Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Apr 1;339(Pt 1):167–175.

Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases.

B D Sudbeck 1, P Baumann 1, G J Ryan 1, K Breitkopf 1, R Nischt 1, T Krieg 1, C Mauch 1
PMCID: PMC1220141  PMID: 10085241

Abstract

Many cell types, including fibroblasts and primary keratinocytes, increase matrix metalloproteinase 1 (MMP-1) production in response to agonists such as growth factors and phorbol esters. However, the spontaneously transformed human keratinocyte cell line HaCaT, although it increases MMP-1 production in response to epidermal growth factor (EGF), does not respond similarly to stimulation with PMA. This phenomenon occurs even though HaCaT cells remain proliferatively responsive to both agonists, suggesting a HaCaT-specific defect in a PMA-mediated signal transduction pathway. Using an inside-out approach to elucidate the source of this defect, we found that EGF, but not PMA, stimulated MMP-1 promoter activity in transiently transfected HaCaT keratinocytes. In addition, an assessment of fibroblast and HaCaT c-fos and c-jun gene expression after exposure to EGF and PMA showed that although both agonists increased the expression of c-fos and c-jun mRNA in fibroblasts, only EGF did so in HaCaT keratinocytes. Finally, we looked at the activation of mitogen-activated protein (MAP) family kinases after stimulation with EGF or PMA and found that both agonists increased the phosphorylation and activation of fibroblast extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but only EGF activated the same kinase activities in HaCaT cells. Further, the EGF-mediated increase in MMP-1 gene expression was inhibited by the MAP kinase/ERK kinase (MEK)-specific inhibitor PD98059 and the p38 kinase-specific inhibitor SB203580. Our evidence indicates that although HaCaT MAP kinases are functional, they are not properly regulated in response to the activation of protein kinase C, and that the defect that bars HaCaT MMP-1 expression in response to stimulation with PMA lies before MAP kinase activation.

Full Text

The Full Text of this article is available as a PDF (282.3 KB).

