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. 1995 Dec;15(12):6813–6819. doi: 10.1128/mcb.15.12.6813

Tyrosine dephosphorylation of nuclear proteins mimics transforming growth factor beta 1 stimulation of alpha 2(I) collagen gene expression.

P Greenwel 1, W Hu 1, R A Kohanski 1, F Ramirez 1
PMCID: PMC230935  PMID: 8524247

Abstract

Transforming growth factor beta 1 (TGF-beta 1) exerts a positive effect on the transcription of genes coding for several extracellular matrix-related products, including collagen I. We have previously identified a strong TGF-beta 1-responsive element (TbRE) in the upstream promoter sequence of the alpha 2(I) collagen (COL1A2) gene. Our experiments have shown that TGF-beta 1 stimulates COL1A2 transcription by increasing binding of an Sp1-containing complex (TbRC) to the TbRE. They have also suggested that the change occurs via posttranslational modification of a protein(s) directly or indirectly interacting with Sp1. Here, we provide evidence showing that tyrosine dephosphorylation of nuclear proteins mimics the stimulation of COL1A2 transcription by the TGF-beta 1-activated signaling pathway. Preincubation of nuclear extracts with protein tyrosine phosphatase (PTPase) but not with protein phosphatase type 2A (PP2A), a serine/threonine phosphatase, enhanced binding of the TbRC to the same degree as culturing cells in TGF-beta 1. Consistent with these in vitro findings, genistein, a tyrosine kinase inhibitor, led to markedly increased COL1A2 gene expression, whereas sodium orthovanadate, a tyrosine phosphatase inhibitor, decreased it substantially. These results were supported by transfection experiments showing that genistein and sodium orthovanadate have opposite effects on TbRE-mediated transcription. Moreover, nuclear proteins isolated from genistein-treated cells were found to interact with the TbRE significantly more than those from untreated cells. Furthermore, pretreatment of cells with sodium orthovanadate virtually abrogated nuclear protein binding to the TbRE, but not to a neighboring cis-acting element unresponsive to TGF-beta 1. The results of this study, therefore, provide the first correlation between tyrosine dephosphorylation, increased binding of a transcriptional complex, and TGF-beta 1 stimulation of gene expression.

