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. 1996 Mar 1;97(5):1173–1183. doi: 10.1172/JCI118531

Thrombin receptor activation elicits rapid protein tyrosine phosphorylation and stimulation of the raf-1/MAP kinase pathway preceding delayed mitogenesis in cultured rat aortic smooth muscle cells: evidence for an obligate autocrine mechanism promoting cell proliferation induced by G-protein-coupled receptor agonist.

C J Molloy 1, J E Pawlowski 1, D S Taylor 1, C E Turner 1, H Weber 1, M Peluso 1
PMCID: PMC507169  PMID: 8636428

Abstract

Treatment of quiescent rat aortic smooth muscle cells with either alpha-thrombin or a thrombin receptor-derived agonist peptide (SFLLRNP) resulted in pronounced increases in [3H]thymidine incorporation that were concentration dependent and reached a maximum of approximately 15-fold above serum-starved controls. However, in contrast to FBS, PDGF-BB, or basic fibroblast growth factor (bFGF), that initiated DNA synthesis promptly after 16-19 h, thymidine incorporation in response to thrombin was delayed by an additional 3-6 h. Delayed mitogenesis correlated with the appearance of a potent mitogenic activity in conditioned media samples obtained from thrombin-stimulated rat aortic smooth muscle cells, as assayed using Swiss 3T3 fibroblasts. This activity was not inhibited by neutralizing antibodies directed against PDGF or bFGF. Furthermore, in the Swiss 3T3 cells, simple addition of either alpha-thrombin or SFLLRNP failed to elicit a significant mitogenic response. In signal transduction studies, both thrombin and SFLLRNP treatment led to rapid tyrosine phosphorylation of proteins with apparent molecular masses of 42, 44, 75, 120, and 190 kD, respectively, as assessed by antiphosphotyrosine immunoblotting. The overall pattern of protein tyrosine phosphorylation was distinct from that observed after PDGF-BB addition. Activation of Raf-1 and the mitogen-activated protein (MAP) kinases p44mapk and p42mapk was also observed. However, the time course and duration of Raf-1/MAP kinase activation after thrombin stimulation were similar to those elicited by PDGF-BB. Taken together, our results indicate that thrombin-stimulated vascular smooth muscle proliferation is delayed and requires the de novo expression of one or more autocrine mitogens. In addition, the rapid induction of discrete intracellular signaling mechanisms by thrombin, including the Raf-1/MAP kinase pathway, appears to be insufficient alone to promote vascular smooth muscle cell mitogenesis.

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Selected References

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  1. Aaronson S. A. Growth factors and cancer. Science. 1991 Nov 22;254(5035):1146–1153. doi: 10.1126/science.1659742. [DOI] [PubMed] [Google Scholar]
  2. Berk B. C., Taubman M. B., Griendling K. K., Cragoe E. J., Jr, Fenton J. W., Brock T. A. Thrombin-stimulated events in cultured vascular smooth-muscle cells. Biochem J. 1991 Mar 15;274(Pt 3):799–805. doi: 10.1042/bj2740799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blenis J. Growth-regulated signal transduction by the MAP kinases and RSKs. Cancer Cells. 1991 Nov;3(11):445–449. [PubMed] [Google Scholar]
  4. Boulton T. G., Nye S. H., Robbins D. J., Ip N. Y., Radziejewska E., Morgenbesser S. D., DePinho R. A., Panayotatos N., Cobb M. H., Yancopoulos G. D. ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell. 1991 May 17;65(4):663–675. doi: 10.1016/0092-8674(91)90098-j. [DOI] [PubMed] [Google Scholar]
