Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Sep 1;134(5):1301–1311. doi: 10.1083/jcb.134.5.1301

PDGF induction of alpha 2 integrin gene expression is mediated by protein kinase C-zeta

PMCID: PMC2120983  PMID: 8794869

Abstract

Platelet-derived growth factor (PDGF) stimulates fibroblasts to move over collagen and contract three-dimensional collagen gels, processes important in wound repair and fibrocontractive diseases. These processes depend on alpha 2 beta 1 integrin ligation of collagen and PDGF induces the expression of this integrin. Several lines of evidence presented here suggest that PKC-zeta plays a role in alpha 2 integrin gene expression. The induction was blocked by chemical inhibitors for protein tyrosine kinases (PTK), genistein, and protein kinase C (PKC), chelerythrine, and bisindolylmaleimide GF 109203X. Cells depleted of phorbol 12-myristate 13-acetate (PMA)-inducible PKCs by chronic treatment with PMA still demonstrated an alpha 2 response to PDGF indicating that a non-PMA-sensitive PKC isoform was required. PDGF induced kinase activity in PKC-zeta immunoprecipitates. Antisense oligonucleotides complementary to 5' end of PKC-zeta mRNA sequences blocked the PDGF-induced increase of alpha 2 mRNA levels up to 70%, indicating PKC-zeta, a non-PMA-sensitive PKC isoform, is a component of the PDGF stimulatory pathway for alpha 2 mRNA synthesis. A 961-base pair (bp) upstream region of alpha 2 gene/CAT construct transfected into human dermal fibroblasts was positively regulated by PDGF as judged by CAT enzymatic levels. Both PTK and PKC inhibitors blocked PDGF-stimulation of the alpha 2 promoter fragment/CAT construct, indicating that the phosphorylation requirement occurred at alpha 2 promoter-directed transcription level. Therefore, we propose that PDGF- stimulatory pathway of alpha 2 integrin gene expression involves multiple cellular protein kinases, one of which is PKC-zeta.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahlén K., Rubin K. Platelet-derived growth factor-BB stimulates synthesis of the integrin alpha 2-subunit in human diploid fibroblasts. Exp Cell Res. 1994 Dec;215(2):347–353. doi: 10.1006/excr.1994.1351. [DOI] [PubMed] [Google Scholar]
  2. Akiyama T., Ogawara H. Use and specificity of genistein as inhibitor of protein-tyrosine kinases. Methods Enzymol. 1991;201:362–370. doi: 10.1016/0076-6879(91)01032-w. [DOI] [PubMed] [Google Scholar]
  3. Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Herrlich P., Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987 Jun 19;49(6):729–739. doi: 10.1016/0092-8674(87)90611-8. [DOI] [PubMed] [Google Scholar]
  4. Auger K. R., Serunian L. A., Soltoff S. P., Libby P., Cantley L. C. PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell. 1989 Apr 7;57(1):167–175. doi: 10.1016/0092-8674(89)90182-7. [DOI] [PubMed] [Google Scholar]
  5. Barbee J. L., Deutscher S. L., Loomis C. R., Burns D. J. The cDNA sequence encoding human protein kinase C-zeta. Gene. 1993 Oct 15;132(2):305–306. doi: 10.1016/0378-1119(93)90213-m. [DOI] [PubMed] [Google Scholar]
  6. Berra E., Diaz-Meco M. T., Dominguez I., Municio M. M., Sanz L., Lozano J., Chapkin R. S., Moscat J. Protein kinase C zeta isoform is critical for mitogenic signal transduction. Cell. 1993 Aug 13;74(3):555–563. doi: 10.1016/0092-8674(93)80056-k. [DOI] [PubMed] [Google Scholar]
  7. Bignon C., Daniel N., Djiane J. Beta-galactosidase and chloramphenicol acetyltransferase assays in 96-well plates. Biotechniques. 1993 Aug;15(2):243–246. [PubMed] [Google Scholar]
