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• W2078862807 abstract "Both the vitamin D receptor (VDR) and hairless (hr) genes play a role in the mammalian hair cycle, as inactivating mutations in either result in total alopecia. VDR is a nuclear receptor that functions as a ligand-activated transcription factor, whereas the hairless gene product (Hr) acts as a corepressor of both the thyroid hormone receptor (TR) and the orphan nuclear receptor, RORα. In the present study, we show that VDR-mediated transactivation is strikingly inhibited by coexpression of rat Hr. The repressive effect of Hr is observed on both synthetic and naturally occurring VDR-responsive promoters and also when VDR-mediated transactivation is augmented by overexpression of its heterodimeric partner, retinoid X receptor. Utilizing in vitro pull down methods, we find that Hr binds directly to VDR but insignificantly to nuclear receptors that are not functionally repressed by Hr. Coimmunoprecipitation data demonstrate that Hr and VDR associate in a cellular milieu, suggesting in vivo interaction. The Hr contact site in human VDR is localized to the central portion of the ligand binding domain, a known corepressor docking region in other nuclear receptors separate from the activation function-2 domain. Coimmunoprecipitation and functional studies of Hr deletants reveal that VDR contacts a C-terminal region of Hr that includes motifs required for TR and RORα binding. Finally, in situ hybridization analysis of hr and VDR mRNAs in mouse skin demonstrates colocalization in cells of the hair follicle, consistent with a hypothesized intracellular interaction between these proteins to repress VDR target gene expression, in vivo. Both the vitamin D receptor (VDR) and hairless (hr) genes play a role in the mammalian hair cycle, as inactivating mutations in either result in total alopecia. VDR is a nuclear receptor that functions as a ligand-activated transcription factor, whereas the hairless gene product (Hr) acts as a corepressor of both the thyroid hormone receptor (TR) and the orphan nuclear receptor, RORα. In the present study, we show that VDR-mediated transactivation is strikingly inhibited by coexpression of rat Hr. The repressive effect of Hr is observed on both synthetic and naturally occurring VDR-responsive promoters and also when VDR-mediated transactivation is augmented by overexpression of its heterodimeric partner, retinoid X receptor. Utilizing in vitro pull down methods, we find that Hr binds directly to VDR but insignificantly to nuclear receptors that are not functionally repressed by Hr. Coimmunoprecipitation data demonstrate that Hr and VDR associate in a cellular milieu, suggesting in vivo interaction. The Hr contact site in human VDR is localized to the central portion of the ligand binding domain, a known corepressor docking region in other nuclear receptors separate from the activation function-2 domain. Coimmunoprecipitation and functional studies of Hr deletants reveal that VDR contacts a C-terminal region of Hr that includes motifs required for TR and RORα binding. Finally, in situ hybridization analysis of hr and VDR mRNAs in mouse skin demonstrates colocalization in cells of the hair follicle, consistent with a hypothesized intracellular interaction between these proteins to repress VDR target gene expression, in vivo. Nuclear receptors comprise a family of ligand-activated transcription factors that coordinate physiological and developmental processes by regulating specific changes in gene expression (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6002) Google Scholar, 2Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2805) Google Scholar). The vitamin D receptor (VDR) 1The abbreviations used are: VDR, vitamin D receptor; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; RXR, retinoid X receptor; VDRE, vitamin D-responsive element; CYP24, vitamin D 24-hydroxylase; N-CoR, nuclear receptor corepressor; SMRT, silencing mediator for retinoic acid and thyroid hormone receptors; hr, hairless; Hr, hairless gene product; TR, thyroid hormone receptor; RAR, retinoic acid receptor; ROR, RAR-related orphan receptor; h, human; m, mouse; GR, glucocorticoid receptor; r, rat; HRE, hormone-responsive element; DR, direct repeat; tk, thymidine kinase; GH, growth hormone; GST, glutathione S-transferase; LCA, lithocholic acid; CA, cholic acid; CoIP, coimmunoprecipitation; AF-2, activation function-2; LBD, ligand binding domain; DBD, DNA binding domain; PTHrP, parathyroid hormone related peptide; luc, luciferase.1The abbreviations used are: VDR, vitamin D receptor; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; RXR, retinoid X receptor; VDRE, vitamin D-responsive element; CYP24, vitamin D 24-hydroxylase; N-CoR, nuclear receptor corepressor; SMRT, silencing mediator for retinoic acid and thyroid hormone receptors; hr, hairless; Hr, hairless gene product; TR, thyroid hormone receptor; RAR, retinoic acid receptor; ROR, RAR-related orphan receptor; h, human; m, mouse; GR, glucocorticoid receptor; r, rat; HRE, hormone-responsive element; DR, direct repeat; tk, thymidine kinase; GH, growth hormone; GST, glutathione S-transferase; LCA, lithocholic acid; CA, cholic acid; CoIP, coimmunoprecipitation; AF-2, activation function-2; LBD, ligand binding domain; DBD, DNA binding domain; PTHrP, parathyroid hormone related peptide; luc, luciferase. mediates signaling by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and is a member of the thyroid hormone/retinoic acid receptor subfamily of nuclear receptors that heterodimerize with the retinoid X receptor (RXR) on direct repeat hormone-responsive elements in the promoters of regulated genes (3Haussler M.R. Whitfield G.K. Haussler C.A. Hsieh J.-C. Thompson P.D. Selznick S.H. Encinas Dominguez C. Jurutka P.W. J. Bone Miner. Res. 1998; 13: 325-349Crossref PubMed Scopus (1197) Google Scholar, 4Jones G. Strugnell S.A. DeLuca H.F. Physiol. Rev. 1998; 78: 1193-1231Crossref PubMed Scopus (999) Google Scholar). Binding of liganded VDR·RXR to a vitamin D-responsive element (VDRE) in target genes such as osteocalcin, osteopontin, and vitamin D 24-hydroxylase (CYP24) is accompanied by the recruitment of coactivator proteins (5Jurutka P.W. Whitfield G.K. Hsieh J.-C. Thompson P.D. Haussler C.A. Haussler M.R. Rev. Endocr. Metab. Disord. 2001; 2: 203-216Crossref PubMed Scopus (235) Google Scholar). VDR coactivators such as steroid receptor coactivator-1 (6Gill R.K. Atkins L.M. Hollis B.W. Bell N.H. Mol. Endocrinol. 1998; 12: 57-65Crossref PubMed Scopus (48) Google Scholar), NCoA-62 (7Baudino T.A. Kraichely D.M. Jefcoat Jr., S.C. Winchester S.K. Partridge N.C. MacDonald P.N. J. Biol. 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A. 1996; 93: 7567-7571Crossref PubMed Scopus (220) Google Scholar), evidence suggests that VDR does not associate strongly with these corepressors (9Hörlein A.J. Näär 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 (1688) Google Scholar, 10Chen J.D. Umesono K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7567-7571Crossref PubMed Scopus (220) Google Scholar, 11Wong C.W. Privalsky M.L. Mol. Cell. Biol. 1998; 18: 5724-5733Crossref PubMed Scopus (67) Google Scholar, 12Tagami T. Lutz W.H. Kumar R. Jameson J.L. Biochem. Biophys. Res. Commun. 