Dexamethasone Enhances 1α,25-Dihydroxyvitamin D3 Effects by Increasing Vitamin D Receptor Transcription

Abstract

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner. Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner. Introduction1α,25-Dihydroxycholecalciferol (calcitriol), the most active metabolite of vitamin D, is a seco-steroid hormone that regulates calcium homeostasis, bone development, and bone maintenance (1Dhawan P. Christakos S. J. Cell. Biochem. 2010; 110: 1314-1323Crossref PubMed Scopus (54) Google Scholar, 2Kallay E. Pietschmann P. Toyokuni S. Bajna E. Hahn P. Mazzucco K. Bieglmayer C. Kato S. Cross H.S. Carcinogenesis. 2001; 22: 1429-1435Crossref PubMed Scopus (157) Google Scholar). Calcitriol has potent anti-proliferative effects in a number of human and murine cancer cell types in vitro and in in vivo xenograft tumor models. Calcitriol inhibits growth in breast, colon, squamous cell carcinoma, and prostate cancer models (3Trump D.L. Potter D.M. Muindi J. Brufsky A. Johnson C.S. Cancer. 2006; 106: 2136-2142Crossref PubMed Scopus (94) Google Scholar). More recently, in support of calcitriol as an anti-angiogenesis therapy, our laboratory has described that tumor-derived endothelial cells are more sensitive to the anti-proliferative effect of calcitriol compared with non-tumor-derived endothelial cells (4Chung I. Karpf A.R. Muindi J.R. Conroy J.M. Nowak N.J. Johnson C.S. Trump D.L. J. Biol. Chem. 2007; 282: 8704-8714Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 5Chung I. Yu W.D. Karpf A.R. Flynn G. Bernardi R.J. Modzelewski R.A. Johnson C.S. Trump D.L. J. Steroid Biochem. Mol. Biol. 2007; 103: 768-770Crossref PubMed Scopus (22) Google Scholar, 6Flynn G. Chung I. Yu W.D. Romano M. Modzelewski R.A. Johnson C.S. Trump D.L. Oncology. 2006; 70: 447-457Crossref PubMed Scopus (38) Google Scholar).The mechanism by which calcitriol exerts its anti-proliferative action through cell cycle arrest, cell differentiation, and induction of apoptosis varies between different tumor cell types (7Deeb K.K. Trump D.L. Johnson C.S. Nat. Rev. Cancer. 2007; 7: 684-700Crossref PubMed Scopus (1108) Google Scholar). The anti-proliferative effect of calcitriol depends on the levels of the vitamin D receptor (VDR), 3The abbreviations used are: VDRvitamin D receptorDexdexamethasoneSCCsquamous cell carcinomaGRglucocorticoid receptorGREglucocorticoid response elementActDactinomycin DCHXcycloheximide. a member of the steroid receptor super family. VDR acts as a ligand-inducible transcription factor. Upon binding to its ligand, calcitriol, the VDR-calcitriol complex binds to DNA sequences called vitamin D response elements located in regulatory regions of target genes to either increase or decrease transcription (7Deeb K.K. Trump D.L. Johnson C.S. Nat. Rev. Cancer. 2007; 7: 684-700Crossref PubMed Scopus (1108) Google Scholar, 8Mangelsdorf 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 (6026) Google Scholar). VDR is expressed in many tissues and cell types. Calcitriol plays an important role in regulating VDR at both the transcriptional and post-transcriptional levels. In support of a direct transcriptional mechanism, functional vitamin D response elements have been found within intronic regions of Vdr gene (9Bagamasbad P. Denver R.J. Gen. Comp. Endocrinol. 2011; 170: 3-17Crossref PubMed Scopus (81) Google Scholar, 10Zella L.A. Kim S. Shevde N.K. Pike J.W. Mol. Endocrinol. 