  5. Burridge K., Turner C. E., Romer L. H. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol. 1992 Nov;119(4):893–903. doi: 10.1083/jcb.119.4.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  7. Carney D. H., Scott D. L., Gordon E. A., LaBelle E. F. Phosphoinositides in mitogenesis: neomycin inhibits thrombin-stimulated phosphoinositide turnover and initiation of cell proliferation. Cell. 1985 Sep;42(2):479–488. doi: 10.1016/0092-8674(85)90105-9. [DOI] [PubMed] [Google Scholar]
  8. Chambard J. C., Paris S., L'Allemain G., Pouysségur J. Two growth factor signalling pathways in fibroblasts distinguished by pertussis toxin. Nature. 1987 Apr 23;326(6115):800–803. doi: 10.1038/326800a0. [DOI] [PubMed] [Google Scholar]
  9. Chen Y. H., Pouysségur J., Courtneidge S. A., Van Obberghen-Schilling E. Activation of Src family kinase activity by the G protein-coupled thrombin receptor in growth-responsive fibroblasts. J Biol Chem. 1994 Nov 4;269(44):27372–27377. [PubMed] [Google Scholar]
  10. Clemmons D. R., Van Wyk J. J. Evidence for a functional role of endogenously produced somatomedinlike peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells. J Clin Invest. 1985 Jun;75(6):1914–1918. doi: 10.1172/JCI111906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Crews C. M., Erikson R. L. Extracellular signals and reversible protein phosphorylation: what to Mek of it all. Cell. 1993 Jul 30;74(2):215–217. doi: 10.1016/0092-8674(93)90411-i. [DOI] [PubMed] [Google Scholar]
  12. Di Marco E., Pierce J. H., Fleming T. P., Kraus M. H., Molloy C. J., Aaronson S. A., Di Fiore P. P. Autocrine interaction between TGF alpha and the EGF-receptor: quantitative requirements for induction of the malignant phenotype. Oncogene. 1989 Jul;4(7):831–838. [PubMed] [Google Scholar]
  13. Fantl W. J., Johnson D. E., Williams L. T. Signalling by receptor tyrosine kinases. Annu Rev Biochem. 1993;62:453–481. doi: 10.1146/annurev.bi.62.070193.002321. [DOI] [PubMed] [Google Scholar]
  14. Ferrell J. E., Jr, Martin G. S. Platelet tyrosine-specific protein phosphorylation is regulated by thrombin. Mol Cell Biol. 1988 Sep;8(9):3603–3610. doi: 10.1128/mcb.8.9.3603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gardner A. M., Vaillancourt R. R., Johnson G. L. Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase by G protein and tyrosine kinase oncoproteins. J Biol Chem. 1993 Aug 25;268(24):17896–17901. [PubMed] [Google Scholar]
  16. Giachelli C., Bae N., Lombardi D., Majesky M., Schwartz S. Molecular cloning and characterization of 2B7, a rat mRNA which distinguishes smooth muscle cell phenotypes in vitro and is identical to osteopontin (secreted phosphoprotein I, 2aR). Biochem Biophys Res Commun. 1991 Jun 14;177(2):867–873. doi: 10.1016/0006-291x(91)91870-i. [DOI] [PubMed] [Google Scholar]
  17. Glusa E. Vascular effects of thrombin. Semin Thromb Hemost. 1992;18(3):296–304. doi: 10.1055/s-2007-1002568. [DOI] [PubMed] [Google Scholar]
  18. Golden A., Brugge J. S. Thrombin treatment induces rapid changes in tyrosine phosphorylation in platelets. Proc Natl Acad Sci U S A. 1989 Feb;86(3):901–905. doi: 10.1073/pnas.86.3.901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hedin U., Frebelius S., Sanchez J., Dryjski M., Swedenborg J. Antithrombin III inhibits thrombin-induced proliferation in human arterial smooth muscle cells. Arterioscler Thromb. 1994 Feb;14(2):254–260. doi: 10.1161/01.atv.14.2.254. [DOI] [PubMed] [Google Scholar]
  20. Huang C. L., Ives H. E. Growth inhibition by protein kinase C late in mitogenesis. 1987 Oct 29-Nov 4Nature. 329(6142):849–850. doi: 10.1038/329849a0. [DOI] [PubMed] [Google Scholar]