  8. Brooks S. F., Herget T., Broad S., Rozengurt E. The expression of 80K/MARCKS, a major substrate of protein kinase C (PKC), is down-regulated through both PKC-dependent and -independent pathways. Effects of bombesin, platelet-derived growth factor, and cAMP. J Biol Chem. 1992 Jul 15;267(20):14212–14218. [PubMed] [Google Scholar]
  9. Chen Q., Kinch M. S., Lin T. H., Burridge K., Juliano R. L. Integrin-mediated cell adhesion activates mitogen-activated protein kinases. J Biol Chem. 1994 Oct 28;269(43):26602–26605. [PubMed] [Google Scholar]
  10. 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]
  11. Choudhury G. G., Biswas P., Grandaliano G., Abboud H. E. Involvement of PKC-alpha in PDGF-mediated mitogenic signaling in human mesangial cells. Am J Physiol. 1993 Nov;265(5 Pt 2):F634–F642. doi: 10.1152/ajprenal.1993.265.5.F634. [DOI] [PubMed] [Google Scholar]
  12. Circolo A., Welgus H. G., Pierce G. F., Kramer J., Strunk R. C. Differential regulation of the expression of proteinases/antiproteinases in fibroblasts. Effects of interleukin-1 and platelet-derived growth factor. J Biol Chem. 1991 Jul 5;266(19):12283–12288. [PubMed] [Google Scholar]
  13. Claesson-Welsh L. Platelet-derived growth factor receptor signals. J Biol Chem. 1994 Dec 23;269(51):32023–32026. [PubMed] [Google Scholar]
  14. Diaz-Meco M. T., Dominguez I., Sanz L., Dent P., Lozano J., Municio M. M., Berra E., Hay R. T., Sturgill T. W., Moscat J. zeta PKC induces phosphorylation and inactivation of I kappa B-alpha in vitro. EMBO J. 1994 Jun 15;13(12):2842–2848. doi: 10.1002/j.1460-2075.1994.tb06578.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Diaz-Meco M. T., Lozano J., Municio M. M., Berra E., Frutos S., Sanz L., Moscat J. Evidence for the in vitro and in vivo interaction of Ras with protein kinase C zeta. J Biol Chem. 1994 Dec 16;269(50):31706–31710. [PubMed] [Google Scholar]
  16. Diaz-Meco M. T., Quiñones S., Municio M. M., Sanz L., Bernal D., Cabrero E., Saus J., Moscat J. Protein kinase C-independent expression of stromelysin by platelet-derived growth factor, ras oncogene, and phosphatidylcholine-hydrolyzing phospholipase C. J Biol Chem. 1991 Nov 25;266(33):22597–22602. [PubMed] [Google Scholar]
  17. Dominguez I., Diaz-Meco M. T., Municio M. M., Berra E., García de Herreros A., Cornet M. E., Sanz L., Moscat J. Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes. Mol Cell Biol. 1992 Sep;12(9):3776–3783. doi: 10.1128/mcb.12.9.3776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Finkenzeller G., Marmé D., Weich H. A., Hug H. Platelet-derived growth factor-induced transcription of the vascular endothelial growth factor gene is mediated by protein kinase C. Cancer Res. 1992 Sep 1;52(17):4821–4823. [PubMed] [Google Scholar]
  19. Finkenzeller G., Totzke F., Fitzke E., Marmé D., Dieter P. Over-expression of protein kinase C-alpha enhances platelet-derived growth factor- and phorbol ester- but not calcium ionophore-induced formation of prostaglandins in NIH 3T3 fibroblasts. FEBS Lett. 1993 Apr 19;321(1):11–14. doi: 10.1016/0014-5793(93)80610-7. [DOI] [PubMed] [Google Scholar]
  20. Freter R. R., Alberta J. A., Lam K. K., Stiles C. D. A new platelet-derived growth factor-regulated genomic element which binds a serine/threonine phosphoprotein mediates induction of the slow immediate-early gene MCP-1. Mol Cell Biol. 1995 Jan;15(1):315–325. doi: 10.1128/mcb.15.1.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fujii K., Dousaka-Nakajima N., Imamura S. Epidermal growth factor enhancement of HSC-1 human cutaneous squamous carcinoma cell adhesion and migration on type I collagen involves selective up-regulation of alpha 2 beta 1 integrin expression. Exp Cell Res. 1995 Jan;216(1):261–272. doi: 10.1006/excr.1995.1032. [DOI] [PubMed] [Google Scholar]