2006; 20: 1231-1247Crossref PubMed Scopus (140) Google Scholar). Calcitriol up-regulation of VDR is also observed in cells where calcitriol does not induce transcription of VDR gene; however, the mechanisms by which calcitriol increases VDR protein in those cells remain unknown. Calcitriol induces phosphorylation of VDR at serine residues −51 and −208 to maintain VDR in a transcriptionally active state. In addition, phosphorylated serine 208 has been implicated in the recruitment of co-activators to increase gene expression (11Arriagada G. Paredes R. Olate J. van Wijnen A. Lian J.B. Stein G.S. Stein J.L. Onate S. Montecino M. J. Steroid Biochem. Mol. Biol. 2007; 103: 425-429Crossref PubMed Scopus (31) Google Scholar, 12Hsieh 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 (191) Google Scholar, 13Jurutka P.W. Hsieh J.C. MacDonald P.N. Terpening C.M. Haussler C.A. Haussler M.R. Whitfield G.K. J. Biol. Chem. 1993; 268: 6791-6799Abstract Full Text PDF PubMed Google Scholar).VDR expression is tightly regulated at the transcriptional level. In addition, the complexity of VDR gene regulatory regions makes its study challenging because of a complex 5′ exonic region, which has partial homology between human and mouse (14Halsall J.A. Osborne J.E. Hutchinson P.E. Pringle J.H. J. Steroid Biochem. Mol. Biol. 2007; 103: 352-356Crossref PubMed Scopus (11) Google Scholar, 15Zella L.A. Meyer M.B. Nerenz R.D. Lee S.M. Martowicz M.L. Pike J.W. Mol. Endocrinol. 2010; 24: 128-147Crossref PubMed Scopus (117) Google Scholar). Expression of Vdr is regulated by several transcription factors including VDR itself. Our group and others have shown that treatment with glucocorticoids increases VDR levels in adipocytes, SCC, and kidney (2Kallay E. Pietschmann P. Toyokuni S. Bajna E. Hahn P. Mazzucco K. Bieglmayer C. Kato S. Cross H.S. Carcinogenesis. 2001; 22: 1429-1435Crossref PubMed Scopus (157) Google Scholar, 16Bernardi R.J. Trump D.L. Yu W.D. McGuire T.F. Hershberger P.A. Johnson C.S. Clin. Cancer Res. 2001; 7: 4164-4173PubMed Google Scholar, 17Sun X. Zemel M.B. Int. J. Obes. 2008; 32: 1305-1311Crossref PubMed Scopus (32) Google Scholar, 18Yu W.D. McElwain M.C. Modzelewski R.A. Russell D.M. Smith D.C. Trump D.L. Johnson C.S. J. Natl. Cancer Inst. 1998; 90: 134-141Crossref PubMed Scopus (92) Google Scholar). Glucocorticoids are a class of steroid hormones that exert their actions though binding to the glucocorticoid receptor (GR). GR is also a member of the ligand-activated nuclear receptor group of transcription factors. GR binds with high affinity to glucocorticoids, as and Dex, to increase or transcription of target genes Proc. Natl. Acad. Sci. U.S.A. 106: PubMed Scopus Google Scholar). GR a and to the where binds to sequences glucocorticoids response elements (GREs) in regulatory regions of target genes to either increase or decrease expression (9Bagamasbad P. Denver R.J. Gen. Comp. Endocrinol. 2011; 170: 3-17Crossref PubMed Scopus (81) Google Scholar, E. R. K. J. J.A. R. PubMed Scopus Google Scholar).The in the of calcitriol as a cancer in has been effects PubMed Scopus (92) Google Scholar). as have effects that decrease calcium and of calcium Lee G.S. Sci. PubMed Scopus (32) Google Scholar, Lee G.S. PubMed Scopus Google Scholar, R.D. E. J. Clin. PubMed Scopus Google Scholar, S. R. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). In of Dex expression of calcium regulatory genes including expression of receptor member and Lee G.S. Sci. PubMed Scopus (32) Google of the effects of Dex, our laboratory has the effects of calcitriol and Dex combination to calcitriol and the effects of calcitriol (3Trump D.L. Potter D.M. Muindi J. Brufsky A. Johnson C.S. Cancer. 