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Activation of the p38 Mitogen-activated Protein Kinase Pathway by Estrogen or by 4-Hydroxytamoxifen Is Coupled to Estrogen Receptor-induced Apoptosis*

  • Cheng Cheng Zhang
    Affiliations
    From the Department of Biochemistry, University of Illinois, Urbana, Illinois 61801
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  • David J. Shapiro
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, 413 RAL, University of Illinois, 600 S. Mathews Ave., Urbana, IL 61801. Tel.: 217-333-1788; Fax: 217-244-5858;
    Affiliations
    From the Department of Biochemistry, University of Illinois, Urbana, Illinois 61801
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant HD16720.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:January 07, 2000DOI:https://doi.org/10.1074/jbc.275.1.479
      17β-Estradiol (E2) or the antiestrogen, 4-hydroxytamoxifen (OHT), induce apoptosis in stably transfected estrogen receptor (ER)-positive HeLa-ER5 cells. p38 mitogen-activated protein kinase is implicated in cellular processes involving apoptosis. The p38 kinase inhibitor, SB203580, partially protects HeLa-ER5 cells against apoptosis induced by E2 or by OHT. E2 induces the p38 pathway 12–36-fold in ER-positive cell lines, while OHT induces p38 activity 2–5-fold. In an ER-positive cell line selected for resistance to E2-induced apoptosis, E2 no longer induced p38, and the ER no longer bound to the estrogen response element, while OHT induced both p38 and apoptosis. In cells selected for resistance to OHT-induced apoptosis, OHT no longer induced p38, while E2 induced p38 and apoptosis, and transactivated an estrogen response element-containing reporter gene. In MCF-7 cells, whose growth is stimulated by estrogen, E2 did not induce p38 or apoptosis, while OHT induced both p38 and apoptosis, and SB203580 protected against OHT-induced apoptosis. This work shows that E2 and OHT activate the p38 pathway, suggests that they use different pathways for p38 activation, and links activation of the p38 pathway to apoptosis induced by E2 and by OHT.
      MAP
      mitogen-activated protein
      MAPK
      mitogen-activated protein kinase
      ERK
      extracellular signal-regulated kinase
      JNK
      Jun N-terminal kinase
      E2
      17β-estradiol
      ER
      estrogen receptor
      OHT
      4-hydroxytamoxifen
      CD
      charcoal-dextran-treated
      FBS
      fetal bovine serum
      hERα
      human estrogen receptor α
      ERE
      estrogen response element
      AD
      activation domain
      MKK
      mitogen activated protein kinase kinase
      Several mitogen-activated protein (MAP)1 kinase pathways have been described in mammalian cells, including the extracellular signal-regulated kinases (ERKs) (
      • Schaeffer H.J.
      • Weber M.J.
      ), the 3 Jun N-terminal kinases (JNKs) (
      • Minden A.
      • Karin M.
      ), and the p38 MAP kinases (
      • Xia Z.
      • Dickens M.
      • Raingeaud J.
      • Davis R.J.
      • Greenberg M.E.
      ) (also termed stress-activated protein kinase 2). The p38 MAP kinase pathway is activated by proinflammatory cytokines and by environmental stress (
      • Paul A.
      • Wilson S.
      • Belham C.M.
      • Robinson C.J.
      • Scott P.H.
      • Gould G.W.
      • Plevin R.
      ). While the ERK kinases are activated by mitogenic stimuli, activation of the p38 pathway is strongly correlated with apoptosis (
      • Xia Z.
      • Dickens M.
      • Raingeaud J.
      • Davis R.J.
      • Greenberg M.E.
      ). Although the relationship between p38 activation and apoptosis is not well understood, a recent report links activation of p38 and the induction of apoptosis. p38 kinase, activated by UV light, was shown to phosphorylate the p53 tumor suppresser (
      • Huang C.
      • Ma W.-Y.
      • Maxiner A.
      • Sun Y.
      • Dong Z.
