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CCAAT Enhancer-binding Protein α Is a Molecular Target of 1,25-Dihydroxyvitamin D3 in MCF-7 Breast Cancer Cells

2008; Elsevier BV; Volume: 284; Issue: 5 Linguagem: Inglês

10.1074/jbc.m803602200

ISSN

1083-351X

Autores

Puneet Dhawan, Robert Weider, Sylvia Christakos,

Tópico(s)

Estrogen and related hormone effects

Resumo

Numerous studies have shown that the active form of vitamin D, 1,25(OH)2D3, can exert growth inhibitory effects on human breast cancer cells and mammary tumor growth. However, the molecular mechanisms remain to be fully delineated. This study demonstrates for the first time that CCAAT enhancer-binding protein α (C/EBPα), a member of the C/EBP family of transcription factors, is induced by 1,25(OH)2D3 and is a potent enhancer of VDR transcription in MCF-7 breast cancer cells. 1,25(OH)2D3 was found to induce C/EBPα as well as VDR expression in MCF-7 cells. C/EBPα was not detected in MDA-MB-231 cells that are poorly responsive to 1,25(OH)2D3. Antiproliferative effects of 1,25(OH)2D3 and induction of VDR were observed in MDA-MB-231 cells transfected with C/EBPα, and knockdown of C/EBPα suppressed VDR and antiproliferative effects of 1,25(OH)2D3 in MCF-7 cells. Transfection of C/EBPα in MCF-7 cells resulted in a dose-dependent enhancement of hVDR transcription. Our studies show that C/EBPα can bind to Brahma (Brm), an ATPase that is a component of the SWI/SNF complex, and cooperate with Brm in the regulation of hVDR transcription in MCF-7 cells. Because the levels of VDR in MCF-7 breast cancer cells correlate with the antiproliferative effects of 1,25(OH)2D3 and because C/EBPα has been suggested as a potential tumor suppressor in breast cancer, these findings provide important mechanisms whereby 1,25(OH)2D3 may act to inhibit growth of breast cancer cells. These findings also identify C/EBPα as a 1,25(OH)2D3 target in breast cancer cells and provide evidence for C/EBPα as a candidate for breast cancer treatment. Numerous studies have shown that the active form of vitamin D, 1,25(OH)2D3, can exert growth inhibitory effects on human breast cancer cells and mammary tumor growth. However, the molecular mechanisms remain to be fully delineated. This study demonstrates for the first time that CCAAT enhancer-binding protein α (C/EBPα), a member of the C/EBP family of transcription factors, is induced by 1,25(OH)2D3 and is a potent enhancer of VDR transcription in MCF-7 breast cancer cells. 1,25(OH)2D3 was found to induce C/EBPα as well as VDR expression in MCF-7 cells. C/EBPα was not detected in MDA-MB-231 cells that are poorly responsive to 1,25(OH)2D3. Antiproliferative effects of 1,25(OH)2D3 and induction of VDR were observed in MDA-MB-231 cells transfected with C/EBPα, and knockdown of C/EBPα suppressed VDR and antiproliferative effects of 1,25(OH)2D3 in MCF-7 cells. Transfection of C/EBPα in MCF-7 cells resulted in a dose-dependent enhancement of hVDR transcription. Our studies show that C/EBPα can bind to Brahma (Brm), an ATPase that is a component of the SWI/SNF complex, and cooperate with Brm in the regulation of hVDR transcription in MCF-7 cells. Because the levels of VDR in MCF-7 breast cancer cells correlate with the antiproliferative effects of 1,25(OH)2D3 and because C/EBPα has been suggested as a potential tumor suppressor in breast cancer, these findings provide important mechanisms whereby 1,25(OH)2D3 may act to inhibit growth of breast cancer cells. These findings also identify C/EBPα as a 1,25(OH)2D3 target in breast cancer cells and provide evidence for C/EBPα as a candidate for breast cancer treatment. Vitamin D, to exert its effects, must be metabolized to its most active form, 1,25-dihydroxyvitaminD3 (1,25(OH)2D3) 2The abbreviations used are: 1,25(OH)2d3, 1,25-dihydroxyvitamin D3; ER, estrogen receptor; VDR, vitamin D receptor; hVDR, human VDR; C/EBPα, CCAAT enhancer-binding protein α; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; DN, dominant negative; CMV, cytomegalovirus; siRNA, small interference RNA; ChIP, chromatin immunoprecipitation.2The abbreviations used are: 1,25(OH)2d3, 1,25-dihydroxyvitamin D3; ER, estrogen receptor; VDR, vitamin D receptor; hVDR, human VDR; C/EBPα, CCAAT enhancer-binding protein α; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; DN, dominant negative; CMV, cytomegalovirus; siRNA, small interference RNA; ChIP, chromatin immunoprecipitation. (1Christakos S. Dhawan P. Liu Y. Peng X. Porta A. J. Cell Biochem... 2003; 88: 695-705Google Scholar). The actions of 1,25(OH)2D3 include not only maintenance of calcium homeostasis but also effects on numerous other systems, including effects on the immune system and the growth and differentiation of a number of malignant cells including breast cancer cells (1Christakos S. Dhawan P. Liu Y. Peng X. Porta A. J. Cell Biochem... 