2006; 106: 2136-2142Crossref PubMed Scopus (94) Google Scholar). We have shown that Dex the squamous cell cells to the effect of calcitriol, increases and increases VDR protein R.J. Trump D.L. Yu W.D. McGuire T.F. Hershberger P.A. Johnson C.S. Clin. Cancer Res. 2001; 7: 4164-4173PubMed Google Scholar, 18Yu W.D. McElwain M.C. Modzelewski R.A. Russell D.M. Smith D.C. Trump D.L. Johnson C.S. J. Natl. Cancer Inst. 1998; 90: 134-141Crossref PubMed Scopus (92) Google Scholar). Treatment with in a decrease of and treatment with either calcitriol or Dex of apoptosis was observed by of and combination treatment of and compared with treatment with either calcitriol or Dex R.J. Trump D.L. Yu W.D. McGuire T.F. Hershberger P.A. Johnson C.S. Clin. Cancer Res. 2001; 7: 4164-4173PubMed Google that treatment with glucocorticoids increase VDR protein expression G. Chung I. Yu W.D. Romano M. Modzelewski R.A. Johnson C.S. Trump D.L. Oncology. 2006; 70: 447-457Crossref PubMed Scopus (38) Google Scholar, 16Bernardi R.J. Trump D.L. Yu W.D. McGuire T.F. Hershberger P.A. Johnson C.S. Clin. Cancer Res. 2001; 7: 4164-4173PubMed Google Scholar, 18Yu W.D. McElwain M.C. Modzelewski R.A. Russell D.M. Smith D.C. Trump D.L. Johnson C.S. J. Natl. Cancer Inst. 1998; 90: 134-141Crossref PubMed Scopus (92) Google however, the mechanisms by which glucocorticoids VDR expression to L.A. Meyer M.B. Nerenz R.D. Lee S.M. Martowicz M.L. Pike J.W. Mol. Endocrinol. 2010; 24: 128-147Crossref PubMed Scopus (117) Google used chromatin immunoprecipitation by to in regulatory sequences of the mouse Vdr In the we that VDR is a target of the of Vdr we the glucocorticoid dexamethasone induces expression of mouse Vdr gene in the cell We Dex anti-proliferative and VDR-mediated effects of We also Dex treatment increases expression of Vdr at a transcriptional and expression of VDR target we Dex induces Vdr gene expression in a GR-dependent through binding to a GRE located upstream of transcription start site of are to in a number of including C.S. Muindi J.R. Hershberger P.A. Trump D.L. Res. 2006; Google Scholar). their glucocorticoids are used to with and and prostate (3Trump D.L. Potter D.M. Muindi J. Brufsky A. Johnson C.S. Cancer. 2006; 106: 2136-2142Crossref PubMed Scopus (94) Google Scholar, J. S. I. R. B. J. PubMed Google Scholar, X. S. J.E. Cancer. 2010; PubMed Scopus Google Scholar, A. Y. S. A. E. C. M. J. Clin. PubMed Scopus Google Scholar). The glucocorticoid dexamethasone increases VDR and the anti-proliferative effects of calcitriol R.J. Trump D.L. Yu W.D. McGuire T.F. Hershberger P.A. Johnson C.S. Clin. Cancer Res. 2001; 7: 4164-4173PubMed Google Scholar, 18Yu W.D. McElwain M.C. Modzelewski R.A. Russell D.M. Smith D.C. Trump D.L. Johnson C.S. J. Natl. Cancer Inst. 1998; 90: 134-141Crossref PubMed Scopus (92) Google Scholar). In this we the mechanisms by which Dex increases the levels of VDR and VDR-mediated effects in the and Zemel X. Zemel M.B. Int. J. Obes. 2008; 32: 1305-1311Crossref PubMed Scopus (32) Google the increase of Vdr mRNA with Dex in Dex increases expression of VDR in Lee G.S. Sci. PubMed Scopus (32) Google Scholar). Our study shows de novo transcription of Vdr by Dex that in VDR increases to calcitriol, and potentiates VDR-mediated transcription of VDR and many VDR target genes including and response to calcitriol was observed in and the because of R.A. C.S. M. J. PubMed Scopus Google Scholar, K. Cancer Google Scholar, E. G. G. Int. 1993; PubMed Scopus (11) Google Scholar). of calcitriol in prostate cancer where the increase in of a of of prostate was by treatment in or in others D.M. J. Cell. Biochem. 88: PubMed Scopus Google Scholar, D.M. J. Steroid Biochem. Mol. Biol. PubMed Scopus Google Scholar). our laboratory used high calcitriol Dex in cancer and that treatment is and has an effect as by a decrease in (3Trump D.L. Potter D.M. Muindi J. Brufsky A. Johnson C.S. Cancer. 2006; 106: 2136-2142Crossref PubMed Scopus (94) Google Scholar).The effects of calcitriol are through binding to the The complex expression of target including the Vdr gene itself. up-regulation or is observed in a number of cell models including colon, adipocytes, and (7Deeb K.K. Trump D.L. Johnson C.S. Nat. Rev. Cancer. 2007; 7: 684-700Crossref PubMed Scopus (1108) Google Scholar, P. Denver R.J. Gen. Comp. Endocrinol. 2011; 170: 3-17Crossref PubMed Scopus (81) Google Scholar, L.A. Kim S. Shevde N.K. Pike J.W. J. Steroid Biochem. Mol. Biol. 2007; 103: PubMed Scopus Google Scholar). active vitamin D response elements have been within intronic sequences of the mouse Vdr gene L.A. Kim S. Shevde N.K. Pike J.W. Mol. Endocrinol. 2006; 20: 1231-1247Crossref PubMed Scopus (140) Google Scholar). In addition, a number of transcription factors have been found to VDR including and Biochem. PubMed Scopus Google Scholar, R. M. Y. K. M. Y. K. Y. Cancer Res. 2006; PubMed Scopus Google Scholar, J.W. Meyer M.B. Martowicz M.L. Lee S.M. Nerenz R.D. J. Steroid Biochem. Mol. Biol. 2010; PubMed Scopus Google Scholar). of VDR with Dex treatment has been in several models G. Chung I. Yu W.D. Romano M. Modzelewski R.A. Johnson C.S. Trump D.L. Oncology. 2006; 70: 447-457Crossref PubMed Scopus (38) Google Scholar, 16Bernardi R.J. Trump D.L. Yu W.D. McGuire T.F. Hershberger P.A. Johnson C.S. Clin. Cancer Res. 2001; 7: 4164-4173PubMed Google Scholar, 17Sun X. Zemel M.B. Int. J. Obes. 2008; 32: 1305-1311Crossref PubMed Scopus (32) Google Scholar, Lee G.S. Sci. PubMed Scopus (32) Google Scholar). We several upstream of the Vdr transcription start site and within a luciferase reporter to active of the putative was active Trump D.L. Johnson C.S. J. Steroid Biochem. Mol. Biol. 2011; Scopus Google Scholar). by using high L.A. Meyer M.B. Nerenz R.D. Lee S.M. Martowicz M.L. Pike J.W. Mol. Endocrinol. 2010; 24: 128-147Crossref PubMed Scopus (117) Google DNA regions that GR within the of Vdr the the was to induce transcription with Dex of luciferase reporter compared with in a luciferase reporter in cells L.A. Meyer M.B. Nerenz R.D. Lee S.M. Martowicz M.L. Pike J.W. Mol. Endocrinol. 2010; 24: 128-147Crossref PubMed Scopus (117) Google Scholar). of the fragment that two putative in the fragment We of the putative luciferase reporter and and that the distal GRE is transcription and In our the transcription of with Dex was to compared with our not of GR as by L.A. Meyer M.B. Nerenz R.D. Lee S.M. Martowicz M.L. Pike J.W. Mol. Endocrinol. 2010; 24: 128-147Crossref PubMed Scopus (117) Google Scholar). Treatment with Dex or in combination with calcitriol GR by Y. S. J.A. 2010; PubMed Scopus Google Scholar, J.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google which transcription and induction of Vdr mRNA is as compared with the induction in luciferase reporter We that the of the active GRE in fragment to increase Vdr transcription is by an of regulatory elements within the of the Vdr response to glucocorticoids is by GR in the of glucocorticoids binds to and expression of target The in the distal GRE of Vdr gene was within the and the by with GR J. Steroid Biochem. Mol. Biol. PubMed Scopus Google Scholar, B. S. J.A. Cell. Full Text PDF PubMed Scopus Google Scholar, R. G. Beato M. Cell. Full Text PDF PubMed Scopus Google Scholar, B. M. R. G. Cell. Full Text PDF PubMed Scopus Google Scholar, R. A. G. Beato M. G. Cell. Full Text PDF PubMed Scopus Google Scholar, P.A. M. Google Scholar, Cell. Full Text PDF PubMed Scopus Google Scholar, K. J. S. J. Mol. Endocrinol. PubMed Scopus Google Scholar, M. Proc. Natl. Acad. Sci. U.S.A. 2008; PubMed Scopus Google Scholar, M. G. M. J. Steroid Biochem. 