Selected References

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

  1. 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]
  2. Angel P., Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta. 1991 Dec 10;1072(2-3):129–157. doi: 10.1016/0304-419x(91)90011-9. [DOI] [PubMed] [Google Scholar]
  3. Armelin H. A. Pituitary extracts and steroid hormones in the control of 3T3 cell growth. Proc Natl Acad Sci U S A. 1973 Sep;70(9):2702–2706. doi: 10.1073/pnas.70.9.2702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benbow U., Brinckerhoff C. E. The AP-1 site and MMP gene regulation: what is all the fuss about? Matrix Biol. 1997 Mar;15(8-9):519–526. doi: 10.1016/s0945-053x(97)90026-3. [DOI] [PubMed] [Google Scholar]
  5. Birkedal-Hansen H. Proteolytic remodeling of extracellular matrix. Curr Opin Cell Biol. 1995 Oct;7(5):728–735. doi: 10.1016/0955-0674(95)80116-2. [DOI] [PubMed] [Google Scholar]
  6. Borden P., Heller R. A. Transcriptional control of matrix metalloproteinases and the tissue inhibitors of matrix metalloproteinases. Crit Rev Eukaryot Gene Expr. 1997;7(1-2):159–178. doi: 10.1615/critreveukargeneexpr.v7.i1-2.90. [DOI] [PubMed] [Google Scholar]
  7. Boukamp P., Petrussevska R. T., Breitkreutz D., Hornung J., Markham A., Fusenig N. E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol. 1988 Mar;106(3):761–771. doi: 10.1083/jcb.106.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brinckerhoff C. E. Regulation of metalloproteinase gene expression: implications for osteoarthritis. Crit Rev Eukaryot Gene Expr. 1992;2(2):145–164. [PubMed] [Google Scholar]
  9. Broberg A., Heino J. Integrin alpha2beta1-dependent contraction of floating collagen gels and induction of collagenase are inhibited by tyrosine kinase inhibitors. Exp Cell Res. 1996 Oct 10;228(1):29–35. doi: 10.1006/excr.1996.0295. [DOI] [PubMed] [Google Scholar]
  10. Burleson F. G., Simeonova P. P., Germolec D. R., Luster M. I. Dermatotoxic chemical stimulate of c-jun and c-fos transcription and AP-1 DNA binding in human keratinocytes. Res Commun Mol Pathol Pharmacol. 1996 Aug;93(2):131–148. [PubMed] [Google Scholar]
  11. Chao T. S., Byron K. L., Lee K. M., Villereal M., Rosner M. R. Activation of MAP kinases by calcium-dependent and calcium-independent pathways. Stimulation by thapsigargin and epidermal growth factor. J Biol Chem. 1992 Oct 5;267(28):19876–19883. [PubMed] [Google Scholar]
  12. Chao T. S., Foster D. A., Rapp U. R., Rosner M. R. Differential Raf requirement for activation of mitogen-activated protein kinase by growth factors, phorbol esters, and calcium. J Biol Chem. 1994 Mar 11;269(10):7337–7341. [PubMed] [Google Scholar]
  13. Chou S. Y., Baichwal V., Ferrell J. E., Jr Inhibition of c-Jun DNA binding by mitogen-activated protein kinase. Mol Biol Cell. 1992 Oct;3(10):1117–1130. doi: 10.1091/mbc.3.10.1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Crawford H. C., Matrisian L. M. Mechanisms controlling the transcription of matrix metalloproteinase genes in normal and neoplastic cells. Enzyme Protein. 1996;49(1-3):20–37. doi: 10.1159/000468614. [DOI] [PubMed] [Google Scholar]
  15. Delany A. M., Brinckerhoff C. E. Post-transcriptional regulation of collagenase and stromelysin gene expression by epidermal growth factor and dexamethasone in cultured human fibroblasts. J Cell Biochem. 1992 Dec;50(4):400–410. doi: 10.1002/jcb.240500409. [DOI] [PubMed] [Google Scholar]
  16. Deng T., Karin M. c-Fos transcriptional activity stimulated by H-Ras-activated protein kinase distinct from JNK and ERK. Nature. 1994 Sep 8;371(6493):171–175. doi: 10.1038/371171a0. [DOI] [PubMed] [Google Scholar]
  17. Dicker P., Rozengurt E. Stimulation of DNA synthesis by transient exposure of cell cultures to TPA or polypeptide mitogens: induction of competence or incomplete removal? J Cell Physiol. 1981 Oct;109(1):99–109. doi: 10.1002/jcp.1041090112. [DOI] [PubMed] [Google Scholar]
  18. Dudley D. T., Pang L., Decker S. J., Bridges A. J., Saltiel A. R. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7686–7689. doi: 10.1073/pnas.92.17.7686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Franklin C. C., Kraft A. S. Protein kinase C-independent activation of c-jun and c-fos transcription by epidermal growth factor. Biochim Biophys Acta. 1992 Mar 16;1134(2):137–142. doi: 10.1016/0167-4889(92)90036-b. [DOI] [PubMed] [Google Scholar]
