Differential Mechanisms of Plasminogen Activator Inhibitor-1 Gene Activation by Transforming Growth Factor-β and Tumor Necrosis Factor-α in Endothelial Cells

Summary

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (SERPIN) specific for tissue-type and urokinase-like plasminogen activators. High plasma PAI-1 activity is a risk factor for thrombotic diseases. Due to the short half-life of PAI-1, regulation of PAI-1 gene expression and secretion of active PAI-1 into the blood stream is important for hemostatic balance. We have investigated transcriptional control of PAI-1 gene expression in bovine aortic endothelial cells (BAECs) and human cell lines using PAI-1 5’ promoter-luciferase reporter assays. Contrary to the cytokine-induced up-regulation of PAI-1 mRNA and protein levels, we found that only transforming growth factor-β (TGF-β) was efficient in inducing PAI-1 promoter activation. Tissue necrosis factor-α (TNF-α) induced a small luciferase activity with the 2.5 kb PAI-1 promoter, but not with the PAI-800/4G/5G and p3TP-lux promoters. Next we investigated whether a lack of response to TNF-α was due to deficient signaling pathways. BAECs responded to TNF-α with robust NFκB promoter activation. TGF-β activated the p38 MAP kinase, while TNF-α activated both the SAPK/JNK and p38 MAP kinases. The ERK1/2 MAP kinases were constitutively activated in BAECs. BAEC therefore responded to TNF-α stimulation with activation of the MAP kinases and the NFκB transcriptional factors. We further measured the messenger RNA stability under the influence by TGF-β and TNF-α and found no difference. PAI-1 gene activation by TNF-α apparently is yet to be defined for the location of the response element and/or the signaling pathway, while TGF-β is the most important cytokine for PAI-1 transcriptional activation through its 5’ proximal promoter.

Keywords

Plasminogen activator inhibitor-1 - transforming growth factor-β - tumor necrosis factor-α - MAP kinase - endothelium