32: PubMed Scopus Google Scholar). of a number of GR binding sequences in genes that are regulated by glucocorticoids a GRE which has an with a of have found that of GR binding sequences to the GRE as of the of GR binding sequences the and the of genes are genes J. Steroid Biochem. Mol. Biol. PubMed Scopus Google Scholar). The active GRE within the regulatory regions of the Vdr gene we the the GRE described is an containing a of hormone and with nuclear an important role in to different between nuclear or and expression of nuclear at both the transcriptional or post-transcriptional levels (9Bagamasbad P. Denver R.J. Gen. Comp. Endocrinol. 2011; 170: 3-17Crossref PubMed Scopus (81) Google Scholar). In our we found that Dex increases VDR by increasing transcription of Vdr gene in a GR-dependent manner. VDR levels with Dex have also been shown in and kidney and endothelial cells G. Chung I. Yu W.D. Romano M. Modzelewski R.A. Johnson C.S. Trump D.L. Oncology. 2006; 70: 447-457Crossref PubMed Scopus (38) Google Scholar, 17Sun X. Zemel M.B. Int. J. Obes. 2008; 32: 1305-1311Crossref PubMed Scopus (32) Google Scholar, Lee G.S. Sci. PubMed Scopus (32) Google Scholar). In support of between VDR and GR was that calcitriol glucocorticoid in human through increasing which the of the glucocorticoid to Zemel M.B. Obes. Res. PubMed Scopus Google Scholar). More recently, the group an increase in Vdr expression with Dex treatment and of glucocorticoids with calcitriol treatment using the X. Zemel M.B. Int. J. Obes. 2008; 32: 1305-1311Crossref PubMed Scopus (32) Google Scholar). and our a between GR and where glucocorticoids to GR to directly increase Vdr transcription. In we that dexamethasone increases VDR at the transcriptional in a GR-dependent and that GR with Dex binds to an on the distal regulatory regions of mouse the between VDR and GR is of to the of that calcitriol and of Vdr expression by Dex has been in the where Dex treatment to a in expression of Vdr Lee G.S. Sci. PubMed Scopus (32) Google Scholar). with the role of Dex calcitriol Dex VDR-mediated expression of calcium genes in the mouse effects of glucocorticoids and its role in by calcium in the Dex potentiates calcitriol anti-proliferative effects in Introduction1α,25-Dihydroxycholecalciferol (calcitriol), the most active metabolite of vitamin D, is a seco-steroid hormone that regulates calcium homeostasis, bone development, and bone maintenance (1Dhawan P. Christakos S. J. Cell. Biochem. 2010; 110: 1314-1323Crossref PubMed Scopus (54) Google Scholar, 2Kallay E. Pietschmann P. Toyokuni S. Bajna E. Hahn P. Mazzucco K. Bieglmayer C. Kato S. Cross H.S. Carcinogenesis. 2001; 22: 1429-1435Crossref PubMed Scopus (157) Google Scholar). Calcitriol has potent anti-proliferative effects in a number of human and murine cancer cell types in vitro and in in vivo xenograft tumor models. Calcitriol inhibits growth in breast, colon, squamous cell carcinoma, and prostate cancer models (3Trump D.L. Potter D.M. Muindi J. Brufsky A. Johnson C.S. Cancer. 2006; 106: 2136-2142Crossref PubMed Scopus (94) Google Scholar). More recently, in support of calcitriol as an anti-angiogenesis therapy, our laboratory has described that tumor-derived endothelial cells are more sensitive to the anti-proliferative effect of calcitriol compared with non-tumor-derived endothelial cells (4Chung I. Karpf A.R. Muindi J.R. Conroy J.M. Nowak N.J. Johnson C.S. Trump D.L. J. Biol. Chem. 2007; 282: 8704-8714Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 5Chung I. Yu W.D. Karpf A.R. Flynn G. Bernardi R.J. Modzelewski R.A. Johnson C.S. Trump D.L. J. Steroid Biochem. Mol. Biol. 2007; 103: 768-770Crossref PubMed Scopus (22) Google Scholar, 6Flynn G. Chung I. Yu W.D. Romano M. Modzelewski R.A. Johnson C.S. Trump D.L. Oncology. 