  20. Fürstenberger G., Berry D. L., Sorg B., Marks F. Skin tumor promotion by phorbol esters is a two-stage process. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7722–7726. doi: 10.1073/pnas.78.12.7722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gaire M., Barro C. D., Kerr L. D., Carlisle F., Matrisian L. M. Protein kinase C isotypes required for phorbol-ester induction of stromelysin-1 in rat fibroblasts. Mol Carcinog. 1996 Feb;15(2):124–133. doi: 10.1002/(SICI)1098-2744(199602)15:2<124::AID-MC5>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  22. Gallagher T. F., Seibel G. L., Kassis S., Laydon J. T., Blumenthal M. J., Lee J. C., Lee D., Boehm J. C., Fier-Thompson S. M., Abt J. W. Regulation of stress-induced cytokine production by pyridinylimidazoles; inhibition of CSBP kinase. Bioorg Med Chem. 1997 Jan;5(1):49–64. doi: 10.1016/s0968-0896(96)00212-x. [DOI] [PubMed] [Google Scholar]
  23. Geiges D., Marks F., Gschwendt M. Loss of protein kinase C delta from human HaCaT keratinocytes upon ras transfection is mediated by TGF alpha. Exp Cell Res. 1995 Jul;219(1):299–303. doi: 10.1006/excr.1995.1231. [DOI] [PubMed] [Google Scholar]
  24. Goldberg G. I., Wilhelm S. M., Kronberger A., Bauer E. A., Grant G. A., Eisen A. Z. Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein. J Biol Chem. 1986 May 15;261(14):6600–6605. [PubMed] [Google Scholar]
  25. Gupta S., Campbell D., Dérijard B., Davis R. J. Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science. 1995 Jan 20;267(5196):389–393. doi: 10.1126/science.7824938. [DOI] [PubMed] [Google Scholar]
  26. Gutman A., Wasylyk B. The collagenase gene promoter contains a TPA and oncogene-responsive unit encompassing the PEA3 and AP-1 binding sites. EMBO J. 1990 Jul;9(7):2241–2246. doi: 10.1002/j.1460-2075.1990.tb07394.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hai T., Curran T. Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3720–3724. doi: 10.1073/pnas.88.9.3720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hawley-Nelson P., Stanley J. R., Schmidt J., Gullino M., Yuspa S. H. The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis. Exp Cell Res. 1982 Jan;137(1):155–167. doi: 10.1016/0014-4827(82)90017-9. [DOI] [PubMed] [Google Scholar]
  29. Hernández-Sotomayor S. M., Carpenter G. Epidermal growth factor receptor: elements of intracellular communication. J Membr Biol. 1992 Jun;128(2):81–89. doi: 10.1007/BF00231881. [DOI] [PubMed] [Google Scholar]
  30. Janknecht R., Ernst W. H., Pingoud V., Nordheim A. Activation of ternary complex factor Elk-1 by MAP kinases. EMBO J. 1993 Dec 15;12(13):5097–5104. doi: 10.1002/j.1460-2075.1993.tb06204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Karin M., Liu Z. g., Zandi E. AP-1 function and regulation. Curr Opin Cell Biol. 1997 Apr;9(2):240–246. doi: 10.1016/s0955-0674(97)80068-3. [DOI] [PubMed] [Google Scholar]
  32. Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem. 1995 Jul 14;270(28):16483–16486. doi: 10.1074/jbc.270.28.16483. [DOI] [PubMed] [Google Scholar]
  33. Knall C., Young S., Nick J. A., Buhl A. M., Worthen G. S., Johnson G. L. Interleukin-8 regulation of the Ras/Raf/mitogen-activated protein kinase pathway in human neutrophils. J Biol Chem. 1996 Feb 2;271(5):2832–2838. doi: 10.1074/jbc.271.5.2832. [DOI] [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. Korzus E., Nagase H., Rydell R., Travis J. The mitogen-activated protein kinase and JAK-STAT signaling pathways are required for an oncostatin M-responsive element-mediated activation of matrix metalloproteinase 1 gene expression. J Biol Chem. 1997 Jan 10;272(2):1188–1196. doi: 10.1074/jbc.272.2.1188. [DOI] [PubMed] [Google Scholar]
  36. Kyriakis J. M., Banerjee P., Nikolakaki E., Dai T., Rubie E. A., Ahmad M. F., Avruch J., Woodgett J. R. The stress-activated protein kinase subfamily of c-Jun kinases. Nature. 1994 May 12;369(6476):156–160. doi: 10.1038/369156a0. [DOI] [PubMed] [Google Scholar]
  37. Langholz O., Roeckel D., Petersohn D., Broermann E., Eckes B., Krieg T. Cell-matrix interactions induce tyrosine phosphorylation of MAP kinases ERK1 and ERK2 and PLCgamma-1 in two-dimensional and three-dimensional cultures of human fibroblasts. Exp Cell Res. 1997 Aug 25;235(1):22–27. doi: 10.1006/excr.1997.3640. [DOI] [PubMed] [Google Scholar]
  38. Lenormand P., Sardet C., Pagès G., L'Allemain G., Brunet A., Pouysségur J. Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts. J Cell Biol. 1993 Sep;122(5):1079–1088. doi: 10.1083/jcb.122.5.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Moses M. A. The regulation of neovascularization of matrix metalloproteinases and their inhibitors. Stem Cells. 1997;15(3):180–189. doi: 10.1002/stem.150180. [DOI] [PubMed] [Google Scholar]
  40. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  41. Ohuchi E., Imai K., Fujii Y., Sato H., Seiki M., Okada Y. Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J Biol Chem. 1997 Jan 24;272(4):2446–2451. doi: 10.1074/jbc.272.4.2446. [DOI] [PubMed] [Google Scholar]
  42. Pulverer B. J., Hughes K., Franklin C. C., Kraft A. S., Leevers S. J., Woodgett J. R. Co-purification of mitogen-activated protein kinases with phorbol ester-induced c-Jun kinase activity in U937 leukaemic cells. Oncogene. 1993 Feb;8(2):407–415. [PubMed] [Google Scholar]
  43. Ray J. M., Stetler-Stevenson W. G. The role of matrix metalloproteases and their inhibitors in tumour invasion, metastasis and angiogenesis. Eur Respir J. 1994 Nov;7(11):2062–2072. [PubMed] [Google Scholar]
  44. Robinson L. J., Razzack Z. F., Lawrence J. C., Jr, James D. E. Mitogen-activated protein kinase activation is not sufficient for stimulation of glucose transport or glycogen synthase in 3T3-L1 adipocytes. J Biol Chem. 1993 Dec 15;268(35):26422–26427. [PubMed] [Google Scholar]
  45. Ryder K., Lau L. F., Nathans D. A gene activated by growth factors is related to the oncogene v-jun. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1487–1491. doi: 10.1073/pnas.85.5.1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Salamonsen L. A., Woolley D. E. Matrix metalloproteinases in normal menstruation. Hum Reprod. 1996 Oct;11 (Suppl 2):124–133. doi: 10.1093/humrep/11.suppl_2.124. [DOI] [PubMed] [Google Scholar]
  47. Schaap D., van der Wal J., Howe L. R., Marshall C. J., van Blitterswijk W. J. A dominant-negative mutant of raf blocks mitogen-activated protein kinase activation by growth factors and oncogenic p21ras. J Biol Chem. 1993 Sep 25;268(27):20232–20236. [PubMed] [Google Scholar]
  48. Schönthal A., Herrlich P., Rahmsdorf H. J., Ponta H. Requirement for fos gene expression in the transcriptional activation of collagenase by other oncogenes and phorbol esters. Cell. 1988 Jul 29;54(3):325–334. doi: 10.1016/0092-8674(88)90195-x. [DOI] [PubMed] [Google Scholar]
  49. Sudbeck B. D., Parks W. C., Welgus H. G., Pentland A. P. Collagen-stimulated induction of keratinocyte collagenase is mediated via tyrosine kinase and protein kinase C activities. J Biol Chem. 1994 Nov 25;269(47):30022–30029. [PubMed] [Google Scholar]
  50. Troppmair J., Bruder J. T., Munoz H., Lloyd P. A., Kyriakis J., Banerjee P., Avruch J., Rapp U. R. Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation by oncogenes, serum, and 12-O-tetradecanoylphorbol-13-acetate requires Raf and is necessary for transformation. J Biol Chem. 1994 Mar 4;269(9):7030–7035. [PubMed] [Google Scholar]
  51. Ueda Y., Hirai S. i., Osada S. i., Suzuki A., Mizuno K., Ohno S. Protein kinase C activates the MEK-ERK pathway in a manner independent of Ras and dependent on Raf. J Biol Chem. 1996 Sep 20;271(38):23512–23519. doi: 10.1074/jbc.271.38.23512. [DOI] [PubMed] [Google Scholar]
  52. Westwick J. K., Weitzel C., Minden A., Karin M., Brenner D. A. Tumor necrosis factor alpha stimulates AP-1 activity through prolonged activation of the c-Jun kinase. J Biol Chem. 1994 Oct 21;269(42):26396–26401. [PubMed] [Google Scholar]
  53. Whitmarsh A. J., Davis R. J. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med (Berl) 1996 Oct;74(10):589–607. doi: 10.1007/s001090050063. [DOI] [PubMed] [Google Scholar]
  54. Whitmarsh A. J., Yang S. H., Su M. S., Sharrocks A. D., Davis R. J. Role of p38 and JNK mitogen-activated protein kinases in the activation of ternary complex factors. Mol Cell Biol. 1997 May;17(5):2360–2371. doi: 10.1128/mcb.17.5.2360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Xu J., Clark R. A. A three-dimensional collagen lattice induces protein kinase C-zeta activity: role in alpha2 integrin and collagenase mRNA expression. J Cell Biol. 1997 Jan 27;136(2):473–483. doi: 10.1083/jcb.136.2.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yang Y., Farese R. V. Insulin activates myelin basic protein (p42 MAP) kinase by a protein kinase C-independent pathway in rat adipocytes. Dissociation from glucose transport. FEBS Lett. 1993 Nov 1;333(3):287–290. doi: 10.1016/0014-5793(93)80672-h. [DOI] [PubMed] [Google Scholar]
  57. van Straaten F., Müller R., Curran T., Van Beveren C., Verma I. M. Complete nucleotide sequence of a human c-onc gene: deduced amino acid sequence of the human c-fos protein. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3183–3187. doi: 10.1073/pnas.80.11.3183. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES