Rho GTPases as Modulators of the Estrogen Receptor Transcriptional Response

The estrogen receptor α (ER) is a ligand-dependent transcription factor that plays a critical role in the development and progression of breast cancer, in part, by regulating target genes involved in cellular proliferation. To identify novel components that affect the ER transcriptional response, we performed a genetic screen in yeast and identified RDI1, a Rho guanine nucleotide dissociation inhibitor (Rho GDI), as a positive regulator of ER transactivation. Overexpression of the human homologue of RDI1, Rho GDIα, increases ERα, ERβ, androgen receptor, and glucocorticoid receptor transcriptional activation in mammalian cells but not activation by the unrelated transcription factors serum response factor and Sp1. In contrast, expression of constitutively active forms of RhoA, Rac1, and Cdc42 decrease ER transcriptional activity, suggesting that Rho GDI increases ER transactivation by antagonizing Rho function. Inhibition of RhoA by expression of either the Clostridium botulinum C3 transferase or a dominant negative RhoA resulted in enhanced ER transcriptional activation, thus phenocopying the effect of Rho GDI expression on ER transactivation. Together, these findings establish the Rho GTPases as important modulators of ER transcriptional activation. Since Rho GTPases regulate actin polymerization, our findings suggest a link between the major regulators of cellular architecture and steroid receptor transcriptional response. The estrogen receptor α (ER) is a ligand-dependent transcription factor that plays a critical role in the development and progression of breast cancer, in part, by regulating target genes involved in cellular proliferation. To identify novel components that affect the ER transcriptional response, we performed a genetic screen in yeast and identified RDI1, a Rho guanine nucleotide dissociation inhibitor (Rho GDI), as a positive regulator of ER transactivation. Overexpression of the human homologue of RDI1, Rho GDIα, increases ERα, ERβ, androgen receptor, and glucocorticoid receptor transcriptional activation in mammalian cells but not activation by the unrelated transcription factors serum response factor and Sp1. In contrast, expression of constitutively active forms of RhoA, Rac1, and Cdc42 decrease ER transcriptional activity, suggesting that Rho GDI increases ER transactivation by antagonizing Rho function. Inhibition of RhoA by expression of either the Clostridium botulinum C3 transferase or a dominant negative RhoA resulted in enhanced ER transcriptional activation, thus phenocopying the effect of Rho GDI expression on ER transactivation. Together, these findings establish the Rho GTPases as important modulators of ER transcriptional activation. Since Rho GTPases regulate actin polymerization, our findings suggest a link between the major regulators of cellular architecture and steroid receptor transcriptional response. estrogen receptor α estrogen receptor β guanine nucleotide dissociation inhibitor wild type guanine nucleotide exchange factor GTPase-activating protein glucocorticoid receptor androgen receptor serum response factor estrogen response element 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside relative luminescence units The estrogen receptor α (ER)1 is a ligand-dependent transcription factor that transduces the estrogen signal (1Parker M.G. Biochem. Soc. Symp. 1998; 63: 45-50PubMed Google Scholar). Activation of ER is responsible for female sexual development and maintenance of bone density (2Warner M. Nilsson S. Gustafsson J.A. Curr. Opin. Obstet. Gynecol. 1999; 11: 249-254Crossref PubMed Scopus (115) Google Scholar, 3Bland R. Clin. Sci. 2000; 98: 217-240Crossref PubMed Google Scholar). In addition, ER plays a critical role in the development and progression of breast cancer by regulating genes and signaling pathways involved in cellular proliferation (4Jordan V.C. J. Lab. Clin. Med. 1999; 133: 408-414Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Regulation of gene expression by the ER requires the coordinate activity of ligand binding, phosphorylation, and cofactor interactions, with particular combinations probably resulting in the tissue-specific responses elicited by the receptor (5Robyr D. Wolffe A.P. Wahli W. Mol. Endocrinol. 