2006; 70: 447-457Crossref PubMed Scopus (38) Google Scholar).The mechanism by which calcitriol exerts its anti-proliferative action through cell cycle arrest, cell differentiation, and induction of apoptosis varies between different tumor cell types (7Deeb K.K. Trump D.L. Johnson C.S. Nat. Rev. Cancer. 2007; 7: 684-700Crossref PubMed Scopus (1108) Google Scholar). The anti-proliferative effect of calcitriol depends on the levels of the vitamin D receptor (VDR), 3The abbreviations used are: VDRvitamin D receptorDexdexamethasoneSCCsquamous cell carcinomaGRglucocorticoid receptorGREglucocorticoid response elementActDactinomycin DCHXcycloheximide. a member of the steroid receptor super family. VDR acts as a ligand-inducible transcription factor. Upon binding to its ligand, calcitriol, the VDR-calcitriol complex binds to DNA sequences called vitamin D response elements located in regulatory regions of target genes to either increase or decrease transcription (7Deeb K.K. Trump D.L. Johnson C.S. Nat. Rev. Cancer. 2007; 7: 684-700Crossref PubMed Scopus (1108) Google Scholar, 8Mangelsdorf 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 (6026) Google Scholar). VDR is expressed in many tissues and cell types. Calcitriol plays an important role in regulating VDR at both the transcriptional and post-transcriptional levels. In support of a direct transcriptional mechanism, functional vitamin D response elements have been found within intronic regions of Vdr gene (9Bagamasbad P. Denver R.J. Gen. Comp. Endocrinol. 2011; 170: 3-17Crossref PubMed Scopus (81) Google Scholar, 10Zella L.A. Kim S. Shevde N.K. Pike J.W. Mol. Endocrinol. 2006; 20: 1231-1247Crossref PubMed Scopus (140) Google Scholar). Calcitriol up-regulation of VDR is also observed in cells where calcitriol does not induce transcription of VDR gene; however, the mechanisms by which calcitriol increases VDR protein in those cells remain unknown. Calcitriol induces phosphorylation of VDR at serine residues −51 and −208 to maintain VDR in a transcriptionally active state. In addition, phosphorylated serine 208 has been implicated in the recruitment of co-activators to increase gene expression (11Arriagada G. Paredes R. Olate J. van Wijnen A. Lian J.B. Stein G.S. Stein J.L. Onate S. Montecino M. J. Steroid Biochem. Mol. Biol. 2007; 103: 425-429Crossref PubMed Scopus (31) Google Scholar, 12Hsieh 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 (191) Google Scholar, 13Jurutka P.W. Hsieh J.C. MacDonald P.N. Terpening C.M. Haussler C.A. Haussler M.R. Whitfield G.K. J. Biol. Chem. 1993; 268: 6791-6799Abstract Full Text PDF PubMed Google Scholar).VDR expression is tightly regulated at the transcriptional level. In addition, the complexity of VDR gene regulatory regions makes its study challenging because of a complex 5′ exonic region, which has partial homology between human and mouse (14Halsall J.A. Osborne J.E. Hutchinson P.E. Pringle J.H. J. Steroid Biochem. Mol. Biol. 2007; 103: 352-356Crossref PubMed Scopus (11) Google Scholar, 15Zella L.A. Meyer M.B. Nerenz R.D. Lee S.M. Martowicz M.L. Pike J.W. Mol. Endocrinol. 2010; 24: 128-147Crossref PubMed Scopus (117) Google Scholar). Expression of Vdr is regulated by several transcription factors including VDR itself. Our group and others have shown that treatment with glucocorticoids increases VDR levels in adipocytes, SCC, and kidney (2Kallay E. Pietschmann P. Toyokuni S. Bajna E. Hahn P. Mazzucco K. Bieglmayer C. Kato S. Cross H.S. Carcinogenesis. 