Skip to main content

Advertisement

SpringerLink
Log in

Okadaic acid-induced transcriptional downregulation of type I collagen gene expression is mediated by protein phosphatase 2A

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Expression of type I collagen genes is highly regulated and becomes abnormal in various pathological conditions, from excessive collagen production in fibrotic diseases to their downregulation in transformed cells. Some inflammatory cytokines and other ligands, capable of eliciting intracellular phosphorylation, can profoundly alter collagen gene expression. We investigated the role of serine/threonine protein phosphatases (PP) in the regulation of collagen gene expression. Biosynthesis of the endogenous type I procollagen, and expression of Proal(I) promoter-luciferase (Luc) constructs transfected in NIH3T3 fibroblasts, were evaluated in response to PP2A and PP I inhibitor okadaic acid (OA) and exogenously expressed PP catalytic subunits. OA suppressed type I collagen gene expression as judged by reduced rates of protein synthesis, steady state levels of Proα 1(I) collagen mRNA and expression of Luc driven by Proa 1(I) collagen promoter in OA-treated cells. Co-transfection of Proα 1(I)-Luc with expression vectors containing PP2A, but not PPI, stimulated collagen promoter activity. These results strongly suggest that OA acts via PP2A-mediated dephosphorylation of an unidentified transcription factor(s) or cofactor(s) needed to activate Proαl(I) collagen promoter.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

OA:

okadaic acid

PP2A and PPI:

protein phosphatases 2A and 1

Luc:

luciferase

CAT:

chloramphenicol acetyltransferase

DMSO:

dimethyl sulfoxide

PCR:

polymerise chain reaction

bp:

base pair(s)

DMEM:

Dulbecco's modified Eagle's medium

FBS:

fetal bovine serum

PAGE:

polyacrylamide gel electrophoresis

References

  1. Raghow R, Thompson JP: Molecular mechanisms of collagen gene expression. Mol Cell Biochem 86: 5–18, 1989

    Article  CAS  Google Scholar 

  2. Vuorio E, de Crombrugghe B: The family of collagen genes. Annu Rev Biochem 59: 837–872, 1990

    Article  CAS  Google Scholar 

  3. Raghow R: The role of extracellular matrix in postinflammatory wound healing and fibrosis. FASEB J 8: 823–831, 1994

    Article  CAS  Google Scholar 

  4. Slack JL, Parker MI, Robinson VR, Bornstein P: Regulation of collagen I gene expression by ras. Mol Cell Biol 12: 4714–4723, 1992

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Santala P, Larjava H, Nissinen L, Riikonen T, Maatta A, Heino J: Suppressed collagen gene expression and induction of a2(31 integrin-type collagen receptor in tumorigenic derivatives of human osteogenic sarcoma (HOS) cell line. J Biol Chem 269: 1276–1283, 1994

    Article  CAS  Google Scholar 

  6. Wang Q, Hyde DM, Giri SN: Abatement of bleomycin-induced increases in vascular permeability, inflammatory cell infiltration, and fibrotic lesions in hamster lungs by combined treatment with taurine and niacin. Lab Invest 67: 234–242, 1992

    CAS  PubMed  Google Scholar 

  7. Slack JL, Liska DJ, Bornstein P: Regulation of expression of the type I collagen genes. Am J Med Genet 45: 140–151, 1993

    Article  CAS  Google Scholar 

  8. Katai H, Stephenson JD, Simkevich CP, Thompson JP, Raghow R: An AP-1-like motif in the first intron of human Proal(I) collagen gene is a critical determinant of its transcriptional activity. Mol Cell Biochem 118: 119–129, 1992

    Article  CAS  Google Scholar 

  9. Armendariz-Borunda J, Simkevich CP, Roy N, Raghow R, Kang AH, Seyer JM: Activation of Ito cells involves regulation of AP-1 binding proteins and induction of type I collagen gene expression. Biochem J 304: 817–824, 1994