1998; 253: 358-363Crossref PubMed Scopus (87) Google Scholar). Targeted gene deletion studies in mice (13Yoshizawa T. Handa Y. Uematsu Y. Takeda S. Sekine K. Yoshihara Y. Kawakami T. Arioka K. Sato H. Uchiyama Y. Masushige S. Fukamizu A. Matsumoto T. Kato S. Nat. Genet. 1997; 16: 391-396Crossref PubMed Scopus (942) Google Scholar, 14Li Y.C. Pirro A.E. Amling M. Delling G. Baron R. Bronson R. Demay M.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9831-9835Crossref PubMed Scopus (780) Google Scholar) and inactivating mutations in humans (15Malloy P.J. Pike J.W. Feldman D. Endocr. Rev. 1999; 20: 156-188Crossref PubMed Scopus (338) Google Scholar) have revealed multiple biological consequences of VDR signaling. VDR is required primarily for the following: (i) stimulation of calcium and phosphate absorption from the intestine to prevent rickets, (ii) induction of the CYP24 enzyme that initiates the catabolism of 1,25(OH)2D3, and (iii) progression of the normal hair cycle in mammalian skin. Although VDR gene ablation in mice elicits both rickets and hair loss (13Yoshizawa T. Handa Y. Uematsu Y. Takeda S. Sekine K. Yoshihara Y. Kawakami T. Arioka K. Sato H. Uchiyama Y. Masushige S. Fukamizu A. Matsumoto T. Kato S. Nat. Genet. 1997; 16: 391-396Crossref PubMed Scopus (942) Google Scholar, 14Li Y.C. Pirro A.E. Amling M. Delling G. Baron R. Bronson R. Demay M.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9831-9835Crossref PubMed Scopus (780) Google Scholar), point mutations in human VDR that specifically compromise either 1,25(OH)2D3 ligand (16Kristjansson K. Rut A.R. Hewison M. O'Riordan J.L.H. Hughes M.R. J. Clin. Invest. 1993; 92: 12-16Crossref PubMed Scopus (129) Google Scholar, 17Whitfield G.K. Selznick S.H. Haussler C.A. Hsieh J.-C. Galligan M.A. Jurutka P.W. Thompson P.D. Lee S.M. Zerwekh J.E. Haussler M.R. Mol. Endocrinol. 1996; 10: 1617-1631Crossref PubMed Scopus (113) Google Scholar) or coactivator (18Malloy P.J. Xu R. Peng L. Clark P.A. Feldman D. Mol. Endocrinol. 2002; 16: 2538-2546Crossref PubMed Scopus (81) Google Scholar) contacts confer rickets without disruption of the hair cycle. Loss of function mutations in human VDR that do result in both rickets and congenital hair loss (alopecia or atrichia) abolish either VDR DNA binding (19Hirst M.A. Hochman H.I. Feldman D. J. Clin. Endocrinol Metab. 1985; 60: 490-495Crossref PubMed Scopus (75) Google Scholar, 20Hughes M.R. Malloy P.J. Kieback D.G. Kesterson R.A. Pike J.W. Feldman D. O'Malley B.W. Science. 1988; 242: 1702-1705Crossref PubMed Scopus (409) Google Scholar, 21Rut A.R. Hewison M. Kristjansson K. Luisi B. Hughes M.R. O'Riordan J.L.H. Clin. Endocrinol. 1994; 41: 581-590Crossref PubMed Scopus (55) Google Scholar) or VDR·RXR heterodimerization (17Whitfield G.K. Selznick S.H. Haussler C.A. Hsieh J.-C. Galligan M.A. Jurutka P.W. Thompson P.D. Lee S.M. Zerwekh J.E. Haussler M.R. Mol. Endocrinol. 1996; 10: 1617-1631Crossref PubMed Scopus (113) Google Scholar). Interaction of VDR with its heterodimeric partner RXR is likely relevant to hair cycling, as the conditional inactivation of RXRα in mouse skin (22Li M. Indra A.K. Warot X. Brocard J. Messaddeq N. Kato S. Metzger D. Chambon P. Nature. 2000; 407: 633-636Crossref PubMed Scopus (270) Google Scholar) results in alopecia resembling that in VDR-null mice (14Li Y.C. Pirro A.E. Amling M. Delling G. Baron R. Bronson R. Demay M.