A Forkhead/Winged Helix-related Transcription Factor Mediates Insulin-increased Plasminogen Activator Inhibitor-1 Gene Transcription
2002; Elsevier BV; Volume: 277; Issue: 23 Linguagem: Inglês
10.1074/jbc.m112073200
ISSN1083-351X
AutoresAnthony I. Vulin, Frederick M. Stanley,
Tópico(s)Autophagy in Disease and Therapy
ResumoPlasminogen activator inhibitor-1 (PAI-1) is an important regulator of fibrinolysis by its inhibition of both tissue-type and urokinase plasminogen activators. PAI-1 levels are elevated in type II diabetes and this elevation correlates with macro- and microvascular complications of diabetes. Insulin increases PAI-1 production in several experimental systems, but the mechanism of insulin-activated PAI-1 transcription remains to be determined. Deletion analysis of the PAI-1 promoter revealed that the insulin response element is between −117 and −7. Mutation of the AT-rich site at −52/−45 abolished the insulin responsiveness of the PAI-1 promoter. This sequence is similar to the inhibitory sequence found in the phosphoenolpyruvate carboxylkinase/insulin-like growth factor-I-binding protein I promoters. Gel-mobility shift assays demonstrated that the forkhead bound to the PAI-1 promoter insulin response element. Expression of the DNA-binding domain of FKHR acted as a dominant negative to block insulin-increased PAI-1-CAT expression. A LexA-FKHR construct was also insulin responsive. These data suggested that a member of the Forkhead/winged helix family of transcription factors mediated the effect of insulin on PAI-1 transcription. Inhibition of phosphatidylinositol 3-kinase reduced the effect of insulin on PAI-1 gene expression, a result consistent with activation through FKHR. However, it was likely that a different member of the FKHR family (not FKHR) mediated this effect since FKHR was present in both insulin-responsive and non-responsive cell lines. Plasminogen activator inhibitor-1 (PAI-1) is an important regulator of fibrinolysis by its inhibition of both tissue-type and urokinase plasminogen activators. PAI-1 levels are elevated in type II diabetes and this elevation correlates with macro- and microvascular complications of diabetes. Insulin increases PAI-1 production in several experimental systems, but the mechanism of insulin-activated PAI-1 transcription remains to be determined. Deletion analysis of the PAI-1 promoter revealed that the insulin response element is between −117 and −7. Mutation of the AT-rich site at −52/−45 abolished the insulin responsiveness of the PAI-1 promoter. This sequence is similar to the inhibitory sequence found in the phosphoenolpyruvate carboxylkinase/insulin-like growth factor-I-binding protein I promoters. Gel-mobility shift assays demonstrated that the forkhead bound to the PAI-1 promoter insulin response element. Expression of the DNA-binding domain of FKHR acted as a dominant negative to block insulin-increased PAI-1-CAT expression. A LexA-FKHR construct was also insulin responsive. These data suggested that a member of the Forkhead/winged helix family of transcription factors mediated the effect of insulin on PAI-1 transcription. Inhibition of phosphatidylinositol 3-kinase reduced the effect of insulin on PAI-1 gene expression, a result consistent with activation through FKHR. However, it was likely that a different member of the FKHR family (not FKHR) mediated this effect since FKHR was present in both insulin-responsive and non-responsive cell lines. PAI-1 1The abbreviations used are: PAI-1plasminogen activator inhibitor-1FKHRForkheadCATchloramphenicol acetyltransferasedbdDNA-binding domainRTreverse transcriptaseHUVEChuman umbilical