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Selective Activation of the Glucocorticoid Receptor by Steroid Antagonists in Human Breast Cancer and Osteosarcoma Cells*

  • Christy J. Fryer
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  • H.Karimi Kinyamu
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  • Inez Rogatsky
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  • Michael J. Garabedian
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  • Trevor K. Archer
    Correspondence
    To whom correspondence should be addressed: Chromatin and Gene Expression Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, 111 Alexander Dr., P. O. Box 12233 (MD E4-06), Research Triangle Park, NC 27709. Tel.: 919-316-4565; Fax: 919-316-4566
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  • Author Footnotes
    * This work was supported in part by grants from the National Cancer Institute of Canada (to T. K. A.) and Canadian Breast Cancer Research Initiative of Canada.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.
    § Recipient of a Medical Research of Canada Studentship award. Present address: The Salk Inst., Regulatory Biology Laboratory, La Jolla, CA, 92037-1099.
    ** Recipient of a Parker B. Francis Fellowship for Pulmonary Research. Present address, Dept. of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143.
Open AccessPublished:June 09, 2000DOI:https://doi.org/10.1074/jbc.M908729199
      Steroid hormones regulate the transcription of numerous genes via high affinity receptors that act in concert with chromatin remodeling complexes, coactivators and corepressors. We have compared the activities of a variety of glucocorticoid receptor (GR) antagonists in breast cancer and osteosarcoma cell lines engineered to stably maintain the mouse mammary tumor virus promoter. In both cell types, GR activation by dexamethasone occurs via the disruption of mouse mammary tumor virus chromatin structure and the recruitment of receptor coactivator proteins. However, when challenged with a variety of antagonists the GR displays differential ability to activate transcription within the two cell types. For the breast cancer cells, the antagonists fail to activate the promoter and do not promote the association of the GR with either remodeling or coactivator proteins. In contrast, in osteosarcoma cells, the antiglucocorticoids, RU486 and RU43044, exhibit partial agonist activity. The capacity of these antagonists to stimulate transcription in the osteosarcoma cells is reflected in the ability of the RU486-bound receptor to remodel chromatin and associate with chromatin-remodeling proteins. Similarly, the observation that the RU486-bound receptor does not fully activate transcription is consistent with its inability to recruit receptor coactivator proteins.
      SR
      steroid receptors
      GR
      glucocorticoid receptor
      PR
      progesterone receptor
      MMTV
      mouse mammary tumor virus
      PAGE
      polyacrylamide gel electrophoresis
      Dex
      dexamethasone
      NF1
      nuclear factor 1
      BAFs
      hBRG1-associated factors
      Clinically, steroids and steroid antagonists are widely used in the treatment of endocrine disorders, cardiovascular disease, and cancers, including those of the breast and ovary (
      • Fuhrmann U.
      • Parczyk K.
      • Klotzbucher M.
      • Klocker H.
      • Cato A.C.
      ). Steroids regulate growth and development through binding to a superfamily of high affinity steroid receptors (SR)1 that regulate the transcription of target genes (
      • Mangelsdorf D.J.
      • Thummel C.
      • Beato M.
      • Herrlich P.
      • Schutz G.
      • Umesono K.
      • Blumberg B.
      • Kastner P.
      • Mark M.
      • Chambon P.
      • Evans R.M.
      ). Upon binding ligand the SR undergoes a conformational change that allows the receptor to dimerize and bind to the hormone response element within target genes (
      • Mangelsdorf D.J.
      • Thummel C.
      • Beato M.
      • Herrlich P.
      • Schutz G.
      • Umesono K.
      • Blumberg B.
      • Kastner P.
      • Mark M.
      • Chambon P.
      • Evans R.M.
      ). This multistep pathway, which ultimately results in changes in gene transcription, can be manipulated pharmacologically by steroid hormone antagonists. In the case of the glucocorticoid receptor (GR), antiglucocorticoids have been shown to either block the capacity of the GR to interact with the hormone response element or interfere with the subsequent processes linked to transcriptional activation (
      • Mymryk J.S.
      • Archer T.K.
      ,
      • Fryer C.J.
      • Archer T.K.
      ).
      Differences in the activity of hormones and antihormones may reflect the ability with which the cellular transcriptional machinery discriminates between structurally distinct receptor conformations at the carboxyl-terminal transcriptional activation domain (
      • Brzozowski A.M.
      • Pike A.C.
      • Dauter Z.
      • Hubbard R.E.
      • Bonn T.
      • Engstrom O.
      • Ohman L.
      • Greene G.L.
      • Gustafsson J.A.
      • Carlquist M.