  22. Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
  23. Gómez J., Pitton C., García A., Martinez de Aragón A., Silva A., Rebollo A. The zeta isoform of protein kinase C controls interleukin-2-mediated proliferation in a murine T cell line: evidence for an additional role of protein kinase C epsilon and beta. Exp Cell Res. 1995 May;218(1):105–113. doi: 10.1006/excr.1995.1136. [DOI] [PubMed] [Google Scholar]
  24. Haas T. A., Plow E. F. Integrin-ligand interactions: a year in review. Curr Opin Cell Biol. 1994 Oct;6(5):656–662. doi: 10.1016/0955-0674(94)90091-4. [DOI] [PubMed] [Google Scholar]
  25. Hagiwara M., Uchida C., Usuda N., Nagata T., Hidaka H. zeta-Related protein kinase C in nuclei of nerve cells. Biochem Biophys Res Commun. 1990 Apr 16;168(1):161–168. doi: 10.1016/0006-291x(90)91688-o. [DOI] [PubMed] [Google Scholar]
  26. Hansen S. K., Nerlov C., Zabel U., Verde P., Johnsen M., Baeuerle P. A., Blasi F. A novel complex between the p65 subunit of NF-kappa B and c-Rel binds to a DNA element involved in the phorbol ester induction of the human urokinase gene. EMBO J. 1992 Jan;11(1):205–213. doi: 10.1002/j.1460-2075.1992.tb05043.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Herbert J. M., Augereau J. M., Gleye J., Maffrand J. P. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun. 1990 Nov 15;172(3):993–999. doi: 10.1016/0006-291x(90)91544-3. [DOI] [PubMed] [Google Scholar]
  28. Herget T., Rozengurt E. Bombesin, endothelin and platelet-derived growth factor induce rapid translocation of the myristoylated alanine-rich C-kinase substrate in Swiss 3T3 cells. Eur J Biochem. 1994 Oct 15;225(2):539–548. doi: 10.1111/j.1432-1033.1994.00539.x. [DOI] [PubMed] [Google Scholar]
  29. Homma Y., Sakamoto H., Tsunoda M., Aoki M., Takenawa T., Ooyama T. Evidence for involvement of phospholipase C-gamma 2 in signal transduction of platelet-derived growth factor in vascular smooth-muscle cells. Biochem J. 1993 Mar 15;290(Pt 3):649–653. doi: 10.1042/bj2900649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  31. Inui H., Kitami Y., Tani M., Kondo T., Inagami T. Differences in signal transduction between platelet-derived growth factor (PDGF) alpha and beta receptors in vascular smooth muscle cells. PDGF-BB is a potent mitogen, but PDGF-AA promotes only protein synthesis without activation of DNA synthesis. J Biol Chem. 1994 Dec 2;269(48):30546–30552. [PubMed] [Google Scholar]
  32. Kelly K., Cochran B. H., Stiles C. D., Leder P. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell. 1983 Dec;35(3 Pt 2):603–610. doi: 10.1016/0092-8674(83)90092-2. [DOI] [PubMed] [Google Scholar]
  33. Klein C. E., Dressel D., Steinmayer T., Mauch C., Eckes B., Krieg T., Bankert R. B., Weber L. Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils. J Cell Biol. 1991 Dec;115(5):1427–1436. doi: 10.1083/jcb.115.5.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kolch W., Heidecker G., Kochs G., Hummel R., Vahidi H., Mischak H., Finkenzeller G., Marmé D., Rapp U. R. Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature. 1993 Jul 15;364(6434):249–252. doi: 10.1038/364249a0. [DOI] [PubMed] [Google Scholar]