      ). Phosphorylation of p53 is coupled to its ability to activate transcription of genes involved in control of cell cycle progression and apoptosis (
      • Martinez J.D.
      • Page G.
      • Lohrum M.
      • Sweiland S.
      • Scheidtmann K.-H.
      ,
      • Mine D.M.
      • Campbell D.G.
      • Caudwell F.B.
      • Meek D.W.
      ).
      The estrogen, 17β-estradiol (E2), can stimulate proliferation, or induce differentiation or death depending on the cell context. In estrogen receptor (ER)-positive MCF-7 human breast cancer cells, E2 strongly stimulates cell proliferation and does not induce cell death. In contrast, in several types of stably transfected cell lines expressing significant levels of ER, E2 is highly toxic and induces cell death (
      • Kushner P.J.
      • Hort E.
      • Shine J.
      • Baxter J.D.
      • Greene G.L.
      ,
      • Zhang C.C.
      • Krieg S.
      • Shapiro D.J.
      ). The clinically important antiestrogen, tamoxifen, or its active metabolite, 4-hydroxytamoxifen (OHT), also induces cell death in ER-positive cell lines (
      • Coradini D.
      • Biffi A.
      • Cappelletti V.
      • Di Fronzo G.
      ,
      • Perry R.R.
      • Kang Y.
      • Greaves B.
      ,
      • Chen H.
      • Tritton T.R.
      • Kenny N.
      • Absher M.
      • Chiu J.-F.
      ,
      • Fattman C.L.
      • An B.
      • Sussman L.
      • Dou Q.P.
      ). While most studies of ER, and of the other steroid/nuclear receptors, have centered on their ability to act as ligand-regulated nuclear transcription factors, recent studies demonstrate that ER also exerts non-genomic effects (
      • Morey A.K.
      • Pedram A.
      • Razandi M.
      • Prins B.A.
      • Hu R-M.
      • Biesiada E.
      • Levin E.R.
      ,
      • Watters J.J.
      • Campbell J.S.
      • Cunningham M.J.
      • Krebs E.G.
      • Dorsa D.M.
      ,
      • Endoh H.
      • Sasaki H.
      • Maruyama K.
      • Takeyama K.
      • Waga I.
      • Shimizu T.
      • Kato S.
      • Kawashima H.
      ,
      • Razandi M.
      • Pedram A.
      • Greene G.L.
      • Levin E.R.
      ). Some non-genomic effects of ER may be related to its ability to activate signaling pathways, such as nitric-oxide synthase in endothelial cells (
      • Chen Z.
      • Yuhanna I.S.
      • Galcheva-Gargova Z.
      • Karas R.H.
      • Mendelsohn M.E.
      • Shaul P.W.
      ) and the p21 ras /MAP kinase pathway (
      • Migliaccio A.
      • Piccolo D.
      • Castoria G.
      • Di Domenico M.
      • Bilancio A.
      • Lombardi M.
      • Gong W.
      • Beato M.
      • Auricchio F.
      ).
      We recently described several lines of HeLa cells stably transfected to express ER. These HeLa-ER cell lines are killed by E2 or by OHT (
      • Zhang C.C.
      • Krieg S.
      • Shapiro D.J.
      ). In this work, we show that the specific inhibitor of the p38 MAP kinase pathway SB203580 (
      • Guenda A.
      • Rouse J.
      • Doza Y.N.
      • Meier R.
      • Cohen P.
      • Gallagher T.F.
      • Young P.R.
      • Lee J.C.
      ) partially protects the HeLa-ER cells against E2 or OHT-induced apoptosis. We show that E2 and OHT induce the p38 pathway. To analyze the role of the p38 pathway in E2- or OHT-induced apoptosis, we used human breast cancer cell lines and cell lines selected for resistance to killing by E2 or by OHT. In each of these cell lines, we observed an excellent correlation between the ability to activate the p38 pathway and the ability of E2 or OHT to induce apoptosis. Activation of the p38 signal transduction pathway represents a novel ER-dependent activity of E2 and OHT.

      ACKNOWLEDGEMENTS

      We are grateful to Dr. R. Davis for the gift of MKK3(Ala), to Dr. G. Greene for the gift of the D547 antibody, and to Dr. R. Dodson for many helpful comments on the manuscript.

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