2001; 22: 1429-1435Crossref PubMed Scopus (157) Google Scholar, 16Bernardi R.J. Trump D.L. Yu W.D. McGuire T.F. Hershberger P.A. Johnson C.S. Clin. Cancer Res. 2001; 7: 4164-4173PubMed Google Scholar, 17Sun X. Zemel M.B. Int. J. Obes. 2008; 32: 1305-1311Crossref PubMed Scopus (32) Google Scholar, 18Yu W.D. McElwain M.C. Modzelewski R.A. Russell D.M. Smith D.C. Trump D.L. Johnson C.S. J. Natl. Cancer Inst. 1998; 90: 134-141Crossref PubMed Scopus (92) Google Scholar). Glucocorticoids are a class of steroid hormones that exert their actions though binding to the glucocorticoid receptor (GR). GR is also a member of the ligand-activated nuclear receptor group of transcription factors. GR binds with high affinity to glucocorticoids, as and Dex, to increase or transcription of target genes Proc. Natl. Acad. Sci. U.S.A. 106: PubMed Scopus Google Scholar). GR a and to the where binds to sequences glucocorticoids response elements (GREs) in regulatory regions of target genes to either increase or decrease expression (9Bagamasbad P. Denver R.J. Gen. Comp. Endocrinol. 2011; 170: 3-17Crossref PubMed Scopus (81) Google Scholar, E. R. K. J. J.A. R. PubMed Scopus Google Scholar).The in the of calcitriol as a cancer in has been effects PubMed Scopus (92) Google Scholar). as have effects that decrease calcium and of calcium Lee G.S. Sci. PubMed Scopus (32) Google Scholar, Lee G.S. PubMed Scopus Google Scholar, R.D. E. J. Clin. PubMed Scopus Google Scholar, S. R. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). In of Dex expression of calcium regulatory genes including expression of receptor member and Lee G.S. Sci. PubMed Scopus (32) Google of the effects of Dex, our laboratory has the effects of calcitriol and Dex combination to calcitriol and the effects of calcitriol (3Trump D.L. Potter D.M. Muindi J. Brufsky A. Johnson C.S. Cancer. 2006; 106: 2136-2142Crossref PubMed Scopus (94) Google Scholar). We have shown that Dex the squamous cell cells to the effect of calcitriol, increases and increases VDR protein R.J. Trump D.L. Yu W.D. McGuire T.F. Hershberger P.A. Johnson C.S. Clin. Cancer Res. 2001; 7: 4164-4173PubMed Google Scholar, 18Yu W.D. McElwain M.C. Modzelewski R.A. Russell D.M. Smith D.C. Trump D.L. Johnson C.S. J. Natl. Cancer Inst. 1998; 90: 134-141Crossref PubMed Scopus (92) Google Scholar). Treatment with in a decrease of and treatment with either calcitriol or Dex of apoptosis was observed by of and combination treatment of and compared with treatment with either calcitriol or Dex R.J. Trump D.L. Yu W.D. McGuire T.F. Hershberger P.A. Johnson C.S. Clin. Cancer Res. 2001; 7: 4164-4173PubMed Google that treatment with glucocorticoids increase VDR protein expression G. Chung I. Yu W.D. Romano M. Modzelewski R.A. Johnson C.S. Trump D.L. Oncology. 2006; 70: 447-457Crossref PubMed Scopus (38) Google Scholar, 16Bernardi R.J. Trump D.L. Yu W.D. McGuire T.F. Hershberger P.A. Johnson C.S. Clin. Cancer Res. 2001; 7: 4164-4173PubMed Google Scholar, 18Yu W.D. McElwain M.C. Modzelewski R.A. Russell D.M. Smith D.C. Trump D.L. Johnson C.S. J. Natl. Cancer Inst. 1998; 90: 134-141Crossref PubMed Scopus (92) Google however, the mechanisms by which glucocorticoids VDR expression to L.A. Meyer M.B. Nerenz R.D. Lee S.M. Martowicz M.L. Pike J.W. Mol. Endocrinol. 2010; 24: 128-147Crossref PubMed Scopus (117) Google used chromatin immunoprecipitation by to in regulatory sequences of the mouse Vdr In the we that VDR is a target of the of Vdr we the glucocorticoid dexamethasone induces expression of mouse Vdr gene in the cell We Dex anti-proliferative and VDR-mediated effects of We also Dex treatment increases expression of Vdr at a transcriptional and expression of VDR target we Dex induces Vdr gene expression in a GR-dependent through binding to a GRE located upstream of transcription start site of Vdr.