vein endothelial cellsHIRhuman insulin receptorHAhemagglutininAEBSF4-(2-aminoethyl)-benzenesulfonylfluoride-HClCHOChinese hamster ovaryGSTglutathione S-transferaseIGF-1insulin-like growth factor-1RSVRous sarcoma virusβGalβ-galactosidasePI 3-kinasephosphatidylinositol 3-kinase1The abbreviations used are: PAI-1plasminogen activator inhibitor-1FKHRForkheadCATchloramphenicol acetyltransferasedbdDNA-binding domainRTreverse transcriptaseHUVEChuman umbilical vein endothelial cellsHIRhuman insulin receptorHAhemagglutininAEBSF4-(2-aminoethyl)-benzenesulfonylfluoride-HClCHOChinese hamster ovaryGSTglutathione S-transferaseIGF-1insulin-like growth factor-1RSVRous sarcoma virusβGalβ-galactosidasePI 3-kinasephosphatidylinositol 3-kinase is a major regulator of fibrinolysis. It inhibits both tissue-type and urokinase plasminogen activators and serves an essential role in wound healing where it is required to maintain the fibrin clot. Abnormal expression of PAI-1 is observed in obesity (1Samad F. Loskutoff D.J. Mol. Med. 1996; 2: 568-582Crossref PubMed Google Scholar), inflammation (2Agrenius V. Chmielewska J. Widstrom O. Blomback M. Am. Rev. Respir. Dis. 1989; 140: 1381-1385Crossref PubMed Scopus (42) Google Scholar), and diabetes (3Marutsuka K. Woodcock-Mitchell J. Sakamoto T. Sobel B.E. Fujii S. Coron. Artery Dis. 1998; 9: 177-184Crossref PubMed Scopus (14) Google Scholar) and increased PAI-1 has been correlated to the higher risk or cardiovascular disease seen in these syndromes (4Padro T. Emeis J.J. Steins M. Schmid K.W. Kienast J. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 893-902Crossref PubMed Scopus (111) Google Scholar).PAI-1 was found to be expressed in virtually all of the tissue types studied. These included pancreas (5Friess H. Duarte R. Kleeff J. Fukuda A. Tang W.H. Graber H. Schilling M. Zimmermann A. Korc M. Buchler M.W. Surgery. 1998; 124: 79-86Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), liver (6Simpson A.J. Booth N.A. Moore N.R. Bennett B. J. Clin. Pathol. 1991; 44: 139-143Crossref PubMed Scopus (126) Google Scholar), spleen (6Simpson A.J. Booth N.A. Moore N.R. Bennett B. J. Clin. Pathol. 1991; 44: 139-143Crossref PubMed Scopus (126) Google Scholar), kidney (7Yamamoto K. Loskutoff D.J. Am. J. Pathol. 1997; 151: 725-734PubMed Google Scholar), brain (8Dietzmann K. von Bossanyi P. Krause D. Wittig H. Mawrin C. Kirches E. Pathol. Res. Pract. 2000; 196: 15-21Crossref PubMed Scopus (50) Google Scholar), adipocytes (1Samad F. Loskutoff D.J. Mol. Med. 1996; 2: 568-582Crossref PubMed Google Scholar), synovial membranes (9Busso N. Peclat V., So, A. Sappino A.P. Ann. Rheum. Dis. 1997; 56: 550-557Crossref PubMed Scopus (104) Google Scholar), platelets monocytes and other blood cells (6Simpson A.J. Booth N.A. Moore N.R. Bennett B. J. Clin. Pathol. 1991; 44: 139-143Crossref PubMed Scopus (126) Google Scholar), heart (6Simpson A.J. Booth N.A. Moore N.R. Bennett B. J. Clin. Pathol. 1991; 44: 139-143Crossref PubMed Scopus (126) Google Scholar), and the smooth muscle and endothelial cells of the vasculature (1Samad F. Loskutoff D.J. Mol. Med. 1996; 2: 568-582Crossref PubMed Google Scholar). Numerous studies suggest that PAI-1 is elevated due to stress or injury in these tissues (10Tamaki K. Okuda S. Nakayama M. Yanagida T. Fujishima M. J. Am. Soc. Nephrol. 1996; 7: 2578-2589PubMed Google Scholar, 11Hasenstab D. Forough R. Clowes A.W. Circ. Res. 1997; 80: 490-496Crossref PubMed Scopus (76) Google Scholar, 12Sawa H. Fujii S. Sobel B.E. Arterioscler. Thromb. 1992; 12: 1507-1515Crossref PubMed Google Scholar, 13Sawa H. Sobel B.E. Fujii S. Circ. Res. 1993; 73: 671-680Crossref PubMed Scopus (56) Google Scholar, 14Lang I.M. Marsh J.J. Olman M.A. Moser K.M. Loskutoff D.J. Schleef R.R. Circulation. 