Negative Feedback at the Level of Nuclear Receptor Coregulation

Nuclear receptor-mediated gene expression is proposed to be regulated by the ordered recruitment of large protein complexes in which activity depends on mutual interactions and posttranslational modifications. In contrast, relatively little attention has been given to mechanisms regulating the expression of the coregulator proteins themselves. Previously we have shown that the ligand-dependent corepressor, RIP140, is a direct transcriptional target of all-trans retinoic acid (RA). Here we demonstrate that RA induction of RIP140 constitutes a rate-limiting step in the regulation of retinoic acid receptor signaling. Silencing of the RA induction of RIP140 dramatically enhances and accelerates retinoid receptor transactivation, endogenous expression of other RA target genes, and RA-induced neuronal differentiation and cell cycle arrest in human embryonal carcinoma cells. The data suggest that RA induction of RIP140 constitutes a functional negative feedback loop that limits activation of retinoid receptors in the continued presence of RA and that acutely regulated expression of coregulators may be a general regulatory mechanism in hormonal signaling. Nuclear receptor-mediated gene expression is proposed to be regulated by the ordered recruitment of large protein complexes in which activity depends on mutual interactions and posttranslational modifications. In contrast, relatively little attention has been given to mechanisms regulating the expression of the coregulator proteins themselves. Previously we have shown that the ligand-dependent corepressor, RIP140, is a direct transcriptional target of all-trans retinoic acid (RA). Here we demonstrate that RA induction of RIP140 constitutes a rate-limiting step in the regulation of retinoic acid receptor signaling. Silencing of the RA induction of RIP140 dramatically enhances and accelerates retinoid receptor transactivation, endogenous expression of other RA target genes, and RA-induced neuronal differentiation and cell cycle arrest in human embryonal carcinoma cells. The data suggest that RA induction of RIP140 constitutes a functional negative feedback loop that limits activation of retinoid receptors in the continued presence of RA and that acutely regulated expression of coregulators may be a general regulatory mechanism in hormonal signaling. Retinoids are ligands for retinoid receptors, which are members of the steroid receptor superfamily (1Mangelsdorf D.J. Umesono K. Evans R.M. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. Raven Press, Ltd., New York1994: 319-349Google Scholar, 2Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2606) Google Scholar). All-trans retinoic acid (RA) 1The abbreviations used are: RA, all-trans retinoic acid; RARE, retinoic acid response element; RIP140, receptor-interacting protein 140; siRNA, small interfering RNA; hn-PCR, heteronuclear-PCR; RT-PCR, reverse transcriptase-PCR; FACS, fluorescence-activated cell sorter; N-CoR, nuclear receptor co-repressor; SMRT, silencing mediator for retinoid and thyroid hormone receptors; SRC-1, steroid receptor co-activator 1. regulates gene expression primarily through activation of retinoid receptors bound to consensus retinoic acid response elements (RAREs) (1Mangelsdorf D.J. Umesono K. Evans R.M. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. Raven Press, Ltd., New York1994: 319-349Google Scholar, 2Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2606) Google Scholar). Detailed molecular mechanisms have been elucidated whereby acetylation, methylation, and phosphorylation of specific histone tail residues by coregulatory complexes regulate the transcriptional activity of nuclear receptors (3Gamble M.J. Freedman L P. Trends Biochem. Sci. 2002; 27: 165-167Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 4McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Abstract Full Text Full Text PDF PubMed Scopus (1255) Google Scholar, 5Hermanson O. Glass C.K. Rosenfeld M.G. Trends Endocrinol. Metab. 