Plasmin triggers a switch-like decrease in thrombospondin-dependent activation of TGF-β1.

Transforming growth factor-β1 (TGF-β1) is a potent regulator of extracellular matrix production, wound healing, differentiation, and immune response, and is implicated in the progression of fibrotic diseases and cancer. Extracellular activation of TGF-β1 from its latent form provides spatiotemporal control over TGF-β1 signaling, but the current understanding of TGF-β1 activation does not emphasize cross talk between activators. Plasmin (PLS) and thrombospondin-1 (TSP1) have been studied individually as activators of TGF-β1, and in this work we used a systems-level approach with mathematical modeling and in vitro experiments to study the interplay between PLS and TSP1 in TGF-β1 activation. Simulations and steady-state analysis predicted a switch-like bistable transition between two levels of active TGF-β1, with an inverse correlation between PLS and TSP1. In particular, the model predicted that increasing PLS breaks a TSP1-TGF-β1 positive feedback loop and causes an unexpected net decrease in TGF-β1 activation. To test these predictions in vitro, we treated rat hepatocytes and hepatic stellate cells with PLS, which caused proteolytic cleavage of TSP1 and decreased activation of TGF-β1. The TGF-β1 activation levels showed a cooperative dose response, and a test of hysteresis in the cocultured cells validated that TGF-β1 activation is bistable. We conclude that switch-like behavior arises from natural competition between two distinct modes of TGF-β1 activation: a TSP1-mediated mode of high activation and a PLS-mediated mode of low activation. This switch suggests an explanation for the unexpected effects of the plasminogen activation system on TGF-β1 in fibrotic diseases in vivo, as well as novel prognostic and therapeutic approaches for diseases with TGF-β dysregulation.

[1]  D. Lauffenburger,et al.  Quantitative analysis of pathways controlling extrinsic apoptosis in single cells. , 2008, Molecular cell.

[2]  C. Legrand,et al.  Proteolysis of subendothelial adhesive glycoproteins (fibronectin, thrombospondin, and von Willebrand factor) by plasmin, leukocyte cathepsin G, and elastase. , 2000, Thrombosis research.

[3]  D. Lauffenburger,et al.  Physicochemical modelling of cell signalling pathways , 2006, Nature Cell Biology.

[4]  H. Moses,et al.  Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin , 1990, The Journal of cell biology.

[5]  Justin P. Annes,et al.  Making sense of latent TGFβ activation , 2003, Journal of Cell Science.

[6]  H. Lodish,et al.  Role of transforming growth factor beta in human disease. , 2000, The New England journal of medicine.

[7]  D. Rifkin,et al.  7 TGF-β Bioavailability: Latency, Targeting, and Activation , 2008 .

[8]  S. Friedman,et al.  Modulation of transforming growth factor beta receptors of rat lipocytes during the hepatic wound healing response. Enhanced binding and reduced gene expression accompany cellular activation in culture and in vivo. , 1994, The Journal of biological chemistry.

[9]  L. Sottrup-jensen,et al.  Mechanism of alpha 2-macroglobulin-proteinase interactions. Studies with trypsin and plasmin. , 1984, Biochemistry.

[10]  Jacob K. White,et al.  Steady states and dynamics of urokinase-mediated plasmin activation in silico and in vitro. , 2011, Biophysical journal.

[11]  P. Carmeliet,et al.  Plasminogen mediates liver regeneration and angiogenesis after experimental partial hepatectomy , 2003, The British journal of surgery.

[12]  Y. Takada,et al.  The concentration of tissue plasminogen activator and urokinase in plasma and tissues of patients with ovarian and uterine tumors. , 1990, Thrombosis research.

[13]  M. Scully,et al.  Plasminogen activation by single-chain urokinase in functional isolation. A kinetic study. , 1987, The Journal of biological chemistry.

[14]  U. Bhalla,et al.  Emergent properties of networks of biological signaling pathways. , 1999, Science.

[15]  G. Salvesen,et al.  Human plasma proteinase inhibitors. , 1983, Annual review of biochemistry.