1994; 89: 2715-2721Crossref PubMed Scopus (89) Google Scholar). This could be secondary to an increase in cytokines or growth factors in these areas.PAI-1 transcription was increased by numerous factors including platelet-derived growth factor (15Lau H.K. Cardiovasc. Res. 1999; 43: 1049-1059Crossref PubMed Scopus (25) Google Scholar), β-fibroblast growth factor (15Lau H.K. Cardiovasc. Res. 1999; 43: 1049-1059Crossref PubMed Scopus (25) Google Scholar), interleukin-1β, transforming growth factor β (16Sato Y. Tsuboi R. Lyons R. Moses H. Rifkin D.B. J. Cell Biol. 1990; 111: 757-763Crossref PubMed Scopus (363) Google Scholar), angiotensin II (17Brown N.J. Kim K.S. Chen Y.Q. Blevins L.S. Nadeau J.H. Meranze S.G. Vaughan D.E. J. Clin. Endocrinol. Metab. 2000; 85: 336-344Crossref PubMed Scopus (168) Google Scholar), tumor necrosis factor-α (18Samad F. Yamamoto K. Loskutoff D.J. J. Clin. Invest. 1996; 97: 37-46Crossref PubMed Scopus (283) Google Scholar), thrombin (19Cockell K.A. Ren S. Sun J. Angel A. Shen G.X. Thromb. Res. 1995; 77: 119-131Abstract Full Text PDF PubMed Scopus (27) Google Scholar), and oxidation products (20Dichtl W. Stiko A. Eriksson P. Goncalves I. Calara F. Banfi C. Ares M.P. Hamsten A. Nilsson J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 3025-3032Crossref PubMed Scopus (48) Google Scholar), while interferon-γ (21Gallicchio M. Hufnagl P. Wojta J. Tipping P. J. Immunol. 1996; 157: 2610-2617PubMed Google Scholar) inhibited PAI-1 production. Several specific response elements were defined in the PAI-1 promoter. A paired Sp1 element at −73 and −42 mediated responses to glucose and angiotensin II (22Chen Y.Q., Su, M. Walia R.R. Hao Q. Covington J.W. Vaughan D.E. J. Biol. Chem. 1998; 273: 8225-8231Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 23Motojima M. Ando T. Yoshioka T. Biochem. J. 2000; 349: 435-441Crossref PubMed Scopus (44) Google Scholar). An Ap-1-like element at −59/−52 was reported to mediate the response of the PAI-1 promoter to D dimer, a proteolytic fragment of fibrin (24Olman M.A. Hagood J.S. Simmons W.L. Fuller G.M. Vinson C. White K.E. Blood. 1999; 94: 2029-2038Crossref PubMed Google Scholar), and also to mediate effects from PKC and PKA (25Arts J. Grimbergen J. Toet K. Kooistra T. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 39-46Crossref PubMed Scopus (34) Google Scholar, 26Knudsen H. Olesen T. Riccio A. Ungaro P. Christensen L. Andreasen P.A. Eur. J. Biochem. 1994; 220: 63-74Crossref PubMed Scopus (40) Google Scholar). The response element for transforming growth factor β was sought by a number of groups with conflicting results. One group found an element at −732/−721 that was transforming growth factor β responsive when 6 copies were cloned in front of a heterologous promoter (27Song C.Z. Siok T.E. Gelehrter T.D. J. Biol. Chem. 1998; 273: 29287-29290Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Another group found that duplicate E box sequences between −740/−528 mediated the response to transforming growth factor β (28Hua X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (257) Google Scholar). A glucocorticoid response element was identified at −1212 (29Bruzdzinski C.J. Johnson M.R. Goble C.A. Winograd S.S. Gelehrter T.D. Mol. Endocrinol. 1993; 7: 1169-1177Crossref PubMed Scopus (33) Google Scholar). Two hypoxia response elements were identified at −175/−158 whose mutation eliminated the 3-fold response to hypoxia (30Kietzmann T. Roth U. Jungermann K. Blood. 