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9831-9835Crossref PubMed Scopus (780) Google Scholar). Like mutations in the gene encoding VDR, mutations in the mammalian hairless (hr) gene result in congenital hair loss in both mice (23Stoye J.P. Fenner S. Greenoak G.E. Moran C. Coffin J.M. Cell. 1988; 54: 383-391Abstract Full Text PDF PubMed Scopus (154) Google Scholar) and humans (24Ahmad W. Faiyaz ul Haque M. Brancolini V. Tsou H.C. ul Haque S. Lam H. Aita V.M. Owen J. deBlaquiere M. Frank J. Cserhalmi-Friedman P.B. Leask A. McGrath J.A. Peacocke M. Ahmad M. Ott J. Christiano A.M. Science. 1998; 279: 720-724Crossref PubMed Scopus (352) Google Scholar, 25Cichon S. Anker M. Vogt I.R. Rohleder H. Putzstuck M. Hillmer A. Farooq S.A. Al-Dhafri K.S. Ahmad M. Haque S. Rietschel M. Propping P. Kruse R. Nothen M.M. Hum. Mol. Genet. 1998; 7: 1671-1679Crossref PubMed Scopus (128) Google Scholar). Remarkably, the hair loss phenotype caused by specific mutations in the human VDR gene resembles the generalized atrichia caused by mutations in the hr gene (26Miller J. Djabali K. Chen T. Liu Y. Ioffreda M. Lyle S. Christiano A.M. Holick M. Cotsarelis G. J. Invest. Dermatol. 2001; 117: 612-617Abstract Full Text Full Text PDF PubMed Google Scholar). The shared hair loss phenotype of hr and VDR mutant animals and humans suggests that both proteins impact a common signaling pathway. In addition to genetic evidence of a potential relationship between hr and VDR, the function of the hr gene product (Hr) may be relevant to VDR signaling as well. Hr is a nuclear protein with a molecular mass of 130 kDa that is expressed primarily in skin and brain (27Cachon-Gonzalez M.B. Fenner S. Coffin J.M. Moran C. Best S. Stoye J.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7717-7721Crossref PubMed Scopus (164) Google Scholar, 28Thompson C.C. J. Neurosci. 1996; 16: 7832-7840Crossref PubMed Google Scholar, 29Potter G.B. Zarach J.M. Sisk J.M. Thompson C.C. Mol. Endocrinol. 2002; 16: 2547-2560Crossref PubMed Scopus (92) Google Scholar) and was shown recently (30Potter G.B. Beaudoin III, G.M. DeRenzo C.L. Zarach J.M. Chen S.H. Thompson C.C. Genes Dev. 2001; 15: 2687-2701Crossref PubMed Scopus (149) Google Scholar) to function as a potent nuclear receptor corepressor. Despite a lack of sequence identity with other corepressors (N-CoR and SMRT), Hr functions in a similar manner; Hr was shown to mediate repression via unliganded thyroid hormone receptor (TR), bind to TR through conserved hydrophobic motifs, and interact with histone deacetylases (29Potter G.B. Zarach J.M. Sisk J.M. Thompson C.C. Mol. Endocrinol. 2002; 16: 2547-2560Crossref PubMed Scopus (92) Google Scholar, 30Potter G.B. Beaudoin III, G.M. DeRenzo C.L. Zarach J.M. Chen S.H. Thompson C.C. Genes Dev. 2001; 15: 2687-2701Crossref PubMed Scopus (149) Google Scholar). Unlike N-CoR and SMRT, Hr interacts with TR but not retinoic acid receptor (RAR) and can also inhibit transcriptional activation by the RAR-related orphan receptor, ROR (31Moraitis A.N. Giguere V. Thompson C.C. Mol. Cell. Biol. 2002; 22: 6831-6841Crossref PubMed Scopus (80) Google Scholar). Based on genetic and biochemical evidence, we postulate that a network of interactions between Hr and VDR exists in the skin to drive the progression of the hair cycle. Because VDR and TR are similar in structure, we tested the hypothesis that Hr interacts with VDR as a corepressor. In the present report we show the following: (i) VDR interacts physically with Hr, (ii) this association dramatically represses VDR-mediated transactivation, and (iii) Hr and VDR are coexpressed in cells of the hair follicle. Our results are consistent with the presence of VDR and Hr in a signal transduction cascade in the hair follicle that represses gene expression, possibly silencing a gene that codes for an inhibitor of the hair cycle. Plasmid Constructions—Cloned cDNAs encoding human VDR (hVDR) (32Baker A.R. McDonnell D.P. Hughes M.R. Crisp T.M. Mangelsdorf D.J. Haussler M.R. Pike J.W. Shine J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3294-3298Crossref PubMed Scopus (841) Google Scholar), mouse RXRβ (mRXRβ) (33Hamada K. Gleason S.L. Levi B.