  35. Larrodera P., Cornet M. E., Diaz-Meco M. T., Lopez-Barahona M., Diaz-Laviada I., Guddal P. H., Johansen T., Moscat J. Phospholipase C-mediated hydrolysis of phosphatidylcholine is an important step in PDGF-stimulated DNA synthesis. Cell. 1990 Jun 15;61(6):1113–1120. doi: 10.1016/0092-8674(90)90074-o. [DOI] [PubMed] [Google Scholar]
  36. Lee B. A., Donoghue D. J. Membrane-anchored form of v-sis/PDGF-B induces mitogenesis without detectable PDGF receptor autophosphorylation. J Cell Biol. 1991 Apr;113(2):361–370. doi: 10.1083/jcb.113.2.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lee Y. H., Kim H. S., Pai J. K., Ryu S. H., Suh P. G. Activation of phospholipase D induced by platelet-derived growth factor is dependent upon the level of phospholipase C-gamma 1. J Biol Chem. 1994 Oct 28;269(43):26842–26847. [PubMed] [Google Scholar]
  38. Lopata M. A., Cleveland D. W., Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res. 1984 Jul 25;12(14):5707–5717. doi: 10.1093/nar/12.14.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ma Y. H., Reusch H. P., Wilson E., Escobedo J. A., Fantl W. J., Williams L. T., Ives H. E. Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C gamma. J Biol Chem. 1994 Dec 2;269(48):30734–30739. [PubMed] [Google Scholar]
  40. Marquardt B., Frith D., Stabel S. Signalling from TPA to MAP kinase requires protein kinase C, raf and MEK: reconstitution of the signalling pathway in vitro. Oncogene. 1994 Nov;9(11):3213–3218. [PubMed] [Google Scholar]
  41. Mundschau L. J., Faller D. V. Platelet-derived growth factor signal transduction through the interferon-inducible kinase PKR. Immediate early gene induction. J Biol Chem. 1995 Feb 17;270(7):3100–3106. doi: 10.1074/jbc.270.7.3100. [DOI] [PubMed] [Google Scholar]
  42. Mundschau L. J., Forman L. W., Weng H., Faller D. V. Platelet-derived growth factor (PDGF) induction of egr-1 is independent of PDGF receptor autophosphorylation on tyrosine. J Biol Chem. 1994 Jun 10;269(23):16137–16142. [PubMed] [Google Scholar]
  43. Nakanishi H., Brewer K. A., Exton J. H. Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1993 Jan 5;268(1):13–16. [PubMed] [Google Scholar]
  44. Nakanishi H., Exton J. H. Purification and characterization of the zeta isoform of protein kinase C from bovine kidney. J Biol Chem. 1992 Aug 15;267(23):16347–16354. [PubMed] [Google Scholar]
  45. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992 Oct 23;258(5082):607–614. doi: 10.1126/science.1411571. [DOI] [PubMed] [Google Scholar]
  46. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 1995 Apr;9(7):484–496. [PubMed] [Google Scholar]
  47. Rauscher F. J., 3rd, Cohen D. R., Curran T., Bos T. J., Vogt P. K., Bohmann D., Tjian R., Franza B. R., Jr Fos-associated protein p39 is the product of the jun proto-oncogene. Science. 1988 May 20;240(4855):1010–1016. doi: 10.1126/science.3130660. [DOI] [PubMed] [Google Scholar]
  48. Riikonen T., Koivisto L., Vihinen P., Heino J. Transforming growth factor-beta regulates collagen gel contraction by increasing alpha 2 beta 1 integrin expression in osteogenic cells. J Biol Chem. 1995 Jan 6;270(1):376–382. doi: 10.1074/jbc.270.1.376. [DOI] [PubMed] [Google Scholar]
  49. Rodriguez-Viciana P., Warne P. H., Dhand R., Vanhaesebroeck B., Gout I., Fry M. J., Waterfield M. D., Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 1994 Aug 18;370(6490):527–532. doi: 10.1038/370527a0. [DOI] [PubMed] [Google Scholar]
  50. Rupprecht H. D., Sukhatme V. P., Lacy J., Sterzel R. B., Coleman D. L. PDGF-induced Egr-1 expression in rat mesangial cells is mediated through upstream serum response elements. Am J Physiol. 1993 Sep;265(3 Pt 2):F351–F360. doi: 10.1152/ajprenal.1993.265.3.F351. [DOI] [PubMed] [Google Scholar]