  21. Hung D. T., Vu T. K., Wheaton V. I., Ishii K., Coughlin S. R. Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation. J Clin Invest. 1992 Apr;89(4):1350–1353. doi: 10.1172/JCI115721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kanthou C., Parry G., Wijelath E., Kakkar V. V., Demoliou-Mason C. Thrombin-induced proliferation and expression of platelet-derived growth factor-A chain gene in human vascular smooth muscle cells. FEBS Lett. 1992 Dec 14;314(2):143–148. doi: 10.1016/0014-5793(92)80961-f. [DOI] [PubMed] [Google Scholar]
  23. LaMorte V. J., Kennedy E. D., Collins L. R., Goldstein D., Harootunian A. T., Brown J. H., Feramisco J. R. A requirement for Ras protein function in thrombin-stimulated mitogenesis in astrocytoma cells. J Biol Chem. 1993 Sep 15;268(26):19411–19415. [PubMed] [Google Scholar]
  24. Li P., Wood K., Mamon H., Haser W., Roberts T. Raf-1: a kinase currently without a cause but not lacking in effects. Cell. 1991 Feb 8;64(3):479–482. doi: 10.1016/0092-8674(91)90228-q. [DOI] [PubMed] [Google Scholar]
  25. Liu L. W., Vu T. K., Esmon C. T., Coughlin S. R. The region of the thrombin receptor resembling hirudin binds to thrombin and alters enzyme specificity. J Biol Chem. 1991 Sep 15;266(26):16977–16980. [PubMed] [Google Scholar]
  26. McNamara C. A., Sarembock I. J., Gimple L. W., Fenton J. W., 2nd, Coughlin S. R., Owens G. K. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor. J Clin Invest. 1993 Jan;91(1):94–98. doi: 10.1172/JCI116206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Molloy C. J., Bottaro D. P., Fleming T. P., Marshall M. S., Gibbs J. B., Aaronson S. A. PDGF induction of tyrosine phosphorylation of GTPase activating protein. Nature. 1989 Dec 7;342(6250):711–714. doi: 10.1038/342711a0. [DOI] [PubMed] [Google Scholar]
  28. Molloy C. J., Taylor D. S., Weber H. Angiotensin II stimulation of rapid protein tyrosine phosphorylation and protein kinase activation in rat aortic smooth muscle cells. J Biol Chem. 1993 Apr 5;268(10):7338–7345. [PubMed] [Google Scholar]
  29. Nakano T., Raines E. W., Abraham J. A., Wenzel F. G., 4th, Higashiyama S., Klagsbrun M., Ross R. Glucocorticoid inhibits thrombin-induced expression of platelet-derived growth factor A-chain and heparin-binding epidermal growth factor-like growth factor in human aortic smooth muscle cells. J Biol Chem. 1993 Oct 25;268(30):22941–22947. [PubMed] [Google Scholar]
  30. Nakashima S., Chatani Y., Nakamura M., Miyoshi N., Kohno M., Nozawa Y. Tyrosine phosphorylation and activation of mitogen-activated protein kinases by thrombin in human platelets: possible involvement in late arachidonic acid release. Biochem Biophys Res Commun. 1994 Jan 28;198(2):497–503. doi: 10.1006/bbrc.1994.1073. [DOI] [PubMed] [Google Scholar]
  31. Nelken N. A., Soifer S. J., O'Keefe J., Vu T. K., Charo I. F., Coughlin S. R. Thrombin receptor expression in normal and atherosclerotic human arteries. J Clin Invest. 1992 Oct;90(4):1614–1621. doi: 10.1172/JCI116031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ngaiza J. R., Jaffe E. A. A 14 amino acid peptide derived from the amino terminus of the cleaved thrombin receptor elevates intracellular calcium and stimulates prostacyclin production in human endothelial cells. Biochem Biophys Res Commun. 1991 Sep 30;179(3):1656–1661. doi: 10.1016/0006-291x(91)91765-5. [DOI] [PubMed] [Google Scholar]