2002; 13: 55-60Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Coregulators associated with histone acetylation/deacetylation (coactivators and corepressors) associate with nuclear receptors in a ligand-dependent fashion (3Gamble M.J. Freedman L P. Trends Biochem. Sci. 2002; 27: 165-167Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 4McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Abstract Full Text Full Text PDF PubMed Scopus (1255) Google Scholar, 5Hermanson O. Glass C.K. Rosenfeld M.G. Trends Endocrinol. Metab. 2002; 13: 55-60Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Corepressors N-CoR and SMRT, through direct interaction with the AF-2 domain of nuclear receptors, actively repress transcription in the absence of ligand (6Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1714) Google Scholar, 7Chen J.D. Evans R M. Nature. 1995; 377: 454-457Crossref PubMed Scopus (1715) Google Scholar). In the presence of hormone, corepressor complexes disassociate allowing interaction of coactivator complexes such as SRC-1/NCoA-1, TIF2/GRIP1/NCoA-2, and ACTR/pCIP/AIB1/RAC3 with the AF-2 domain of receptors (3Gamble M.J. Freedman L P. Trends Biochem. Sci. 2002; 27: 165-167Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 4McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Abstract Full Text Full Text PDF PubMed Scopus (1255) Google Scholar, 5Hermanson O. Glass C.K. Rosenfeld M.G. Trends Endocrinol. Metab. 2002; 13: 55-60Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). LXXLL motifs present in coactivators SRC-1, TIF2, and AIB1 are essential for hormone-dependent interaction with nuclear receptors (8Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1778) Google Scholar). Receptor-interacting protein 140 (RIP140) is one of the most enigmatic nuclear receptor coregulators (9Parker M.G. Leonardsson G. White R. Steel J. Milligan S. FEBS Lett. 2003; 546: 149-153Crossref PubMed Scopus (11) Google Scholar). RIP140 contains 10 LXXLL motifs and interacts preferentially with nuclear receptors in the presence of ligand (10Cavailles V. Dauvois S. L'Horset F. Lopez G. Hoare S. Kushner P.J. Parker M.G. EMBO J. 1995; 14: 3741-3751Crossref PubMed Scopus (673) Google Scholar). However, RIP140 inhibits the transactivation function of ligand bound receptors, including retinoid receptors (11Lee C.H. Wei L.N. J. Biol. Chem. 1999; 274: 31320-31326Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 12Treuter E. Albrektsen T. Johansson L. Leers J. Gustafsson J.A. Mol. Endocrinol. 1998; 12: 864-881Crossref PubMed Scopus (0) Google Scholar, 13Miyata K.S. McCaw S.E. Meertens L.M. Patel H.V. Rachubinski R.A. Capone J.P. Mol. Cell. Endocrinol. 1998; 146: 69-76Crossref PubMed Scopus (62) Google Scholar, 14Eng F.C. Barsalou A. Akutsu N. Mercier I. Zechel C. Mader S. White J.H. J. Biol. Chem. 1998; 273: 28371-28377Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 15Teyssier C. Belguise K. Galtier F. Cavailles V. Chalbos D. Mol. Endocrinol. 2003; 17: 287-299Crossref PubMed Scopus (33) Google Scholar, 16Wei L.N. Hu X. Chandra D. Seto E. Farooqui M. J. Biol. Chem. 2000; 275: 40782-40787Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 17Vo N. Fjeld C. Goodman R.H. Mol. Cell. Biol. 2001; 21: 6181-6188Crossref PubMed Scopus (130) Google Scholar). This property sets RIP140 apart from classic corepressors such as N-CoR and SMRT, which repress apo-receptors, as well as classic coactivators, such as SRC-1, which bind and activate holo-receptors (3Gamble M.J. Freedman L P. Trends Biochem. Sci. 2002; 27: 165-167Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 4McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Abstract Full Text Full Text PDF PubMed Scopus (1255) Google Scholar, 5Hermanson O. Glass C.K. Rosenfeld M.G. Trends Endocrinol. Metab. 2002; 13: 55-60Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). RIP140 has intrinsic repression activity when fused to the Gal4-DNA binding domain and directly recruits histone deacetylases and the transcriptional repressor, CtBP (C-terminal-binding protein), to nuclear receptors (16Wei L.N. Hu X. Chandra D. Seto E. Farooqui M. J. Biol. Chem. 2000; 275: 40782-40787Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 17Vo N. Fjeld C. Goodman R.H. Mol. Cell. Biol. 2001; 21: 6181-6188Crossref PubMed Scopus (130) Google Scholar). Thus RIP140 is predicted to suppress target gene expression of agonist-bound hormone receptors. Utilizing expression array profiling we discovered that RIP140 is itself an immediate RA target gene (18Freemantle S.J. Kerley J.S. Olsen S.L. Gross R.H. Spinella M.J. Oncogene. 