-Z. Hirschfeld S. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8289-8293Crossref PubMed Scopus (247) Google Scholar), human RXRα (hRXRα) (34Mangelsdorf D.J. Ong E.S. Dyck J.A. Evans R.M. Nature. 1990; 345: 224-229Crossref PubMed Scopus (1246) Google Scholar), and mouse glucocorticoid receptor (mGR) (35Danielsen M. Hinck L. Ringold G.M. Cancer Res. 1989; 49: 2286-2291Google Scholar) were subcloned into the expression plasmid pSG5 (36Green S. Isseman I. Sheer E. Nucleic Acids Res. 1988; 16: 369Crossref PubMed Scopus (543) Google Scholar) as described (37Hsieh J.-C. Jurutka P.W. Galligan M.A. Terpening C.M. Haussler C.A. Samuels D.S. Shimizu Y. Shimizu N. Haussler M.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9315-9319Crossref PubMed Scopus (186) Google Scholar). Truncations of hVDR in pSG5 (receptor fragments Δ1–88, Δ134, Δ202, Δ304, and Δ403) were generated as described (38Nakajima S. Hsieh J.-C. MacDonald P.N. Galligan M.A. Haussler C.A. Whitfield G.K. Haussler M.R. Mol. Endocrinol. 1994; 8: 159-172Crossref PubMed Scopus (87) Google Scholar, 39Hsieh J.-C. Nakajima S. Galligan M.A. Jurutka P.W. Haussler C.A. Whitfield G.K. Haussler M.R. J. Steroid Biochem. Mol. Biol. 1995; 53: 583-594Crossref PubMed Scopus (18) Google Scholar); the expression plasmid for a point mutant hVDR (E420A) is detailed elsewhere (40Jurutka P.W. Hsieh J.-C. Remus L.S. Whitfield G.K. Thompson P.D. Haussler C.A. Blanco J.C.G. Ozato K. Haussler M.R. J. Biol. Chem. 1997; 272: 14592-14599Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). pCMX-hVDR and pCMX-hRXRα were kindly provided by Dr. R. Evans (The Salk Institute, San Diego, CA). Expression plasmids for epitope (Myc)-tagged Hr (pRK5myc-rhr) and rat Hr (rHr) deletion derivatives have been described (30Potter G.B. Beaudoin III, G.M. DeRenzo C.L. Zarach J.M. Chen S.H. Thompson C.C. Genes Dev. 2001; 15: 2687-2701Crossref PubMed Scopus (149) Google Scholar). pCMX-mSMRT-αFL (41Ordentlich P. Downes M. Xie W. Genin A. Spinner N.B. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2639-2644Crossref PubMed Scopus (138) Google Scholar) was kindly provided by Drs. M. Downes and R. Evans (The Salk Institute, San Diego, CA). Hormone-responsive element (HRE) reporter plasmids were constructed as follows. Synthetic oligonucleotides containing four copies of the rat osteocalcin direct repeat 3 (DR3) VDRE (GGGTGAATGAGGACA) (42Terpening C.M. Haussler C.A. Jurutka P.W. Galligan M.A. Komm B.S. Haussler M.R. Mol. Endocrinol. 1991; 5: 373-385Crossref PubMed Scopus (118) Google Scholar), four copies of the rat CRBPII direct repeat 1 (DR1) RXR-responsive element (AGGTCACAGGTCA) (43Mangelsdorf D.J. Umesono K. Kliewer S.A. Borgmeyer U. Ong E.S. Evans R.M. Cell. 1991; 66: 555-561Abstract Full Text PDF PubMed Scopus (523) Google Scholar), or three copies of the rat tyrosine aminotransferase indirect repeat 3 glucocorticoid-responsive element (TGTACAGGATGTTCT) (44Tsai S.Y. Carlstedt-Duke J. Weigel N.L. Dahlman K. Gustafsson J.-Å. Tsai M.-J. O'Malley B.W. Cell. 1988; 55: 361-369Abstract Full Text PDF PubMed Scopus (410) Google Scholar) were each cloned into the HindIII site of ptkGH. Each construct includes a herpes simplex virus thymidine kinase (tk) promoter directing basal transcription of a human growth hormone (hGH) reporter gene. DR3x2 tk-luc was constructed by insertion of synthetic oligonucleotides containing two copies of the consensus AGGTCA DR3 VDRE upstream of the minimal tk promoter in tk-luc. p24-OHaseLuc was constructed by subcloning 5.5 kb of the promoter region (45Jin C.H. Kerner S.A. Hong M.H. Pike J.W. Mol. Endocrinol. 