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EMBO J. Jun 1, 1998; 17(11): 3091–3100.
PMCID: PMC1170648

Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.

Abstract

Smad proteins play a key role in the intracellular signalling of transforming growth factor beta (TGF beta), which elicits a large variety of cellular responses. Upon TGF beta receptor activation, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. These complexes translocate to the nucleus where they control expression of target genes. However, the mechanism by which Smads mediate transcriptional regulation is largely unknown. Human plasminogen activator inhibitor-1 (PAI-1) is a gene that is potently induced by TGF beta. Here we report the identification of Smad3/Smad4 binding sequences, termed CAGA boxes, within the promoter of the human PAI-1 gene. The CAGA boxes confer TGF beta and activin, but not bone morphogenetic protein (BMP) stimulation to a heterologous promoter reporter construct. Importantly, mutation of the three CAGA boxes present in the PAI-1 promoter was found to abolish TGF beta responsiveness. Thus, CAGA elements are essential and sufficient for the induction by TGF beta. In addition, TGFbeta induces the binding of a Smad3/Smad4-containing nuclear complex to CAGA boxes. Furthermore, bacterially expressed Smad3 and Smad4 proteins, but not Smad1 nor Smad2 protein, bind directly to this sequence in vitro. The presence of this box in TGF beta-responsive regions of several other genes suggests that this may be a widely used motif in TGF beta-regulated transcription.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abdollah S, Macías-Silva M, Tsukazaki T, Hayashi H, Attisano L, Wrana JL. TbetaRI phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling. J Biol Chem. 1997 Oct 31;272(44):27678–27685. [PubMed]
  • Baker JC, Harland RM. A novel mesoderm inducer, Madr2, functions in the activin signal transduction pathway. Genes Dev. 1996 Aug 1;10(15):1880–1889. [PubMed]
  • Chen X, Rubock MJ, Whitman M. A transcriptional partner for MAD proteins in TGF-beta signalling. Nature. 1996 Oct 24;383(6602):691–696. [PubMed]
  • Chen X, Weisberg E, Fridmacher V, Watanabe M, Naco G, Whitman M. Smad4 and FAST-1 in the assembly of activin-responsive factor. Nature. 1997 Sep 4;389(6646):85–89. [PubMed]
  • Chen Y, Lebrun JJ, Vale W. Regulation of transforming growth factor beta- and activin-induced transcription by mammalian Mad proteins. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12992–12997. [PMC free article] [PubMed]
  • Chen Y, Bhushan A, Vale W. Smad8 mediates the signaling of the ALK-2 [corrected] receptor serine kinase. Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12938–12943. [PMC free article] [PubMed]
  • Datto MB, Hu PP, Kowalik TF, Yingling J, Wang XF. The viral oncoprotein E1A blocks transforming growth factor beta-mediated induction of p21/WAF1/Cip1 and p15/INK4B. Mol Cell Biol. 1997 Apr;17(4):2030–2037. [PMC free article] [PubMed]
  • de Caestecker MP, Hemmati P, Larisch-Bloch S, Ajmera R, Roberts AB, Lechleider RJ. Characterization of functional domains within Smad4/DPC4. J Biol Chem. 1997 May 23;272(21):13690–13696. [PubMed]
  • de Winter JP, Roelen BA, ten Dijke P, van der Burg B, van den Eijnden-van Raaij AJ. DPC4 (SMAD4) mediates transforming growth factor-beta1 (TGF-beta1) induced growth inhibition and transcriptional response in breast tumour cells. Oncogene. 1997 Apr 24;14(16):1891–1899. [PubMed]
  • Derynck R. TGF-beta-receptor-mediated signaling. Trends Biochem Sci. 1994 Dec;19(12):548–553. [PubMed]
  • Elbendary A, Berchuck A, Davis P, Havrilesky L, Bast RC, Jr, Iglehart JD, Marks JR. Transforming growth factor beta 1 can induce CIP1/WAF1 expression independent of the p53 pathway in ovarian cancer cells. Cell Growth Differ. 1994 Dec;5(12):1301–1307. [PubMed]
  • Eppert K, Scherer SW, Ozcelik H, Pirone R, Hoodless P, Kim H, Tsui LC, Bapat B, Gallinger S, Andrulis IL, et al. MADR2 maps to 18q21 and encodes a TGFbeta-regulated MAD-related protein that is functionally mutated in colorectal carcinoma. Cell. 1996 Aug 23;86(4):543–552. [PubMed]
  • Graff JM, Bansal A, Melton DA. Xenopus Mad proteins transduce distinct subsets of signals for the TGF beta superfamily. Cell. 1996 May 17;85(4):479–487. [PubMed]
  • Hata A, Lo RS, Wotton D, Lagna G, Massagué J. Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4. Nature. 1997 Jul 3;388(6637):82–87. [PubMed]
  • Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone MA, Jr, Wrana JL, et al. The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. Cell. 1997 Jun 27;89(7):1165–1173. [PubMed]
  • Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997 Dec 4;390(6659):465–471. [PubMed]
  • Hoodless PA, Haerry T, Abdollah S, Stapleton M, O'Connor MB, Attisano L, Wrana JL. MADR1, a MAD-related protein that functions in BMP2 signaling pathways. Cell. 1996 May 17;85(4):489–500. [PubMed]
  • Imamura T, Takase M, Nishihara A, Oeda E, Hanai J, Kawabata M, Miyazono K. Smad6 inhibits signalling by the TGF-beta superfamily. Nature. 1997 Oct 9;389(6651):622–626. [PubMed]
  • Inagaki Y, Truter S, Ramirez F. Transforming growth factor-beta stimulates alpha 2(I) collagen gene expression through a cis-acting element that contains an Sp1-binding site. J Biol Chem. 1994 May 20;269(20):14828–14834. [PubMed]
  • Keeton MR, Curriden SA, van Zonneveld AJ, Loskutoff DJ. Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor beta. J Biol Chem. 1991 Dec 5;266(34):23048–23052. [PubMed]
  • Kim J, Johnson K, Chen HJ, Carroll S, Laughon A. Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic. Nature. 1997 Jul 17;388(6639):304–308. [PubMed]
  • Kim SJ, Jeang KT, Glick AB, Sporn MB, Roberts AB. Promoter sequences of the human transforming growth factor-beta 1 gene responsive to transforming growth factor-beta 1 autoinduction. J Biol Chem. 1989 Apr 25;264(12):7041–7045. [PubMed]
  • Kim SJ, Denhez F, Kim KY, Holt JT, Sporn MB, Roberts AB. Activation of the second promoter of the transforming growth factor-beta 1 gene by transforming growth factor-beta 1 and phorbol ester occurs through the same target sequences. J Biol Chem. 1989 Nov 15;264(32):19373–19378. [PubMed]
  • Kretzschmar M, Massagué J. SMADs: mediators and regulators of TGF-beta signaling. Curr Opin Genet Dev. 1998 Feb;8(1):103–111. [PubMed]
  • Kretzschmar M, Liu F, Hata A, Doody J, Massagué J. The TGF-beta family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase. Genes Dev. 1997 Apr 15;11(8):984–995. [PubMed]
  • Lagna G, Hata A, Hemmati-Brivanlou A, Massagué J. Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways. Nature. 1996 Oct 31;383(6603):832–836. [PubMed]
  • Li JM, Nichols MA, Chandrasekharan S, Xiong Y, Wang XF. Transforming growth factor beta activates the promoter of cyclin-dependent kinase inhibitor p15INK4B through an Sp1 consensus site. J Biol Chem. 1995 Nov 10;270(45):26750–26753. [PubMed]
  • Lin YC, Stavnezer J. Regulation of transcription of the germ-line Ig alpha constant region gene by an ATF element and by novel transforming growth factor-beta 1-responsive elements. J Immunol. 1992 Nov 1;149(9):2914–2925. [PubMed]
  • Liu F, Hata A, Baker JC, Doody J, Cárcamo J, Harland RM, Massagué J. A human Mad protein acting as a BMP-regulated transcriptional activator. Nature. 1996 Jun 13;381(6583):620–623. [PubMed]
  • Liu F, Pouponnot C, Massagué J. Dual role of the Smad4/DPC4 tumor suppressor in TGFbeta-inducible transcriptional complexes. Genes Dev. 1997 Dec 1;11(23):3157–3167. [PMC free article] [PubMed]
  • Macías-Silva M, Abdollah S, Hoodless PA, Pirone R, Attisano L, Wrana JL. MADR2 is a substrate of the TGFbeta receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell. 1996 Dec 27;87(7):1215–1224. [PubMed]
  • Massagué J, Weis-Garcia F. Serine/threonine kinase receptors: mediators of transforming growth factor beta family signals. Cancer Surv. 1996;27:41–64. [PubMed]
  • Meersseman G, Verschueren K, Nelles L, Blumenstock C, Kraft H, Wuytens G, Remacle J, Kozak CA, Tylzanowski P, Niehrs C, et al. The C-terminal domain of Mad-like signal transducers is sufficient for biological activity in the Xenopus embryo and transcriptional activation. Mech Dev. 1997 Jan;61(1-2):127–140. [PubMed]
  • Nakao A, Afrakhte M, Morén A, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin NE, Heldin CH, et al. Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling. Nature. 1997 Oct 9;389(6651):631–635. [PubMed]
  • Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin CH, Miyazono K, et al. TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J. 1997 Sep 1;16(17):5353–5362. [PMC free article] [PubMed]
  • Newfeld SJ, Chartoff EH, Graff JM, Melton DA, Gelbart WM. Mothers against dpp encodes a conserved cytoplasmic protein required in DPP/TGF-beta responsive cells. Development. 1996 Jul;122(7):2099–2108. [PubMed]
