Advertisement

Green Tea Polyphenol Stimulates a Ras, MEKK1, MEK3, and p38 Cascade to Increase Activator Protein 1 Factor-dependent Involucrin Gene Expression in Normal Human Keratinocytes*

  • Sivaprakasam Balasubramanian
    Affiliations
    Search for articles by this author
  • Tatiana Efimova
    Affiliations
    Search for articles by this author
  • Richard L. Eckert
    Correspondence
    To whom correspondence should be addressed: Dept. of Physiology/Biophysics, Case Western Reserve University School of Medicine, 2109 Adelbert Rd., Cleveland, OH 44106-4970. Tel.: 216-368-5530; Fax: 216-368-5586
    Affiliations
    Search for articles by this author
  • Author Footnotes
    * This work was supported by grants from the National Institutes of Health (to R. L. E.) and by a Dermatology Foundation research fellowship (to T. E.) and used the facilities of the Skin Diseases Research Center of Northeast Ohio, National Institutes of Health Grant AR39750.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:November 06, 2001DOI:https://doi.org/10.1074/jbc.M110376200
      (−)-Epigallocatechin-3-gallate (EGCG) is an important bioactive constituent of green tea that efficiently reduces epidermal cancer cell proliferation. This inhibition is associated with a reduction in activator protein 1 (AP1) transcription factor level and activity. However, its effects on AP1 function in normal epidermal cells have not been extensively explored. Our present studies show that EGCG regulates normal keratinocyte function. To understand the mechanism of action, we examined the effects of EGCG on AP1 factor activity, MAPK signal transduction, and expression of the AP1 factor-regulated human involucrin (hINV) gene. EGCG increases hINV promoter activity in a concentration-dependent manner that requires the presence of an intact hINV promoter AP1 factor binding site. This response appears to be physiologic, as endogenous hINV gene expression is also increased. Fra-1, Fra-2, FosB, JunB, JunD, c-Jun, and c-Fos levels are increased by EGCG treatment, as is AP1 factor binding to hINV promoter AP1 site. Gel mobility shift studies show that this complex contains Fra-1 and JunD. Signal transduction analysis indicates that the EGCG response requires Ras, MEKK1, MEK3, and p38 kinases. Kinase assays and inhibitor studies suggest that p38δ is the p38 isoform responsible for the regulation. These changes are also associated with a cessation of cell proliferation and enhanced cornified envelope formation. These studies show that in normal human keratinocytes EGCG markedly increases, via a MAPK signaling mechanism, AP1 factor-associated responses.
      EGCG
      (−)-epigallocatechin-3-gallate
      hINV
      human involucrin
      TPA
      12-O-tetradecanoylphorbol-13-acetate
      AP1
      activator protein 1
      KSFM
      keratinocyte serum-free medium
      dn
      dominant-negative
      MAPK
      mitogen-activated protein kinase
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
      MEKK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase
      The polyphenol constituents of green tea inhibit carcinogenesis in a variety of tissues (
      • Mukhtar H.
      • Ahmad N.
      ,
      • Lin J.K.
      • Liang Y.C.
      • Lin-Shiau S.Y.
      ,
      • Mukhtar H.
      • Ahmad N.
      ,
      • Kuroda Y.
      • Hara Y.
      ); however, their mechanism of action is not well understood. (−)-Epigallocatechin-3-gallate (EGCG)1 is the major polyphenol isolated from green tea. AP1 transcription factors and AP1 factor-associated signal transduction are important targets of EGCG action (
      • Nomura M.
      • Ma W.Y.
      • Huang C.
      • Yang C.S.
      • Bowden G.T.
      • Miyamoto K.
      • Dong Z.
      ,
      • Chen W.
      • Dong Z.
      • Valcic S.
      • Timmermann B.N.
      • Bowden G.T.
      ,
      • Barthelman M.
      • Bair III, W.B.
      • Stickland K.K.
      • Chen W.
      • Timmermann B.N.
      • Valcic S.
      • Dong Z.
      • Bowden G.T.
      ). AP1 proteins consist of homodimers of Jun proteins (c-Jun, JunB, and JunD) and heterodimers of Jun and Fos (c-Fos, FosB, Fra-1, and Fra-2) factors (
      • Angel P.
      • Karin M.
      ). These proteins regulate transcription, differentiation, and cell proliferation by binding to specific recognition motifs in target genes (
      • Kovary K.
      • Bravo R.
      ,
      • Boise L.H.
      • Petryniak B.
      • Mao X.
      • June C.H.
      • Wang C.Y.
      • Lindsten T.
      • Bravo R.
      • Kovary K.
      • Leiden J.M.
      • Thompson C.B.
      ,
      • Redner R.L.
      • Lee A.W.