      ,
      • Nichols M.
      • Rientjes J.M.
      • Stewart A.F.
      ,
      • Shiau A.K.
      • Barstad D.
      • Loria P.M.
      • Cheng L.
      • Kushner P.J.
      • Agard D.A.
      • Greene G.L.
      ). This region of the receptor is thought to form a protein interaction surface for steroid receptor coactivators (
      • Darimont B.D.
      • Wagner R.L.
      • Apriletti J.W.
      • Stallcup M.R.
      • Kushner P.J.
      • Baxter J.D.
      • Fletterick R.J.
      • Yamamoto K.R.
      ,
      • vom Baur E.
      • Zechel C.
      • Heery D.
      • Heine M.J.
      • Garnier J.M.
      • Vivat V.
      • Le Douarin B.
      • Gronemeyer H.
      • Chambon P.
      • Losson R.
      ). Accordingly, interactions between steroid antagonists and the receptor would block the coactivator recruitment site, and this may then prevent transactivation.
      Steroid antagonists exhibit a range of activity between “pure” antagonists that efficiently antagonize receptor function and “mixed” antagonists that may selectively stimulate receptor action depending on cell type and/or promoter context (
      • Nordeen S.K.
      • Bona B.J.
      • Moyer M.L.
      ,
      • Sartorius C.A.
      • Tung L.
      • Takimoto G.S.
      • Horwitz K.B.
      ). For example, with the estrogen receptor, ICI 164 384 is a pure antiestrogen, andtrans-4-hydroxytamoxifen is a mixed antagonist (
      • Dhingra K.
      ). Similarly, a class of progesterone receptor (PR) antagonists have been shown to function as mixed agonists (
      • Meyer M.E.
      • Pornon A.
      • Ji J.W.
      • Bocquel M.T.
      • Chambon P.
      • Gronemeyer H.
      ). These include compounds such as RU486 that induce a conformational change in the PR that is different from that induced by agonists such as progesterone or antagonists such as ZK98299 (
      • Meyer M.E.
      • Pornon A.
      • Ji J.W.
      • Bocquel M.T.
      • Chambon P.
      • Gronemeyer H.
      ,
      • Allan G.F.
      • Lombardi E.
      • Haynes-Johnson D.
      • Palmer S.
      • Kiddoe M.
      • Kraft P.
      • Campen C.
      • Rybczynski P.
      • Combs D.W.
      • Phillips A.
      ). Other studies suggest that the extreme carboxyl terminus of the PR may bind a corepressor that normally suppresses RU486 agonist activity resulting in partial agonist activity (
      • Xu J.
      • Nawaz Z.
      • Tsai S.Y.
      • Tsai M.J.
      • O'Malley B.W.
      ). Alternatively, the partial agonist activity of RU486 is enhanced in the presence of 8-bromo-cyclic AMP (
      • Nordeen S.K.
      • Bona B.J.
      • Moyer M.L.
      ,
      • Sartorius C.A.
      • Tung L.
      • Takimoto G.S.
      • Horwitz K.B.
      ). Thus multiple factors including receptor structure, corepressor expression, and activation of protein kinase A signaling may influence the transactivation properties of an antagonist-bound receptor.
      Transactivation of steroid-responsive genes may also be regulated by the chromatin structure of the promoter (
      • Archer T.K.
      • Deroo B.J.
      • Fryer C.J.
      ,
      • Wolffe A.P.
      ). In eukaryotic cells the DNA is intimately associated with histone and non-histone proteins, and the architecture of the DNA as chromatin can modulate gene expression (
      • Hayes J.J.
      • Wolffe A.P.
      ). In general, the packaging of DNA into nucleosomes prevents the access of transcription factors to their binding sites and has a repressive effect on transcription (
      • Hayes J.J.
      • Wolffe A.P.
      ,
      • Workman J.L.
      • Buchman A.R.
      ). Indeed, hormone-induced chromatin remodeling is a hallmark of activated transcription for steroid-regulated genes (
      • Wolffe A.P.
      ). The mouse mammary tumor virus (MMTV) promoter is a well defined model system to study glucocorticoid receptor activation of transcription (
      • Archer T.K.
      • Fryer C.J.
      • Lee H.L.
      • Zaniewski E.
      • Liang T.
      • Mymryk J.S.
      ). When stably introduced into cells, the MMTV promoter reproducibly acquires a phased array of nucleosomes. Glucocorticoid treatment induces remodeling of the chromatin, the binding of transcription factors, and concomitant transcriptional activation (
      • Richard-Foy H.
      • Hager G.L.
      ,
      • Cordingley M.G.
      • Riegel A.T.
      • Hager G.L.
      ).
      In this study we examined the ability of antiglucocorticoids to induce transcription, chromatin remodeling, and transcription factor binding at the MMTV promoter in breast cancer and osteosarcoma cell lines. The antiglucocorticoids ORG31710 (Org) and ZK98299 (ZK98) displayed no agonist activity in either breast or osteosarcoma cells. In contrast, RU486 and RU43044 (RU43) exhibited partial agonist activity in osteosarcoma cells but not in breast cancer cells. This partial agonist activity of RU486 was consistent with its ability to induce GR-mediated chromatin remodeling and transcription factor loading and reflected the capacity of the RU486-bound receptor to recruit components of the chromatin remodeling complex to the promoter in osteosarcoma cells.