1999; 94: 4177-4185Crossref PubMed Google Scholar) and an E box at −165/−160 was shown to bind USF-1 and increase basal transcription of the PAI-1 promoter (31White L.A. Bruzdzinski C. Kutz S.M. Gelehrter T.D. Higgins P.J. Exp. Cell Res. 2000; 260: 127-135Crossref PubMed Scopus (20) Google Scholar).Insulin increases PAI-1 mRNA under a number of conditions. PAI-1 is increased in patients with type 2 diabetes (32Sobel B. Woodcock-Mitchell J. Schneider D. Holt R. Marutsuka K. Gold H. Circulation. 1998; 97: 2213-2221Crossref PubMed Scopus (245) Google Scholar). Insulin or proinsulin infusion can cause local elevation of PAI-1 detected by analysis of PAI-1 protein levels (33Samad F. Pandey M. Bell P. Loskutoff D. Mol. Med. 2000; 6: 680-692Crossref PubMed Google Scholar, 34Nordt T. Sawa H. Fuji S. Sobel B. Circulation. 1995; 91: 764-770Crossref PubMed Google Scholar, 35Carmassi F. Morale M. Ferrini L. Del'Omo G. Ferdeghini M. Pedrinelli R. De Negri F. Am. J. Med. 1999; 107: 344-350Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) or by in situ hybridization (36Nordt T. Sawa H. Fujii S. Bode C. Sobel B. J. Mol. Cell. Cardiol. 1998; 30: 1535-1543Abstract Full Text PDF PubMed Scopus (54) Google Scholar). Insulin also increases expression of the endogenous PAI-1 gene in HepG2 cells (37Nordt T. Schneider D. Sobel B. Circulation. 1994; 89: 321-330Crossref PubMed Scopus (156) Google Scholar) and the transcription of a luciferase reporter plasmid under control of the PAI-1 promoter in human umbilical vein endothelial cells (HUVEC) in culture (38Grenett H. Benza R. Fless G., Li, X.-N. Davis G. Booyse F. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1803-1809Crossref PubMed Scopus (29) Google Scholar, 39Grenett H. Benza R., Li, X.-N. Akens M. Grammer J. Brown S. Booyse F. Thromb. Haemostasis. 1999; 82: 1504-1509Crossref PubMed Scopus (16) Google Scholar). The insulin response element was suggested to be in the region −98/−62, but this was never confirmed by mutational analysis of the PAI-1 promoter (40Banfi C. Eriksson P. Giandomenico G. Mussoni L. Sironi L. Hamsten A. Tremoli E. Diabetes. 2001; 50: 1522-1530Crossref PubMed Scopus (60) Google Scholar). Thus, the location of the insulin response element of the PAI-1 promoter has not been well defined.A number of potential transcription factor-binding sites in the proximal PAI-1 promoter were mutated to determine which of these mediated the insulin response. Mutation of a sequence resembling the negative insulin response elements found in the phosphoenolpyruvate carboxylkinase/IGF-I-binding protein promoters eliminated insulin activation of PAI-1. Gel mobility shifts demonstrated that GST fusion proteins with the Forkhead DNA-binding domain bound to this element, but did not bind to a non-functional mutant of this sequence. Expression of the DNA-binding domain of Forkhead acted as a dominant negative inhibitor of insulin-increased PAI-1 gene expression. Insulin-increased expression of a LexA-CAT reporter in cells expressing a LexA-Forkhead fusion protein. Finally, PI 3-kinase inhibition abrogated insulin-increased PAI-1 gene transcription. This is consistent with activation through a Forkhead-related protein and with data on insulin activation of PAI-1 (40Banfi C. Eriksson P. Giandomenico G. Mussoni L. Sironi L. Hamsten A. Tremoli E. Diabetes. 