  33. Okazaki H., Majesky M. W., Harker L. A., Schwartz S. M. Regulation of platelet-derived growth factor ligand and receptor gene expression by alpha-thrombin in vascular smooth muscle cells. Circ Res. 1992 Dec;71(6):1285–1293. doi: 10.1161/01.res.71.6.1285. [DOI] [PubMed] [Google Scholar]
  34. Pagès G., Lenormand P., L'Allemain G., Chambard J. C., Meloche S., Pouysségur J. Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8319–8323. doi: 10.1073/pnas.90.18.8319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Papkoff J., Chen R. H., Blenis J., Forsman J. p42 mitogen-activated protein kinase and p90 ribosomal S6 kinase are selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation. Mol Cell Biol. 1994 Jan;14(1):463–472. doi: 10.1128/mcb.14.1.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rasmussen U. B., Vouret-Craviari V., Jallat S., Schlesinger Y., Pagès G., Pavirani A., Lecocq J. P., Pouysségur J., Van Obberghen-Schilling E. cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca2+ mobilization. FEBS Lett. 1991 Aug 19;288(1-2):123–128. doi: 10.1016/0014-5793(91)81017-3. [DOI] [PubMed] [Google Scholar]
  37. Sakurai T., Abe Y., Kasuya Y., Takuwa N., Shiba R., Yamashita T., Endo T., Goto K. Activin A stimulates mitogenesis in Swiss 3T3 fibroblasts without activation of mitogen-activated protein kinases. J Biol Chem. 1994 May 13;269(19):14118–14122. [PubMed] [Google Scholar]
  38. Sarembock I. J., Gertz S. D., Gimple L. W., Owen R. M., Powers E. R., Roberts W. C. Effectiveness of recombinant desulphatohirudin in reducing restenosis after balloon angioplasty of atherosclerotic femoral arteries in rabbits. Circulation. 1991 Jul;84(1):232–243. doi: 10.1161/01.cir.84.1.232. [DOI] [PubMed] [Google Scholar]
  39. Schuler G., Hambrecht R., Schlierf G., Niebauer J., Hauer K., Neumann J., Hoberg E., Drinkmann A., Bacher F., Grunze M. Regular physical exercise and low-fat diet. Effects on progression of coronary artery disease. Circulation. 1992 Jul;86(1):1–11. doi: 10.1161/01.cir.86.1.1. [DOI] [PubMed] [Google Scholar]
  40. Schwartz R. S. Neointima and arterial injury: dogs, rats, pigs, and more. Lab Invest. 1994 Dec;71(6):789–791. [PubMed] [Google Scholar]
  41. Seiler S. M., Goldenberg H. J., Michel I. M., Hunt J. T., Zavoico G. B. Multiple pathways of thrombin-induced platelet activation differentiated by desensitization and a thrombin exosite inhibitor. Biochem Biophys Res Commun. 1991 Dec 16;181(2):636–643. doi: 10.1016/0006-291x(91)91238-8. [DOI] [PubMed] [Google Scholar]
  42. Simonson M. S., Herman W. H. Protein kinase C and protein tyrosine kinase activity contribute to mitogenic signaling by endothelin-1. Cross-talk between G protein-coupled receptors and pp60c-src. J Biol Chem. 1993 May 5;268(13):9347–9357. [PubMed] [Google Scholar]
  43. Stiles C. D., Capone G. T., Scher C. D., Antoniades H. N., Van Wyk J. J., Pledger W. J. Dual control of cell growth by somatomedins and platelet-derived growth factor. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1279–1283. doi: 10.1073/pnas.76.3.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stouffer G. A., Sarembock I. J., McNamara C. A., Gimple L. W., Owens G. K. Thrombin-induced mitogenesis of vascular SMC is partially mediated by autocrine production of PDGF-AA. Am J Physiol. 1993 Sep;265(3 Pt 1):C806–C811. doi: 10.1152/ajpcell.1993.265.3.C806. [DOI] [PubMed] [Google Scholar]
  45. Turner C. E. Paxillin: a cytoskeletal target for tyrosine kinases. Bioessays. 1994 Jan;16(1):47–52. doi: 10.1002/bies.950160107. [DOI] [PubMed] [Google Scholar]