2002; 21: 2880-2889Crossref PubMed Scopus (82) Google Scholar, 19Kerley J.S. Olsen S.L. Freemantle S.J. Spinella M.J. Biochem. Biophys. Res. Commun. 2001; 285: 969-975Crossref PubMed Scopus (48) Google Scholar). Induction of RIP140 mRNA occurs within 3 h and induction of its protein within 6 h (19Kerley J.S. Olsen S.L. Freemantle S.J. Spinella M.J. Biochem. Biophys. Res. Commun. 2001; 285: 969-975Crossref PubMed Scopus (48) Google Scholar). The RA induction of RIP140 mRNA does not require de novo protein synthesis, consistent with RIP140 being a direct transcriptional target of retinoid receptors (19Kerley J.S. Olsen S.L. Freemantle S.J. Spinella M.J. Biochem. Biophys. Res. Commun. 2001; 285: 969-975Crossref PubMed Scopus (48) Google Scholar). A promoter fragment of RIP140 contains a near consensus DR5 RARE and is activated by RA (19Kerley J.S. Olsen S.L. Freemantle S.J. Spinella M.J. Biochem. Biophys. Res. Commun. 2001; 285: 969-975Crossref PubMed Scopus (48) Google Scholar). The current study utilizes the embryonal carcinoma cell line NT2/D1 as a model of RA-induced differentiation (20Spinella M.J. Kerley J.S White K.A. Curtin J.C. J. Nutr. 2003; 133: 273S-276SCrossref PubMed Google Scholar) and proposes that RA induction of RIP140 serves as a negative feedback signal limiting RA activity at the level of nuclear receptor coregulation. Transient Transfection Reporter Assays—NT2/D1 is a clonal line derived from a xenograft of Tera-2 cells (21Andrews P.W. Acta Pathol. Microbiol. Immunol. Scand. 1998; 106: 158-168Crossref PubMed Scopus (122) Google Scholar). The reporters, RARE-TK-Luc and RARβ2-Luc were described previously (22Curtin J.C. Dragnev K.H. Sekula D. Christie A.J Dmitrovsky E. Spinella M.J. Oncogene. 2001; 20: 2559-2569Crossref PubMed Scopus (48) Google Scholar). 1.75 × 10 5 cells/well of a 6-well plate were transfected with 2.75 μg of DNA including 0.75 μg of reporter plasmid, 1.5 μg of expression plasmid, and 0.5 μg of a β-actin-β-galactosidase expression plasmid using a modified calcium phosphate precipitation technique (22Curtin J.C. Dragnev K.H. Sekula D. Christie A.J Dmitrovsky E. Spinella M.J. Oncogene. 2001; 20: 2559-2569Crossref PubMed Scopus (48) Google Scholar). Cells were exposed to precipitates for 14–16 h and then washed and cultured with or without 1 μm RA for an additional 48 h before harvesting. For siRNA, 0.75 μg of RARE-TK-Luc, 0.5 μg of β-actin-β-galactosidase expression plasmid, and indicated amounts of siRNA were co-transfected using Polyfect (Qiagen). Luciferase activities were measured and normalized to β-galactosidase activity as described (22Curtin J.C. Dragnev K.H. Sekula D. Christie A.J Dmitrovsky E. Spinella M.J. Oncogene. 2001; 20: 2559-2569Crossref PubMed Scopus (48) Google Scholar). Data points represent the average of triplicate transfections with similar results obtained in at least three independent experiments. SiRNA—The sequence of the custom siRNA duplex for RIP140 corresponds to the human RIP140 cDNA starting 331 bp downstream of the ATG start codon (Dharmacon). The siRNA duplex control used was the Scramble II sequence from Dharmacon. Transfection of siRNA was performed with OligofectAMINE Reagent (Invitrogen) according to the manufacturer's instructions. The final concentration of siRNA used in each experiment was ∼150 nm. In independent experiments, transfection efficiency was assessed to be greater than 90% using fluorescein-labeled siRNA (Dharmacon). Immunophenotypic and Cell Cycle Analysis—Indirect fluorescence-activated cell sorter (FACS) analysis to evaluate RA-induced neuronal differentiation of NT2/D1 cells was performed using established techniques (23Spinella M.J. Freemantle S.J. Sekula D. Chang J.H. Christie A.J. Dmitrovsky E. J. Biol. Chem. 1999; 274: 