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• W2003980854 abstract "Because sex steroids regulate the life span of bone cells by modulating cytoplasmic kinase activity via a nongenotropic action of their classical receptors, we have explored the possibility that the vitamin D nuclear receptor (VDR) might exhibit similar nongenotropic actions. We report that the conformationally flexible full VDR agonist, 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3), and the 6-s-cis-locked 1α,25(OH)2-lumisterol3 (JN) analog, also acting through the VDR but with poor transcriptional activity, protected murine osteoblastic or osteocytic cells from apoptosis. This effect was reproduced in HeLa cells transiently transfected with either wild type VDR or a mutant consisting of only the VDR ligand binding domain. The VDR ligand binding domain bound [3H]1α,25(OH)2D3 as effectively as wild type VDR but did not induce vitamin D response element-mediated transcription. The anti-apoptotic effects of 1α,25(OH)2D3 and the 6-s-cis-locked 1α,25(OH)2-lumisterol3 analog in calvaria cells were blocked by three cytoplasmic kinase inhibitors: Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), phosphatidylinositol 3 kinase inhibitor Wortmannin, and the JNK kinase inhibitor SP600125. However, inhibition of p38 with SB203580 or ERK with either U0126 or a transfected dominant negative MEK did not interfere with these anti-apoptotic actions. Further, 1α,25(OH)2D3 induced rapid (5 min) association of VDR with Src kinase in OB-6 cells. Finally, actinomycin D or cycloheximide prevented the anti-apoptotic effect of 1α,25(OH)2D3, indicating that transcriptional events are also required. These findings suggest that nongenotropic modulation of kinase activity is also a general property of the VDR and that ligands that activate nongenotropic signals, but lack transcriptional activity, display different biological profiles from the steroid hormone 1α,25(OH)2D3. Because sex steroids regulate the life span of bone cells by modulating cytoplasmic kinase activity via a nongenotropic action of their classical receptors, we have explored the possibility that the vitamin D nuclear receptor (VDR) might exhibit similar nongenotropic actions. We report that the conformationally flexible full VDR agonist, 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3), and the 6-s-cis-locked 1α,25(OH)2-lumisterol3 (JN) analog, also acting through the VDR but with poor transcriptional activity, protected murine osteoblastic or osteocytic cells from apoptosis. This effect was reproduced in HeLa cells transiently transfected with either wild type VDR or a mutant consisting of only the VDR ligand binding domain. The VDR ligand binding domain bound [3H]1α,25(OH)2D3 as effectively as wild type VDR but did not induce vitamin D response element-mediated transcription. The anti-apoptotic effects of 1α,25(OH)2D3 and the 6-s-cis-locked 1α,25(OH)2-lumisterol3 analog in calvaria cells were blocked by three cytoplasmic kinase inhibitors: Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), phosphatidylinositol 3 kinase inhibitor Wortmannin, and the JNK kinase inhibitor SP600125. However, inhibition of p38 with SB203580 or ERK with either U0126 or a transfected dominant negative MEK did not interfere with these anti-apoptotic actions. Further, 1α,25(OH)2D3 induced rapid (5 min) association of VDR with Src kinase in OB-6 cells. Finally, actinomycin D or cycloheximide prevented the anti-apoptotic effect of 1α,25(OH)2D3, indicating that transcriptional events are also required. These findings suggest that nongenotropic modulation of kinase activity is also a general property of the VDR and that ligands that activate nongenotropic signals, but lack transcriptional activity, display different biological profiles from the steroid hormone 1α,25(OH)2D3. Changes in the birth or death of osteoblasts and/or osteoclasts represent fundamental pathophysiologic changes in most acquired metabolic bone diseases, including the osteoporosis that results from sex steroid deficiency, glucocorticoid excess, or old age (1.Manolagas S.C. Endocr. Rev. 2000; 21: 115-137Crossref PubMed Scopus (2008) Google Scholar, 2.Kousteni S. Chen J.