  • Newfeld SJ, Mehra A, Singer MA, Wrana JL, Attisano L, Gelbart WM. Mothers against dpp participates in a DDP/TGF-beta responsive serine-threonine kinase signal transduction cascade. Development. 1997 Aug;124(16):3167–3176. [PubMed]
  • Riccio A, Pedone PV, Lund LR, Olesen T, Olsen HS, Andreasen PA. Transforming growth factor beta 1-responsive element: closely associated binding sites for USF and CCAAT-binding transcription factor-nuclear factor I in the type 1 plasminogen activator inhibitor gene. Mol Cell Biol. 1992 Apr;12(4):1846–1855. [PMC free article] [PubMed]
  • Roberts AB, Sporn MB. Physiological actions and clinical applications of transforming growth factor-beta (TGF-beta). Growth Factors. 1993;8(1):1–9. [PubMed]
  • Sadowski HB, Gilman MZ. Cell-free activation of a DNA-binding protein by epidermal growth factor. Nature. 1993 Mar 4;362(6415):79–83. [PubMed]
  • Savage C, Das P, Finelli AL, Townsend SR, Sun CY, Baird SE, Padgett RW. Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components. Proc Natl Acad Sci U S A. 1996 Jan 23;93(2):790–794. [PMC free article] [PubMed]
  • Sekelsky JJ, Newfeld SJ, Raftery LA, Chartoff EH, Gelbart WM. Genetic characterization and cloning of mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster. Genetics. 1995 Mar;139(3):1347–1358. [PMC free article] [PubMed]
  • Shi Y, Hata A, Lo RS, Massagué J, Pavletich NP. A structural basis for mutational inactivation of the tumour suppressor Smad4. Nature. 1997 Jul 3;388(6637):87–93. [PubMed]
  • Souchelnytskyi S, Tamaki K, Engström U, Wernstedt C, ten Dijke P, Heldin CH. Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-beta signaling. J Biol Chem. 1997 Oct 31;272(44):28107–28115. [PubMed]
  • Suzuki A, Chang C, Yingling JM, Wang XF, Hemmati-Brivanlou A. Smad5 induces ventral fates in Xenopus embryo. Dev Biol. 1997 Apr 15;184(2):402–405. [PubMed]
  • Thomsen GH. Xenopus mothers against decapentaplegic is an embryonic ventralizing agent that acts downstream of the BMP-2/4 receptor. Development. 1996 Aug;122(8):2359–2366. [PubMed]
  • Westerhausen DR, Jr, 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 Jan 15;266(2):1092–1100. [PubMed]
  • Wiersdorff V, Lecuit T, Cohen SM, Mlodzik M. Mad acts downstream of Dpp receptors, revealing a differential requirement for dpp signaling in initiation and propagation of morphogenesis in the Drosophila eye. Development. 1996 Jul;122(7):2153–2162. [PubMed]
  • Wieser R, Wrana JL, Massagué J. GS domain mutations that constitutively activate T beta R-I, the downstream signaling component in the TGF-beta receptor complex. EMBO J. 1995 May 15;14(10):2199–2208. [PMC free article] [PubMed]
  • Wrana JL, Attisano L, Cárcamo J, Zentella A, Doody J, Laiho M, Wang XF, Massagué J. TGF beta signals through a heteromeric protein kinase receptor complex. Cell. 1992 Dec 11;71(6):1003–1014. [PubMed]
  • Wrana JL, Attisano L, Wieser R, Ventura F, Massagué J. Mechanism of activation of the TGF-beta receptor. Nature. 1994 Aug 4;370(6488):341–347. [PubMed]
  • Wu RY, Zhang Y, Feng XH, Derynck R. Heteromeric and homomeric interactions correlate with signaling activity and functional cooperativity of Smad3 and Smad4/DPC4. Mol Cell Biol. 1997 May;17(5):2521–2528. [PMC free article] [PubMed]
  • Yamashita H, ten Dijke P, Huylebroeck D, Sampath TK, Andries M, Smith JC, Heldin CH, Miyazono K. Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects. J Cell Biol. 1995 Jul;130(1):217–226. [PMC free article] [PubMed]
  • Yingling JM, Das P, Savage C, Zhang M, Padgett RW, Wang XF. Mammalian dwarfins are phosphorylated in response to transforming growth factor beta and are implicated in control of cell growth. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8940–8944. [PMC free article] [PubMed]
  • 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. Mol Cell Biol. 1997 Dec;17(12):7019–7028. [PMC free article] [PubMed]
  • Zawel L, Dai JL, Buckhaults P, Zhou S, Kinzler KW, Vogelstein B, Kern SE. Human Smad3 and Smad4 are sequence-specific transcription activators. Mol Cell. 1998 Mar;1(4):611–617. [PubMed]
  • Zhang Y, Feng X, We R, Derynck R. Receptor-associated Mad homologues synergize as effectors of the TGF-beta response. Nature. 1996 Sep 12;383(6596):168–172. [PubMed]
  • Zhang Y, Musci T, Derynck R. The tumor suppressor Smad4/DPC 4 as a central mediator of Smad function. Curr Biol. 1997 Apr 1;7(4):270–276. [PubMed]

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