[16]  Eduardo Sontag,et al.  Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2 , 2003, Nature Cell Biology.

[17]  A. Vaheri,et al.  Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants , 1989, The Journal of cell biology.

[18]  Rui Wang,et al.  Modeling of the role of a Bax-activation switch in the mitochondrial apoptosis decision. , 2007, Biophysical journal.

[19]  Tingzhe Sun,et al.  Two Independent Positive Feedbacks and Bistability in the Bcl-2 Apoptotic Switch , 2008, PloS one.

[20]  C. Hugo.  The thrombospondin 1–TGF‐β axis in fibrotic renal disease , 2003 .

[21]  P. Bornstein,et al.  Thrombospondins as matricellular modulators of cell function. , 2001, The Journal of clinical investigation.

[22]  D. Collen,et al.  The mechanism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro. , 1989, Blood.

[23]  P. Hogg,et al.  Thrombospondin is a slow tight-binding inhibitor of plasmin. , 1992, Biochemistry.

[24]  J. Armendáriz-Borunda,et al.  Plasmin plays a key role in the regulation of profibrogenic molecules in hepatic stellate cells , 2010, Liver international : official journal of the International Association for the Study of the Liver.

[25]  S H Teoh,et al.  Development of dual-compartment perfusion bioreactor for serial coculture of hepatocytes and stellate cells in poly(lactic-co-glycolic acid)-collagen scaffolds. , 2008, Journal of biomedical materials research. Part B, Applied biomaterials.

[26]  S. L. Gonias,et al.  Characterization of the antiplasmin activity of human thrombospondin-1 in solution. , 1993, The Biochemical journal.

[27]  P. McKeown-Longo,et al.  TGF-beta1-induced PAI-1 gene expression requires MEK activity and cell-to-substrate adhesion. , 2001, Journal of cell science.

[28]  H. Moses,et al.  Mechanism of Activation of Latent Recombinant Transforming Growth Factor by Plasmin , 2002 .

[29]  S. Friedman,et al.  An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitro. , 2000, Journal of lipid research.

[30]  W. B. Reeves,et al.  Transforming growth factor β contributes to progressive diabetic nephropathy , 2000 .

[31]  G. Stouffer,et al.  Autocrine thrombospondin partially mediates TGF-beta1- induced proliferation of vascular smooth muscle cells. , 2000, American journal of physiology. Heart and circulatory physiology.

[32]  D. Roulot,et al.  Transforming growth factor β and the liver , 2001 .

[33]  Tae J. Lee,et al.  A bistable Rb–E2F switch underlies the restriction point , 2008, Nature Cell Biology.

[34]  G. Tytgat,et al.  Metabolism of plasminogen in healthy subjects: effect of tranexamic acid. , 1972, The Journal of clinical investigation.

[35]  Wei Zhang,et al.  Robustness analysis identifies the plausible model of the Bcl‐2 apoptotic switch , 2007, FEBS letters.

[36]  Jacob K. White,et al.  Cell-delivery therapeutics for liver regeneration. , 2010, Advanced drug delivery reviews.

[37]  G. Leipnitz,et al.  Half-life of single-chain urokinase-type plasminogen activator (scu-PA) and two-chain urokinase-type plasminogen activator (tcu-PA) in patients with acute myocardial infarction. , 1991, Thrombosis research.

[38]  P. McKee,et al.  The effects of fibrinogen and its cleavage products on the kinetics of plasminogen activation by urokinase and subsequent plasmin activity. , 1983, The Journal of biological chemistry.

[39]  H. Lijnen.  Pleiotropic functions of plasminogen activator inhibitor‐1 , 2005, Journal of thrombosis and haemostasis : JTH.

[40]  Wei-Shou Hu,et al.  Three-dimensional co-culture of hepatocytes and stellate cells , 2004, Cytotechnology.

[41]  Chris Sander,et al.  Signal Processing in the TGF-β Superfamily Ligand-Receptor Network , 2005, PLoS Comput. Biol..

[42]  Nils Blüthgen,et al.  Mathematical Modeling Identifies Inhibitors of Apoptosis as Mediators of Positive Feedback and Bistability , 2006, PLoS Comput. Biol..