      • Osawa G.A.
      • Nienhuis A.W.
      ,
      • Suzuki T.
      • Okuno H.
      • Yoshida T.
      • Endo T.
      • Nishina H.
      • Iba H.
      ,
      • Castellazzi M.
      • Spyrou G.
      • La Vista N.
      • Dangy J.P.
      • Piu F.
      • Yaniv M.
      • Brun G.
      ). EGCG produces specific changes in AP1 factor function in immortalized and transformed keratinocytes. These changes include an EGCG-dependent reduction in phorbol ester-dependent mitogen-activated protein kinase (MAPK) activity (
      • Chen W.
      • Dong Z.
      • Valcic S.
      • Timmermann B.N.
      • Bowden G.T.
      ), and reduced AP1 factor level and activity (
      • Barthelman M.
      • Bair III, W.B.
      • Stickland K.K.
      • Chen W.
      • Timmermann B.N.
      • Valcic S.
      • Dong Z.
      • Bowden G.T.
      ,
      • Chung J.Y.
      • Huang C.
      • Meng X.
      • Dong Z.
      • Yang C.S.
      ). EGCG also inhibits ultraviolet light-associated activation of c-Fos gene expression and the accumulation of c-Fos protein (
      • Barthelman M.
      • Bair III, W.B.
      • Stickland K.K.
      • Chen W.
      • Timmermann B.N.
      • Valcic S.
      • Dong Z.
      • Bowden G.T.
      ). Based on these studies, it has been suggested that the cancer-preventive role of EGCG may be due, in part, to its ability to reduce AP1 factor-related responses (
      • Chen W.
      • Dong Z.
      • Valcic S.
      • Timmermann B.N.
      • Bowden G.T.
      ,
      • Barthelman M.
      • Bair III, W.B.
      • Stickland K.K.
      • Chen W.
      • Timmermann B.N.
      • Valcic S.
      • Dong Z.
      • Bowden G.T.
      ,
      • Chung J.Y.
      • Huang C.
      • Meng X.
      • Dong Z.
      • Yang C.S.
      ). However, very little information is available regarding how EGCG effects AP1 factor-regulated responses in normal keratinocytes. Our present studies show that EGCG increases AP1 factor levels in normal keratinocytes, and increases human involucrin gene expression: an AP1 factor-controlled marker of keratinocyte differentiation (
      • Rice R.H.
      • Green H.
      ). EGCG also increases cornified envelope formation. In addition, our study suggests that this EGCG-dependent regulation is mediated via a MAPK cascade that includes Ras, MEKK1, MEK3, and p38δ. Thus, in marked contrast to the EGCG-dependent decrease in AP1 factor function that is observed in cancer cells, EGCG increases AP1 factor activity and AP1 factor-dependent gene expression in normal keratinocytes. These results suggest that the mechanism of EGCG action is markedly different in normal and transformed cells.

      REFERENCES

        • Mukhtar H.
        • Ahmad N.
        Toxicol. Sci. 1999; 52: 111-117
        • Lin J.K.
        • Liang Y.C.
        • Lin-Shiau S.Y.
        Biochem. Pharmacol. 1999; 58: 911-915
        • Mukhtar H.
        • Ahmad N.
        Am. J. Clin. Nutr. 2000; 71: 1698S-1702S
        • Kuroda Y.
        • Hara Y.
        Mutat. Res. 1999; 436: 69-97
        • Nomura M.
        • Ma W.Y.
        • Huang C.
        • Yang C.S.
        • Bowden G.T.
        • Miyamoto K.
        • Dong Z.
        Mol. Carcinog. 2000; 28: 148-155
        • Chen W.
        • Dong Z.
        • Valcic S.
        • Timmermann B.N.
        • Bowden G.T.
        Mol. Carcinog. 1999; 24: 79-84
        • Barthelman M.
        • Bair III, W.B.
        • Stickland K.K.
        • Chen W.
        • Timmermann B.N.
        • Valcic S.
        • Dong Z.
        • Bowden G.T.
        Carcinogenesis. 1998; 19: 2201-2204
        • Angel P.
        • Karin M.
        Biochim. Biophys. Acta. 1991; 1072: 129-157
        • Kovary K.
        • Bravo R.
        Mol. Cell. Biol. 1992; 12: 5015-5023
        • Boise L.H.
        • Petryniak B.
        • Mao X.
        • June C.H.
        • Wang C.Y.
        • Lindsten T.
        • Bravo R.
        • Kovary K.
        • Leiden J.M.
        • Thompson C.B.
        Mol. Cell. Biol. 1993; 13: 1911-1919
        • Redner R.L.
        • Lee A.W.
        • Osawa G.A.
        • Nienhuis A.W.
        Oncogene. 1992; 7: 43-50
        • Suzuki T.
        • Okuno H.
        • Yoshida T.
        • Endo T.
        • Nishina H.
        • Iba H.
        Nucleic Acids Res. 1991; 19: 5537-5542
        • Castellazzi M.
        • Spyrou G.
        • La Vista N.
        • Dangy J.P.
        • Piu F.
        • Yaniv M.
        • Brun G.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8890-8894
        • Chung J.Y.
        • Huang C.
        • Meng X.
        • Dong Z.
        • Yang C.S.
        Cancer Res. 1999; 59: 4610-4617
        • Rice R.H.
        • Green H.
        Cell. 1979; 18: 681-694
        • LaCelle P.T.
        • Lambert A.
        • Ekambaram M.C.
        • Robinson N.A.
        • Eckert R.L.
        Skin Pharmacol. Appl. Skin Physiol. 1998; 11: 214-226
        • Efimova T.
        • LaCelle P.
        • Welter J.F.
        • Eckert R.L.
        J. Biol. Chem. 1998; 273: 24387-24395
        • Welter J.F.
        • Crish J.F.
        • Agarwal C.
        • Eckert R.L.
        J. Biol. Chem. 1995; 270: 12614-12622
        • Robbins D.J.
        • Zhen E.
        • Owaki H.
        • Vanderbilt C.A.
        • Ebert D.
        • Geppert T.D.
        • Cobb M.H.
        J. Biol. Chem. 1993; 268: 5097-5106
        • Minden A.
        • Lin A.
        • McMahon M.
        • Lange Carter C.
        • Derijard B.
        • Davis R.J.
        • Johnson G.L.
        • Karin M.
        Science. 1994; 266: 1719-1723
        • Yan M.
        • Templeton D.J.
        J. Biol. Chem. 1994; 269: 19067-19073
        • Yan M.
        • Dai T.
        • Deak J.C.
        • Kyriakis J.M.
        • Zon L.I.
        • Woodgett J.R.
        • Templeton D.J.
        Nature. 1994; 372: 798-800
        • Mahalingam M.
        • Templeton D.J.
        Mol. Cell. Biol. 1996; 16: 405-413
        • Moriguchi T.
        • Toyoshima F.
        • Masuyama N.
        • Hanafusa H.
        • Gotoh Y.
        • Nishida E.
        EMBO J. 1997; 16: 7045-7053
        • Derijard B.
        • Raingeaud J.
        • Barrett T.
        • Wu I.H.
        • Han J.
        • Ulevitch R.J.
        • Davis R.J.
        Science. 1995; 267: 682-685
        • Derijard B.
        • Hibi M.
        • Wu I.H.
        • Barrett T.
        • Su B.
        • Deng T.
        • Karin M.
        • Davis R.J.
        Cell. 1994; 76: 1025-1037
        • Raingeaud J.
        • Whitmarsh A.J.
        • Barrett T.
        • Derijard B.
        • Davis R.J.
        Mol. Cell. Biol. 1996; 16: 1247-1255
        • Dashti S.R.
        • Efimova T.
        • Eckert R.L.
        J. Biol. Chem. 2001; 276: 8059-8063
        • Wang Y.
        • Su B.
        • Sah V.P.
        • Brown J.H.
        • Han J.
        • Chien K.R.
        J. Biol. Chem. 1998; 273: 5423-5426
        • Wang Y.
        • Huang S.
        • Sah V.P.
        • Ross Jr., J.
        • Brown J.H.
        • Han J.
        • Chien K.R.
        J. Biol. Chem. 1998; 273: 2161-2168
        • Schreiber E.
        • Matthias P.
        • Muller M.M.
        • Schaffner W.
        Nucleic Acids Res. 1989; 176419
        • Agarwal C.
        • Rorke E.A.
        • Irwin J.C.
        • Eckert R.L.
        Cancer Res. 1991; 51: 3982-3989
        • Gorodeski G.I.
        • Eckert R.L.
        • Utian W.H.
        • Sheean L.
        • Rorke E.A.
        Differentiation. 1989; 42: 75-80
        • Efimova T.
        • Eckert R.L.
        J. Biol. Chem. 2000; 275: 1601-1607
        • Agarwal C.
        • Efimova T.
        • Welter J.F.
        • Crish J.F.
        • Eckert R.L.
        J. Biol. Chem. 1999; 274: 6190-6194
        • Robinson M.J.
        • Cobb M.H.
        Curr. Opin. Cell Biol. 1997; 9: 180-186
        • Dashti S.R.
        • Efimova T.
        • Eckert R.L.
        J. Biol. Chem. 2001; 276: 27214-27220
        • Itin P.H.
        • Pittelkow M.R.
        • Kumar R.
