins and the subsequent detachment of these integrins from the underlying extracellular matrix (ECM). This inhibitor-mediated inactivation of integrins requires direct interaction between uPAR and those integrins since cells attached to the ECM through integrins incapable of binding uPAR do not respond to the presence of either PAI-1 of PN-1. Although both inhibitors initiate the clearance of uPAR, only PAI-1 triggers the internalization of integrins. However, cell detachment by PAI-1 or PN-1 does not depend on the endocytosis of these integrins since cell detachment was also observed when clearance of these integrins was blocked. Thus, PAI-1 and PN-1 induce cell detachment through two slightly different mechanisms that affect integrin metabolism. These differences may be important for distinct cellular processes that require controlled changes in the subcellular localization of these receptors.
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Affiliation(s)
- Ralf-Peter Czekay
- Albany Medical College, Center for Cell Biology & Cancer Research, 47 New Scotland Avenue, Albany, NY 12208, USA.
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15
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Sakai K, Tiebel O, Ljungberg MC, Sullivan M, Lee HJ, Terashima T, Li R, Kobayashi K, Lu HC, Chan L, Oka K. A neuronal VLDLR variant lacking the third complement-type repeat exhibits high capacity binding of apoE containing lipoproteins. Brain Res 2009; 1276:11-21. [PMID: 19393635 DOI: 10.1016/j.brainres.2009.04.030] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2009] [Revised: 03/30/2009] [Accepted: 04/12/2009] [Indexed: 12/31/2022]
Abstract
Very-low-density lipoprotein receptor (VLDLR) is a multi ligand apolipoprotein E (apoE) receptor and is involved in brain development through Reelin signaling. Different forms of VLDLR can be generated by alternative splicing. VLDLR-I contains all exons. VLDLR-II lacks an O-linked sugar domain encoded by exon 16, while VLDLR-III lacks the third complement-type repeat in the ligand binding domain encoded by exon 4. We quantitatively compared lipoprotein binding to human VLDLR variants and analyzed their mRNA expression in both human cerebellum and mouse brain. VLDLR-III exhibited the highest capacity in binding to apoE enriched beta-VLDL in vitro and was more effective in removing apoE containing lipoproteins from the circulation than other variants in vivo. In human cerebellum, the major species was VLDLR-II, while the second most abundant species was a newly identified VLDLR-IV which lacks both exon 4 and 16. VLDLR-I was present at low levels. In adult mice, exon 4 skipping varied between 30 and 47% in different brain regions, while exon 16 skipping ranged by 51-76%. Significantly higher levels of VLDLR proteins were found in mouse cerebellum and cerebral cortex than other regions. The deletions of exon 4 and exon 16 frequently occurred in primary neurons, indicating that newly identified variant VLDLR-IV is abundant in neurons. In contrast, VLDLR mRNA lacking exon 4 was not detectable in primary astrocytes. Such cell type-specific splicing patterns were found in both mouse cerebellum and cerebral cortex. These results suggest that a VLDLR variant lacking the third complement-type repeat is generated by neuron-specific alternative splicing. Such differential splicing may result in different lipid uptake in neurons and astrocytes.
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Affiliation(s)
- Keiko Sakai
- Department of Neurology, Baylor College of Medicine, Houston, TX 77030, USA
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16
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Rasch MG, Pass J, Illemann M, Høyer-Hansen G, Lund IK. Discrimination of different forms of the murine urokinase plasminogen activator receptor on the cell surface using monoclonal antibodies. J Immunol Methods 2008; 339:55-65. [PMID: 18761343 DOI: 10.1016/j.jim.2008.08.002] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2008] [Revised: 08/04/2008] [Accepted: 08/05/2008] [Indexed: 11/18/2022]
Abstract
The urokinase plasminogen activator receptor (uPAR) is a versatile three-domain GPI-anchored protein, which binds urokinase plasminogen activator (uPA) and thereby focalises plasminogen activation on the cell surface. Generation of a proteolytic potential is essential in both normal physiological and pathological extracellular tissue remodelling processes. uPA can also cleave uPAR, resulting in liberation of the amino-terminal domain I, which encompasses binding sites for both uPA and the adhesion molecule, vitronectin. In order to localise the different uPAR forms on the plasma membrane of murine monocyte macrophage-like P388D.1 cells, we have now generated and characterised two high-affinity murine mAbs, mR3 and mR4, raised against murine uPAR. mR3 was found to recognise an epitope located in domain I of uPAR. Surface plasmon resonance analyses and cell binding studies revealed that this mAb was able to bind preformed complexes of murine pro-uPA and murine uPAR. In contrast, mR4 recognises domains II-III in uPAR and does not bind preformed pro-uPA-uPAR complexes in similar analyses. Immunofluorescence microscopy of P388D.1 cells revealed that mR3 stained the cells equally well in the presence or absence of saturation with the amino-terminal fragment of uPA, ATF. However, the signal intensity obtained using another uPAR domain I specific mAb, mR1, was significantly reduced upon ATF saturation. Furthermore, when adding ATF, mR4 selectively stained the cleaved receptor. Applying these newly generated mAbs, we additionally demonstrated that cleaved and intact uPAR was evenly distributed on the surface of these cells.
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Affiliation(s)
- Morten G Rasch
- Finsen Laboratory, Rigshospitalet section 3735, Copenhagen Biocenter, Copenhagen N, Denmark
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17
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Lillis AP, Van Duyn LB, Murphy-Ullrich JE, Strickland DK. LDL receptor-related protein 1: unique tissue-specific functions revealed by selective gene knockout studies. Physiol Rev 2008; 88:887-918. [PMID: 18626063 DOI: 10.1152/physrev.00033.2007] [Citation(s) in RCA: 531] [Impact Index Per Article: 31.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The LDL receptor-related protein (originally called LRP, but now referred to as LRP1) is a large endocytic receptor that is widely expressed in several tissues. LRP1 is a member of the LDL receptor family that plays diverse roles in various biological processes including lipoprotein metabolism, degradation of proteases, activation of lysosomal enzymes, and cellular entry of bacterial toxins and viruses. Deletion of the LRP1 gene leads to lethality in mice, revealing a critical, but as of yet, undefined role in development. Tissue-specific gene deletion studies reveal an important contribution of LRP1 in the vasculature, central nervous system, macrophages, and adipocytes. Three important properties of LRP1 dictate its diverse role in physiology: 1) its ability to recognize more than 30 distinct ligands, 2) its ability to bind a large number of cytoplasmic adaptor proteins via determinants located on its cytoplasmic domain in a phosphorylation-specific manner, and 3) its ability to associate with and modulate the activity of other transmembrane receptors such as integrins and receptor tyrosine kinases.
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Affiliation(s)
- Anna P Lillis
- Center for Vascular and Inflammatory Diseases and Department of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
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18
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Montel V, Gaultier A, Lester RD, Campana WM, Gonias SL. The low-density lipoprotein receptor-related protein regulates cancer cell survival and metastasis development. Cancer Res 2007; 67:9817-24. [PMID: 17942912 DOI: 10.1158/0008-5472.can-07-0683] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. In this study, we show that LRP-1 is abundantly expressed in severe combined immunodeficient (SCID) mouse xenografts by various human cancer cell lines that express very low or undetectable levels of LRP-1 when cultured in 21% O2 in vitro (standard cell culture conditions). To test whether LRP-1 expression in vivo may be explained by hypoxia in the xenografts, CL16 cells, which are derived from the MDA-MB-435 cell line, were cultured in 1.0% O2. A substantial increase in LRP-1 expression was observed. To test the activity of LRP-1 in cancer progression in vivo, LRP-1 expression was silenced in CL16 cells with short hairpin RNA. These cells formed tumors in SCID mice, in which LRP-1 expression remained silenced. Although LRP-1 gene silencing did not inhibit CL16 cell dissemination from the primary tumors to the lungs, the pulmonary metastases failed to enlarge, suggesting compromised survival or growth at the implantation site. In cell culture experiments, significantly increased cell death was observed when LRP-1-silenced CL16 cells were exposed to CoCl2, which models changes that occur in hypoxia. Furthermore, LRP-1-silenced cells expressed decreased levels of vascular endothelial growth factor in response to 1.0% O2. These results suggest mechanisms by which LRP-1 may facilitate the development and growth of cancer metastases in vivo.
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Affiliation(s)
- Valérie Montel
- Department of Pathology, University of California San Diego School of Medicine, La Jolla, California 92093-0612, USA
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19
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Croucher D, Saunders D, Stillfried G, Ranson M. A structural basis for differential cell signalling by PAI-1 and PAI-2 in breast cancer cells. Biochem J 2007; 408:203-10. [PMID: 17696882 PMCID: PMC2267350 DOI: 10.1042/bj20070767] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
PAI-1 and PAI-2 (plasminogen-activator inibitor types 1 and 2) are inhibitors of cell surface uPA (urokinase plasminogen activator). However, tumour expression of PAI-1 and PAI-2 correlates with poor compared with good patient prognosis in breast cancer respectively. This biological divergence may be related to additional functional roles of PAI-1. For example, the inhibition of uPA by PAI-1 reveals a cryptic high-affinity site within the PAI-1 moiety for the VLDLr (very-low-density-lipoprotein receptor), which sustains cell signalling events initiated by binding of uPA to its receptor. These interactions and subsequent signalling events promote proliferation of breast cancer cells. Biochemical and structural analyses show that, unlike PAI-1, the PAI-2 moiety of uPA-PAI-2 does not contain a high-affinity-binding site for VLDLr, although uPA-PAI-2 is still efficiently endocytosed via this receptor in breast cancer cells. Furthermore, global protein tyrosine phosphorylation events were not sustained by uPA-PAI-2 and cell proliferation was not affected. We thus propose a structurally based mechanism for these differences between PAI-1 and PAI-2 and suggest that PAI-2 is able to inhibit and clear uPA activity without initiating mitogenic signalling events through VLDLr.
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Affiliation(s)
- David R. Croucher
- *School of Biological Sciences, University of Wollongong, NSW 2522, Australia
| | - Darren N. Saunders
- †Cancer Research Program, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia
- ‡Department of Pathology and Laboratory Medicine, University of British Columbia and Department of Molecular Oncology, BC Cancer Research Centre, Vancouver, BC, Canada V5Z 1L3
| | | | - Marie Ranson
- *School of Biological Sciences, University of Wollongong, NSW 2522, Australia
- To whom correspondence should be addressed (email )
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20
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Skeldal S, Larsen JV, Pedersen KE, Petersen HH, Egelund R, Christensen A, Jensen JK, Gliemann J, Andreasen PA. Binding areas of urokinase-type plasminogen activator?plasminogen activator inhibitor-1 complex for endocytosis receptors of the low-density lipoprotein receptor family, determined by site-directed mutagenesis. FEBS J 2006; 273:5143-59. [PMID: 17042782 DOI: 10.1111/j.1742-4658.2006.05511.x] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Some endocytosis receptors related to the low-density lipoprotein receptor, including low-density lipoprotein receptor-related protein-1A, very-low-density lipoprotein receptor, and sorting protein-related receptor, bind protease-inhibitor complexes, including urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and the uPA-PAI-1 complex. The unique capacity of these receptors for high-affinity binding of many structurally unrelated ligands renders mapping of receptor-binding surfaces of serpin and serine protease ligands a special challenge. We have mapped the receptor-binding area of the uPA-PAI-1 complex by site-directed mutagenesis. Substitution of a cluster of basic residues near the 37-loop and 60-loop of uPA reduced the receptor-binding affinity of the uPA-PAI-1 complex approximately twofold. Deletion of the N-terminal growth factor domain of uPA reduced the affinity 2-4-fold, depending on the receptor, and deletion of both the growth factor domain and the kringle reduced the affinity sevenfold. The binding affinity of the uPA-PAI-1 complex to the receptors was greatly reduced by substitution of basic and hydrophobic residues in alpha-helix D and alpha-helix E of PAI-1. The localization of the implicated residues in the 3D structures of uPA and PAI-1 shows that they form a continuous receptor-binding area spanning the serpin as well as the A-chain and the serine protease domain of uPA. Our results suggest that the 10-100-fold higher affinity of the uPA-PAI-1 complex compared with the free components depends on the bonus effect of bringing the binding areas on uPA and PAI-1 together on the same binding entity.
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Affiliation(s)
- Sune Skeldal
- Department of Molecular Biology, University of Aarhus, Denmark
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21
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Croucher D, Saunders DN, Ranson M. The urokinase/PAI-2 complex: a new high affinity ligand for the endocytosis receptor low density lipoprotein receptor-related protein. J Biol Chem 2006; 281:10206-13. [PMID: 16459332 DOI: 10.1074/jbc.m513645200] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
The efficient inactivation of urokinase plasminogen activator (uPA) by plasminogen activator inhibitor type 2 (PAI-2) at the surface of carcinoma cells is followed by rapid endocytosis of the uPA-PAI-2 complex. We now show that one pathway of this receptor-mediated endocytosis is mediated via the low density lipoprotein receptor-related protein (LRP) in prostate cancer cells. Detailed biochemical analyses using ligand binding assays and surface plasmon resonance revealed a novel and distinct interaction mechanism between native, human LRP and uPA-PAI-2. As reported previously for PAI-1, inhibition of uPA by PAI-2 significantly increased the affinity of the complex for LRP (K(D) of 36 nm for uPA-PAI-2 versus 200 nm for uPA). This interaction was maintained in the presence of uPAR, confirming the validity of this interaction at the cell surface. However, unlike PAI-1, no interaction was observed between LRP and PAI-2 in either the stressed or the relaxed conformation. This suggests that the uPA-PAI-2-LRP interaction is mediated by site(s) within the uPA molecule alone. Thus, as inhibition of uPA by PAI-2 resulted in accelerated clearance of uPA from the cell surface possibly via its increased affinity for LRP, this represents a mechanism through which PAI-2 can clear proteolytic activity from the cell surface. Furthermore, lack of a direct interaction between PAI-2 and LRP implies that downstream signaling events initiated by PAI-1 may not be activated by PAI-2.
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Affiliation(s)
- David Croucher
- School of Biological Sciences, University of Wollongong, New South Wales 2522
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22
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Chen T, Wu F, Chen FM, Tian J, Qu S. Variations of very low-density lipoprotein receptor subtype expression in gastrointestinal adenocarcinoma cells with various differentiations. World J Gastroenterol 2005; 11:2817-21. [PMID: 15884130 PMCID: PMC4305924 DOI: 10.3748/wjg.v11.i18.2817] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: This study is aimed at investigating the expression and possible significances of very low-density lipoprotein receptor (VLDLR) subtypes in gastroenteric adenocarcinoma tissues and cells with various differentiations.
METHODS: Thirty-one cases of gastroenteric carcinoma/adjacent normal tissues were enrolled in the study, which were diagnosed and classified by the clinicopathological diagnosis. The expression of VLDLR subtypes was detected in gastroenteric carcinoma/adjacent normal tissues and three various differentiated human gastric adenocarcinoma cell lines (MKN28, SGC7901 and MKN45) by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis.
RESULTS: Two VLDLR subtypes, namely, type II VLDLR and type I VLDLR, were found to express changes in gastroenteric carcinoma tissues, their adjacent normal tissue, and gastric adenocarcinoma cell lines as well. Type II VLDLR is predominantly expressed in poorly- or moderately-differentiated gastroenteric carcinoma tissues and gastric adenocarcinoma cell lines, whereas type I VLDLR is mainly detected in well-differentiated intestinal carcinoma tissues and gastric adenocarcinoma cells compared with the adjacent normal tissues.
CONCLUSION: The results suggested that the variations of the VLDLR subtype expression might be correlated with the progress and differentiation of gastroenteric carcinoma.
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Affiliation(s)
- Tao Chen
- Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
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23
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Jossin Y. Neuronal migration and the role of reelin during early development of the cerebral cortex. Mol Neurobiol 2005; 30:225-51. [PMID: 15655250 DOI: 10.1385/mn:30:3:225] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2004] [Accepted: 04/29/2004] [Indexed: 11/11/2022]
Abstract
During development, neurons migrate to the cortex radially from periventricular germinative zones as well as tangentially from ganglionic eminences. The vast majority of cortical neurons settle radially in the cortical plate. Neuronal migration requires an exquisite regulation of leading edge extension, nuclear translocation (nucleokinesis), and retraction of trailing processes. During the past few years, several genes and proteins have been identified that are implicated in neuronal migration. Many have been characterized by reference to known mechanisms of neuronal and non-neuronal cell migration in culture; however, probably the most interesting have been identified by gene inactivation or modification in mice and by positional cloning of brain malformation genes in humans and mice. Although it is impossible to provide a fully integrated view, some patterns clearly emerge and are the subject of this article. Specific emphasis is placed on three aspects: first, the role of the actin treadmill, with cyclic formation of filopodial and lamellipodial extensions, in relation to surface events that occur at the leading edge of radially migrating neurons; second, the regulation of microtubule dynamics, which seems to play a key role in nucleokinesis; and third, the mechanisms by which the extracellular protein Reelin regulates neuronal positioning at the end of migration.
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Affiliation(s)
- Yves Jossin
- Developmental Neurobiology Unit, University of Louvain Medical School, Brussels, Belgium.
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24
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Na YR, Im H. The length of the reactive center loop modulates the latency transition of plasminogen activator inhibitor-1. Protein Sci 2005; 14:55-63. [PMID: 15576554 PMCID: PMC2253313 DOI: 10.1110/ps.041063705] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2004] [Revised: 09/13/2004] [Accepted: 09/14/2004] [Indexed: 10/26/2022]
Abstract
Plasminogen activator inhibitor-1 (PAI-1) belongs to the serine protease inhibitor (serpin) protein family, which has a common tertiary structure consisting of three beta-sheets and several alpha-helices. Despite the similarity of its structure with those of other serpins, PAI-1 is unique in its conformational lability, which allows the conversion of the metastable active form to a more stable latent conformation under physiological conditions. For the conformational conversion to occur, the reactive center loop (RCL) of PAI-1 must be mobilized and inserted into the major beta-sheet, A sheet. In an effort to understand how the structural conversion is regulated in this conformationally labile serpin, we modulated the length of the RCL of PAI-1. We show that releasing the constraint on the RCL by extension of the loop facilitates a conformational transition of PAI-1 to a stable state. Biochemical data strongly suggest that the stabilization of the transformed conformation is owing to the insertion of the RCL into A beta-sheet, as in the known latent form. In contrast, reducing the loop length drastically retards the conformational change. The results clearly show that the constraint on the RCL is a factor that regulates the conformational transition of PAI-1.
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Affiliation(s)
- Yu-Ran Na
- Department of Molecular Biology, Research Center for Conformational Degenerative Diseases, Sejong University, 98 Gunja-dong, Kwangjin-gu, Seoul 143-747, Korea
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25
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Gliemann J, Hermey G, Nykjaer A, Petersen CM, Jacobsen C, Andreasen PA. The mosaic receptor sorLA/LR11 binds components of the plasminogen-activating system and platelet-derived growth factor-BB similarly to LRP1 (low-density lipoprotein receptor-related protein), but mediates slow internalization of bound ligand. Biochem J 2004; 381:203-12. [PMID: 15053742 PMCID: PMC1133778 DOI: 10.1042/bj20040149] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2004] [Revised: 03/24/2004] [Accepted: 03/30/2004] [Indexed: 11/17/2022]
Abstract
The type-1 receptor sorLA/LR11, a member of the Vps10p-domain receptor family that also contains domains characterizing members of the LDL (low-density lipoprotein) receptor family, has been shown to induce increased uPAR (urokinase receptor) expression as well as enhanced migration and invasion activities in smooth muscle cells in the presence of PDGF-BB (platelet-derived growth factor-BB). Here we show that sorLA interacts with both components of the plasminogen activating system and PDGF-BB similarly to LRP1 (LDL receptor-related protein/alpha2-macroglobulin receptor), which is an important clearance receptor with established functions in controlling uPAR expression as well as PDGF-BB signalling. In contrast with LRP1, sorLA does not interact with alpha2-macroglobulin, which is a binding protein for several growth factors, including PDGF-BB. By using LRP1-deficient cells transfected with sorLA, we demonstrate that sorLA-bound ligand is internalized at a much lower rate than LRP1-bound ligand, and that sorLA is inefficient in regulating cell surface uPAR expression, which depends on rapid internalization of the ternary complex between urokinase-type plasminogen activator, its type-1 inhibitor, and uPAR. Thus, although overlapping with regard to binding profiles, sorLA is substantially less efficient as a clearance receptor than LRP1. We propose that sorLA can divert ligands away from LRP1 and thereby inhibit both their clearance and signalling events mediated by LRP1.
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Affiliation(s)
- Jørgen Gliemann
- Institute of Medical Biochemistry, University of Aarhus, Ole Worms Allé bldg 170, DK-8000 Arhus C, Denmark.
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26
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Behrendt N. The urokinase receptor (uPAR) and the uPAR-associated protein (uPARAP/Endo180): membrane proteins engaged in matrix turnover during tissue remodeling. Biol Chem 2004; 385:103-36. [PMID: 15101555 DOI: 10.1515/bc.2004.031] [Citation(s) in RCA: 71] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
The breakdown of the barriers formed by extracellular matrix proteins is a pre-requisite for all processes of tissue remodeling. Matrix degradation reactions take part in specific physiological events in the healthy organism but also represent a crucial step in cancer invasion. These degradation processes involve a highly organized interplay between proteases and their cellular binding sites as well as specific substrates and internalization receptors. This review article is focused on two components, the urokinase plasminogen activator receptor (uPAR) and the uPAR-associated protein (uPARAP, also designated Endo180), that are considered crucially engaged in matrix degradation. uPAR and uPARAP have highly diverse functions, but on certain cell types they interact with each other in a process that is still incompletely understood. uPAR is a glycosyl-phosphatidylinositol-anchored glycoprotein on the surface of various cell types that serves to bind the urokinase plasminogen activator and localize the activation reactions in the proteolytic cascade system of plasminogen activation. uPARAP is an integral membrane protein with a pronounced role in the internalization of collagen for intracellular degradation. Both receptors have additional functions that are currently being unraveled. The present discussion of uPAR and uPARAP is centered on their protein structure and molecular and cellular function.
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Affiliation(s)
- Niels Behrendt
- Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, Bldg. 7.2, DK-2100 Copenhagen O, Denmark
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27
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Chung NS, Wasan KM. Potential role of the low-density lipoprotein receptor family as mediators of cellular drug uptake. Adv Drug Deliv Rev 2004; 56:1315-34. [PMID: 15109771 DOI: 10.1016/j.addr.2003.12.003] [Citation(s) in RCA: 103] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2003] [Accepted: 12/20/2003] [Indexed: 12/12/2022]
Abstract
We highlight the importance of the low-density lipoprotein (LDL) receptor family and its pharmaceutical implications in the field of drug delivery. The members of the LDL receptor family are a group of cell surface receptors that transport a number of macromolecules into cells through a process called receptor-mediated endocytosis. This process involves the receptor recognizing a ligand from the extracellular membrane (ECM), internalizing it through clathrin-coated pits and degrading it upon fusion with lysosomes. There are nine members of the receptor family, which include the LDL receptor, low-density lipoprotein-related protein (LRP), megalin, very low-density lipoprotein (VLDL) receptor, apoER2 and sorLA/LRP11, LRP1b, MEGF7, LRP5/6; the former six having been identified in humans. Each member is expressed in a number of different tissues and has a wide range of different ligands, not specific to the recognition of the LDL particle. Thus, rather than the original hypothesis that the receptor is only a mediator of cholesterol uptake, it may also be involved in a number of other physiological functions, including the progression of certain disease states and, potentially, cellular drug uptake. A number of studies have suggested that the LDL receptors are involved in endocytosis of drugs and drug formulations including aminoglycosides, anionic liposomes and cyclosporine A (CsA). This article reviews the importance of lipoproteins as a drug delivery system and how LDL receptors are relevant to the design and targeting of specific drugs.
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Affiliation(s)
- Nancy S Chung
- Faculty of Pharmaceutical Sciences, Division of Pharmaceutics and Biopharmaceutics, University of British Columbia, 2146 East Mall Ave., Vancouver, BC, Canada V6T 1Z3
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28
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Takahashi S, Sakai J, Fujino T, Hattori H, Zenimaru Y, Suzuki J, Miyamori I, Yamamoto TT. The Very Low-density Lipoprotein (VLDL) Receptor: Characterization and Functions as a Peripheral Lipoprotein Receptor. J Atheroscler Thromb 2004; 11:200-8. [PMID: 15356379 DOI: 10.5551/jat.11.200] [Citation(s) in RCA: 116] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
The very low-density lipoprotein (VLDL) receptor is a member of the low-density lipoprotein (LDL) receptor family. In vitro and in vivo studies have shown that VLDL receptor binds triglyceride (TG)-rich lipoproteins but not LDL, and functions as a peripheral remnant lipoprotein receptor. VLDL receptor is expressed abundantly in fatty acid-active tissues (heart, skeletal muscle and fat), the brain and macrophages. It is likely that VLDL receptor functions in concert with lipoprotein lipase (LPL), which hydrolyses TG in VLDL and chylomicron. In contrast to the LDL receptor, VLDL receptor binds apolipoprotein (apo) E2/2 VLDL particles as well as apoE3/3 VLDL, and the expression is not down-regulated by intracellular lipoproteins. Recently, various functions of the VLDL receptor have been reported in lipoprotein metabolism, metabolic syndrome/atherosclerosis, cardiac fatty acid metabolism, neuronal migration and angiogenesis/tumor growth. Gene therapy of VLDL receptor into the liver showed a benefit effect for lipoprotein metabolism in both LDL receptor knockout and apoE mutant mice. Beyond its function as a peripheral lipoprotein receptor, possibilities of its physiological function have been extended to include signal transduction, angiogenesis and tumor growth.
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Affiliation(s)
- Sadao Takahashi
- The Third Department of Internal Medicine, University of Fukui, 23-3, Matsuokacho, Fukui, 910-1193, Japan.
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29
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Glunde K, Guggino SE, Solaiyappan M, Pathak AP, Ichikawa Y, Bhujwalla ZM. Extracellular acidification alters lysosomal trafficking in human breast cancer cells. Neoplasia 2003; 5:533-45. [PMID: 14965446 PMCID: PMC1502575 DOI: 10.1016/s1476-5586(03)80037-4] [Citation(s) in RCA: 218] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2003] [Revised: 10/06/2003] [Accepted: 10/08/2003] [Indexed: 11/24/2022]
Abstract
Cancer cells invade by secreting degradative enzymes, which are sequestered in lysosomal vesicles. In this study, the impact of an acidic extracellular environment on lysosome size, number, and distance from the nucleus in human mammary epithelial cells (HMECs) and breast cancer cells of different degrees of malignancy was characterized because the physiological microenvironment of tumors is frequently characterized by extracellular acidity. An acidic extracellular pH (pH(e)) resulted in a distinct shift of lysosomes from the perinuclear region to the cell periphery irrespective of the HMECs' degree of malignancy. With decreasing pH, larger lysosomal vesicles were observed more frequently in highly metastatic breast cancer cells, whereas smaller lysosomes were observed in poorly metastatic breast cancer cells and HMECs. The number of lysosomes decreased with acidic pH values. The displacement of lysosomes to the cell periphery driven by extracellular acidosis may facilitate exocytosis of these lysosomes and increase secretion of degradative enzymes. Filopodia formations, which were observed more frequently in highly metastatic breast cancer cells maintained at acidic pH(e), may also contribute to invasion.
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Affiliation(s)
- Kristine Glunde
- Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Sandra E. Guggino
- Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Meiyappan Solaiyappan
- Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Arvind P. Pathak
- Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Yoshitaka Ichikawa
- Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Zaver M. Bhujwalla
- Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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30
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Strickland DK, Ranganathan S. Diverse role of LDL receptor-related protein in the clearance of proteases and in signaling. J Thromb Haemost 2003; 1:1663-70. [PMID: 12871303 DOI: 10.1046/j.1538-7836.2003.00330.x] [Citation(s) in RCA: 87] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that participates in several biological pathways and plays prominent roles in lipoprotein metabolism and in the catabolism of proteinases involved in coagulation and fibrinolysis. LRP also mediates the cellular entry of certain viruses and toxins and facilitates the activation of various lysosomal enzymes. Deletion of the LRP gene in mice is lethal, confirming an important role for this receptor in development, although its exact function in development is still not known. In addition to its role in the endocytosis of numerous ligands, recent studies are emerging that describe a signaling role for this receptor as well.
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Affiliation(s)
- D K Strickland
- Department of Vascular Biology, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, MD, USA.
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31
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Takahashi S, Sakai J, Fujino T, Miyamori I, Yamamoto TT. The very low density lipoprotein (VLDL) receptor--a peripheral lipoprotein receptor for remnant lipoproteins into fatty acid active tissues. Mol Cell Biochem 2003; 248:121-7. [PMID: 12870663 DOI: 10.1023/a:1024184201941] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
The VLDL (very low density lipoprotein) receptor is a member of the LDL (low density lipoprotein) receptor family. The VLDL receptor binds apolipoprotein (apo) E but not apo B, and is expressed in fatty acid active tissues (heart, muscle, adipose) and macrophages abundantly. Lipoprotein lipase (LPL) modulates the binding of triglyceride (TG)-rich lipoprotein particles to the VLDL receptor. By the unique ligand specificity, VLDL receptor practically appeared to function as IDL (intermediate density lipoprotein) and chylomicron remnant receptor in peripheral tissues in concert with LPL. In contrast to LDL receptor, the VLDL receptor expression is not down regulated by lipoproteins. Recently several possible functions of the VLDL receptor have been reported in lipoprotein metabolism, atherosclerosis, obesity/insulin resistance, cardiac fatty acid metabolism and neuronal migration. The gene therapy of VLDL receptor into the LDL receptor knockout mice liver showed a benefit effect for lipoprotein metabolism and atherosclerosis. Further researches about the VLDL receptor function will be needed in the future.
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Affiliation(s)
- Sadao Takahashi
- The Third Department of Internal Medicine, Fukui Medical University, Fukui, Japan.
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32
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Nykjaer A, Willnow TE. The low-density lipoprotein receptor gene family: a cellular Swiss army knife? Trends Cell Biol 2002; 12:273-80. [PMID: 12074887 DOI: 10.1016/s0962-8924(02)02282-1] [Citation(s) in RCA: 152] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
The low-density lipoprotein receptor gene family is an evolutionarily conserved group of cell-surface receptors produced by mammals and other organisms. Initially thought to be endocytic receptors that mediate the uptake of lipoproteins, recent findings have shown that these receptors have other roles in a range of cellular processes. Among other activities, members of this family act as signal transducers in neuronal migration processes, regulate synaptic plasticity or control vitamin homeostasis. Such multifunctionality is achieved by interaction with diverse cell-surface proteins including glycolipid-anchored receptors, G-protein-coupled receptors and ion channels. Here, we review the molecular interactions of this protein family with other cell-surface proteins that provide specificity and versatility - a versatility that may be reminiscent of a cellular Swiss army knife.
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Affiliation(s)
- Anders Nykjaer
- Dept of Medical Biochemistry, University of Aarhus, DK-8000, Aarhus C, Denmark.
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33
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Kjøller L. The urokinase plasminogen activator receptor in the regulation of the actin cytoskeleton and cell motility. Biol Chem 2002; 383:5-19. [PMID: 11928822 DOI: 10.1515/bc.2002.002] [Citation(s) in RCA: 70] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Cell migration is a complex process requiring tight control of several mechanisms including dynamic reorganization of the actin cytoskeleton and adhesion to the extracellular matrix. The GPI-anchored urokinase plasminogen activator receptor (uPAR) has an important role in the regulation of cell motility in many cell types. This is partly due to the localization of proteolytic activity on the cell surface by binding of the serine protease uPA. Results accumulated over the last decade suggest that uPAR is also involved in motility control through other mechanisms. These include induction of signal transduction events after ligation with uPA, binding to the extracellular matrix molecule vitronectin (VN), and association with integrins and other transmembrane partners. In this review these mechanisms will be discussed with a special emphasis on how the GPI-linked receptor transmits signals to the intracellular milieu and how uPAR participates in the regulation of actin cytoskeleton reorganization and cell adhesion during cell migration.
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Affiliation(s)
- Lars Kjøller
- The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark
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34
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Chazaud B, Ricoux R, Christov C, Plonquet A, Gherardi RK, Barlovatz-Meimon G. Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations. THE AMERICAN JOURNAL OF PATHOLOGY 2002; 160:237-46. [PMID: 11786417 PMCID: PMC1867133 DOI: 10.1016/s0002-9440(10)64367-2] [Citation(s) in RCA: 82] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The urokinase-type plasminogen activator (uPA) system is a dynamic complex in which the membrane receptor uPAR binds uPA that binds the plasminogen activator inhibitor (PAI)-1 localized in the extracellular matrix, resulting in endocytosis of the whole complex by the low-density lipoprotein receptor-related protein (LRP). High expression of PAI-1 is paradoxically associated with marked tumor spreading and poor prognosis. We previously reported a nonproteolytic role of the [uPAR:uPA:PAI-1:LRP] complex operative in cell migration. Here we explored whether matrix PAI-1 could be used as a migration support by human breast cancer cells. We showed that the uPA system and LRP are localized at filopodia of invasive cells, and that formation/internalization of the [uPAR:uPA:PAI-1:LRP] complex is required for attachment and migration of cancer cells on plastic and on a PAI-1 coat. PAI-1 increased both filopodia formation and migration of cancer cells suggesting a chemokine-like activity. Migration velocity, expression of the uPA system, use of the [uPAR:uPA:PAI-1:LRP] complex to migrate, and promigratory effects of PAI-1 paralleled cancer cell invasiveness. Phenotyping and functional analysis of invasive cancer cell subclones indicated that different cell subpopulations may use different strategies to migrate depending on both the environment and their expression of the uPA system, some of them taking advantage of abundant available PAI-1.
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Affiliation(s)
- Bénédicte Chazaud
- GERMEN, INSERM EMI-0011, Université Paris XII Val-De-Marne, Créteil, France
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35
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Ma Z, Webb DJ, Jo M, Gonias SL. Endogenously produced urokinase-type plasminogen activator is a major determinant of the basal level of activated ERK/MAP kinase and prevents apoptosis in MDA-MB-231 breast cancer cells. J Cell Sci 2001; 114:3387-96. [PMID: 11591826 DOI: 10.1242/jcs.114.18.3387] [Citation(s) in RCA: 95] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Urokinase-type plasminogen activator (uPA) binds to the uPA receptor (uPAR) and activates the Ras-extracellular signal-regulated kinase (ERK) signaling pathway in many different cell types. In this study, we demonstrated that endogenously produced uPA functions as a major determinant of the basal level of activated ERK in MDA-MB-231 breast cancer cells. When these cells were cultured in the presence of antibodies that block the binding of uPA to uPAR, the level of phosphorylated ERK decreased substantially. Furthermore, conditioned medium from MDA-MB-231 cells activated ERK in MCF-7 cells and this response was blocked by uPA-specific antibody. The mitogen-activated protein kinase kinase inhibitor, PD098059, decreased expression of uPA and uPAR in MDA-MB-231 cells. Thus, uPA and the uPAR-ERK signaling pathway form a positive feedback loop in these cells. When this feedback loop was disrupted with uPA- or uPAR-specific antibody, uPA mRNA-specific antisense oligodeoxynucleotides or PD098059, cell growth was inhibited and apoptosis was promoted, as determined by the increase in cytoplasmic nucleosomes and caspase-3 activity. Treating the cells simultaneously with PD098059 and uPA- or uPAR-specific antibody did not further promote apoptosis, compared with either reagent added separately, supporting the hypothesis that uPAR and ERK are components of the same cell growth/survival-regulatory pathway. The ability of uPA to signal through uPAR, maintain an elevated basal level of activated ERK and inhibit apoptosis represents a novel mechanism whereby the uPA-uPAR system may affect breast cancer progression in vivo.
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Affiliation(s)
- Z Ma
- Department of Pathology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA
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36
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Degryse B, Sier CF, Resnati M, Conese M, Blasi F. PAI-1 inhibits urokinase-induced chemotaxis by internalizing the urokinase receptor. FEBS Lett 2001; 505:249-54. [PMID: 11566185 DOI: 10.1016/s0014-5793(01)02797-1] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
PAI-1 (plasminogen activator inhibitor-1) binds the urokinase-type plasminogen activator (uPA) and causes its degradation via its receptor uPAR and low-density lipoprotein receptor-related protein (LRP). While both uPA and PAI-1 are chemoattractants, we find that a preformed uPA-PAI-1 complex has no chemotactic activity and that PAI-1 inhibits uPA-induced chemotaxis. The inhibitory effect of PAI-1 on uPA-dependent chemotaxis is reversed when uPAR internalization is inhibited by the 39 kDa receptor-associated protein or by anti-LRP antibodies. Under the same conditions, the uPA-PAI-1 complex is turned into a chemoattractant causing cytoskeleton reorganization and extracellular-regulated kinase/mitogen-activated protein kinases activation. Thus, uPAR internalization by PAI-1 regulates cell migration.
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Affiliation(s)
- B Degryse
- Molecular Genetics Unit, DIBIT, Department of Cell Biology and Functional Genetics, University Vita-Salute San Raffaele, Via Olgettina 58, 20132 Milan, Italy
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37
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Duggan A, Paolucci M, Tercyak A, Gigliotti M, Small D, Callard I. Seasonal variation in plasma lipids, lipoproteins, apolipoprotein A-I and vitellogenin in the freshwater turtle, Chrysemys picta. Comp Biochem Physiol A Mol Integr Physiol 2001; 130:253-69. [PMID: 11544071 DOI: 10.1016/s1095-6433(01)00364-6] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
An analysis of plasma lipids and lipoprotein fractions was performed over the course of the annual ovarian cycle of the female turtle, Chrysemys picta. Determinations of total plasma triglycerides, cholesterol, vitellogenin and apolipoprotein A-I (apoA-I) were made. The lipid and protein composition of the lipoprotein fractions [very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL) and very high density lipoprotein (VHDL)] were also observed over the same period. Plasma triglyceride and vitellogenin levels were significantly increased in the spring preovulatory period and fall recrudescent phase. Total plasma cholesterol levels were significantly elevated only at the onset of the fall recrudescent phase and apoA-I levels were highest during the postoviposition/ovarian arrest phase. The triglyceride content of VLDL was highest in preovulatory animals and there were apparent seasonal changes in the expression of apoA-I and apoE of HDL/VHDL. We conclude that the coordinate regulation of lipids and protein contributes to seasonal ovarian growth and clearance of lipids from plasma, both of which are most likely under hormonal control.
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Affiliation(s)
- A Duggan
- Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA.
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38
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Andersen OM, Petersen HH, Jacobsen C, Moestrup SK, Etzerodt M, Andreasen PA, Thøgersen HC. Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein. Biochem J 2001; 357:289-96. [PMID: 11415462 PMCID: PMC1221954 DOI: 10.1042/0264-6021:3570289] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.
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Affiliation(s)
- O M Andersen
- Laboratory of Gene Expression, Department of Molecular and Structural Biology, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark
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39
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Korschineck I, Ziegler S, Breuss J, Lang I, Lorenz M, Kaun C, Ambros PF, Binder BR. Identification of a novel exon in apolipoprotein E receptor 2 leading to alternatively spliced mRNAs found in cells of the vascular wall but not in neuronal tissue. J Biol Chem 2001; 276:13192-7. [PMID: 11152697 DOI: 10.1074/jbc.m011795200] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Novel members of the low density lipoprotein receptor family were identified in human endothelial and vascular smooth muscle cells utilizing a homology-cloning strategy. Four novel mRNA transcripts could be identified as isoforms of the apolipoprotein E receptor 2 (apoEr2): one form lacking three ligand binding repeats (nucleotides 497-883) but containing a novel ligand binding repeat adjacent to a unique cysteine-rich domain preceding the epidermal growth factor precursor domain of apoEr2, forms lacking the O-linked sugar domain, and forms containing a 59-amino acid deletion within the cytoplasmic tail. By fluorescence in situ hybridization for chromosome mapping, we could confirm that the novel alternative forms of apoEr2 are splice variants of transcripts from a single copy gene on chromosome 1p34. To analyze whether the different splice variants of apoEr2 mRNA are expressed in a splice variant-specific pattern, we concentrated on the central nervous system, where high expression of apoEr2 has been described originally. By means of splice variant-specific in situ hybridization, we could confirm that apoEr2 mRNA is abundantly expressed in brain tissue and, with exception of the newly identified ligand binding domain, all mRNA splice variants exhibited a similar expression pattern. The mRNA of the newly identified ligand binding domain, however, was expressed in brain only in cells of the vascular wall, confirming data from Northern blotting, where the mRNA of the newly identified ligand binding domain was found in several tissues but was absent in brain tissue.
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Affiliation(s)
- I Korschineck
- Department of Vascular Biology and Thrombosis Research, University of Vienna, Schwarzspanierstrasse 17, Austria
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40
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Webb DJ, Thomas KS, Gonias SL. Plasminogen activator inhibitor 1 functions as a urokinase response modifier at the level of cell signaling and thereby promotes MCF-7 cell growth. J Cell Biol 2001; 152:741-52. [PMID: 11266465 PMCID: PMC2195772 DOI: 10.1083/jcb.152.4.741] [Citation(s) in RCA: 84] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2000] [Accepted: 01/11/2001] [Indexed: 11/22/2022] Open
Abstract
Plasminogen activator inhibitor 1 (PAI-1) is a major inhibitor of urokinase-type plasminogen activator (uPA). In this study, we explored the role of PAI-1 in cell signaling. In MCF-7 cells, PAI-1 did not directly activate the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and ERK2, but instead altered the response to uPA so that ERK phosphorylation was sustained. This effect required the cooperative function of uPAR and the very low density lipoprotein receptor (VLDLr). When MCF-7 cells were treated with uPA-PAI-1 complex in the presence of the VLDLr antagonist, receptor-associated protein, or with uPA-PAI-1(R76E) complex, which binds to the VLDLr with greatly decreased affinity, transient ERK phosphorylation (<5 min) was observed, mimicking the uPA response. ERK phosphorylation was not induced by tissue-type plasminogen activator-PAI-1 complex or by uPA-PAI-1 complex in the presence of antibodies that block uPA binding to uPAR. uPA-PAI-1 complex induced tyrosine phosphorylation of focal adhesion kinase and Shc and sustained association of Sos with Shc, whereas uPA caused transient association of Sos with Shc. By sustaining ERK phosphorylation, PAI-1 converted uPA into an MCF-7 cell mitogen. This activity was blocked by receptor-associated protein and not observed with uPA-PAI-1(R76E) complex, demonstrating the importance of the VLDLr. uPA promoted the growth of other cells in which ERK phosphorylation was sustained, including beta3 integrin overexpressing MCF-7 cells and HT 1080 cells. The MEK inhibitor, PD098059, blocked the growth-promoting activity of uPA and uPA-PAI-1 complex in these cells. Our results demonstrate that PAI-1 may regulate uPA-initiated cell signaling by a mechanism that requires VLDLr recruitment. The kinetics of ERK phosphorylation in response to uPAR ligation determine the function of uPA and uPA-PAI-1 complex as growth promoters.
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Affiliation(s)
- D J Webb
- Department of Pathology, and Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA
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41
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Sakthivel R, Zhang JC, Strickland DK, Gåfvels M, McCrae KR. Regulation of the ligand binding activity of the human very low density lipoprotein receptor by protein kinase C-dependent phosphorylation. J Biol Chem 2001; 276:555-62. [PMID: 11010963 DOI: 10.1074/jbc.m003953200] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The very low density lipoprotein receptor (VLDL-R) binds and internalizes several ligands, including very low density lipoprotein (VLDL), urokinase-type plasminogen activator:plasminogen activator inhibitor type 1 complexes, lipoprotein lipase, and the 39-kDa receptor-associated protein that copurifies with the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor. Although several agonists regulate VLDL-R mRNA and/or protein expression, post-transcriptional regulation of receptor activity has not been described. Here, we report that the ligand binding activity of the VLDL-R in THP-1 monocytic cells, endothelial cells, smooth muscle cells, and VLDL-R-transfected HEK 293 cells is diminished after treatment with phorbol 12-myristate 13-acetate. This response was blocked by inhibitors of protein kinase C (PK-C), including a specific inhibitor of the PK-C beta II isoform, and was associated with phosphorylation of serine residues in the cytoplasmic domain of the receptor. Culture of endothelial cells in the presence of high glucose concentrations, which stimulate diacylglycerol synthesis and PK-C beta II activation, also induced a PK-C-dependent loss of VLDL-R ligand binding activity. Taken together, these studies demonstrate that the ligand binding activity of the VLDL-R is regulated by PK-C-dependent phosphorylation and that hyperglycemia may diminish VLDL-R activity.