-R. Bellido T. Han L. Ali A.A. O'Brien C. Plotkin L.I. Fu Q. Mancino A.T. Wen Y. Vertino A.M. Powers C.C. Stewart S.A. Ebert R. Parfit A.M. Weinstein R.S. Jilka R.L. Manolagas S.C. Science. 2002; 298: 843-846Crossref PubMed Scopus (388) Google Scholar, 3.Jilka R.L. Hangoc G. Girasole G. Passeri G. Williams D.C. Abrams J.S. Boyce B. Broxmeyer H. Manolagas S.C. Science. 1992; 257: 88-91Crossref PubMed Scopus (1287) Google Scholar, 4.Weinstein R.S. Jilka R.L. Parfitt A.M. Manolagas S.C. J. Clin. Investig. 1998; 102: 274-282Crossref PubMed Scopus (1408) Google Scholar, 5.Weinstein R.S. Chen J.R. Powers C.C. Stewart S.A. Landes R.D. Bellido T. Jilka R.L. Parfitt A.M. Manolagas S.C. J. Clin. Investig. 2002; 109: 1041-1048Crossref PubMed Scopus (332) Google Scholar, 6.Jilka R.L. Weinstein R.S. Bellido T. Roberson P. Parfitt A.M. Manolagas S.C. J. Clin. Investig. 1999; 104: 439-446Crossref PubMed Scopus (895) Google Scholar). Furthermore, pharmacotherapeutics used commonly for the treatment of metabolic bone diseases exert their beneficial effects on bone by regulating the rate of birth of new osteoclasts or osteoblasts or their apoptosis (6.Jilka R.L. Weinstein R.S. Bellido T. Roberson P. Parfitt A.M. Manolagas S.C. J. Clin. Investig. 1999; 104: 439-446Crossref PubMed Scopus (895) Google Scholar, 7.Plotkin L.I. Weinstein R.S. Parfitt A.M. Roberson P.K. Manolagas S.C. Bellido T. J. Clin. Investig. 1999; 104: 1363-1374Crossref PubMed Scopus (784) Google Scholar, 8.Bellido T. Huening M. Raval-Pandya M. Manolagas S.C. Christakos S. J. Biol. Chem. 2000; 275: 26328-26332Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). We have recently shown that estrogens and androgens, acting via their classical nuclear receptors (ERα, 1The abbreviations used are: ER, estrogen receptor; AR, androgen receptor; PI3K, phosphatidylinositol 3-kinase; VDR, vitamin D receptor; wtVDR, wild type VDR; hVDR, human VDR; VDRE, vitamin D-responsive element; RXR, retinoic acid receptor; PPI, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; LBD, ligand binding domain; GFP, green fluorescent protein; ECFP, enhanced cyan fluorescent protein; JN, 6-s-cis-locked 1α,25(OH)2-lumisterol3; JM, 1α,25(OH)2-7-dehydrocholesterol; JB, 1α,25(OH)-dihydrotachysterol3; JD, 1α,25(OH)2-trans-isotachysterol3; HL, 1α,25(OH)2-lumisterol (JN), 1β,25(OH)2D3; MK, (23S)-25-dehydro-1α-dihydroxyvitamin D3-26,23-lactone; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; E2, 17β estradiol; SH, Src homology. ERβ, or AR), attenuate the apoptosis of several different cell types, including osteoblasts and osteocytes, by rapidly activating the Src/Shc/ERK and phosphatidylinositol 3-kinase (PI3K) and down-regulating the JNK signaling pathways. This effect requires only the ligand binding, not the DNA binding, domain of the receptor, and, unlike its classical transcriptional action, it is eliminated by nuclear targeting of the receptor (9.Kousteni S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han K. DiGregorio G. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jilka R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar). Activation of ERKs leads to the rapid translocation of the kinases into the nucleus where they phosphorylate common transcription factors like Elk-1, CCAAT enhancer-binding protein-β, and cAMP-response element-binding protein. These transcription factors in turn up-regulate gene expression, as exemplified by the up-regulation of the early growth response-1 protein gene, an ERK/serum response element target gene. Likewise, suppression of the JNK signaling cascade by sex steroids leads to down-regulation of c-Jun expression (10.Kousteni S. Han L. Chen J.-R. Almeida M. Plotkin L.I. Bellido T. Manolagas S.C. J. Clin. Investig. 2003; 111: 1651-1664Crossref PubMed Scopus (245) Google Scholar). We have earlier used a ligand that potently and selectively activates nongenotropic actions of the classical ER or AR and thereby activates kinases and their downstream transcription factors and target genes with only minimal effects on classical estrogen response element-mediated genotropic transcription. Furthermore, we have demonstrated that, although such classical genotropic actions of sex steroid receptors are essential for their effects on reproductive tissues, they are dispensable for their bone protective effects (2.Kousteni S. Chen J.-R. Bellido T. Han L. Ali A.A. O'Brien C. Plotkin L.I. Fu Q. Mancino A.T. Wen Y. Vertino A.M. Powers C.C. Stewart S.A. Ebert R. Parfit A.M. Weinstein R.S. Jilka R.L. Manolagas S.C. Science. 2002; 298: 843-846Crossref PubMed Scopus (388) Google Scholar). 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3) regulates gene transcription (genomic responses) acting through its classical nuclear vitamin D receptor (VDR) and also elicits a variety of nongenotropic rapid responses acting through a membrane-associated vitamin D receptor (11.Norman A.W. Bishop J.E. Bula C.M. Olivera C.J. Mizwicki M.T. Zanello L.P. Ishida H. Okamura W.H. Steroids. 2002; 67: 457-466Crossref PubMed Scopus (75) Google Scholar, 12.Huhtakangas J.A. Olivera C.J. Bishop J.E. Zanello L.P. Norman A.W. Mol. Endocrinol. 2004; 18: 2660-2671Crossref PubMed Scopus (298) Google Scholar). Notable rapid responses include activation of mitogen-activated protein kinase in human leukemia NB4 cells (13.Song X. Bishop J.E. Okamura W.H. Norman A.W. Endocrinology. 1998; 139: 457-465Crossref PubMed Scopus (113) Google Scholar) and in growth plate chondrocytes, release of insulin from pancreatic β-cells (14.Kajikawa M. Ishida H. Fujimoto S. Mukai E. Nishimura M. Fujita J. Tsuura Y. Okamoto Y. Norman A.W. Seino Y. Endocrinology. 1999; 140: 4706-4712Crossref PubMed Google Scholar), and stimulation of intestinal calcium transport (transcaltachia) (15.Schwartz Z. Ehland H. Sylvia V.L. Larsson D. Hardin R.R. Bingham V. Lopez D. Dean D.D. Boyan B.D. Endocrinology. 2002; 143: 2775-2786Crossref PubMed Scopus (68) Google Scholar, 16.Nemere I. Yoshimoto Y. Norman A.W. Endocrinology. 1984; 115: 1476-1483Crossref PubMed Scopus (240) Google Scholar). In bone, 1α,25(OH)2D3 elicits physiological responses at both the genomic level (de novo production of the bone matrix proteins osteocalcin and osteopontin) and the nongenotropic level (modulation of the electrical activity of CI− and Ca2+ channels in osteoblasts or protein kinase C activation in chondrocytes) (17.Zanello L.P. Norman A.W. J. Biol. Chem. 1997; 272: 22617-22622Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 18.Zanello L.P. Norman A.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1589-1594Crossref PubMed Scopus (137) Google Scholar, 19.Boyan B.D. Jennings E.G. Wang L. Schwartz Z. J. Steroid Biochem. Mol. Biol. 2004; 89-90: 309-315Crossref PubMed Scopus (20) Google Scholar, 20.Boyan B.D. Schwartz Z. Steroids. 