        Endocrinology. 1994; 135: 1793-1798
        • Hanley K.
        • Jiang Y.
        • He S.S.
        • Friedman M.
        • Elias P.M.
        • Bikle D.D.
        • Williams M.L.
        • Feingold K.R.
        J. Invest. Dermatol. 1998; 110: 368-375
        • Eckert R.L.
        • Crish J.F.
        • Robinson N.A.
        Physiol. Rev. 1997; 77: 397-424
        • Rice R.H.
        • Green H.
        Cell. 1977; 11: 417-422
        • Green H.
        Harvey Lect. 1980; 74: 101-139
        • Steinert P.M.
        Cell Death Differ. 1995; 2: 33-40
        • Dong Z.
        • Ma W.
        • Huang C.
        • Yang C.S.
        Cancer Res. 1997; 57: 4414-4419
        • Guo W.
        • Liu X.
        • Lee S.
        • Park N.H.
        Int. J Oncol. 1999; 15: 817-821
        • Enslen H.
        • Raingeaud J.
        • Davis R.J.
        J. Biol. Chem. 1998; 273: 1741-1748
        • Enslen H.
        • Brancho D.M.
        • Davis R.J.
        EMBO J. 2000; 19: 1301-1311
        • Jiang Y.
        • Chen C.
        • Li Z.
        • Guo W.
        • Gegner J.A.
        • Lin S.
        • Han J.
        J. Biol. Chem. 1996; 271: 17920-17926
        • Jiang Y.
        • Gram H.
        • Zhao M.
        • New L.
        • Gu J.
        • Di Feng L.
        • Padova F.
        • Ulevitch R.J.
        • Han J.
        J. Biol. Chem. 1997; 272: 30122-30128
        • Wang X.S.
        • Diener K.
        • Manthey C.L.
        • Wang S.
        • Rosenzweig B.
        • Bray J.
        • Delaney J.
        • Cole C.N.
        • Chan-Hui P.Y.
        • Mantlo N.
        • Lichenstein H.S.
        • Zukowski M.
        • Yao Z.
        J. Biol. Chem. 1997; 272: 23668-23674
        • Cuenda A.
        • Cohen P.
        • Buee Scherrer V.
        • Goedert M.
        EMBO J. 1997; 16: 295-305
        • Suganuma M.
        • Okabe S.
        • Sueoka E.
        • Iida N.
        • Komori A.
        • Kim S.J.
        • Fujiki H.
        Cancer Res. 1996; 56: 3711-3715
        • Yu R.
        • Jiao J.J.
        • Duh J.L.
        • Gudehithlu K.
        • Tan T.H.
        • Kong A.N.
        Carcinogenesis. 1997; 18: 451-456
        • Karin M.
        • Liu Z.
        • Zandi E.
        Curr. Opin. Cell Biol. 1997; 9: 240-246
        • Wang H.
        • Scott R.E.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4649-4653
        • Wang H.
        • Xie Z.
        • Scott R.E.
        J. Cell Biol. 1996; 135: 1151-1162
        • Pfarr C.M.
        • Mechta F.
        • Spyrou G.
        • Lallemand D.
        • Carillo S.
        • Yaniv M.
        Cell. 1994; 76: 747-760
        • Rutberg S.E.
        • Saez E.
        • Glick A.
        • Dlugosz A.A.
        • Spiegelman B.M.
        • Yuspa S.H.
        Oncogene. 1996; 13: 167-176
        • Rutberg S.E.
        • Saez E.
        • Lo S.
        • Jang S.I.
        • Markova N.
        • Spiegelman B.M.
        • Yuspa S.H.
        Oncogene. 1997; 15: 1337-1346
        • Saez E.
        • Rutberg S.E.
        • Mueller E.
        • Oppenheim H.
        • Smoluk J.
        • Yuspa S.H.
        • Spiegelman B.M.
        J. Cell. Biochem. 1995; 82: 721-732
        • Welter J.F.
        • Eckert R.L.
        Oncogene. 1995; 11: 2681-2687
        • Ren F.
        • Zhang S.
        • Mitchell S.H.
        • Butler R.
        • Young C.Y.
        Oncogene. 2000; 19: 1924-1932
        • Ahmad N.
        • Gupta S.
        • Mukhtar H.
        Arch. Biochem. Biophys. 2000; 376: 338-346
        • Chen Z.P.
        • Schell J.B.
        • Ho C.T.
        • Chen K.Y.
        Cancer Lett. 1998; 129: 173-179
        • Ahmad N.
        • Feyes D.K.
        • Nieminen A.L.
        • Agarwal R.
        • Mukhtar H.
        J. Natl. Cancer Inst. 1997; 89: 1881-1886
        • Laemmli U.K.
        Nature. 1970; 227: 680-685