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Affiliation(s)
- R Sakthivel
- Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA
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42
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Chazaud B, Bonavaud S, Plonquet A, Pouchelet M, Gherardi RK, Barlovatz-Meimon G. Involvement of the [uPAR:uPA:PAI-1:LRP] complex in human myogenic cell motility. Exp Cell Res 2000; 258:237-44. [PMID: 10896774 DOI: 10.1006/excr.2000.4934] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The urokinase-type plasminogen activator system is a proteolytic system involved in tissue remodeling and cell migration. At the cell surface, receptor (uPAR)-bound urokinase (uPA) binds its inhibitor PAI-1, localized in the matrix, and the complex is internalized by endocytic receptors, such as the low-density lipoprotein receptor-related protein (LRP). We previously proposed a nonproteolytic role for the uPA system in human myogenic cell differentiation in vitro, i.e., cell fusion, and showed that myogenic cells can use PAI-1 as an adhesion matrix molecule. The aim of this study was to define the role of the uPA system in myogenic cell migration that is necessary for fusion. Using a two-dimensional motility assay and microcinematography, we showed that any interference with the [uPAR:uPA:PAI-1] complex formation, and interference with LRP binding to this complex, markedly decreased myogenic cell motility. This phenomenon was reversible and independent of plasmin activity. Inhibition of cell motility was associated with suppression of both filopodia and membrane ruffling activity. [uPAR:uPA:PAI-1:LRP] complex formation involves high-affinity molecular interactions and results in quick internalization of the complex. It is likely that this complex supports the membrane ruffling activity involved in the guidance of the migrating cell toward appropriate sites for attachment.
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Affiliation(s)
- B Chazaud
- GERMEN, Faculté de Médecine, Université Paris XII Val-de-Marne, 8 Rue du Général Sarrail, Créteil Cedex, 94010, France
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43
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Li Y, Wood N, Grimsley P, Yellowlees D, Donnelly PK. In vitro invasiveness of human breast cancer cells is promoted by low density lipoprotein receptor-related protein. INVASION & METASTASIS 2000; 18:240-51. [PMID: 10729769 DOI: 10.1159/000024517] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor (LRP) is a surface membrane endocytic receptor, one of whose many functions is the regulation of plasminogen activator-mediated cell migration. LRP is known to have a role in migration and invasion, but its direct involvement has been demonstrated only in non-tumour cells. We investigated six breast cancer cell lines and a normal mammary epithelial cell clone for surface and total cellular LRP expression, and confirmed that its presence corresponds to the ability to invade and migrate in vitro. We showed that LRP in the tumour cell lines is expressed at a wide range of levels: from approximately 300 to approximately 6,300 sites per cell. Four of the breast cancer cell lines expressed LRP at over 1,000 sites/cell and were markedly invasive in our assay, the remainder of the cell lines and the normal clone having far fewer LRP sites and lacking invasive ability. We further showed that the migratory and invasive abilities of a highly invasive breast cancer cell line are both inhibited by receptor-associated protein, a unique LRP ligand which normally has a solely intracellular distribution but which, when added to culture medium, can inhibit all other ligand interactions with this receptor.
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Affiliation(s)
- Y Li
- Department of Surgery, North Queensland Clinical School, University of Queensland, Townsville, Australia
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44
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Ippoliti R, Lendaro E, Benedetti PA, Torrisi MR, Belleudi F, Carpani D, Soria MR, Fabbrini MS. Endocytosis of a chimera between human pro‐urokinase and the plant toxin saporin: an unusual internalization mechanism. FASEB J 2000. [DOI: 10.1096/fasebj.14.10.1335] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Affiliation(s)
- Rodolfo Ippoliti
- Department of Biochemical Sciences ‘A. Rossi Fanelli’University of Rome La Sapienza Rome Italy
| | - Eugenio Lendaro
- Department of Biochemical Sciences ‘A. Rossi Fanelli’University of Rome La Sapienza Rome Italy
| | | | - Maria Rosaria Torrisi
- Department of Experimental Medicine and PathologyUniversity of Rome La Sapienza Rome Italy
| | - Francesca Belleudi
- Department of Experimental Medicine and PathologyUniversity of Rome La Sapienza Rome Italy
| | - Daniela Carpani
- DIBITDepartment of Biological and Technological Research‐DIBITS. Raffaele Scientific Institute Milano Italy
| | - Marco Raffaello Soria
- DIBITDepartment of Biological and Technological Research‐DIBITS. Raffaele Scientific Institute Milano Italy
| | - Maria Serena Fabbrini
- DIBITDepartment of Biological and Technological Research‐DIBITS. Raffaele Scientific Institute Milano Italy
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45
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Lametsch R, Rasmussen JT, Johnsen LB, Purup S, Sejrsen K, Petersen TE, Heegaard CW. Structural characterization of the fibroblast growth factor-binding protein purified from bovine prepartum mammary gland secretion. J Biol Chem 2000; 275:19469-74. [PMID: 10867016 DOI: 10.1074/jbc.m002550200] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
A novel heparin-binding protein was purified to homogeneity from bovine prepartum mammary gland secretion using heparin-Sepharose chromatography and reverse-phase high performance liquid chromatography successively. Structural information obtained by N-terminal amino acid sequencing of a series of proteolytically generated peptides permitted the cloning of the corresponding cDNA. The isolated cDNA was 1170 base pairs long and consisted of an 83-base pair 5'-untranslated region followed by a 702-base pair coding region and a 385-base pair 3'-untranslated region. The open reading frame resulted in a protein comprising 234- amino acid residues, including a signal sequence. Instead of Lys(24) as the predicted N terminus, Edman degradation of the native protein revealed N-terminal processing at two sites as follows: a primary site between Arg(31)-Gly(32) and a secondary site between Arg(51)-Ser(52). The amino acid sequence showed a significant similarity with that of human (60%) and mouse (53%) fibroblast growth factor-binding protein (FGF-BP). Accordingly, ligand blotting experiments revealed that bovine FGF-BP bound FGF-2. The theoretical mass of the protein predicted from the cDNA sequence is 22.5 kDa. However, the molecular mass of the purified protein was estimated to 28.6 kDa by mass spectrometry and 36 kDa by electrophoresis. The apparent molecular weight differences are most likely due to post-transcriptional modifications, shown to involve N- and O-glycosylation of Asn(155) and Ser(172), respectively. All 10 cysteine residues in the protein participated in disulfide bonds, and the pattern was identified as Cys(71)-Cys(88), Cys(97)-Cys(130), Cys(106)-Cys(142), Cys(198)-Cys(234), and Cys(214)-Cys(222). As the 10 cysteines of the three known FGF-BPs are positionally conserved, the disulfide bond pattern of bovine FGF-BP may be regarded as representative for the FGF-BP family.
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Affiliation(s)
- R Lametsch
- Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Science Park, Gustav Wieds Vej 10, DK-8000 Aarhus
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Drapkin PT, O'Riordan CR, Yi SM, Chiorini JA, Cardella J, Zabner J, Welsh MJ. Targeting the urokinase plasminogen activator receptor enhances gene transfer to human airway epithelia. J Clin Invest 2000; 105:589-96. [PMID: 10712430 PMCID: PMC292456 DOI: 10.1172/jci8858] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Developing gene therapy for cystic fibrosis has been hindered by limited binding and endocytosis of vectors by human airway epithelia. Here we show that the apical membrane of airway epithelia express the urokinase plasminogen activator receptor (uPAR). Urokinase plasminogen activator (uPA), or a 7-residue peptide derived from this protein (u7-peptide), bound the receptor and stimulated apical endocytosis. Both ligands enhanced gene transfer by nonspecifically bound adenovirus and adeno-associated virus vectors and by a modified adenovirus vector that had been coupled to the u7-peptide. These data provide the first evidence that targeting an apical receptor can circumvent the two most important barriers to gene transfer in airway epithelia. Thus, the uPA/uPAR system may offer significant advantages for delivering genes and other pharmaceuticals to airway epithelia.