2004; 69: 591-597Crossref PubMed Scopus (36) Google Scholar). In our earlier studies, we demonstrated that 1α,25(OH)2D3 prevented apoptosis of HeLa cells that were transfected either with the VDR or the retinoic acid receptor (RXR), but not in untransfected cells or cells transfected with the ERα or the AR (9.Kousteni S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han K. DiGregorio G. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jilka R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar). 1α,25(OH)2D3 has also been shown to inhibit ultraviolet B-induced apoptosis and Jun kinase activation in human keratinocytes (21.De Haes P. Garmyn M. Degreef H. Vantieghem K. Bouillon R. Segaert S. J. Cell. Biochem. 2003; 89: 663-673Crossref PubMed Scopus (117) Google Scholar) and also to up-regulate Bcl-2 expression and protect thyrocytes from apoptosis (22.Wang S.H. Koenig R.J. Giordano T.J. Myc A. Thompson N.W. Baker Jr., J.R. Endocrinology. 1999; 140: 1649-1656Crossref PubMed Scopus (41) Google Scholar). In the present study, we have followed up these preliminary observations and show that 1α,25(OH)2D3, as well as other natural metabolites of vitamin D and synthetic analogs of 1α,25(OH)2D3 incapable of inducing vitamin D-responsive element (VDRE)-mediated transcription, attenuates osteoblast and osteocyte apoptosis. Chemicals—1α,25(OH)2D3 and the vitamin D3 metabolites 24R,25(OH)2D3 and 25(OH)D3 were obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). The 1α,25(OH)2D3 analogs 1α,25(OH)-dihydrotachysterol3 (JB), 1α,25(OH)2-trans-isotachysterol3 (JD), 1α,25(OH)2-7-dehydrocholesterol (JM), 1α,25(OH)2-lumisterol (JN), 1β,25(OH)2D3 (HL), and (23S)-25-dehydro-1α-dihydroxyvitamin D3-26,23-lactone (MK) were obtained from Dr. William H. Okamura University of California, Riverside, CA. 17β estradiol (E2), etoposide, and cycloheximide were purchased from Sigma. PP1, SB-203580 Wortmannin, and actinomycin D were purchased from A.G. Scientific, Inc. (San Diego, CA). U0126 was purchased from Promega (Madison, WI). Cell Cultures—Osteoblastic cells were isolated from neonatal murine calvaria and cultured as previously described (3.Jilka R.L. Hangoc G. Girasole G. Passeri G. Williams D.C. Abrams J.S. Boyce B. Broxmeyer H. Manolagas S.C. Science. 1992; 257: 88-91Crossref PubMed Scopus (1287) Google Scholar). OB-6 cells, HeLa cells, and osteocytic MLOY-4 cells were cultured as previously described (2.Kousteni S. Chen J.-R. Bellido T. Han L. Ali A.A. O'Brien C. Plotkin L.I. Fu Q. Mancino A.T. Wen Y. Vertino A.M. Powers C.C. Stewart S.A. Ebert R. Parfit A.M. Weinstein R.S. Jilka R.L. Manolagas S.C. Science. 2002; 298: 843-846Crossref PubMed Scopus (388) Google Scholar, 3.Jilka R.L. Hangoc G. Girasole G. Passeri G. Williams D.C. Abrams J.S. Boyce B. Broxmeyer H. Manolagas S.C. Science. 1992; 257: 88-91Crossref PubMed Scopus (1287) Google Scholar, 4.Weinstein R.S. Jilka R.L. Parfitt A.M. Manolagas S.C. J. Clin. Investig. 1998; 102: 274-282Crossref PubMed Scopus (1408) Google Scholar, 10.Kousteni S. Han L. Chen J.-R. Almeida M. Plotkin L.I. Bellido T. Manolagas S.C. J. Clin. Investig. 2003; 111: 1651-1664Crossref PubMed Scopus (245) Google Scholar). Osteoblastic and osteocytic cells were treated at 80% confluence with either vehicle or the appropriate kinase inhibitor for 30 min prior to the addition of 1α,25(OH)2D3 or the indicated 1α,25(OH)2D3 metabolites or its synthetic analogs for an additional 1 h in the presence of 10% serum. To induce apoptosis, etoposide was added to the cells (final concentration of 50 and 100 μm for calvaria or MLO-Y4 cells and OB-6 cells, respectively), and the cultures were continued for 6 h. CV-1 monkey kidney cells were seeded at 1.5 × 106 cells/150-mm culture dishes and cultured in minimal Eagle's medium