• References

• 1 Charlton P. The status of plasminogen activator inhibitor-1 as a therapeutic target. Exp Opin Invest Drugs 1997; 6: 539-54.
• 2 Noel A, Bajou K, Masson V, Devy L, Frankenne F, Rakic JM, Lambert V, Carmeliet P, Foidart JM. Regulation of cancer invasion and vascularization by plasminogen activator inhibitor-1. Fibrinolysis & Proteolysis 1999; 13: 220-5.
• 3 Loskutoff DJ. PAI-1 inhibits neointimal formation after arterial injury in mice: a new target for controlling restenosis?. Circulation 1997; 96: 2772-4.
• 4 Margaglione M, Cappucci G, Colaizzo D, Giuliani N, Vecchione G, Grandone E, Pennelli O, Di Minno G. The PAI-1 gene locus 4G/5G polymorphism is associated with a family history of coronary artery disease. Arteriosclerosis. Thromb Vasc Biol 1998; 18: 152-6.
• 5 Westendorp RGJ, Hottenga JJ, Slagboom PE. Variation in plasminogen-activator-inhibitor-1 gene and risk of meningococcal septic shock. Lancet 1999; 354: 561-3.
• 6 Iyer VR, Eisen MB, Ross DT, Schuler G, Moore T, Lee JCF, Trent JM, Staudt LM, Hudson Jr J, Boguski MS, Lashkari D, Shalon D, Botstein D, Brown PO. The transcriptional program in the response of human fibro-blasts to serum. Science 1999; 283: 83-7.
• 7 Gils A, Declerck PJ. Structure-function relationships in serpins: current concepts and controversies. Thromb Haemost 1998; 80: 531-41.
• 8 Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J 1998; 17: 3091-100.
• 9 Hua X, Miller ZA, Wu G, Shi Y, Lodish HF. Specificity in transforming growth factor beta-induced transcription of the plasminogen activator inhibitor-1 gene: interactions of promoter DNA, transcription factor muE3, and Smad proteins. Proc Natl Acad Sci USA 1999; 96: 13130-5.
• 10 Stroschein SL, Wang W, Luo K. Cooperative binding of Smad proteins to two adjacent DNA elements in the plasminogen activator inhibitor-1 promoter mediates transforming growth factor beta-induced smad-dependent transcriptional activation. J Biol Chem 1999; 274: 9431-41.
• 11 Massague J. TGF-beta signal transduction. Ann Rev Biochem 1998; 67: 753-91.
• 12 Attisano L, Wrana JL. Smads as transcriptional co-modulators. Curr Opin Cell Biol 2000; 12: 235-43.
• 13 Hanafusa H, Ninomiya-Tsuji J, Masuyama N, Nishita M, Fujisawa J, Shibuya H, Matsumoto K, Nishida E. Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor-beta-induced gene expression. J Biol Chem 1999; 274: 27161-7.
• 14 Sano Y, Harada J, Tashiro S, Gotoh-Mandeville R, Maekawa T, Ishii S. ATF-2 is a common nuclear target of Smad and TAK1 pathways in transforming growth factor-beta signaling. J Biol Chem 1999; 274: 8949-57.
• 15 Li JM, Shen X, Hu PP, Wang XF. Transforming growth factor beta stimulates the human immunodeficiency virus 1 enhancer and requires NFkappaB activity. Mol Cell Biol 1998; 18: 110-21.
• 16 Sakurai H, Shigemori N, Hasegawa K, Sugita T. TGF-beta-activated kinase 1 stimulates NF-kappa B activation by an NF-kappa B-inducing kinase-independent mechanism. Biochem Biophys Res Comm 1998; 243: 545-9.
• 17 Kyriakis JM. Activation of the AP-1 transcription factor by inflammatory cytokines of the TNF family. Gene Exp 1999; 7: 217-31.
• 18 Mulder KM. Role of Ras and MAPKs in TGF-beta signaling. Cytokine Growth Factor Rev 2000; 11: 23-35.
• 19 Yingling JM, Datto MB, Wong C, Frederick JP, Liberati NT, Wang XF. Tumor suppressor Smad4 is a transforming growth factor beta-inducible DNA binding protein. Mole Cell Biol 1997; 17: 7019-28.
• 20 Sawdey M, Podor TJ, Loskutoff DJ. Regulation of type 1 plasminogen activator inhibitor gene expression in cultured bovine aortic endothelial cells. Induction by transforming growth factor-beta, lipopolysaccharide, and tumor necrosis factor-alpha. J Biol Chem 1989; 264: 10396-401.
• 21 Hocevar BA, Brown TL, Howe PH. TGF-beta induces fibronectin synthesis through a c-Jun N-terminal kinase-dependent, Smad4-independent pathway. EMBO J 1999; 18: 1345-56.
• 22 Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, Trzaskos JM. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 1998; 273: 18623-32.
• 23 Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, Lee JC. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 1995; 364: 229-33.
• 24 Zawel L, Dai JL, Buckhaults P, Zhou S, Kinzler KW, Vogelstein B, Kern SE. Human Smad3 and Smad4 are sequence-specific transcription activators. Mole Cell 1998; 1: 611-7.
• 25 Zhang Y, Feng XH, Derynck R. Smad3 and Smad4 cooperate with c-Jun/cFos to mediate TGF-beta-induced transcription. Nature 1998; 394: 909-13.
• 26 Chen YQ, Su M, Walia RR, Hao Q, Covington JW, Vaughan DE. Sp1 sites mediate activation of the plasminogen activator inhibitor-1 promoter by glucose in vascular smooth muscle cells. J Biol Chem 1998; 273: 8225-31.
• 27 Bitzer M, von Gersdorff G, Liang D, Dominguez-Rosales A, Beg AA, Rojkind M, Bottinger EP. A mechanism of suppression of TGF-beta/ SMAD signaling by NF-kappa B/RelA. Genes & Development 2000; 14: 187-97.
• 28 Winzen R, Kracht M, Ritter B, Wilhelm A, Chen CY, Shyu AB, Muller M, Gaestel M, Resch K, Holtmann H. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J 1999; 18: 4969-80.
• 29 Heaton JH, Tillmann-Bogush M, Leff NS, Gelehrter TD. Cyclic nucleotide regulation of type-1 plasminogen activator-inhibitor mRNA stability in rat hepatoma cells. Identification of cis-acting sequences. J Biol Chem 1998; 273: 14261-8.
• 30 Westerhausen DR, Hopkins WE, Billadello JJ. Multiple transforming growth factor-beta-inducible elements regulate expression of the plasminogen activator inhibitor type-1 gene in Hep G2 cells. J Biol Chem 1991; 266: 1092-100.