22013-22018Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Briefly, NT2/D1 cells were incubated with a monoclonal antibody to the cell surface antigen A2B5 or an isotype-matched monoclonal control antibody. Cells were assayed indirectly with fluorescein isothiocyanate-conjugated goat-anti-mouse antibody, and fluorescence was measured as described (23Spinella M.J. Freemantle S.J. Sekula D. Chang J.H. Christie A.J. Dmitrovsky E. J. Biol. Chem. 1999; 274: 22013-22018Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Mean peak fluorescent values and the percentage of positive cells were measured. For cell cycle phase analysis, cells were fixed with 70% ethanol and stained with propidium iodide for 16 h. The percentages of cells in G1, S, and G2/M were then determined on a BD Biosciences FACscan flow cytometer using Modfit LT software and established techniques (23Spinella M.J. Freemantle S.J. Sekula D. Chang J.H. Christie A.J. Dmitrovsky E. J. Biol. Chem. 1999; 274: 22013-22018Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Northern, RT-PCR, and Heteronuclear-PCR (hn-PCR) Analyses— Total RNA was isolated using TriReagent (Invitrogen). Northern hybridizations were performed as described previously (19Kerley J.S. Olsen S.L. Freemantle S.J. Spinella M.J. Biochem. Biophys. Res. Commun. 2001; 285: 969-975Crossref PubMed Scopus (48) Google Scholar). Expression levels of RA targets genes were also measured by semiquantitative RT-PCR. The cDNA was synthesized from 5 μg total RNA using Superscript II reverse transcriptase (Invitrogen), as described previously (18Freemantle S.J. Kerley J.S. Olsen S.L. Gross R.H. Spinella M.J. Oncogene. 2002; 21: 2880-2889Crossref PubMed Scopus (82) Google Scholar, 22Curtin J.C. Dragnev K.H. Sekula D. Christie A.J Dmitrovsky E. Spinella M.J. Oncogene. 2001; 20: 2559-2569Crossref PubMed Scopus (48) Google Scholar). Amplification of genes and β-actin was performed using Taq polymerase as described (18Freemantle S.J. Kerley J.S. Olsen S.L. Gross R.H. Spinella M.J. Oncogene. 2002; 21: 2880-2889Crossref PubMed Scopus (82) Google Scholar, 22Curtin J.C. Dragnev K.H. Sekula D. Christie A.J Dmitrovsky E. Spinella M.J. Oncogene. 2001; 20: 2559-2569Crossref PubMed Scopus (48) Google Scholar). Experiments were performed to determine the optimal number of cycles that would yield linear phase of amplification, and reverse transcriptase was omitted to control for genomic DNA contamination. For certain products, detection in the linear range required transfer to nitrocellulose and probing with a radiolabeled internal primer. The sequence of PCR primers is available upon request. To detect primary, pre-spliced transcripts, hn-PCR was performed. Total RNA was first treated with with the from was then performed for cycles as described with primers an of human and human RIP140 a of neuronal differentiation and cell cycle arrest in NT2/D1 cells (21Andrews P.W. Acta Pathol. Microbiol. Immunol. Scand. 1998; 106: 158-168Crossref PubMed Scopus (122) Google Scholar, M.J. Freemantle S.J. Sekula D. Chang J.H. Christie A.J. Dmitrovsky E. J. Biol. Chem. 1999; 274: 22013-22018Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). RIP140 as acutely upon RA of NT2/D1 cells (18Freemantle S.J. Kerley J.S. Olsen S.L. Gross R.H. Spinella M.J. Oncogene. 2002; 21: 2880-2889Crossref PubMed Scopus (82) Google Scholar, 19Kerley J.S. Olsen S.L. Freemantle S.J. Spinella M.J. Biochem. Biophys. Res. Commun. 2001; 285: 969-975Crossref PubMed Scopus (48) Google Scholar). RIP140 is a direct transcriptional target of retinoid receptors on induction of RIP140 with RA (18Freemantle S.J. Kerley J.S. Olsen S.L. Gross R.H. Spinella M.J. Oncogene. 2002; 21: 2880-2889Crossref PubMed Scopus (82) Google Scholar, 19Kerley J.S. Olsen S.L. Freemantle S.J. Spinella M.J. Biochem. Biophys. Res. Commun. 2001; 285: 969-975Crossref PubMed Scopus (48) Google Scholar). has been that RIP140 repress retinoid receptors (11Lee C.H. Wei L.N. J. Biol. Chem. 1999; 274: 31320-31326Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 12Treuter E. Albrektsen T. Johansson L. Leers J. Gustafsson J.A. Mol. Endocrinol. 