with Earl's buffered salts and non-essential amino acids (Mediatech Inc., Herndon, VA) with 10% fetal bovine serum (Mediatech Inc., Herndon, VA). Cos-1 monkey kidney cells were seeded at 1 × 106 cells/150-mm culture dishes in Dulbecco's modified Eagle's medium (Mediatech Inc.) with 10% fetal bovine serum. Plasmids—pcDNA plasmid was purchased from Invitrogen. The human VDR and RXR plasmids were obtained from D. McDonnell (Duke University, Durham, NC) and D. Mangelsdorf (University of Texas, Southwestern Medical Center, Dallas, TX), respectively. The nuclear green fluorescent protein (GFP) was obtained by attaching the SV40 large T antigen nuclear localization sequence to the N terminus of the cDNA construct encoding GFP (7.Plotkin L.I. Weinstein R.S. Parfitt A.M. Roberson P.K. Manolagas S.C. Bellido T. J. Clin. Investig. 1999; 104: 1363-1374Crossref PubMed Scopus (784) Google Scholar). Wild type ERα was obtained from B. Katzenellenbogen (University of Illinois, Urbana, IL). The wild type and dominant negative MEK plasmids were donated by Natalie Ahn from the University of Colorado at Boulder (23.Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande W.G. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1260) Google Scholar). The VDRE-secreted alkaline phosphatase (SEAP) reporter was constructed by removing the promoter and untranslated regions from the osteocalcin VDRE-human growth hormone reporter and cloning into the pSEAP2-Basic plasmid (Stratagene, Palo Alto, CA) (24.Liu Y.Y. Collins E.D. Norman A.W. Peleg S. J. Biol. Chem. 1997; 272: 3336-3345Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). This reporter contains the osteocalcin gene VDRE (235-219 bp with respect to the transcription start site) and the calcitonin promoter, having both the Octomer element (167-149 bp) and the Sp1 element (81-76 bp). An N-terminal fluorescent tag was positioned in-frame with the wtVDR or VDR ligand binding domain (LBD-VDR) by using PCR-generated restriction enzyme-flanked inserts included into the pECFP-Nuc plasmid (Stratagene). The full-length wtVDR (residues 4-427) was inserted into pECFP-Nuc, and the nuclear localization signal was removed. The LBD-VDR residues 119-427 were inserted into the pECFP-NUC plasmid, and the nuclear localization signal was also removed. Expression of the plasmid constructs was confirmed by fluorescent microscopy. Transient Transfections—HeLa cells were transiently transfected at 80% confluency with 0.1 μg of nuclear GFP and 0.1 μg of either ERα, wtVDR, VDR 119-C, RXR, or pCDNA using Lipofectamine Plus (Invitrogen). Cells were allowed to recover overnight and then treated with vehicle, 1α,25(OH)2D3, or the indicated 1α,25(OH)2D3 analog 1 h prior to exposure to stimulation of apoptosis by etoposide. Subsequently, etoposide was added to a final concentration of 100 μm, and the cultures were continued (in the presence of the D-compounds or vehicle) for an additional 6 h. Transient transfection of CV-1 cells was performed at 60% confluency and involved 10 min of pretreatment with 0.2 mg/ml DEAE-dextran (Sigma) in phosphate-buffered saline. Pretreated cells were washed in phosphate-buffered saline and incubated for 30 min with phosphate-buffered saline containing 0.1 μg/well pcDNA3, wt hVDR, or the appropriate mutants of hVDR and 0.5 μg/well of the VDRE-Luc reporter. Transfected cells were incubated in 80 μm chloroquine in minimal Eagle's medium with Earl's buffered salts and nonessential amino acids (Mediatech Inc., Herndon, VA) with 4.5% charcoal-stripped fetal bovine serum for 4 h followed by the same culture medium without chloroquine for 24 h. Twenty-eight hours after transfection, the cell medium was replaced with