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Affiliation(s)
- P T Drapkin
- Program in Gene Therapy, Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA
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Nakamura Y, Yamamoto M, Kumamaru E. Very low-density lipoprotein receptor in fetal intestine and gastric adenocarcinoma cells. Arch Pathol Lab Med 2000; 124:119-22. [PMID: 10629142 DOI: 10.5858/2000-124-0119-vldlri] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
BACKGROUND A very low-density lipoprotein receptor (VLDLR) was recently identified. This receptor reportedly binds specifically to very low-density lipoproteins; however, its distribution and functions in vivo have yet to be elucidated. We investigated the expression and regulation of VLDLR in fetal and carcinoma cells. OBJECTIVE The expression of VLDLR was examined by immunohistochemistry and reverse-transcriptase polymerase chain reaction using several specimens, including a fetus of 12 to 15 weeks' gestation, various tumors, AGS cells, and INT407 cells. RESULTS Immunoreactive VLDLR was abundantly present in human fetal intestinal epithelial and gastric adenocarcinoma cells. This receptor was also noted in the intestinal cell line, INT407, and gastric cancer cell line, AGS. In addition, the VLDLR that was expressed in INT407 cells, AGS cells, and gastric adenocarcinoma tissue was present mainly in a variant form lacking the O-linked sugar domain. CONCLUSIONS These data suggest that an important function of VLDLR may be the mediation of cell growth in developing tissues, such as fetal intestinal and cancer cells. The INT407 and AGS cell lines appear to be useful for examining the regulation of VLDLR expression.
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Affiliation(s)
- Y Nakamura
- Department of Pathology, St Mary's Hospital, Tsubukuhon-machi, Kurume, Japan.
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Mikhailenko I, Considine W, Argraves KM, Loukinov D, Hyman BT, Strickland DK. Functional domains of the very low density lipoprotein receptor: molecular analysis of ligand binding and acid-dependent ligand dissociation mechanisms. J Cell Sci 1999; 112 ( Pt 19):3269-81. [PMID: 10504332 DOI: 10.1242/jcs.112.19.3269] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The very low density lipoprotein (VLDL) receptor is closely related in structure to the low density lipoprotein receptor. The ectodomain of these endocytic receptors is composed of modules which include clusters of cysteine-rich class A repeats, epidermal growth factor (EGF)-like repeats, tyrosine-tryptophan-threonine-aspartic acid (YWTD) repeats and an O-linked sugar domain. To identify important functional regions within the ectodomain of the VLDL receptor, we produced a mutant receptor in which the EGF, YWTD and O-linked sugar domains were deleted. Cells transfected with the mutant receptor were able to bind and internalize (125)I-labeled receptor associated protein (RAP). In contrast to the wild-type receptor, however, RAP did not dissociate from the mutant receptor and consequently was not degraded. Immunofluoresence data indicated that once bound to the mutant receptor, fluorescent-labeled RAP co-localized with markers of the endosomal pathway, whereas, in cells expressing the wild-type receptor, RAP fluorescence co-localized with lysosomal markers. Thus this deleted region is responsible for ligand uncoupling within the endosomes. To identify regions responsible for ligand recognition, soluble receptor fragments containing the eight cysteine-rich class A repeats were produced. (125)I-RAP and (125)I-labeled urokinase-type plasminogen activator:plasminogen activator inhibitor type I (uPA:PAI-1) complexes bound to the soluble fragment with K(D, app) values of 0.3 and 14 nM, respectively. Deletion analysis demonstrate that high affinity RAP binding requires the first four cysteine-rich class A repeats (L1-4) in the VLDL receptor while the second repeat (L2) appears responsible for binding uPA:PAI-1 complexes. Together, these results confirm that ligand uncoupling occurs via an allosteric-type mechanism in which pH induced changes in the EGF and/or YWTD repeats alter the ligand binding properties at the amino-terminal portion of the molecule.
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Affiliation(s)
- I Mikhailenko
- Department of Vascular Biology, Holland Laboratory, American Red Cross, Rockville Maryland 20855, USA
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Savonen R, Obermoeller LM, Trausch-Azar JS, Schwartz AL, Bu G. The carboxyl-terminal domain of receptor-associated protein facilitates proper folding and trafficking of the very low density lipoprotein receptor by interaction with the three amino-terminal ligand-binding repeats of the receptor. J Biol Chem 1999; 274:25877-82. [PMID: 10464330 DOI: 10.1074/jbc.274.36.25877] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The 39-kDa receptor-associated protein (RAP) is a specialized antagonist that inhibits all known ligand interactions with receptors that belong to the low density lipoprotein (LDL) receptor gene family. Recent studies have demonstrated a role for RAP as a molecular chaperone for the LDL receptor-related protein during receptor folding and trafficking within the early secretory pathway. In the present study, we investigated a potential role for RAP as a chaperone for the very low density lipoprotein (VLDL) receptor, another member of the LDL receptor gene family. Using intracellular cross-linking techniques, we found that RAP is associated with newly synthesized VLDL receptor. In the absence of RAP co-expression, newly synthesized VLDL receptor exhibited slower trafficking along the early secretory pathway, most likely due to misfolding of the receptor. The role of RAP in the folding of the VLDL receptor was further studied using an anchor-free, soluble VLDL receptor. Metabolic pulse-chase labeling experiments showed that while only 3% of the soluble VLDL receptor was folded and secreted in the absence of RAP co-expression, over 50% of the soluble receptor was secreted in the presence of RAP co-expression. The functions of RAP in VLDL receptor folding and trafficking were mediated by its carboxyl-terminal repeat but not by the amino-terminal and central repeats. Using truncated VLDL receptor constructs, we identified the RAP-binding site within the first three ligand-binding repeats of the VLDL receptor. Thus, our present study demonstrates that RAP serves as a folding and trafficking chaperone for the VLDL receptor via interactions of its carboxyl-terminal repeat with the three amino-terminal ligand-binding repeats of the VLDL receptor.
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Affiliation(s)
- R Savonen
- Departments of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA
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Abstract
The low-density lipoprotein (LDL) receptor (LDL-R) family consists of cell-surface receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. The LDL-R is the prototype of this family, which also contains very-low-density lipoprotein receptors (VLDL-R), apolipoprotein E receptor 2, LRP, and megalin. The family members contain four major structural modules: the cysteine-rich complement-type repeats, epidermal growth factor precursor-like repeats, a transmembrane domain, and a cytoplasmic domain. Each structural module serves distinct and important functions. These receptors bind several structurally dissimilar ligands. It is proposed that instead of a primary sequence, positive electrostatic potential in different ligands constitutes a receptor binding domain. This family of receptors plays crucial roles in various physiologic functions. LDL-R plays an important role in cholesterol homeostasis. Mutations cause familial hypercholesterolemia and premature coronary artery disease. LDL-R-related protein plays an important role in the clearance of plasma-activated alpha 2-macroglobulin and apolipoprotein E-enriched lipoproteins. It is essential for fetal development and has been associated with Alzheimer's disease. Megalin is the major receptor in absorptive epithelial cells of the proximal tubules and an antigenic determinant for Heymann nephritis in rats. Mutations in a chicken homolog of VLDL-R cause female sterility and premature atherosclerosis. This receptor is not expressed in liver tissue; however, transgenic expression of VLDL-R in liver corrects hypercholesterolemia in experiment animals, which suggests that it can be a candidate for gene therapy for various hyperlipidemias. The functional importance of individual receptors may lie in their differential tissue expression. The regulation of expression of these receptors occurs at the transcriptional level. Expression of the LDL-R is regulated by intracellular sterol levels involving novel membrane-bound transcription factors. Other members of the family are not regulated by sterols. All the members are, however, regulated by hormones and growth factors, but the mechanisms of regulation by hormones have not been elucidated. Studies of these receptors have provided important insights into receptor structure-function and mechanisms of ligand removal and catabolism. It is anticipated that increased knowledge about the LDL-R family members will open new avenues for the treatment of many disorders.
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Affiliation(s)
- M M Hussain
- Department of Biochemistry, MCP Hahnemann University, Philadelphia, Pennsylvania 19129, USA.
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