1996; 10: 945-957Crossref PubMed Scopus (72) Google Scholar) of the human CYP24 gene (kindly provided by Drs. S. Christakos and J. W. Pike, New Jersey Medical School and University of Wisconsin, respectively) into a firefly luciferase plasmid, lucp1 (45Jin C.H. Kerner S.A. Hong M.H. Pike J.W. Mol. Endocrinol. 1996; 10: 945-957Crossref PubMed Scopus (72) Google Scholar). Transfection of COS Cells/Transcription Assays—African green monkey kidney cells (COS-1, COS-7) obtained from ATCC were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Omega Scientific, Tarzana, CA or Invitrogen). For transfections with HRE-hGH reporter plasmids, COS-7 cells were plated at 80,000 cells per well in a 24-well plate and transfected 6 h later with 250 ng of reporter plasmid, 250 ng of receptor expression plasmid(s), and either 250 ng of pRK5myc-rhr or pTZ18U carrier DNA. Transfections were performed by calcium phosphate DNA coprecipitation (37Hsieh J.-C. Jurutka P.W. Galligan M.A. Terpening C.M. Haussler C.A. Samuels D.S. Shimizu Y. Shimizu N. Haussler M.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9315-9319Crossref PubMed Scopus (186) Google Scholar). Cells were washed 16 h post-transfection and then treated for 24 h with 1,25(OH)2D3 (10 nm), lithocholic acid (LCA) (10-4m), dexamethasone (1 μm), the rexinoid, LG5004 (5 μm; kindly supplied by Dr. H. Martin Seidel, Ligand Pharmaceuticals, Inc., San Diego, CA), or ethanol vehicle (minus hormone control). Media were assayed by radioimmunoassay for hGH expression (Nichols Institute Diagnostics, San Juan Capistrano, CA). For experiments using DR3x2 tk-luc, COS-1 cells were plated in 12-well plates and transfected the following day using LipofectAMINE 2000 (Invitrogen) with 200 ng of reporter plasmid, 75 ng of receptor and/or Hr expression plasmid, and 300 ng of CMV-β-galactosidase. After 36 h, cells were harvested in passive lysis buffer (Promega, Madison, WI), and extracts were assayed for β-galactosidase and luciferase activity. Luciferase activity was divided by β-galactosidase activity to normalize for transfection efficiency. Experiments were done in duplicate and repeated at least three times with similar results. For transfection assays with p24-OHaseLuc, COS-7 cells were transfected with 37.5 ng/well of p24-OHaseLuc, 0.1 ng/well of pRL-CMV (non-regulated Renilla luciferase control), 25 ng/well of pSG5hVDR, and where indicated, 10 ng/well of pRK5myc-rhr, either in the absence or presence of 10 nm 1,25(OH)2D3. After 48 h, cells were harvested with passive lysis buffer. Firefly and Renilla luciferase activities were measured sequentially from each well using a Sirius Luminometer (Pforzheim, Germany) and dual luciferase reporter assay reagents (Promega, Madison, WI) per the manufacturer's instructions. The ratio of firefly to Renilla luciferase activity was calculated to normalize for transfection efficiency. GST Pull Down Assays—Wild-type hVDR (32Baker A.R. McDonnell D.P. Hughes M.R. Crisp T.M. Mangelsdorf D.J. Haussler M.R. Pike J.W. Shine J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3294-3298Crossref PubMed Scopus (841) Google Scholar), hRXRα (34Mangelsdorf D.J. Ong E.S. Dyck J.A. Evans R.M. Nature. 1990; 345: 224-229Crossref PubMed Scopus (1246) Google Scholar), and rat hr (28Thompson C.C. J. Neurosci. 