  51. Santoro S. A., Zutter M. M. The alpha 2 beta 1 integrin: a collagen receptor on platelets and other cells. Thromb Haemost. 1995 Sep;74(3):813–821. [PubMed] [Google Scholar]
  52. Sanz L., Berra E., Municio M. M., Dominguez I., Lozano J., Johansen T., Moscat J., Diaz-Meco M. T. Zeta PKC plays a critical role during stromelysin promoter activation by platelet-derived growth factor through a novel palindromic element. J Biol Chem. 1994 Apr 1;269(13):10044–10049. [PubMed] [Google Scholar]
  53. Schiro J. A., Chan B. M., Roswit W. T., Kassner P. D., Pentland A. P., Hemler M. E., Eisen A. Z., Kupper T. S. Integrin alpha 2 beta 1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells. Cell. 1991 Oct 18;67(2):403–410. doi: 10.1016/0092-8674(91)90191-z. [DOI] [PubMed] [Google Scholar]
  54. Schwartz M. A., Lechene C. Adhesion is required for protein kinase C-dependent activation of the Na+/H+ antiporter by platelet-derived growth factor. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6138–6141. doi: 10.1073/pnas.89.13.6138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sözeri O., Vollmer K., Liyanage M., Frith D., Kour G., Mark G. E., 3rd, Stabel S. Activation of the c-Raf protein kinase by protein kinase C phosphorylation. Oncogene. 1992 Nov;7(11):2259–2262. [PubMed] [Google Scholar]
  56. Takada Y., Hemler M. E. The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain. J Cell Biol. 1989 Jul;109(1):397–407. doi: 10.1083/jcb.109.1.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Takada Y., Murphy E., Pil P., Chen C., Ginsberg M. H., Hemler M. E. Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen. J Cell Biol. 1991 Oct;115(1):257–266. doi: 10.1083/jcb.115.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Toullec D., Pianetti P., Coste H., Bellevergue P., Grand-Perret T., Ajakane M., Baudet V., Boissin P., Boursier E., Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991 Aug 25;266(24):15771–15781. [PubMed] [Google Scholar]
  59. Treisman R. Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5' element and c-fos 3' sequences. Cell. 1985 Oct;42(3):889–902. doi: 10.1016/0092-8674(85)90285-5. [DOI] [PubMed] [Google Scholar]
  60. Valius M., Kazlauskas A. Phospholipase C-gamma 1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal. Cell. 1993 Apr 23;73(2):321–334. doi: 10.1016/0092-8674(93)90232-f. [DOI] [PubMed] [Google Scholar]
  61. Ventura C., Pintus G., Vaona I., Bennardini F., Pinna G., Tadolini B. Phorbol ester regulation of opioid peptide gene expression in myocardial cells. Role of nuclear protein kinase. J Biol Chem. 1995 Dec 15;270(50):30115–30120. doi: 10.1074/jbc.270.50.30115. [DOI] [PubMed] [Google Scholar]
  62. Wagner B. J., Hayes T. E., Hoban C. J., Cochran B. H. The SIF binding element confers sis/PDGF inducibility onto the c-fos promoter. EMBO J. 1990 Dec;9(13):4477–4484. doi: 10.1002/j.1460-2075.1990.tb07898.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Xu J., Clark R. A. Extracellular matrix alters PDGF regulation of fibroblast integrins. J Cell Biol. 1996 Jan;132(1-2):239–249. doi: 10.1083/jcb.132.1.239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zutter M. M., Santoro S. A., Painter A. S., Tsung Y. L., Gafford A. The human alpha 2 integrin gene promoter. Identification of positive and negative regulatory elements important for cell-type and developmentally restricted gene expression. J Biol Chem. 1994 Jan 7;269(1):463–469. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES