Thrombin Regulates Vascular Smooth Muscle Cell Growth and Heat Shock Proteins via the JAK-STAT Pathway

Abstract

The growth-stimulating effects of thrombin are mediated primarily via activation of a G protein-coupled receptor, PAR-1. Because PAR-1 has no intrinsic tyrosine kinase activity, yet requires tyrosine phosphorylation events to induce mitogenesis, we investigated the role of the Janus tyrosine kinases (JAKs) in thrombin-mediated signaling. JAK2 was activated rapidly in rat vascular smooth muscle cells (VSMC) treated with thrombin, and signal transducers and activators of transcription (STAT1 and STAT3) were phosphorylated and translocated to the nucleus in a JAK2-dependent manner. AG-490, a JAK2-specific inhibitor, and a dominant negative JAK2 mutant inhibited thrombin-induced ERK2 activity and VSMC proliferation suggesting that JAK2 is upstream of the Ras/Raf/MEK/ERK pathway. To elucidate the functional significance of JAK-STAT activation, we studied the effect of thrombin on heat shock protein (Hsp) expression, based upon the following: 1) reports that thrombin stimulates reactive oxygen species production in VSMC; 2) the putative role of Hsps in modulating cellular responses to reactive oxygen species; and 3) the presence of functional STAT1/3-binding sites in Hsp70 and Hsp90β promoters. Indeed, thrombin up-regulated Hsp70 and Hsp90 protein expression via enhanced binding of STATs to cognate binding sites in the Hsp70 and Hsp90 promoters. Together, these results suggest that JAK-STAT pathway activation is necessary for thrombin-induced VSMC growth and Hsp gene expression. The growth-stimulating effects of thrombin are mediated primarily via activation of a G protein-coupled receptor, PAR-1. Because PAR-1 has no intrinsic tyrosine kinase activity, yet requires tyrosine phosphorylation events to induce mitogenesis, we investigated the role of the Janus tyrosine kinases (JAKs) in thrombin-mediated signaling. JAK2 was activated rapidly in rat vascular smooth muscle cells (VSMC) treated with thrombin, and signal transducers and activators of transcription (STAT1 and STAT3) were phosphorylated and translocated to the nucleus in a JAK2-dependent manner. AG-490, a JAK2-specific inhibitor, and a dominant negative JAK2 mutant inhibited thrombin-induced ERK2 activity and VSMC proliferation suggesting that JAK2 is upstream of the Ras/Raf/MEK/ERK pathway. To elucidate the functional significance of JAK-STAT activation, we studied the effect of thrombin on heat shock protein (Hsp) expression, based upon the following: 1) reports that thrombin stimulates reactive oxygen species production in VSMC; 2) the putative role of Hsps in modulating cellular responses to reactive oxygen species; and 3) the presence of functional STAT1/3-binding sites in Hsp70 and Hsp90β promoters. Indeed, thrombin up-regulated Hsp70 and Hsp90 protein expression via enhanced binding of STATs to cognate binding sites in the Hsp70 and Hsp90 promoters. Together, these results suggest that JAK-STAT pathway activation is necessary for thrombin-induced VSMC growth and Hsp gene expression. vascular smooth muscle cells Janus kinases heat shock protein reactive oxygen species signal transducers and activators of transcription mitogen-activated protein extracellular signal-regulated kinase MAP kinase/ERK kinase Dulbecco's modified Eagle's medium chloramphenicol acetyltransferase angiotensin II phorbol 12-myristate 13-acetate platelet-derived growth factor-BB In addition to regulating hemostasis and thrombosis, the serine protease thrombin also promotes the inflammatory response and wound healing (1Coughlin S.R. Thromb. Haemostasis. 1993; 66: 184-187Google Scholar) where it is mitogenic for lymphocytes, fibroblasts, vascular endothelial and smooth muscle cells (2Seuwen K. Kahan C. Hartmann T. Pouyssegur J. J. Biol. Chem. 1990; 265: 22292-22299Abstract Full Text PDF PubMed Google Scholar, 3Graham D.J. Alexander J.J. J. Vasc. 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Thrombin cleaves the N terminus of its PAR-1 receptor between Arg41 and Ser42 to create a new N terminus42SFLLRN47 that acts as a tethered ligand and activates the receptor (5Vu T.K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2654) Google Scholar). Increased Ser/Thr kinase activity in response to the stimulation of PAR-1 and other G protein-coupled receptors is well demonstrated (8Rao G.N. Delafontaine P. Runge M.S. J. Biol. Chem. 1995; 270: 27871-27875Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 9Rao G.N. Katki K.A. Madamanchi N.R. Wu Y. Birrer M.J. J. Biol. Chem. 1999; 274: 6003-6010Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 10Marrero M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Thrombin also stimulates the expression of nuclear proteins that constitute the transcription factor AP-1, which participates in transactivation of several early growth response genes implicated in VSMC1 proliferation (9Rao G.N. Katki K.A. Madamanchi N.R. Wu Y. Birrer M.J. J. Biol. Chem. 1999; 274: 6003-6010Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar,11Grand J.A.R. Turnell A.S. Grabham P.W. Biochem. J. 1996; 313: 353-368Crossref PubMed Scopus (325) Google Scholar). PAR-1, angiotensin II (Ang II) receptor, and other G protein-coupled receptors, which do not themselves possess intrinsic tyrosine kinase activity, require tyrosine kinase activity to induce mitogenesis (8Rao G.N. Delafontaine P. Runge M.S. J. Biol. Chem. 1995; 270: 27871-27875Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 12Weiss R.H. Nuccitelli R. J. Biol. Chem. 1992; 267: 5608-5613Abstract Full Text PDF PubMed Google Scholar, 13Molly C.J. Taylor D.S. Weber H. J. Biol. 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In of protein was with of and of in a of for and chloramphenicol were with and on as were to and the was was were with of and was of were To the role of tyrosine phosphorylation in thrombin-induced VSMC mitogenesis, we investigated the effect of thrombin on the JAK-STAT pathway. rat VSMC were treated with thrombin in the presence and of AG-490, a of in thrombin induced a in as effect that was inhibited in a no effect on a of its effect on the effect of on thrombin-induced inhibited the thrombin-induced in VSMC proliferation in a no effect on VSMC that it is not the in also inhibited VSMC suggesting that the effects of thrombin-induced JAK2 activation are mediated via PAR-1 The effect of on thrombin-induced VSMC proliferation is to its effect on VSMC growth induced G protein-coupled receptor M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). These results suggest that JAK2 a role in VSMC proliferation induced the activation of G protein-coupled receptors and to the activation of of the JAK-STAT pathway in response to thrombin To the of the JAK kinases to thrombin-induced tyrosine we proteins thrombin stimulation and for the presence of phosphorylated JAK proteins we that was phosphorylated stimulation with thrombin In we a in JAK2 tyrosine phosphorylation with thrombin was of with a JAK2 which demonstrated stimulation of JAK2 we also thrombin-induced phosphorylation In to the activation of activation of was of to These results were in which VSMC were with and with the tyrosine phosphorylation was thrombin stimulation protein was not in VSMC which is with reports that the expression of protein is to and cells K. J.J. Nature. PubMed Scopus Google Scholar, C. Nature. PubMed Scopus Google Scholar). The in the of tyrosine phosphorylation of suggest that may in events in tyrosine phosphorylation of JAK2 and activation of in VSMC treated with VSMC were treated with thrombin for the and in of protein were with JAK2 Thrombin no effect on JAK2 protein as in with of JAK2 tyrosine phosphorylation of protein were with and was with or in protein was in the with of tyrosine phosphorylation The in tyrosine phosphorylation of JAK2 and in response to thrombin was not to in the of these protein as that thrombin tyrosine phosphorylation of protein to the of and J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google of VSMC with for inhibited thrombin-induced tyrosine phosphorylation of it no effect on a tyrosine kinase Because JAK2 phosphorylation was with thrombin and of the JAK2 phosphorylation with thrombin-induced proliferation of we to the role of JAK2 in thrombin-induced mitogenesis. To thrombin-induced JAK2 activation to tyrosine phosphorylation of the JAK we tyrosine phosphorylation and nuclear of these VSMC were with and the were with or proteins were in response to thrombin was rapidly effect that was for The in tyrosine phosphorylation of in response to with thrombin was that of To tyrosine phosphorylation of proteins in response to with thrombin was the of nuclear and of VSMC were Thrombin induced nuclear of in that was for no was in the protein in in tyrosine phosphorylation of in response to with thrombin that was the and with also rapidly translocated to the nucleus with thrombin, the of protein in were to nuclear of protein was with thrombin Increased tyrosine phosphorylation and nuclear of was also in VSMC treated with thrombin These that thrombin tyrosine phosphorylation and nuclear of proteins in of JAK2 tyrosine phosphorylation of VSMC with for tyrosine phosphorylation and nuclear of and that JAK2 kinase activity is for phosphorylation of these Because JAK2 has to upstream of the MAP kinase M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, T. S. 1998; PubMed Scopus Google Scholar) and MAP kinase activation is for thrombin-induced in VSMC (8Rao G.N. Delafontaine P. Runge M.S. J. Biol. Chem. 1995; 270: 27871-27875Abstract Full Text Full Text PDF PubMed Scopus (130) Google we investigated JAK2 activation is for thrombin-induced stimulation of activation was in a and also kinase with (8Rao G.N. Delafontaine P. Runge M.S. J. Biol. Chem. 1995; 270: 27871-27875Abstract Full Text Full Text PDF PubMed Scopus (130) Google thrombin activated kinases in a with and and inhibited thrombin-induced activation of in and ERK2 activity in kinase and with and AG-490, no was in the protein with thrombin, in the presence or of or with a of thrombin-induced JAK2 phosphorylation and ERK2 activation and In ERK2 activation induced phorbol 12-myristate 13-acetate and was not activation in VSMC induced and is with the reports (9Rao G.N. Katki K.A. Madamanchi N.R. Wu Y. Birrer M.J. J. Biol. Chem. 1999; 274: 6003-6010Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, J. M.J. 1998; PubMed Scopus Google Scholar). Together, these results suggest that thrombin-induced JAK2 is inhibited AG-490, and thrombin-induced activation is JAK2 kinase in thrombin-induced JAK2 phosphorylation and ERK2 VSMC were with of for and treated with thrombin for of protein were with JAK2 ERK2 activity was of cells treated with thrombin for in the presence and of as for VSMC were with for and treated with thrombin, or for ERK2 activity was as is that may effects on was to activation of induced in cells D. J. Biol. 1999; PubMed Scopus Google Scholar). we not in VSMC with or thrombin To of the role of JAK2 rat cells were with a dominant negative which the kinase S. T. K. J. Biol. 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In we that the JAK-STAT pathway important role in thrombin-induced VSMC In enhanced expression of Hsp70 and Hsp90 via the JAK-STAT pathway that pathway cellular responses to of in VSMC treated with with the on the JAK-STAT pathway in VSMC mitogenesis M.B. Schieffer B. Li B. Sun J. Harp J.B. Ling B.N. J. Biol. Chem. 1997; 272: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, A.S. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google results suggest that pathway a role in the of vascular such as and for