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Selected References

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  1. Boast S., Su M. W., Ramirez F., Sanchez M., Avvedimento E. V. Functional analysis of cis-acting DNA sequences controlling transcription of the human type I collagen genes. J Biol Chem. 1990 Aug 5;265(22):13351–13356. [PubMed] [Google Scholar]
  2. Border W. A., Ruoslahti E. Transforming growth factor-beta in disease: the dark side of tissue repair. J Clin Invest. 1992 Jul;90(1):1–7. doi: 10.1172/JCI115821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang E., Goldberg H. Requirements for transforming growth factor-beta regulation of the pro-alpha 2(I) collagen and plasminogen activator inhibitor-1 promoters. J Biol Chem. 1995 Mar 3;270(9):4473–4477. doi: 10.1074/jbc.270.9.4473. [DOI] [PubMed] [Google Scholar]
  4. Chen F., Weinberg R. A. Biochemical evidence for the autophosphorylation and transphosphorylation of transforming growth factor beta receptor kinases. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1565–1569. doi: 10.1073/pnas.92.5.1565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen L. I., Nishinaka T., Kwan K., Kitabayashi I., Yokoyama K., Fu Y. H., Grünwald S., Chiu R. The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator. Mol Cell Biol. 1994 Jul;14(7):4380–4389. doi: 10.1128/mcb.14.7.4380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Clipstone N. A., Crabtree G. R. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature. 1992 Jun 25;357(6380):695–697. doi: 10.1038/357695a0. [DOI] [PubMed] [Google Scholar]
  8. Furukawa Y., Uenoyama S., Ohta M., Tsunoda A., Griffin J. D., Saito M. Transforming growth factor-beta inhibits phosphorylation of the retinoblastoma susceptibility gene product in human monocytic leukemia cell line JOSK-I. J Biol Chem. 1992 Aug 25;267(24):17121–17127. [PubMed] [Google Scholar]
  9. Ignotz R. A., Endo T., Massagué J. Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta. J Biol Chem. 1987 May 15;262(14):6443–6446. [PubMed] [Google Scholar]
  10. 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] [Google Scholar]
  11. Inagaki Y., Truter S., Tanaka S., Di Liberto M., Ramirez F. Overlapping pathways mediate the opposing actions of tumor necrosis factor-alpha and transforming growth factor-beta on alpha 2(I) collagen gene transcription. J Biol Chem. 1995 Feb 17;270(7):3353–3358. doi: 10.1074/jbc.270.7.3353. [DOI] [PubMed] [Google Scholar]
  12. Jackson S. P. Regulating transcription factor activity by phosphorylation. Trends Cell Biol. 1992 Apr;2(4):104–108. doi: 10.1016/0962-8924(92)90014-e. [DOI] [PubMed] [Google Scholar]
  13. Karsenty G., Golumbek P., de Crombrugghe B. Point mutations and small substitution mutations in three different upstream elements inhibit the activity of the mouse alpha 2(I) collagen promoter. J Biol Chem. 1988 Sep 25;263(27):13909–13915. [PubMed] [Google Scholar]
  14. Kerr L. D., Miller D. B., Matrisian L. M. TGF-beta 1 inhibition of transin/stromelysin gene expression is mediated through a Fos binding sequence. Cell. 1990 Apr 20;61(2):267–278. doi: 10.1016/0092-8674(90)90807-q. [DOI] [PubMed] [Google Scholar]
  15. Kim T. A., Velasquez B. R., Wenner C. E. Okadaic acid regulation of the retinoblastoma gene product is correlated with the inhibition of growth factor-induced cell proliferation in mouse fibroblasts. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5460–5463. doi: 10.1073/pnas.90.12.5460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kramer I. M., Koornneef I., de Laat S. W., van den Eijnden-van Raaij A. J. TGF-beta 1 induces phosphorylation of the cyclic AMP responsive element binding protein in ML-CCl64 cells. EMBO J. 1991 May;10(5):1083–1089. doi: 10.1002/j.1460-2075.1991.tb08048.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kramer I. M., Koornneef I., de Vries C., de Groot R. P., de Laat S. W., van den Eijnden-van Raaij A. J., Kruijer W. Phosphorylation of nuclear protein is an early event in TGF beta 1 action. Biochem Biophys Res Commun. 1991 Mar 29;175(3):816–822. doi: 10.1016/0006-291x(91)91638-s. [DOI] [PubMed] [Google Scholar]
  18. Laiho M., Rönnstrand L., Heino J., Decaprio J. A., Ludlow J. W., Livingston D. M., Massagué J. Control of junB and extracellular matrix protein expression by transforming growth factor-beta 1 is independent of simian virus 40 T antigen-sensitive growth-sensitive growth-inhibitory events. Mol Cell Biol. 1991 Feb;11(2):972–978. doi: 10.1128/mcb.11.2.972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Luckow B., Schütz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 1987 Jul 10;15(13):5490–5490. doi: 10.1093/nar/15.13.5490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. MacKintosh C., MacKintosh R. W. Inhibitors of protein kinases and phosphatases. Trends Biochem Sci. 1994 Nov;19(11):444–448. doi: 10.1016/0968-0004(94)90127-9. [DOI] [PubMed] [Google Scholar]
  21. Massagué J. The transforming growth factor-beta family. Annu Rev Cell Biol. 1990;6:597–641. doi: 10.1146/annurev.cb.06.110190.003121. [DOI] [PubMed] [Google Scholar]
  22. Ohtsuki M., Massagué J. Evidence for the involvement of protein kinase activity in transforming growth factor-beta signal transduction. Mol Cell Biol. 1992 Jan;12(1):261–265. doi: 10.1128/mcb.12.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Penttinen R. P., Kobayashi S., Bornstein P. Transforming growth factor beta increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1105–1108. doi: 10.1073/pnas.85.4.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rizzino A. Transforming growth factor-beta: multiple effects on cell differentiation and extracellular matrices. Dev Biol. 1988 Dec;130(2):411–422. doi: 10.1016/0012-1606(88)90337-5. [DOI] [PubMed] [Google Scholar]
  25. Rossi P., Karsenty G., Roberts A. B., Roche N. S., Sporn M. B., de Crombrugghe B. A nuclear factor 1 binding site mediates the transcriptional activation of a type I collagen promoter by transforming growth factor-beta. Cell. 1988 Feb 12;52(3):405–414. doi: 10.1016/s0092-8674(88)80033-3. [DOI] [PubMed] [Google Scholar]
  26. Sandler M. A., Zhang J. N., Westerhausen D. R., Jr, Billadello J. J. A novel protein interacts with the major transforming growth factor-beta responsive element in the plasminogen activator inhibitor type-1 gene. J Biol Chem. 1994 Aug 26;269(34):21500–21504. [PubMed] [Google Scholar]
  27. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shafit-Zagardo B., Brown F. L., Zavodny P. J., Maio J. J. Transcription of the KpnI families of long interspersed DNAs in human cells. Nature. 1983 Jul 21;304(5923):277–280. doi: 10.1038/304277a0. [DOI] [PubMed] [Google Scholar]
  29. Tamaki T., Ohnishi K., Hartl C., LeRoy E. C., Trojanowska M. Characterization of a GC-rich region containing Sp1 binding site(s) as a constitutive responsive element of the alpha 2(I) collagen gene in human fibroblasts. J Biol Chem. 1995 Mar 3;270(9):4299–4304. doi: 10.1074/jbc.270.9.4299. [DOI] [PubMed] [Google Scholar]
  30. Truter S., Di Liberto M., Inagaki Y., Ramirez F. Identification of an upstream regulatory region essential for cell type-specific transcription of the pro-alpha 2(V) collagen gene (COL5A2). J Biol Chem. 1992 Dec 15;267(35):25389–25395. [PubMed] [Google Scholar]
  31. Wrana J. L., Attisano L., Wieser R., Ventura F., Massagué J. Mechanism of activation of the TGF-beta receptor. Nature. 1994 Aug 4;370(6488):341–347. doi: 10.1038/370341a0. [DOI] [PubMed] [Google Scholar]
  32. Yan Z., Winawer S., Friedman E. Two different signal transduction pathways can be activated by transforming growth factor beta 1 in epithelial cells. J Biol Chem. 1994 May 6;269(18):13231–13237. [PubMed] [Google Scholar]

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