2001; 50: 1522-1530Crossref PubMed Scopus (60) Google Scholar).DISCUSSIONDeletions and mutations of the PAI-1 promoter have identified the sequence −52/−43 as an insulin response element. Deletions from the 5′ direction retained insulin responsiveness until −52 was reached. This is at the 3′ end of the AP-1 site within the PAI-1 promoter, suggesting that the AP-1 element is not the primary insulin response element. Deletion from the 3′ direction to −7 also retained insulin responsiveness. Mutations of the Ets-related elements, of the AP-1 site, or of the Sp-1 sites of the PAI-1 promoter did not affect insulin signaling. Only mutation of the sequence −52/−43 (TCTATTTCCT) eliminated insulin-increased PAI-1-Cat expression. Thus this sequence constitutes the primary insulin response element.The insulin response element of the PAI-1 promoter resembles that found in genes negatively regulated by insulin such as phosphoenolpyruvate carboxylkinase (TGGTGTTTTGAC) (54O'Brien R.M. Bonovich M.T. Forest C.D. Granner D.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6580-6584Crossref PubMed Scopus (70) Google Scholar) or IGF-I binding protein 1 (AACTTATTTTGAA) (55Suwanichkul A. Cubbage M.L. Powell D.R. J. Biol. Chem. 1990; 265: 21185-21193Abstract Full Text PDF PubMed Google Scholar). This was surprising because all of the positive response elements that we had defined previously had been Ets-related-binding sites and Ets-related sites were present in the PAI-1 promoter (56Jacob K.K. Ouyang L. Stanley F.M. J. Biol. Chem. 1995; 270: 27773-27779Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). However, specific mutation of these sites demonstrated that they were not the insulin response element of the PAI-1 promoter. It is possible that the context in which the Ets sites are located is important for their being able to function as insulin response elements. The genes in which we have definitively shown that the Ets site constitutes the insulin response element all have important CAAT/enhancer-binding protein sites. The PAI-1 promoter is without such a site and instead has Sp1 and AP1 sites in the promoter. Alternately, the Sp-1 binding may interfere with Ets factor binding.Sp-1- and AP-1-binding sites are present in the PAI-1 proximal promoter. The Sp-1 sites are at −72/−67 and −45/−40 while the AP-1 site is at −61/−54. The AP-1 site was reported to mediate the effects of both cAMP and phorbol esters on PAI-1 transcription (26Knudsen H. Olesen T. Riccio A. Ungaro P. Christensen L. Andreasen P.A. Eur. J. Biochem. 1994; 220: 63-74Crossref PubMed Scopus (40) Google Scholar). This site was not important for insulin responses since deletion to −52 had only a slight effect on the insulin response and specific mutation of this sequence in the context of the PAI-1 promoter −245/+72 did not decrease insulin-activated transcription of the PAI-1 promoter. However, it is possible that phorbol esters and cAMP may modulate insulin action through effects mediated by this sequence. We have not yet examined these potential interactions that could be important for explaining how insulin and inflammatory responses combine to effect PAI-1 gene transcription.Several experiments suggested that a winged helix/Forkhead-related transcription factor mediated the effects of insulin on the PAI-1 promoter. First, FKHR bound to the PAI-1 promoter (Fig. 5). Expression of the FKHR dbd (Fig. 6) eliminated insulin-increased PAI-1-CAT activity. Finally, a LexA construct containing the C terminus of FKHR was insulin responsive (Fig. 7). However, studies by others (53Nakae J. Park B.C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 57Rena G. Prescott A.R. Guo S. Cohen P. Unterman T.G. Biochem. J. 2001; 354: 605-612Crossref PubMed Scopus (218) Google Scholar,58Tomizawa M. Kumar A. Perrot V. Nakae J. Accili D. Rechler M.M. Kumaro A. J. Biol. Chem. 2000; 275: 7289-7295Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) demonstrated that FKHR phosphorylation in response to insulin caused its exclusion from the nucleus. This