Thrombin activates the p21-activated kinase in pulmonary artery smooth muscle cells

Summary

The p21-activated serine/threonine kinases (PAK) play an important role in a variety of cellular functions. However, their role in the smooth muscle response to thrombin, which is activated upon vascular injury and promotes vascular remodelling processes, is not resolved. Here we investigated the role of PAK in thrombin signalling and regulation of tissue factor (TF), the activator of the extrinsic coagulation cascade, in pulmonary artery smooth muscle cells (PASMC), the main cell type responsible for vascular remodelling in pulmonary hypertension. PAK was rapidly phosphorylated in response to thrombin. Thrombin and active PAKT423E phosphorylated p38 MAP kinase (p38MAPK), ERK1/2, phosphatidylinositol-dependent kinase-1 (PDK1) and protein kinase B/Akt (PKB) whereas kinase-deficient PAK1 prevented activation of these kinases by thrombin. In addition, kinase-deficient MKK3 inhibited activation of PDK1 and PKB by thrombin. Further, thrombin and active PAK1 induced TF expression and promoter activity while kinase-deficient PAK1 diminished thrombin-induced TF upregulation. Moreover, kinase-deficient MKK3, PDK1 and PKB inhibited thrombin- and PAK-dependent TF expression and promoter activity. Together these findings show that PAK is a critical element of thrombin signalling in PASMC which is involved in the regulation of TF expression by sequentially activating MKK3/p38MAPK, PDK1 and PKB. Thus, PAK may play an important role in promoting vascular remodelling processes in pulmonary hypertension.