    Article  CAS  Google Scholar 

  10. Chang E, Goldberg H: Requirements for transforming growth factorbeta regulation of the proa2(I) collagen and plasminogen activator inhibitor-1 promoters. J Biol Chem 270: 4473–4477, 1995

    Article  CAS  Google Scholar 

  11. Philips N, Bashey RI, Jimenez SA: Increased al(I) procollagen gene expression in tight skin (TSK) mice myocardial fibroblasts is due to a reduced interaction of a negative regulatory sequence with AP-1 transcription factor. J Biol Chem 270: 9313–9321, 1995

    Article  CAS  Google Scholar 

  12. Nehls MC, Grapilon ML, Brenner DA: NF- 1/Spl switch elements regulate collagen al (1) gene expression. DNA Cell Biol 11: 443–452, 1992

    Article  CAS  Google Scholar 

  13. Tamaki T, Ohnishi K, Hard C, LeRoy EC, Trojanowska M: Characterization of a GC-rich region containing Spl binding site(s) as a constitutive responsive element of the α2(I) collagen gene in human fibroblasts. J Biol Chem 270: 4299–4304, 1995

    Article  CAS  Google Scholar 

  14. Simkevich CP, Thompson JP, Poppleton H, Raghow R: The transcriptional tissue specificity of the human Proal(l) collagen gene is determined by a negative cis-regulatory element in the promoter. Biochem J 286: 179–185, 1992

    Article  CAS  Google Scholar 

  15. Inagaki Y, Truter S, Tanaka S, Di Liberto M, Ramirez F: Overlapping pathways mediate the opposing actions of tumor necrosis factor-β and transforming growth factor-(3 on α2(I) collagen gene transcription. J Biol Chem 270: 3353–3358, 1995

    Article  CAS  Google Scholar 

  16. Davis RJ: The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 268: 14553–14556, 1993

    Article  CAS  Google Scholar 

  17. Heller RA, Kronke M.: Tumor necrosis factor receptor-mediated signaling pathways. J Cell Biol 126: 5–9, 1994

    Article  CAS  Google Scholar 

  18. Divecha N, Irvine RE, Phospholipid signaling. Cell 80: 269–278, 1995

    Article  CAS  Google Scholar 

  19. Hunter T, Karin M: The regulation of transcription by phosphorylation. Cell 70: 375–387, 1992

    Article  CAS  Google Scholar 

  20. Vlach J, Garcia A, Jacque JM, Rodriguez MS, Michelson S, Virelizier JL: Induction of SpI phosphorylation and NF-kappa B-independent HIV promoter domain activity in T lymphocytes stimulated by okadaic acid. Virology 208: 753–761, 1995

    Article  CAS  Google Scholar 

  21. Cohen P, Holmes CFB, Tsukitani Y: Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci 15: 98–102, 1990

    Article  CAS  Google Scholar 

  22. Mellgren G, Vintermyr OK, Boe R, Doskeland SO: Hepatocyte DNA replication is abolished by inhibitors selecting protein phosphatase 2A rather than phosphatase 1. Exp Cell Res 205: 293–301, 1993

    Article  CAS  Google Scholar 

  23. Mumby MC, Walter G: Protein serine/threonine phosphatases: structure, regulation, and functions in cell growth. Physiol Rev 73: 673–699, 1993

    Article  CAS  Google Scholar 

  24. Walter G, Mumby MC: Protein serine/threonine phosphatases and cell transformation. Biochim Biophys Acta 1155: 207–226, 1993

    CAS  PubMed  Google Scholar 

  25. Raghow R, Gossage D, Kang AH: Pretranslational regulation of type I collagen, fibronectin, and a 50-kilodalton noncollagenous extracellular protein by dexamethasone in rat fibroblasts. J Biol Chem 261: 4677–4684, 1986

    Article  CAS  Google Scholar 

  26. Thompson JP, Simkevich CP, Holness MA, Kang AH, Raghow R: In vitro methylation of the promoter and enhancer of Proα1(1) collagen gene leads to its transcriptional inactivation. J Biol Chem 266: 2549–2556, 1991