prevented FKHR from activating transcription and suggested that FKHR was not itself responsible for insulin-increased PAI-1 transcription. The identification of FKHR mRNA in insulin responsive, as well as non-responsive, cell types (Fig. 8) supported this. Thus, it seemed likely that some other member of the Forkhead family mediated insulin-increased PAI-1 gene expression. The FKHR-related family of winged helix transcription factors is large, containing more than 100 members to date (59Kaestner K.H. Knochel W. Martinez D.E. Genes Dev. 2000; 14: 142-146PubMed Google Scholar). This group contains both cell type-specific as well as ubiquitous transcription factors. The insulin-responsive factor responsible for activating PAI-1 expression could be one of these or a novel factor containing the winged helix DNA-binding domain.The insulin responsiveness of the LexA-FKHR construct indicated that this was plausible. LexA-CAT activity was increased 6-fold by insulin in GH4 cells transfected with LexA-FKHR. This was significantly less than the responses seen with LexA-Elk and LexA-Sap under similar conditions, but it is comparable with insulin-increased PAI-1-CAT expression. The LexA-FKHR construct used here did not include serine 253 that was phosphorylated in response to insulin in a PI 3-kinase/Akt-dependent manner (53Nakae J. Park B.C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Phosphorylation of this serine led to nuclear export of FKRH and inhibition of the activity of the FKHR activated genes. The behavior of the LexA-FKHR fusion protein has important implications for understanding insulin-activated PAI-1 transcription. First, it shows that FKHR and presumably the FKHR related factor that mediates the insulin response can be activated by insulin. This is contrary to previous reports (see below). It also supports the conclusion from experiments with dominant negatives of PKB/AKT that PKB/AKT is not required for insulin-activated PAI-1 transcription.Insulin activation of FKHR was not seen using Gal4-FKHR fusion proteins that contained part of the DNA-binding and the C-terminal activation domains of FKHR (58Tomizawa M. Kumar A. Perrot V. Nakae J. Accili D. Rechler M.M. Kumaro A. J. Biol. Chem. 2000; 275: 7289-7295Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). It is possible that the discrepancy between our results arise from differences in the LexA and Gal4 DNA-binding and expression systems. Insulin may activate FKHR if its promoter binding is strong enough for it to be retained in the nucleus despite signals for its export. Whether insulin activates or represses a gene could depend on the strength of the response element. The LexA system may provide a stronger DNA binding/dimerization domain than Gal4 or the LexA response element of the reporter may be stronger than the Gal4 response element used in those studies (58Tomizawa M. Kumar A. Perrot V. Nakae J. Accili D. Rechler M.M. Kumaro A. J. Biol. Chem. 2000; 275: 7289-7295Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Alternately, an accessory protein may be required for nuclear export of the FKHR fusion proteins and this might not be present in the cell lines that we examined. Studies showing the importance of 14-3-3 proteins for nuclear export of FKHR support this possibility (48Cahill C.M. Tzivion G. Nasrin N. Ogg S. Dore J. Ruvkun G. Alexander-Bridges M. J. Biol. Chem. 2001; 276: 13402-13410Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 57Rena G. Prescott A.R. Guo S. Cohen P. Unterman T.G. Biochem. J. 2001; 354: 605-612Crossref PubMed Scopus (218) Google Scholar).The activation of PAI-1-CAT expression by insulin is inhibited by LY294002 and by overexpression of PTEN (Fig. 9, A and B). This implied that insulin signaled through PI 3-kinase activation. PI 3-kinase was shown to activate PDK-1, which activated PKB/Akt. This suggested that protein kinase B/Akt might phosphorylate the insulin-responsive transcription factor. Expression of mutated Akt that acted as a dominant negative in other systems failed to block insulin-increased PAI-1-CAT expression (Fig. 9C) implying either that PKB/Akt does not phosphorylate the insulin-responsive transcription factor or that the PKB/Akt phosphorylation is not functional in the cell lines tested. If insulin acted through a different kinase to phosphorylate FKHR at a site(s) other than Ser253, then the export signal would not be activated while FKHR transcriptional activity increased, explaining how insulin could activate transcription through FKHR.PAI-1-luciferase was expressed in all of the cell lines tested (Fig. 1), while insulin stimulation was only observed in GH4, HeLa, HepG2, 3T3, and HUVEC cells. The lack of insulin stimulation in Rat-2 and CHO cells might be secondary to an incomplete insulin-signaling pathway in those cells. The lack of an important downstream kinase would make the promoter unresponsive to insulin. However, previous experiments demonstrated that CHO and Rat-2 cells exhibited insulin-increased promoter expression using different reporter genes (50Jacob K.K. Stanley F.M. J. Biol. Chem. 2001; 276: 24931-24936Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Another explanation for the lack of insulin-responsive PAI-1-CAT expression is the lack of the insulin-responsive transcription factor. This could be an insulin modified member of the Forkhead family that activates at the sequence −52/−43. It is intriguing to speculate that this might be a cell type-specific factor since PAI-1-luciferase expression was not insulin activated in all cell lines tested. Finally, it might be an accessory factor for the insulin-modified transcription factor that is not present in all cell types.It is important to conclusively identify the insulin-responsive transcription factor. The experiments presented above suggest that this factor may be a forkhead-related/winged helix transcription factor. The cell type experiments suggested that GH4 cells could be the most useful for isolating this factor. Experiments to determine the size of the factor that binds to the insulin response element of the PAI-1 promoter are currently in progress as a prelude to identifying it from an appropriate cDNA library. PAI-1 1The abbreviations used are: PAI-1plasminogen activator inhibitor-1FKHRForkheadCATchloramphenicol acetyltransferasedbdDNA-binding domainRTreverse transcriptaseHUVEChuman umbilical vein endothelial cellsHIRhuman insulin receptorHAhemagglutininAEBSF4-(2-aminoethyl)-benzenesulfonylfluoride-HClCHOChinese hamster ovaryGSTglutathione S-transferaseIGF-1insulin-like growth factor-1RSVRous sarcoma virusβGalβ-galactosidasePI 3-kinasephosphatidylinositol 3-kinase1The abbreviations used are: PAI-1plasminogen activator inhibitor-1FKHRForkheadCATchloramphenicol acetyltransferasedbdDNA-binding domainRTreverse transcriptaseHUVEChuman umbilical vein endothelial cellsHIRhuman insulin receptorHAhemagglutininAEBSF4-(2-aminoethyl)-benzenesulfonylfluoride-HClCHOChinese hamster ovaryGSTglutathione S-transferaseIGF-1insulin-like growth factor-1RSVRous sarcoma virusβGalβ-galactosidasePI 3-kinasephosphatidylinositol 3-kinase is a major regulator of fibrinolysis. It inhibits both tissue-type and urokinase plasminogen activators and serves an essential role in wound healing where it is required