2000; 14: 329-347Crossref PubMed Scopus (325) Google Scholar, 6Schapira M. Raaka B.M. Samuels H.H. Abagyan R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1008-1013Crossref PubMed Scopus (114) Google Scholar, 7Weigel N.L. Biochem. J. 1996; 319: 657-667Crossref PubMed Scopus (321) Google Scholar). However, the extracellular cues and intracellular signaling pathways modulating these components and regulating ER transcriptional activation are not fully understood. To identify novel proteins that modulate ER transcriptional activation, we have carried out a genetic screen in the yeast Saccharomyces cerevisiae. The ability of the human ER to function within yeast allows a wide variety of genetic approaches to be taken toward further defining the mechanism of ER transcriptional activation given the ease of genetic manipulation and simplicity of gene identification in yeast. In addition, with the large number of orthologous proteins carrying out the same biological functions in both S. cerevisiae and metazoans (8Rubin G.M. Yandell M.D. Wortman J.R. Gabor Miklos G.L. Nelson C.R. Hariharan I.K. Fortini M.E. Li P.W. Apweiler R. Fleischmann W. Cherry J.M. Henikoff S. Skupski M.P. Misra S. Ashburner M. Birney E. Boguski M.S. Brody T. Brokstein P. Celniker S.E. Chervitz S.A. Coates D. Cravchik A. Gabrielian A. Galle R.F. Gelbart W.M. George R.A. Goldstein L.S. Gong F. Guan P. Harris N.L. Hay B.A. Hoskins R.A. Li J. Li Z. Hynes R.O. Jones S.J. Kuehl P.M. Lemaitre B. Littleton J.T. Morrison D.K. Mungall C. O'Farrell P.H. Pickeral O.K. Shue C. Vosshall L.B. Zhang J. Zhao Q. Zheng X.H. Zhong F. Zhong W. Gibbs R. Venter J.C. Adams M.D. Lewis S. Science. 2000; 287: 2204-2215Crossref PubMed Scopus (1362) Google Scholar, 9Chervitz S.A. Aravind L. Sherlock G. Ball C.A. Koonin E.V. Dwight S.S. Harris M.A. Dolinski K. Mohr S. Smith T. Weng S. Cherry J.M. Botstein D. Science. 1998; 282: 2022-2028Crossref PubMed Scopus (361) Google Scholar, 10Botstein D. Chervitz S.A. Cherry J.M. Science. 1997; 277: 1259-1260Crossref PubMed Scopus (288) Google Scholar), it is likely that the yeast factors affecting ER transactivation will have mammalian counterparts, which can be examined in vertebrate systems. The genetic approach we have used to identify factors that affect ER transcriptional activation is dosage suppression analysis. In this technique, a mutant ER protein with a reduced ability to activate transcription is used as a substrate to isolate yeast gene product(s) that are capable of overcoming this mutant phenotype, thus restoring receptor transcriptional activity. The mutant ER derivative used in this screen is defective in transactivation by virtue of serine to alanine mutations in the three major N-terminal phosphorylation sites, serines 104, 106, and 118 (ERAAA). This mutant was selected as the substrate because it has only a modest effect on ER transactivation and therefore has the potential to isolate a broad range of factors that affect receptor activity. We expect to isolate yeast factors that enhance ER transcriptional activity and, importantly, have human homologues that can then be examined in mammalian cells for effects on ER transcriptional response. This approach has proven successful for investigating various aspects of ER signal transduction (11Knoblauch R. Garabedian M.J. Mol. Cell. Biol. 1999; 19: 3748-3759Crossref PubMed Scopus (92) Google Scholar). Using this system, we have isolated RDI1, the yeast Rho guanine nucleotide dissociation inhibitor (Rho GDI), as a gene product that is capable of increasing both ERAAA and WT ER transcriptional activation when overexpressed. This gene product is the yeast homologue of the mammalian Rho GDIα, a cytoplasmic protein originally identified as a negative regulator of the Rho family of GTP-binding proteins (12Fukumoto Y. Kaibuchi K. Hori Y. Fujioka H. Araki S. Ueda T. Kikuchi A. Takai Y. Oncogene. 1990; 5: 1321-1328PubMed Google Scholar, 13Leonard D. Hart M.J. Platko J.V. Eva A. Henzel W. Evans T. Cerione R.A. J. Biol. Chem. 1992; 267: 22860-22868Abstract Full Text PDF PubMed Google Scholar, 14Masuda T. Tanaka K. Nonaka H. Yamochi W. Maeda A. Takai Y. J. Biol. 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This GTP/GDP cycle is tightly regulated in response to extracellular signals by three different classes of proteins. Guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP, GTPase-activating proteins (GAPs) accelerate the intrinsic GTPase activity of the Rho GTPases, and GDIs antagonize their activity by blocking GEFs and GAPs (12Fukumoto Y. Kaibuchi K. Hori Y. Fujioka H. Araki S. Ueda T. Kikuchi A. Takai Y. Oncogene. 1990; 5: 1321-1328PubMed Google Scholar,24Hart M.J. Maru Y. Leonard D. Witte O.N. Evans T. Cerione R.A. Science. 