1998; 12: 864-881Crossref PubMed Scopus (0) Google Scholar, 13Miyata K.S. McCaw S.E. Meertens L.M. Patel H.V. Rachubinski R.A. Capone J.P. Mol. Cell. Endocrinol. 1998; 146: 69-76Crossref PubMed Scopus (62) Google Scholar, 14Eng F.C. Barsalou A. Akutsu N. Mercier I. Zechel C. Mader S. White J.H. J. Biol. Chem. 1998; 273: 28371-28377Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 15Teyssier C. Belguise K. Galtier F. Cavailles V. Chalbos D. Mol. Endocrinol. 2003; 17: 287-299Crossref PubMed Scopus (33) Google Scholar, 16Wei L.N. Hu X. Chandra D. Seto E. Farooqui M. J. Biol. Chem. 2000; 275: 40782-40787Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 17Vo N. Fjeld C. Goodman R.H. Mol. Cell. Biol. 2001; 21: 6181-6188Crossref PubMed Scopus (130) Google the to which RA induction of RIP140 RA was of RIP140 a to in a RIP140 transfection on and control the of RIP140 in the presence of a property not with classic corepressors such as or N-CoR which preferentially bind and repress (3Gamble M.J. Freedman L P. Trends Biochem. Sci. 2002; 27: 165-167Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 4McKenna N.J. O'Malley B.W. Cell. 2002; 108: 465-474Abstract Full Text Full Text PDF PubMed Scopus (1255) Google Scholar, 5Hermanson O. Glass C.K. Rosenfeld M.G. Trends Endocrinol. Metab. 2002; 13: 55-60Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). are to such as siRNA was to repress RIP140 RIP140 siRNA not a control siRNA and RA-induced expression of RIP140 mRNA RIP140 siRNA dramatically to RARE-TK-Luc activity in a RIP140 siRNA on RARE activity and control siRNA to RARE activity RIP140 siRNA also to and control RIP140 and RIP140 siRNA RARE reporter activity in cells not data that RIP140, in a rate-limiting of RA-induced Expression of RIP140 Expression of RA in RIP140 siRNA not control siRNA the endogenous expression of the RA target genes and in NT2/D1 cells (18Freemantle S.J. Kerley J.S. Olsen S.L. Gross R.H. Spinella M.J. Oncogene. 2002; 21: 2880-2889Crossref PubMed Scopus (82) Google Scholar). RIP140 siRNA to the expression of the nuclear receptor coregulators and N-CoR, which as not expression of and in response to RIP140 was at h of RA which is within the of RIP140 protein induction with RA (19Kerley J.S. Olsen S.L. Freemantle S.J. Spinella M.J. Biochem. Biophys. Res. Commun. 2001; 285: 969-975Crossref PubMed Scopus (48) Google Scholar). in target gene expression of was at h of RA in RIP140 siRNA cells as with cells treated with control A similar of expression in response to RIP140 siRNA was for the RA target gene not The of RIP140 expression on RA target genes was which a for RIP140 as a ligand-dependent corepressor on of in This a negative feedback model by which RA induction of RIP140 inhibits retinoid receptor activity in the continued presence of have shown that receptor target gene expression is in the presence of ligand D. Evans R.M. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Hu X. J. M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). For target genes, peak transcription occurs within h in the presence of the h continued D. Evans R.M. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). To RA target genes demonstrate a similar of expression and the of RIP140 on RA target gene the expression of the RA target genes and was measured in the presence of RA in cells. in the of total levels of and mRNA in the continued presence of RA, and was by RIP140 The of and expression was then measured by In control cells the of transcription of and at 1 and 6 h of RA and then the of the peak transcription was by RIP140 siRNA, the was an of the in transcription which in control cells at the In RIP140 siRNA the for at and h of RA the at 6 h was present that other mechanisms of independent of RIP140 at for In RIP140 siRNA cells the transcription for continued to through and h of RA data that the greater of total RA target gene mRNA by RIP140 siRNA is not primarily to an in transcription is to a of peak transcription data that the retinoid receptors also activity in the presence of and a for RIP140 in of RA-induced Expression of RIP140 and Cell Cycle data that RA induction of RIP140 retinoid receptor