the same medium containing vehicle or 1α,25(OH)2D3. Twenty-two hours later, cell medium was harvested to measure secreted alkaline phosphatase using the Phospha-Light™ kit (Tropix, Bedford, MA). The data are presented as dose response curves of the reporter with raw luminometer units plotted versus the log dose of ligand. All experiments were carried out on quadruplicate samples with data expressed as the mean ± S.E. Quantification of Apoptotic Cells in Vitro—HeLa cell apoptosis was quantified by direct visualization of changes in nuclear morphology as previously described (6.Jilka R.L. Weinstein R.S. Bellido T. Roberson P. Parfitt A.M. Manolagas S.C. J. Clin. Investig. 1999; 104: 439-446Crossref PubMed Scopus (895) Google Scholar, 9.Kousteni S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han K. DiGregorio G. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jilka R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar, 10.Kousteni S. Han L. Chen J.-R. Almeida M. Plotkin L.I. Bellido T. Manolagas S.C. J. Clin. Investig. 2003; 111: 1651-1664Crossref PubMed Scopus (245) Google Scholar, 25.Jilka R.L. Weinstein R.S. Bellido T. Parfitt A.M. Manolagas S.C. J. Bone. Miner. Res. 1998; 13: 793-802Crossref PubMed Scopus (471) Google Scholar). Visualization of pyknotic or fragmented nuclei was facilitated by co-transfections of cells plated on glass coverslips with nuclear GFP. The percentage of apoptosis was determined by determining the nuclear morphology in 200-500 transfected (fluorescent) cells. Apoptosis of calvaria cells, OB-6 osteoblastic cells, and MLO-Y4 osteocytes was quantified by trypan blue staining. Equilibrium Saturation Binding Assay—Cos-1 cells were grown to 80% confluency and were then transfected with 3 μg of pcDNA3, wt hVDR, or the appropriate mutants of hVDR as described for CV-1 cells. Sixty-six hours following transfection, cells were washed twice in phosphate-buffered saline and harvested by scraping in TED (10 mm Tris-HCl, 1 mm EDTA, 1 mm dithiothreitol, pH 7.4) hypotonic buffer with Complete EDTA-free protease inhibitor mixture (Roche Diagnostics). Cos-1 cell lysate (0.2 ml) was incubated with increasing concentrations of [3H]1α,25(OH)2D3 106 Ci/mmol in the presence or absence of excess nonradioactive 1α,25(OH)2D3 for 4 h at 0 °C. Samples were performed in triplicate. After equilibrium was established, 200 μl of 50% hydroxyapatite solution in TED was added and washed three times in 1 ml of TED plus 0.5% Triton-X100. Bound [3H]1α,25(OH)2D3 was eluted with 1 ml of ethanol, and the tritium content was determined in 7 ml of Liquiscint™ scintillation mixture using a Beckman LS6500. The dissociation constant KD for binding of 1α,25(OH)2D3 to a given VDR construct was determined by non-linear regression of the curve generated by plotting specific binding versus concentration of [3H]1α,25(OH)2D3 using a one-site binding equation (26.Bula C.M. Bishop J.E. Ishizuka S. Norman A.W. Mol. Endocrinol. 2000; 14: 1788-1796PubMed Google Scholar). Immunoprecipitation of the Src/VDR Complex and Western Blot Analysis—OB-6 osteoblastic cells were treated with 1α,25(OH)2D3 for the indicated periods of time, and cells were lyzed in a buffer containing 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 5 μg/ml leupeptin, 0.14 units/ml aprotinin, 10 mm NaF, 1 mm NA orthovanadate, 1 mm phenylmethylsulfonyl fluoride, and 1% Triton X-100. Cell lysates were collected and were either used to determine total protein concentration with the Bio-Rad 500-0001 protein assay kit or were incubated with a rabbit polyclonal anti-VDR antibody N-20 (SC-1009; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. An equal amount of goat anti-rabbit IgG antibody (SC-2004; Santa Cruz Biotechnology) was added to the lysate for 30 min, followed by the addition of 50% suspension of protein G-Sepharose for 30 min. Samples were centrifuged, and the pellets were size fractionated with SDS-PAGE on a 10% gel. Immunoblotting was performed using a mouse monoclonal anti-v-Src antibody (1:2000, catalogue number OP07; Oncogene Research Products, San Diego, CA). The primary antibody was detected with a horseradish peroxidase-conjugated secondary antibody (1:3000; Santa Cruz Biotechnology) and enhanced chemiluminescence using SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). The phosphorylation status of ERK1/2 in OB-6 cells was analyzed by immunoblotting. The antibodies used were a mouse monoclonal antibody recognizing tyrosine-phosphorylated ERK1/2 and a rabbit polyclonal antibody recognizing total ERK1/2 (Santa Cruz Biotechnology). Statistical Analysis—The data were analyzed by analysis of variance, and the Student-Newman-Kleuss method was used to estimate the level of significance of differences between means. Attenuation of HeLa and Calvaria Cell Apoptosis by 1α,25(OH)2D3 and Other Vitamin D Metabolites—To confirm our earlier observations indicating that 1α,25(OH)2D3 attenuates apoptosis by a VDR- or RXR-dependent mechanism, we performed a dose response study of the effects of 1α,25(OH)2D3 in HeLa cells transiently transfected with VDR or RXR. Pretreatment of the cells for 1 h with the hormone dose dependently attenuated HeLa cell apoptosis induced by 6 h of treatment with etoposide (Fig. 1A). As in our earlier studies, 1α,25(OH)2D3 had no effect on the apoptosis of HeLa cells transfected with an empty vector. In full support of the findings for the HeLa cells, 1α,25(OH)2D3 as well as 24,25(OH)2D3 or 25(OH)D3, but not vehicle, protected primary cultures of osteoblastic cells derived from murine calvaria from apoptosis induced by etoposide. As in the case of the HeLa cells, this effect was dose dependent at concentrations ranging from 10−12 to 10−7 m (Fig. 1B). The anti-apoptotic effect of 1α,25(OH)2D3 (at 10−8 m) was reproduced in both the OB-6 osteoblastic murine cell line and murine MLO-Y4 osteocytic cells, and it was comparable with the anti-apoptotic effect of estradiol in these cell types (Fig. 1C). Synthetic Vitamin D Analogs Exhibit Potent Anti-apoptotic Effects That Are Independent of Their Relative Binding Affinity for the VDR—Numerous synthetic 1α,25(OH)2D3 analogs have been previously classified as genotropic or nongenotropic based on their ability to induce the transcription of VDRE-containing genes like osteocalcin via VDR-VDRE interactions or to induce rapid calcium influxes in the intestine and numerous isolated cell types, via rapid phosphorylation of ERKs (13.Song X. Bishop J.E. Okamura W.H. Norman A.W. Endocrinology. 1998; 139: 457-465Crossref PubMed Scopus (113) Google Scholar). Using six such synthetic analogs (Fig. 2 and Table I), we proceeded to examine whether the anti-apoptotic effects of 1α,25(OH)2D3 result from genotropic or nongenotropic actions of the hormone. As shown in Fig. 3A, the 6-s-cis-locked analog JN prevented etoposide-induced apoptosis in a dose-dependent manner very similar to the dose response curve for 1α,25(OH)2D3 (Fig. 1A). Another 6-s-cis-locked analog, JM, also prevented etoposide-induced apoptosis of calvaria cells as effectively as JN and 1α,25(OH)2D3 (Fig. 3B). Both analogs are potent agonists for the rapid membrane-mediated effects of 1α,25(OH)2D3 but associate poorly with the classical VDR under equilibrium binding conditions (13.Song X. Bishop J.E. Okamura W.H. Norman A.W. Endocrinology. 