1992; 258: 812-815Crossref PubMed Scopus (122) Google Scholar). However, since the cytoplasmic GDP-bound Rho GTPases predominate Rho GDI as a negative regulator of Rho GTPases by blocking the dissociation of In addition, Rho GDI the of the GTPases, their the (12Fukumoto Y. Kaibuchi K. Hori Y. Fujioka H. Araki S. Ueda T. Kikuchi A. Takai Y. Oncogene. 1990; 5: 1321-1328PubMed Google Scholar, N. Cerione R.A. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). D. P. D. L. B. K. K. Gutkind S. J. Oncogene. 1998; PubMed Scopus Google identified which a to the Rho and was to a link between ER signaling and the Rho In this we the findings of D. P. D. L. B. K. K. Gutkind S. J. Oncogene. 1998; PubMed Scopus Google and the effects of human Rho as as the Rho GTPases, RhoA, Rac1, Cdc42, on transcriptional activation by ER and members of the steroid receptor family in mammalian findings that Rho increases the transcriptional activity of and as as the glucocorticoid receptor and androgen receptor but not of the unrelated transcription factors serum response factor and and that this activation is of Rho further establish the signaling as important regulator of and transcriptional activity. The a estrogen response element of a to the gene D. B. Garabedian M.J. M.G. S. Nature. 1990; 348: PubMed Scopus Google Scholar). The yeast was by J. J. Cell. 1990; Full Text PDF PubMed Scopus Google and was by yeast the of the WT ER and ERAAA the in M. D. PubMed Scopus Google Scholar). was by ERAAA J.M. Garabedian M.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google M. D. PubMed Scopus Google Scholar). The ER the of the to the G. D. J. 1996; 15: PubMed Scopus Google Scholar). The is to are for the G. D. J. 1996; 15: PubMed Scopus Google Scholar). The steroid receptor expression are ERα, B. K. E. J. Nilsson S. Gustafsson J.A. 1997; PubMed Scopus Google Scholar), D. J.A. Garabedian M.J. J. 1999; PubMed Scopus Google Scholar), and N.L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of human Rho Hall A. J. PubMed Scopus Google was to a Rho mammalian expression and M. for and have been Hall A. Cell. 1995; 81: Full Text PDF PubMed Scopus Google Scholar). was as C.S. Wynne J. Treisman R. 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Biol. 1999; 19: 3748-3759Crossref PubMed Scopus (92) Google Scholar). performed by and human breast cancer cell the and the human cancer cell was of in with serum of and and and cells in with serum and performed to the with and for and the ER S. R. M.G. J. Sci. PubMed Google or for cells in and in as the activity was in a serum G and as The steroid receptor transcriptional activity is to activity in the of steroid and to protein as by the protein Since cells transcriptional activity of the receptor is to activity in the of ER The the of with the range of the To protein cell for in for protein and for in by to and with GDI or The or and the substrate as the serine to alanine mutations N-terminal phosphorylation 104, 106, and 118 in a in ER transcriptional activity in mammalian cells P. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, S. D. J.M. P. J. PubMed Scopus Google Scholar). To the transcriptional activity of ERAAA is reduced in a expression either WT ER or ERAAA and The transcriptional of WT ER and ERAAA as a function of with WT ERAAA of transcriptional activity suggesting that the ERAAA is in the for transcriptional activation The ERAAA is these yeast WT ER are To screen for ER with with a yeast and for receptor transcriptional activation on the yeast to selected for further the which the of the yeast we identified yeast that enhance ER transcriptional activation genes that enhance ER on of ER ER ER ER GDP dissociation ER to Rho GTPase-activating ER the are yeast homologues of known mammalian regulators that affect ER function. the are factors that to link ER transcriptional activation to signal transduction pathways not known to affect ER function. in a the are yeast homologues of known mammalian regulators that affect ER function. the are factors that to link ER transcriptional activation to signal transduction pathways not known to affect ER function. of the yeast that of the yeast homologues of mammalian proteins to affect ER transactivation. include R. Mol. Biol. PubMed Scopus (92) Google Scholar), a homologue of the mammalian of that ER serine in H. Mol. Endocrinol. 1994; PubMed Scopus Google and A. J.A. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar), a of the transcription factor family that with ER in mammalian cells P. 1997; PubMed Scopus Google Scholar). In to genes known to regulate ER activity, genes not known to affect ER S. J. PubMed Scopus Google Scholar), a with to a protein that with the of the and increases transcriptional activation, was isolated U. H. O.A. Mol. Biol. Cell. 1998; PubMed Scopus Google Scholar). In addition, S.S. A.P. 133: PubMed Google Scholar), a protein with to S.E. M. J.R. PubMed Scopus Google Scholar), was identified We isolated T. Tanaka K. Nonaka H. Yamochi W. Maeda A. Takai Y. J. Biol. Chem. 1994; 269: 19713-19718Abstract Full Text PDF PubMed Google Scholar), the yeast Rho guanine nucleotide dissociation inhibitor (Rho GDI), three L. Xu G. R. W. 1994; PubMed Scopus Google Scholar), a yeast protein that a GTPase-activating protein is to it is to that and are both negative regulators of Rho The of known ER regulators with the of genes that the approach was and that the was probably to Since Rho GDI Rho GTPases, this that the Rho GTPases modulate ER transcriptional activation and is the of this the human Rho is to human Rho GDIα, which the best Rho GTPases, RhoA, Rac1, and To the mammalian Rho GDI ER transcription in mammalian we the ability of human Rho to enhance ER transcriptional activity in the human cell cells with ERα, with increasing of Rho in Rho ER transactivation in a of ER transcriptional activation by Rho was for ERAAA mutant To that this enhanced transcriptional activity was not a of ER protein we protein expression in cell analysis. that ER are not by Rho GDI the of ER with increasing Rho GDI that the effect of Rho on ER activity is that The effect of Rho GDI on ER transactivation is not to cell since Rho enhanced ER transactivation in and cells Rho can as a positive regulator of transcriptional activation in a variety of mammalian cell We the ability of Rho to affect transactivation by members of the steroid receptor ERβ, and that Rho the transcriptional activity of ERβ, and in a To Rho activation is to steroid we the effect of Rho on and transactivation. Rho GTPase signaling has been to enhance transcriptional activation by C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1202) Google we expect Rho GDIα, as a negative regulator of Rho GTPases, to decrease transcriptional activity. with this Rho expression activity a the element transcriptional activity in response to Rho GDI these suggest that Rho GDI increases transactivation by steroid a mechanism suppression of Rho GTPase GDI transcriptional activation by and Sp1. cells as in with of Rho with of or and for activity. in and The GTPases known to with Rho include RhoA, Rac1, and To Rho GDI increases ER transactivation by the Rho GTPases, we constitutively active forms of Rho GTPases and in cells and examined ER transcriptional activation. in expression of and ER transcriptional forms of Rho GTPases ER transactivation in and cells In three cell expression of the constitutively active forms of RhoA, Rac1, and Cdc42 resulted in of as by F. and M. J. are with the that Rho GDI ER transcriptional by antagonizing Rho Rho GTPases ER transcriptional activation in and and cells with the of the constitutively active forms of the Rho GTPases, and and ER transcriptional activity was as in the to is a performed in and three with of the effect of RhoA on ER transcriptional activation, we botulinum C3 a protein that and RhoA but not or Cdc42 S. A. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. M. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with Rho expression of C3 transferase in of ER transcriptional activity but transcriptional activation in cells and not Inhibition of ER transcriptional activation by RhoA, but not or Cdc42, was by C3 expression of C3 transferase ER transcriptional activation in and cells of RhoA by expression of a dominant negative of RhoA in ER transactivation of RhoA in enhanced ER transcriptional activation, that signaling events ER transactivation. We have that Rho the transcriptional activity of the as as ERβ, and but not or Sp1. We that mutant forms of RhoA, Rac1, and Cdc42 of RhoA by C3 transferase or dominant negative RhoA increases ER transcriptional activation. these we that the enhanced ER transactivation Rho is by of Rho GTPases and the Rho family proteins RhoA, Rac1, and Cdc42 in signaling to is the mechanism the of ER transactivation by Since Rho GTPases actin as as the activation of multiple signaling pathways, such as N-terminal and of ER either of these The ability of the Rho GTPase family members to ER transcriptional activity that and signaling to ER a signaling for such a regulator is S. Curr. Biol. 1999; Full Text Full Text PDF PubMed Google Scholar). The GTP-bound forms of RhoA and activate phosphorylation effector and K. K. M. T. S. K. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, G.M. Biol. 1999; PubMed Scopus Google Scholar). 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