    Article  CAS  Google Scholar 

  27. Hagiwara M, Alberts A, Brindle P, Meinkoth J, Feramisco J, Deng T, Karin M, Shenolikar S, Monmiiny M: Transcriptional attenuation following CAMP induction requires PP-I-mediated dephosphorylation of CREB. Cell 70: 105–113, 1992

    Article  CAS  Google Scholar 

  28. Green DD, Yang SI, Mumby MC: Molecular cloning and sequence analysis of the catalytic subunit of bovine type 2A protein phosphatase. Proc Natl Acad Sci USA 84: 4880–4884, 1987

    Article  CAS  Google Scholar 

  29. Sankaran L: A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail. Anal Biochem 200: 180–186, 1992

    Article  CAS  Google Scholar 

  30. Goldstein RH, Fine A, Farnsworth LJ, Poliks C, Polgar P: Phorbol ester-induced inhibition of collagen accumulation by human lung fibroblasts. J Biol Chem 265: 13623–13628, 1990

    Article  CAS  Google Scholar 

  31. Park K, Chung M, Kim S-J: Inhibition of myogenesis by okadaic acid, an inhibitor of protein phosphatases, 1 and 2A, correlates with the induction of AP l. J Biol Chem 267: 10810–10815, 1992

    Article  CAS  Google Scholar 

  32. Chang N-T, Huang LE, Liu AY-C: Okadaic acid markedly potentiates the heat-induced lisp 70 promoter activity. J Biol Chem 268: 1436–1439; 1993

    Article  CAS  Google Scholar 

  33. Fernandez A, Brautigan DL, Mumby MC, Lamb NJC: Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells. J Cell Biol 111: 103–112, 1990

    Article  CAS  Google Scholar 

  34. Mumby MC, Walter G: Protein phosphatases and DNA tumor viruses: transformation through the back door. Cell Regul 2: 589–598, 1991

    Article  CAS  Google Scholar 

  35. Sontag E, Fedorov S, Kamibayashi C, Robbins D, Cobb M, Mumby MC: The interaction of SV40 small tumor antigen with protein phosphatase 2A stimulates the MAP kinase pathway and induces cell proliferation. Cell 75: 887–897, 1993

    Article  CAS  Google Scholar 

  36. Olsen AS, Geddis AE, Prockop DJ: High levels of expression of a minigene version of the human Proαl(I) collagen gene in stably transfected mouse fibroblasts. Effects of deleting putative regulatory sequences in the first intron. J Biol Chem 266: 1117–1121, 1991

    Article  CAS  Google Scholar 

  37. Bedalov A, Breault DT, Sokolov BP, Lichtler AC, Bedalov I, Clark SH, Mack K, Khillan JS, Woody CO, Kream BE, Rowe DW: Regulation of the αl(I) collagen promoter in vascular smooth muscle cells: comparison with other αl(I) collagen-producing cells in transgenic animals and cultured cells. J Biol Chem 269: 4903–4909, 1994

    Article  CAS  Google Scholar 

  38. Slack JL, Liska DJ, Bornstein P: An upstream regulatory region mediates high-level, tissue-specific expression of the human αl(I) collagen gene in transgenic mice. Mol Cell Biol 11: 2066–2074, 1991

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmacology, College of Medicine, University of Tennessee, 38104, Memphis and Veterans Affairs Medical Center, Memphis, TN, USA

    Qingjian Wang & Rajendra Raghow

  2. Research Service (151), VA Medical Center, 1030 Jefferson Avenue, 38104, Memphis, TN, USA

    Rajendra Raghow

Authors
  1. Qingjian Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rajendra Raghow
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This paper was presented in part at the annual meeting of American Association for Cancer Research, Toronto, Canada, March 18–22, 1995 and published as an abstract in Proc Am Assoc Cancer Res 36:531, 1995.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, Q., Raghow, R. Okadaic acid-induced transcriptional downregulation of type I collagen gene expression is mediated by protein phosphatase 2A. Mol Cell Biochem 158, 33–42 (1996). https://doi.org/10.1007/BF00225880

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00225880

Key words

Search

Navigation