      DISCUSSION

      Steroid receptor antagonists have been invaluable tools in the dissection of the molecular mechanisms underlying steroid receptor activation of transcription (
      • Archer T.K.
      • Deroo B.J.
      • Fryer C.J.
      ). The majority of studies examining the mixed antagonist/agonist activity of antihormones have been done with antiestrogens and antiprogestins (
      • Dhingra K.
      ,
      • Horwitz K.B.
      ,
      • Cadepond F.
      • Ulmann A.
      • Baulieu E.E.
      ). In case of estrogen receptor antagonists, some of the compounds have partial agonist effects on the skeletal system and have been used as therapeutic agents in the treatment of bone loss (
      • Love R.R.
      • Mazess R.B.
      • Barden H.S.
      • Epstein S.
      • Newcomb P.A.
      • Jordan V.C.
      • Carbone P.P.
      • DeMets D.L.
      ). In contrast to the protective effects of estrogens on bone, long term use of glucocorticoids in vivo induces bone loss (
      • Lukert B.P.
      • Raisz L.G.
      ). A comparison between the effects of antiglucocorticoids on gene regulation in breast cancer cells and bone-derived osteosarcoma cells would give us an insight on the differences in regulatory mechanisms in the two cell types.
      In this study the antiglucocorticoids RU486 and RU43044 exerted significant agonist activity and activated MMTV-Luc transcription in osteosarcoma cells but not human breast cancer cells. In mouse breast cancer cells, although the GR/type II antagonist has been shown to bind to DNA, it was unable to activate transcription (
      • Mymryk J.S.
      • Archer T.K.
      ). Similar results have been observed with the antiestrogen tamoxifen that exhibits agonist activity in a tissue-dependent manner (
      • Curtis R.E.
      • Boice Jr., J.D.
      • Shriner D.A.
      • Hankey B.F.
      • Fraumeni Jr., J.F.
      ,
      • Rutqvist L.E.
      • Johansson H.
      • Signomklao T.
      • Johansson U.
      • Fornander T.
      • Wilking N.
      ). More directly, we show that the RU486-bound GR can effectively induce the chromatin transition necessary to allow the binding of NF1 to activate the MMTV promoter in osteosarcoma cells. Consistent with the ability to induce chromatin remodeling at Nuc-B, we detected an association of the GR with the hBRG1 complex in the presence of either RU486 or Dex. Although the precise mechanism for this interaction in UL3 cells is unknown, we did detect substantially higher levels of BAF 250, 170, and 155 along with the elevated levels of the GR in the UL3 cells relative to A1-2 cells.
      Although the chromatin transition observed in the presence of either Dex or RU486 was similar in UL3 cells, the resulting transcriptional activation induced by RU486 was at a reduced level in UL3 compared with that induced by Dex. One explanation for this substantially lower activation was provided by an examination of GR association with the coactivators SRC-1/NCoA1, CBP, and p/CIP. Treatment of osteosarcoma cells with Dex resulted in the coimmunoprecipitation of GR and these coactivators. In contrast, RU486 did not promote GR association with any of the coactivators examined. Similarly, in the breast cancer cells none of the antagonists promoted the association with SRC-1/NCoA1, CBP, or p/CIP. The capability of hBRG1, but not coactivators, to associate with the RU486-bound GR provides an explanation for the partial transcriptional activation observed with the antiglucocorticoids in UL3 cells.
      Previous studies revealed a requirement for the hBRG1 chromatin remodeling complex for glucocorticoid-dependent activation of MMTV (
      • Fryer C.J.
      • Archer T.K.
      ). Association of the GR with coactivators in the absence of hBRG1 complex failed to activate transcription from chromatin templates (
      • Fryer C.J.
      • Archer T.K.
      ). The present studies demonstrate that in osteosarcoma cells RU486 induces GR association with hBRG1 to partially activate MMTV transcription. This is consistent with an important role for chromatin remodeling in the activation of transcription, suggesting that there may be an intrinsic transcriptional activation potential in the remodeling process independent of the coactivators (
      • Archer T.K.
      • Deroo B.J.
      • Fryer C.J.
      ). However, this activation by RU486 is only ∼20% that seen with a true agonist, implying that additional coactivators make significant contributions to the fully active GR at genes assembled as chromatin. Given the extensive clinical use of hormone antagonist in the treatment of breast cancer and other endocrine diseases, the capacity to predict if an individual antihormone will block the activity of a specific receptor, in one cell context but not another, may be of significant clinical importance.

      Acknowledgments

      RU486386 and RU43044 were generously provided by Dr. D. Philibert of Roussel, UCLAF (Romainville, France); ZK112993 and ZK9899 were generously provided by Dr. D. Henderson of Schering AG (Berlin, Germany), and Org31710 was generously supplied by Dr. H. J. Kloosterboer of N. V. Organon (Oss, Netherlands). We are especially grateful to Dr. B. Gametchu for the BUGR2 antibody to the GR; Dr. W. Wang for antibodies to BAF 250, 170, 155, and 60a; Drs. I. Imbalzano, G. Schnitzler, and R. Kingston for providing the antibody to hBRG1; and Dr. J. Torchia for the antibodies to NCoA1 and p/CIP.

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