1996; 16: 7832-7840Crossref PubMed Google Scholar) cDNAs were cloned into the EcoRI site of the GST fusion protein vector, pGEX-4T (Amersham Biosciences) to create GST-hVDR, GST-hRXRα, and GST-rHr (residues 31–1207). pGEX fusion constructs were transformed into Escherichia coli (strain BL21 for VDR and Hr; strain DH5α for RXRα). The detailed procedure for overexpression of GST fusion proteins has been described previously (46Jurutka P.W. Remus L.S. Whitfield G.K. Galligan M.A. Haussler C.A. Haussler M.R. Biochem. Biophys. Res. Commun. 2000; 267: 813-819Crossref PubMed Scopus (7) Google Scholar). GST alone was expressed from pGEX-4T in E. coli strain DH5α and linked to glutathione-Sepharose beads to serve as a control for background protein association. For GST pull down assays, expression plasmids (1.0 μg) were used to generate [35S]methionine-labeled proteins by in vitro transcription/translation (TNT Coupled Reticulocyte lysate kit, Promega, Madison, WI). GST-control, GST-hVDR, GST-hVDR-E420A, or GST-RXRα glutathione-Sepharose beads (25 μl each) were incubated in KETZD-0.15 m buffer (47Jurutka P.W. Remus L.S. Whitfield G.K. Thompson P.D. Hsieh J.-C. Zitzer H. Tavakkoli P. Galligan M.A. Dang H.T. Haussler C.A. Haussler M.R. Mol. Endocrinol. 2000; 14: 401-420Crossref PubMed Scopus (260) Google Scholar) at 4 °C for 1.5 h on a rocking platform in the absence or presence of lipophilic ligands: 1,25(OH)2D3 (10–6m), LCA (10–4m), cholic acid (CA) (10-4m), or LG5009 (10-6m; kindly supplied by Dr. H. Martin Seidel, Ligand Pharmaceuticals, Inc., San Diego, CA). Next, the desired 35S-labeled protein(s) was incubated with the beads for 30 min at 4 °C. When Hr beads were employed in pull down assays, the ligand (1,25(OH)2D3 (10-7m) or dexamethasone (10-6m)) was included in the in vitro transcription/translation reaction. The liganded, synthesized protein was then incubated with GST-Hr beads for 30 min at 4 °C as above. In all cases, beads were washed four times with KETZD-0.15 to remove unbound protein(s). The bound proteins were extracted from the beads into loading buffer (4% SDS, 10% β-mercaptoethanol, 125 mm Tris-Cl, pH 6.8, 20% glycerol), boiled 3 min, and separated by gradient (5–20%) SDS-PAGE and visualized by autoradiography. Coimmunoprecipitation—Transfections for coimmunoprecipitation (CoIP) experiments were performed by electroporation of COS-1 cells as described (30Potter G.B. Beaudoin III, G.M. DeRenzo C.L. Zarach J.M. Chen S.H. Thompson C.C. Genes Dev. 2001; 15: 2687-2701Crossref PubMed Scopus (149) Google Scholar). Cells were harvested for immunoprecipitation in IP Buffer (10% glycerol, 150 mm NaCl, 50 mm Tris-Cl, pH 7.4, 1% IGEPAL, and protease inhibitors), and extracts were incubated with 5 μg of either VDR-specific monoclonal antibody (9A7γ) (48Pike J.W. Marion S.L. Donaldson C.A. Haussler M.R. J. Biol. Chem. 1983; 258: 1289-1296Abstract Full Text PDF PubMed Google Scholar) or IgG control (Sigma) overnight at 4 °C. Immunoprecipitates were collected using protein G-Sepharose beads (Amersham Biosciences), and proteins were separated by SDS-PAGE. Hr was detected by Western analysis with either Hr- or Myc-specific antisera as described (30Potter G.B. Beaudoin III, G.M. DeRenzo C.L. Zarach J.M. Chen S.H. Thompson C.C. Genes Dev. 2001; 15: 2687-2701Crossref PubMed Scopus (149) Google Scholar). VDR was detected with VDR-specific antibody 9A7γ (47Jurutka P.W. Remus L.S. Whitfield G.K. Thompson P.D. Hsieh J.-C. Zitzer H. Tavakkoli P. Galligan M.A. Dang H.T. Haussler C.A. Haussler M.R. Mol. Endocrinol. 