[43]  M. Scully,et al.  Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. Potentiation by U937 monocytes. , 1989, The Journal of biological chemistry.

[44]  John J. Tyson,et al.  Irreversible cell-cycle transitions are due to systems-level feedback , 2007, Nature Cell Biology.

[45]  R. Hynes,et al.  Thrombospondin-1 Is a Major Activator of TGF-β1 In Vivo , 1998, Cell.

[46]  K. Robbins,et al.  [10] Human plasminogen and plasmin , 1970 .

[47]  近藤 宏樹.  A blocking peptide for transforming growth factor-β1 activation prevents hepatic fibrosis in vivo , 2004 .

[48]  M. Jinnin,et al.  Constitutive thrombospondin-1 overexpression contributes to autocrine transforming growth factor-beta signaling in cultured scleroderma fibroblasts. , 2005, The American journal of pathology.

[49]  Harish Shankaran,et al.  Smad Signaling Dynamics: Insights from a Parsimonious Model , 2008, Science Signaling.

[50]  J. Donato,et al.  Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay. , 2004, Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas.

[51]  J. Murphy-Ullrich,et al.  Activation of latent TGF-β by thrombospondin-1: mechanisms and physiology , 2000 .

[52]  S. L. Gonias,et al.  Regulation of plasmin, miniplasmin, and streptokinase-plasmin complex by α2-antiplasmin, α2-macroglobulin, and antithrombin III in the presence of heparin1 , 1990 .

[53]  W. Hsueh,et al.  Plasminogen activator inhibitor-1 deficiency retards diabetic nephropathy. , 2005, Kidney international.

[54]  D. Brenner,et al.  Mechanisms of Fibrogenesis , 2008, Experimental biology and medicine.

[55]  M. Cubellis,et al.  Receptor‐mediated internalization and degradation of urokinase is caused by its specific inhibitor PAI‐1. , 1990, The EMBO journal.

[56]  J. F. Bohmfalk,et al.  Plaminogen is synthesized by primary cultures of rat hepatocytes. , 1980, Science.

[57]  S. Thorsen,et al.  Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2. , 1988, European journal of biochemistry.

[58]  G B Ermentrout,et al.  Bistability in apoptosis: roles of bax, bcl-2, and mitochondrial permeability transition pores. , 2006, Biophysical journal.

[59]  David Padua,et al.  Roles of TGFβ in metastasis , 2009, Cell Research.

[60]  Paul A. Bates,et al.  Mathematical modeling identifies Smad nucleocytoplasmic shuttling as a dynamic signal-interpreting system , 2008, Proceedings of the National Academy of Sciences.

[61]  K. Shakesheff,et al.  A simple method for the simultaneous isolation of stellate cells and hepatocytes from rat liver tissue , 2003, Molecular and Cellular Biochemistry.

[62]  Y. Etani,et al.  A blocking peptide for transforming growth factor-beta1 activation prevents hepatic fibrosis in vivo. , 2003, Journal of hepatology.

[63]  Jean S. Campbell,et al.  Liver regeneration. , 2012, Journal of hepatology.

[64]  P. Hogg,et al.  Direct effects of alcohol on hepatic fibrinolytic balance: implications for alcoholic liver disease. , 2008, Journal of hepatology.

[65]  D. Lawrence,et al.  A mutant, noninhibitory plasminogen activator inhibitor type 1 decreases matrix accumulation in experimental glomerulonephritis. , 2003, The Journal of clinical investigation.

[66]  P. Carmeliet,et al.  Type 1 plasminogen activator inhibitor deficiency aggravates the course of experimental glomerulonephritis through overactivation of transforming growth factor β , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[67]  A. Strosberg,et al.  Role of transforming growth factor β type II receptor in hepatic fibrosis: Studies of human chronic hepatitis C and experimental fibrosis in rats , 1999, Hepatology.

[68]  F. Allgöwer,et al.  Bistability Analyses of a Caspase Activation Model for Receptor-induced Apoptosis* , 2004, Journal of Biological Chemistry.