1998; 139: 457-465Crossref PubMed Scopus (113) Google Scholar, 17.Zanello L.P. Norman A.W. J. Biol. Chem. 1997; 272: 22617-22622Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 27.Norman A.W. Okamura W.H. Hammond M.W. Bishop J.E. Dormanen M.C. Bouillon R. Van Baelen H. Ridall A.L. Daane E. Khoury R. Farach-Carson M.C. Mol. Endocrinol. 1997; 11: 1518-1531Crossref PubMed Scopus (123) Google Scholar). Additionally, the analog HL, an epimer of 1α,25(OH)2D3 (28.Norman A.W. Bouillon R. Farach-Carson M.C. Bishop J.E. Zhou L.X. Nemere I. Zhao J. Muralidharan K.R. Okamura W.H. J. Biol. Chem. 1993; 268: 20022-20030Abstract Full Text PDF PubMed Google Scholar), which is an antagonist of many membrane-initiated rapid effects of 1α,25(OH)2D3, but not genomic effects (11.Norman A.W. Bishop J.E. Bula C.M. Olivera C.J. Mizwicki M.T. Zanello L.P. Ishida H. Okamura W.H. Steroids. 2002; 67: 457-466Crossref PubMed Scopus (75) Google Scholar), was able to prevent apoptosis when used either alone or in combination with JM or JN. On the other hand, the analog MK, which is an antagonist of the nuclear actions of the VDR (29.Miura D. Manabe K. Gao Q. Norman A.W. Ishizuka S. FEBS Lett. 1999; 460: 297-302Crossref PubMed Scopus (52) Google Scholar), was ineffective by itself but was able to block the protective effects of analogs JM and JN (Fig. 3C) and 1α,25(OH)2D3(Fig. 3D) in calvarial cells. The 6-s-trans-locked analogs JB and JD, which are weak agonists of both genotropic and nongenotropic actions, also exhibited a modest anti-apoptotic effect in calvaria cells (Fig. 3D) (27.Norman A.W. Okamura W.H. Hammond M.W. Bishop J.E. Dormanen M.C. Bouillon R. Van Baelen H. Ridall A.L. Daane E. Khoury R. Farach-Carson M.C. Mol. Endocrinol. 1997; 11: 1518-1531Crossref PubMed Scopus (123) Google Scholar). Similar to the cis-analogs, HL prevented apoptosis when used alone or in combination with JB, JD, or 1α,25(OH)2D3 (Fig. 3D). The antagonist MK was able to block the anti-apoptotic effect of 1α,25(OH)2D3, but not that of JB or JD (Fig. 3D).Table IClassification of 1α,25(OH)2D3 analogs as genotropic or nongenotropic, based on certain biological assaysNameNotable structural featureVDR binding (RCI)aRCI or Relative Competitive Index is a measure of the relative ability of an analog to compete with [3H]1α,25(OH)2D3 for binding, under equilibrium conditions, to the genotropic ligand binding pocket of the VDRAssayMode of action (genotropic or nongeonotropic)Key reference(s)%1α,25(OH)2D3Conformationally flexible100Transcaltachia Calcium influx ERK phosphorylation Osteocalcin inductionBoth a genotropic and nongenotropic agonist(13.Song X. Bishop J.E. Okamura W.H. Norman A.W. Endocrinology. 1998; 139: 457-465Crossref PubMed Scopus (113) Google Scholar, 16.Nemere I. Yoshimoto Y. Norman A.W. Endocrinology. 1984; 115: 1476-1483Crossref PubMed Scopus (240) Google Scholar, 27.Norman A.W. Okamura W.H. Hammond M.W. Bishop J.E. Dormanen M.C. Bouillon R. Van Baelen H. Ridall A.L. Daane E. Khoury R. Farach-Carson M.C. Mol. Endocrinol. 1997; 11: 1518-1531Crossref PubMed Scopus (123) Google Scholar)HL 1β,25-(OH)2D3β-epimer of 1α,25(OH)2D30.2Transcaltachia Calcium influx ERK phosphorylation Calbindin inductionNongenotropic antagonist(27.Norman A.W. Okamura W.H. Hammond M.W. Bishop J.E. Dormanen M.C. Bouillon R. Van Baelen H. Ridall A.L. Daane E. Khoury R. Farach-Carson M.C. Mol. Endocrinol. 1997; 11: 1518-1531Crossref PubMed Scopus (123) Google Scholar, 28.Norman A.W. Bouillon R. Farach-Carson M.C. Bishop J.E. Zhou L.X. Nemere I. Zhao J. Muralidharan K.R. Okamura W.H. J. Biol. Chem. 1993; 268: 20022-20030Abstract Full Text PDF PubMed Google Scholar)MK (23S)-25-dehydro-1α-OH-D3-26,23-lactoneSide-chain lacto" @default.
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