2000; 14: 401-420Crossref PubMed Scopus (260) Google Scholar, 49Pike J.W. Sleator N.M. Haussler M.R. J. Biol. Chem. 1987; 262: 1305-1311Abstract Full Text PDF PubMed Google Scholar). In Situ Hybridization—For the hr-specific probe, a fragment corresponding to nucleotides 1004–3928 of the mouse hr cDNA (27Cachon-Gonzalez M.B. Fenner S. Coffin J.M. Moran C. Best S. Stoye J.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7717-7721Crossref PubMed Scopus (164) Google Scholar) was subcloned into pBluescript KS+ (Stratagene), and the resulting plasmid was linearized with XhoI and NotI to generate templates for sense and antisense probes, respectively. The VDR-specific probe corresponds to nucleotides 71–1371 of mouse VDR cDNA and was made using reverse transcription of mouse brain total RNA (Thermoscript RT-PCR System; Invitrogen) followed by PCR amplification with specific primers (5′-TCAGGAGATCTCATTGCCAAAC-3′ and 5′-CAGACCAGAGTTCTTTTGGTTG-3′). The mVDR cDNA was ligated into pCR2.1 (Invitrogen) in both orientations, and the resulting plasmids were linearized with BamHI and used as templates to produce sense and antisense probes. Digoxigenin-labeled cRNA probes were made as described (29Potter G.B. Zarach J.M. Sisk J.M. Thompson C.C. Mol. Endocrinol. 2002; 16: 2547-2560Crossref PubMed Scopus (92) Google Scholar). Serial sections (5 μm) of dorsal skin from postnatal day 15 mice were used for in situ hybridization. Sections were fixed in 4% paraformaldehyde and then acetylated/dehydrated in 0.25% acetic anhydride in 0.1 m triethanolamine. Sections were prehybridized with hybridization solution (5× Denhardt's solution, 12.5 mg of yeast tRNA, 5× SSC, 50% formamide) and then incubated with hybridization solution containing ∼200 ng of probe per slide in a humidified chamber overnight at 60 °C. The following day sections were washed at 60 °C with 5× SSC, followed by 2× SSC, 0.2× SSC/50% formamide, and 0.2× SSC. To detect the hybridized probe, sections were incubated with anti-digoxigenin-alkaline phosphatase Fab fragments (Roche Applied Science) diluted 1:5000 in Buffer 1 (0.1 m Tris-Cl, pH 7.5, 0.15 m NaCl). After washing, sections were incubated with color solution (0.34 mg/ml nitro blue tetrazolium, 0.175 mg/ml 5-bromo-4-chloro-3-indolyl phosphate, 4-toluidine salt, 0.24 mg/ml Levamisole in 0.1 m Tris-Cl, pH 9.5, 0.1 m NaCl, 0.005 m MgCl2) overnight in a humidified chamber. The sections were dehydrated and cleared overnight in Citrisolv (Fisher). Digital images of sections were obtained as described previously (29Potter G.B. Zarach J.M. Sisk J.M. Thompson C.C. Mol. Endocrinol. 2002; 16: 2547-2560Crossref PubMed Scopus (92) Google Scholar). Hr Inhibits Transactivation by Liganded VDR—The potential effect of Hr on VDR activity was evaluated in a mammalian cell line (COS) that expresses low levels (≤500 copies/cell) of endogenous VDR. Cells were cotransfected with a hVDR expression plasmid and VDR-responsive reporter constructs, and 1,25(OH)2D3-induced transcriptional activity was measured in the absence and presence of cotransfected Hr. When the rat osteocalcin VDRE reporter construct was employed, as expected, the 1,25(OH)2D3 ligand markedly enhances transcription via VDR (Fig. 1A). Cotransfection of Hr results in a moderate decrease in basal transcription (40%) and a dramatic reduction in transcriptional activation by 1,25(OH)2D3-liganded VDR (∼7-fold). In addition, Hr sharply represses (11-fold) VDR-m" @default.
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• W2078862807 date "2003-10-01" @default.
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• W2078862807 title "Physical and Functional Interaction between the Vitamin D Receptor and Hairless Corepressor, Two Proteins Required for Hair Cycling" @default.
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