to maintain the fibrin clot. Abnormal expression of PAI-1 is observed in obesity (1Samad F. Loskutoff D.J. Mol. Med. 1996; 2: 568-582Crossref PubMed Google Scholar), inflammation (2Agrenius V. Chmielewska J. Widstrom O. Blomback M. Am. Rev. Respir. Dis. 1989; 140: 1381-1385Crossref PubMed Scopus (42) Google Scholar), and diabetes (3Marutsuka K. Woodcock-Mitchell J. Sakamoto T. Sobel B.E. Fujii S. Coron. Artery Dis. 1998; 9: 177-184Crossref PubMed Scopus (14) Google Scholar) and increased PAI-1 has been correlated to the higher risk or cardiovascular disease seen in these syndromes (4Padro T. Emeis J.J. Steins M. Schmid K.W. Kienast J. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 893-902Crossref PubMed Scopus (111) Google Scholar). plasminogen activator inhibitor-1 Forkhead chloramphenicol acetyltransferase DNA-binding domain reverse transcriptase human umbilical vein endothelial cells human insulin receptor hemagglutinin 4-(2-aminoethyl)-benzenesulfonylfluoride-HCl Chinese hamster ovary glutathione S-transferase insulin-like growth factor-1 Rous sarcoma virus β-galactosidase phosphatidylinositol 3-kinase plasminogen activator inhibitor-1 Forkhead chloramphenicol acetyltransferase DNA-binding domain reverse transcriptase human umbilical vein endothelial cells human insulin receptor hemagglutinin 4-(2-aminoethyl)-benzenesulfonylfluoride-HCl Chinese hamster ovary glutathione S-transferase insulin-like growth factor-1 Rous sarcoma virus β-galactosidase phosphatidylinositol 3-kinase PAI-1 was found to be expressed in virtually all of the tissue types studied. These included pancreas (5Friess H. Duarte R. Kleeff J. Fukuda A. Tang W.H. Graber H. Schilling M. Zimmermann A. Korc M. Buchler M.W. Surgery. 1998; 124: 79-86Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), liver (6Simpson A.J. Booth N.A. Moore N.R. Bennett B. J. Clin. Pathol. 1991; 44: 139-143Crossref PubMed Scopus (126) Google Scholar), spleen (6Simpson A.J. Booth N.A. Moore N.R. Bennett B. J. Clin. Pathol. 1991; 44: 139-143Crossref PubMed Scopus (126) Google Scholar), kidney (7Yamamoto K. Loskutoff D.J. Am. J. Pathol. 1997; 151: 725-734PubMed Google Scholar), brain (8Dietzmann K. von Bossanyi P. Krause D. Wittig H. Mawrin C. Kirches E. Pathol. Res. Pract. 2000; 196: 15-21Crossref PubMed Scopus (50) Google Scholar), adipocytes (1Samad F. Loskutoff D.J. Mol. Med. 1996; 2: 568-582Crossref PubMed Google Scholar), synovial membranes (9Busso N. Peclat V., So, A. Sappino A.P. Ann. Rheum. Dis. 1997; 56: 550-557Crossref PubMed Scopus (104) Google Scholar), platelets monocytes and other blood cells (6Simpson A.J. Booth N.A. Moore N.R. Bennett B. J. Clin. Pathol. 1991; 44: 139-143Crossref PubMed Scopus (126) Google Scholar), heart (6Simpson A.J. Booth N.A. Moore N.R. Bennett B. J. Clin. Pathol. 1991; 44: 139-143Crossref PubMed Scopus (126) Google Scholar), and the smooth muscle and endothelial cells of the vasculature (1Samad F. Loskutoff D.J. Mol. Med. 1996; 2: 568-582Crossref PubMed Google Scholar). Numerous studies suggest that PAI-1 is elevated due to stress or injury in these tissues (10Tamaki K. Okuda S. Nakayama M. Yanagida T. Fujishima M. J. Am. Soc. Nephrol. 1996; 7: 2578-2589PubMed Google Scholar, 11Hasenstab D. Forough R. Clowes A.W. Circ. Res. 1997; 80: 490-496Crossref PubMed Scopus (76) Google Scholar, 12Sawa H. Fujii S. Sobel B.E. Arterioscler. Thromb. 1992; 12: 1507-1515Crossref PubMed Google Scholar, 13Sawa H. Sobel B.E. Fujii S. Circ. Res. 1993; 73: 671-680Crossref PubMed Scopus (56) Google Scholar, 14Lang I.M. Marsh J.J. Olman M.A. Moser K.M. Loskutoff D.J. Schleef R.R. 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