  46. Turner C. E., Pietras K. M., Taylor D. S., Molloy C. J. Angiotensin II stimulation of rapid paxillin tyrosine phosphorylation correlates with the formation of focal adhesions in rat aortic smooth muscle cells. J Cell Sci. 1995 Jan;108(Pt 1):333–342. doi: 10.1242/jcs.108.1.333. [DOI] [PubMed] [Google Scholar]
  47. Van Obberghen-Schilling E., Pouysségur J. Signaling pathways of the thrombin receptor. Thromb Haemost. 1993 Jul 1;70(1):163–167. [PubMed] [Google Scholar]
  48. Vogel U. S., Dixon R. A., Schaber M. D., Diehl R. E., Marshall M. S., Scolnick E. M., Sigal I. S., Gibbs J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. doi: 10.1038/335090a0. [DOI] [PubMed] [Google Scholar]
  49. Vouret-Craviari V., Van Obberghen-Schilling E., Rasmussen U. B., Pavirani A., Lecocq J. P., Pouysségur J. Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis. Mol Biol Cell. 1992 Jan;3(1):95–102. doi: 10.1091/mbc.3.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Vu T. K., Hung D. T., Wheaton V. I., Coughlin S. R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991 Mar 22;64(6):1057–1068. doi: 10.1016/0092-8674(91)90261-v. [DOI] [PubMed] [Google Scholar]
  51. Walters T. K., Gorog D. A., Wood R. F. Thrombin generation following arterial injury is a critical initiating event in the pathogenesis of the proliferative stages of the atherosclerotic process. J Vasc Res. 1994 May-Jun;31(3):173–177. doi: 10.1159/000319584. [DOI] [PubMed] [Google Scholar]
  52. Webb M. L., Taylor D. S., Molloy C. J. Effects of thrombin receptor activating peptide on phosphoinositide hydrolysis and protein kinase C activation in cultured rat aortic smooth muscle cells: evidence for "tethered-ligand" activation of smooth muscle cell thrombin receptors. Biochem Pharmacol. 1993 Apr 22;45(8):1577–1582. doi: 10.1016/0006-2952(93)90297-a. [DOI] [PubMed] [Google Scholar]
  53. Weber H., Taylor D. S., Molloy C. J. Angiotensin II induces delayed mitogenesis and cellular proliferation in rat aortic smooth muscle cells. Correlation with the expression of specific endogenous growth factors and reversal by suramin. J Clin Invest. 1994 Feb;93(2):788–798. doi: 10.1172/JCI117033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Weber H., Webb M. L., Serafino R., Taylor D. S., Moreland S., Norman J., Molloy C. J. Endothelin-1 and angiotensin-II stimulate delayed mitogenesis in cultured rat aortic smooth muscle cells: evidence for common signaling mechanisms. Mol Endocrinol. 1994 Feb;8(2):148–158. doi: 10.1210/mend.8.2.8170471. [DOI] [PubMed] [Google Scholar]
  55. Weiss R. H., Maduri M. The mitogenic effect of thrombin in vascular smooth muscle cells is largely due to basic fibroblast growth factor. J Biol Chem. 1993 Mar 15;268(8):5724–5727. [PubMed] [Google Scholar]
  56. Weiss R. H., Nuccitelli R. Inhibition of tyrosine phosphorylation prevents thrombin-induced mitogenesis, but not intracellular free calcium release, in vascular smooth muscle cells. J Biol Chem. 1992 Mar 15;267(8):5608–5613. [PubMed] [Google Scholar]
  57. Wilcox J. N., Rodriguez J., Subramanian R., Ollerenshaw J., Zhong C., Hayzer D. J., Horaist C., Hanson S. R., Lumsden A., Salam T. A. Characterization of thrombin receptor expression during vascular lesion formation. Circ Res. 1994 Dec;75(6):1029–1038. doi: 10.1161/01.res.75.6.1029. [DOI] [PubMed] [Google Scholar]
  58. Zhong C., Hayzer D. J., Corson M. A., Runge M. S. Molecular cloning of the rat vascular smooth muscle thrombin receptor. Evidence for in vitro regulation by basic fibroblast growth factor. J Biol Chem. 1992 Aug 25;267(24):16975–16979. [PubMed] [Google Scholar]

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