Keywords

Thrombin - PAK - protein kinase B/Akt - tissue factor - p38 MAP kinase

• References

• 1 Farber HW, Loscalzo J, Hassell KL. et al Prothrombotic mechanisms in primary pulmonary hypertension. J Lab Clin Med 1999; 134: 561-6.
• 2 Herve P, Humbert M, Sitbon O. et al Pathobiology of pulmonary hypertension. The role of platelets and thrombosis. Clin Chest Med 2001; 22: 451-8
• 3 Ghigliotti G, Waissbluth AR, Speidel C. et al Prolonged activation of prothrombin on the vascular wall after arterial injury. Arterioscler Thromb Vasc Biol 1998; 18: 250-7.
• 4 Rauch U, Nemerson Y. Tissue factor, the blood, and the arterial wall. Trends Cardiovasc Med 2000; 10: 139-43.
• 5 Mackman N, Morrissey JH. Role of tissue factor in hemostasis, thrombosis, and vascular development. Arterioscler Thromb Vasc Biol 2004; 24: 1015-22
• 6 Morrissey JH. Tissue factor: a key molecule in hemostatic and nonhemostatic systems. Int J Hematol 2004; 79: 103-8
• 7 Carmeliet P, Collen D. Tissue factor. Int J Biochem Cell Biol 1998; 30: 661-7
• 8 Patterson C, Stouffer GA, Madamanchi N. et al New tricks for old dogs: nonthrombotic effects of thrombin in vessel wall biology. Circ Res 2001; 88: 987-9
• 9 Coughlin SR. How the protease thrombin talks to cells. Proc Natl Acad Sci U S A 1999; 96: 11023-7
• 10 Mackman N. Regulation of the tissue factor gene. Thromb Haemost 1997; 78: 747-54
• 11 Herkert O, Djordjevic T, BelAiba RS. et al Insights into the redox control of blood coagulation: Role of vascular NADPH oxidase derived reactive oxygen species (ROS) in the thrombogenic cycle. Antioxid Redox Signal 2004; 6: 765-76
• 12 Taubman MB, Marmur JD, Rosenfield CL. et al Agonist-mediated tissue factor expression in cultured vascular smooth muscle cells. Role of Ca2+ mobilization and protein kinase C activation. J Clin Invest 1993; 91: 547-52
• 13 Herkert O, Diebold I, Brandes RP. et al NADPH oxidase mediates tissue factor-dependent surface procoagulant activity by thrombin in human vascular smooth muscle cells. Circulation 2002; 105: 2030-6
• 14 Eto M, Kozai T, Cosentino F. et al Statin prevents tissue factor expression in human endothelial cells: role of Rho/Rho-kinase and Akt pathways. Circulation 2002; 105: 1756-9
• 15 Djordjevic T, Herkert O, Hess J. et al Rac regulates thrombin-induced tissue factor expression in pulmonary artery smooth muscle cells involving the nuclear factor kappa B pathway. Antioxid Redox Signal 2004; 6: 713-20
• 16 Bagrodia S, Cerione RA. Pak to the future. Trends Cell Biol 1999; 9: 350-5
• 17 Bokoch GM. Biology of the p21-activated kinases. Annu Rev Biochem 2003; 72: 743-81
• 18 Buchwald G, Hostinova E, Rudolph MG. et al Conformational switch and role of phosphorylation in PAK activation. Mol Cell Biol 2001; 21: 5179-89
• 19 Chong C, Tan L, Lim L. et al The mechanism of PAK activation. Autophosphorylation events in both regulatory and kinase domains control activity. J Biol Chem 2001; 276: 17347-53
• 20 Parrini MC, Lei M, Harrison SC. et al Pak1 kinase homodimers are autoinhibited in trans and dissociated upon activation by Cdc42 and Rac1. Mol Cell 2002; 9: 73-83
• 21 Tang Y, Chen Z, Ambrose D. et al Kinase-deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts. Mol Cell Biol 1997; 17: 4454-64
• 22 Sells MA, Knaus UG, Bagrodia S. et al Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr Biol 1997; 7: 202-10
• 23 Raingeaud J, Whitmarsh AJ, Barrett T. et al MKK3– and MKK6-regulated gene expression is me- diated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol 1996; 16: 1247-55
• 24 Franke TF, Yang SI, Chan TO. et al The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 1995; 81: 727-36
• 25 Bertrand L, Alessi DR, Deprez J. et al Heart 6-phosphofructo-2-kinase activation by insulin results from Ser-466 and Ser-483 phosphorylation and requires 3-phosphoinositide-dependent kinase-1, but not protein kinase B. J Biol Chem 1999; 274: 30927-33
• 26 Mackman N, Fowler BJ, Edgington TS. et al Functional analysis of the human tissue factor promoter and induction by serum. Proc Natl Acad Sci U S A 1990; 87: 2254-8
• 27 Master Z, Jones N, Tran J. et al Dok-R plays a pivotal role in angiopoietin-1-dependent cell migration through recruitment and activation of Pak. Embo J 2001; 20: 5919-28
• 28 Gorlach A, Brandes RP, Bassus S. et al Oxidative stress and expression of p22phox are involved in the up-regulation of tissue factor in vascular smooth muscle cells in response to activated platelets. Faseb J 2000; 14: 1518-28
• 29 Gorlach A, Diebold I, Schini-Kerth VB. et al Thrombin activates the hypoxia-inducible factor-1 signalling pathway in vascular smooth muscle cells: Role of the p22(phox)-containing NADPH oxidase. Circ Res 2001; 89: 47-54
• 30 Kawabata A, Kawao N. Physiology and pathophysiology of proteinase-activated receptors (PARs): PARs in the respiratory system: cellular signalling and physiological/pathological roles. J Pharmacol Sci 2005; 97: 20-4
• 31 Schmitz U, Thommes K, Beier I. et al Angiotensin II-induced stimulation of p21-activated kinase and c-Jun NH2-terminal kinase is mediated by Rac1 and Nck. J Biol Chem 2001; 276: 22003-10
• 32 Schmitz U, Thommes K, Beier I. et al Lysophosphatidic acid stimulates p21-activated kinase in vascular smooth muscle cells. Biochem Biophys Res Commun 2002; 291: 687-91
• 33 Dechert MA, Holder JM, Gerthoffer WT. p21-activated kinase 1 participates in tracheal smooth muscle cell migration by signalling to p38 Mapk. Am J Physiol Cell Physiol 2001; 281: C123-32
• 34 Vidal C, Geny B, Melle J. et al Cdc42/Rac1-dependent activation of the p21-activated kinase (PAK) regulates human platelet lamellipodia spreading: implication of the cortical-actin binding protein cortactin. Blood 2002; 100: 4462-9
• 35 Eblen ST, Slack JK, Weber MJ. et al Rac-PAK signalling stimulates extracellular signal-regulated kinase (ERK) activation by regulating formation of MEK1-ERK complexes. Mol Cell Biol 2002; 22: 6023-33
• 36 Tang Y, Yu J, Field J. Signals from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts. Mol Cell Biol 1999; 19: 1881-91
• 37 Zhang S, Han J, Sells MA. et al Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1. J Biol Chem 1995; 270: 23934-6
• 38 King CC, Gardiner EM, Zenke FT. et al p21-activated kinase (PAK1) is phosphorylated and activated by 3-phosphoinositide-dependent kinase-1 (PDK1). J Biol Chem 2000; 275: 41201-9
• 39 Tang Y, Zhou H, Chen A. et al The Akt proto-oncogene links Ras to Pak and cell survival signals. J Biol Chem 2000; 275: 9106-9
• 40 Zhou GL, Zhuo Y, King CC. et al Akt phosphorylation of serine 21 on Pak1 modulates Nck binding and cell migration. Mol Cell Biol 2003; 23: 8058-69
• 41 Cabane C, Coldefy AS, Yeow K. et al The p38 pathway regulates Akt both at the protein and transcriptional activation levels during myogenesis. Cell Signal 2004; 16: 1405-15
• 42 Rane MJ, Coxon PY, Powell DW. et al p38 Kinasedependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils. J Biol Chem 2001; 276: 3517-23
• 43 Lali FV, Hunt AE, Turner SJ. et al The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide- dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J Biol Chem 2000; 275: 7395-402
• 44 Taniyama Y, Ushio-Fukai M, Hitomi H. et al Role of p38 MAPK and MAPKAPK-2 in angiotensin II-induced Akt activation in vascular smooth muscle cells. Am J Physiol Cell Physiol 2004; 287: C494-9
• 45 Viswambharan H, Ming XF, Zhu S. et al Reconstituted high-density lipoprotein inhibits thrombin-induced endothelial tissue factor expression through inhibition of RhoA and stimulation of phosphatidylinositol 3-kinase but not Akt/endothelial nitric oxide synthase. Circ Res 2004; 94: 918-25