transactivation and RA target gene The of RIP140 expression on RA-induced differentiation and arrest were RIP140 siRNA not a control siRNA the of RA-induced neuronal differentiation as assayed by the A2B5 A and have previously that 1 μm RA expression of A2B5 within 5 (23Spinella M.J. Freemantle S.J. Sekula D. Chang J.H. Christie A.J. Dmitrovsky E. J. Biol. Chem. 1999; 274: 22013-22018Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). and 3 of RA control siRNA cells were In contrast, RA-induced A2B5 expression was in RIP140 siRNA cells within The of RIP140 siRNA was at 3 of RA values for fluorescence and positive cells were and for RIP140 cells and for control siRNA of expression of the cell was upon RA of RIP140 cells as with not cell cycle arrest is associated with RA-induced differentiation of NT2/D1 cells (23Spinella M.J. Freemantle S.J. Sekula D. Chang J.H. Christie A.J. Dmitrovsky E. J. Biol. Chem. 1999; 274: 22013-22018Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). RIP140 siRNA also RA-induced arrest in NT2/D1 cells at and 3 of RA as with data that RIP140 is rate-limiting for ligand-dependent receptor transactivation, endogenous RA target gene and RA-induced differentiation in NT2/D1 cells. have been proposed to the of transcriptional activation by nuclear receptors. D. Evans R.M. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) that the of receptor target gene expression in response to and h in the presence of This with of co-activator complexes on target gene D. Evans R.M. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Hu X. J. M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). A mechanism proposed for of the of thyroid and retinoic acid which to of the coactivator D. Evans R.M. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus is one mechanism for of nuclear receptor signaling. In contrast, relatively little attention has been given to transcriptional regulation of on the current we that an additional mechanism for nuclear receptors is negative feedback In activated retinoid receptors the of a nuclear receptor corepressor, RIP140 inhibits retinoid receptors in a ligand-dependent fashion is a of RIP140 ligand-dependent RARE promoter of RA induction of RIP140 siRNA dramatically RARE activity and levels of RA target gene RIP140 silencing also RA-induced differentiation and cell cycle This the that RIP140 be a rate-limiting in regulating nuclear receptor RIP140 is and agonist-bound nuclear receptors (9Parker M.G. Leonardsson G. White R. Steel J. Milligan S. FEBS Lett. 2003; 546: 149-153Crossref PubMed Scopus (11) Google Scholar, C.H. C. Wei L.N. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). RIP140 has been to be by S. S. Cavailles V. Mol. Cell. Endocrinol. 1999; PubMed Scopus (82) Google Scholar). be to the regulating the expression of RIP140 and other RIP140, as and by in interaction and transactivation may a number of agonist-bound nuclear receptors, the that RIP140 may in nuclear receptor and to the of hormonal on analysis of the of RIP140 nuclear receptors is the RIP140 and other coregulators in receptor regulation is RIP140 and have been to be at least in binding nuclear receptors E. Albrektsen T. Johansson L. Leers J. Gustafsson J.A. Mol. Endocrinol. 1998; 12: 864-881Crossref PubMed Scopus (0) Google Scholar, 15Teyssier C. Belguise K. Galtier F. Cavailles V. Chalbos D. Mol. Endocrinol. 2003; 17: 287-299Crossref PubMed Scopus (33) Google Scholar, N. E. S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). the RNA and the protein were shown to repress nuclear receptors in a ligand-dependent that of be in cells and J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, I. Y. T. K. R. O. F. N. Mader S. X. White J.H. Mol. Cell. 2000; Full Text Full Text PDF Scopus Google Scholar). In data a model of regulatory control negative feedback by RA induction of a ligand-dependent have shown that of the coregulator RIP140, is itself a direct target of This is of that coregulators are as for RIP140, to be a rate-limiting of nuclear receptor we have that RIP140 has in retinoid receptor activity and on the of to signal and be required to the and of of