2003; 88: 695-705Google Scholar). 1,25(OH)2D3 acts by binding to a high affinity intracellular receptor protein (the vitamin D receptor or VDR). 1,25(OH)2D3 bound to the VDR heterodimerizes with the retinoid X receptor and along with coactivators and additional accessory nuclear proteins interacts with vitamin D response elements in the promoter of target genes and modulates their transcription (1Christakos S. Dhawan P. Liu Y. Peng X. Porta A. J. Cell Biochem... 2003; 88: 695-705Google Scholar). In vivo studies have suggested a role for vitamin D and 1,25(OH)2D3 in breast cancer prevention as well as in treatment of breast cancer. It has been demonstrated that rats fed diets low in vitamin D and calcium develop significantly more mammary tumors when treated with 7,12-dimethylbenz(a)anthracene than rats fed control diets with adequate vitamin D and calcium (2Jacobson E.A. James K.A. Newmark H.L. Carroll K.K. Cancer Res... 1989; 49: 6300-6303Google Scholar). In other in vivo studies using N-methyl-N-nitrosourea-treated rats 1,25(OH)2D3 or analogs of 1,25(OH)2D3 were found to inhibit the progression of mammary tumor growth (3Colston K.W. Chander S.K. Mackay A.G. Coombes R.C. Biochem. Pharmacol... 1992; 44: 693-702Google Scholar, 4Colston K.W. Mackay A.G. James S.Y. Binderup L. Chander S. Coombes R.C. Biochem. Pharmacol... 1992; 44: 2273-2280Google Scholar). When rats were treated with 1,25(OH)2D3 or analogs of 1,25(OH)2D3 prior to treatment with N-methyl-N-nitrosourea, tumor incidence was reduced or prevented (5Anzano M.A. Smith J.M. Uskokovic M.R. Peer C.W. Mullen L.T. Letterio J.J. Welsh M.C. Shrader M.W. Logsdon D.L. Driver C.L. Brown C.C. Roberts A.B. Sporn M.B. Cancer Res... 1994; 54: 1653-1656Google Scholar, 6Mehta R. Hawthorne M. Uselding L. Albinescu D. Moriarty R. Christov K. J. Natl. Cancer Inst.. 2000; 92: 1836-1840Google Scholar). In addition, in patient studies it has been noted that the presence of vitamin D receptors in breast carcinomas is correlated with improved prognosis in breast patients (7Colston K.W. Berger U. Coombes R.C. Lancet.. 1989; 1: 188-191Google Scholar). Also, low serum levels of 1,25(OH)2D3 are associated with increased breast cancer risk or disease progression (8Janowsky E.C. Lester G.E. Weinberg C.R. Millikan R.C. Schildkraut J.M. Garrett P.A. Hulka B.S. Public Health Nutr... 1999; 2: 283-291Google Scholar, 9Mawer E.B. Walls J. Howell A. Davies M. Ratcliffe W.A. Bundred N.J. J. Clin. Endocrinol. Metab... 1997; 82: 118-122Google Scholar). 1,25(OH)2D3 or its analogs also exerts potent growth inhibitory effects on human breast cancer cells (10Abe J. Nakano T. Nishii Y. Matsumoto T. Ogata E. Ikeda K. Endocrinology.. 1991; 129: 832-837Google Scholar, 11Chouvet C. Vicard E. Devonec M. Saez S. J. Steroid Biochem... 1986; 24: 373-376Google Scholar). We have earlier reported that a combination of 1,25(OH)2D3 and retinoic acid lowers the threshold for killing of breast cancer cells by taxol and adriamycin thus suggesting a novel option for the treatment of breast cancer (12Wang Q. Lee D. Sysounthone V. Chandraratna R.A.S. Christakos S. Korah R. Wieder R. Breast Cancer Res. Treat... 2001; 67: 157-168Google Scholar, 13Wang Q. Yang W. Uytingco M.S. Christakos S. Wieder R. Cancer Res... 2000; 60: 2040-2048Google Scholar). Although these studies provide compelling evidence for the use of 1,25(OH)2D3 or less calcemic analogs of 1,25(OH)2D3 that have similar growth-inhibitory effects, little is known about the mechanisms involved in mediating the cellular responsiveness to 1,25(OH)2D3 and the downstream targets involved. Recently, we reported that CCAAT/enhancer-binding protein β (C/EBPβ) is a 1,25(OH)2D3 target gene in kidney and in osteoblastic cells (14Dhawan P. Peng X. Sutton A.L. MacDonald P.N. Croniger C.M. Trautwein C. Centrella M. McCarthy T.L. Christakos S. Mol. Cell Biol... 2005; 25: 472-487Google Scholar). The C/EBP family of transcription factors has been reported to be involved in the regulation of growth, differentiation, and inflammation and the expression of cell type-specific genes (15Hanson R.W. J. Biol. Chem... 1998; 273: 28543Google Scholar, 16Ramji D.P. Foka P. Biochem. J... 2002; 365: 561-575Google Scholar). C/EBPβ has been shown to be essential for the development of the murine mammary gland (17Robinson G.W. Johnson P.F. Hennighausen L. Sterneck E. Genes Dev... 1998; 12: 1907-1916Google Scholar, 18Seagroves T.N. Krnacik S. Raught B. Gay J. Burgess-Beusse B. Darlington G.J. Rosen J.M. Genes Dev... 1998; 12: 1917-1928Google Scholar). C/EBPα has been reported to play a critical role in both proliferation and differentiation in numerous cell types (16Ramji D.P. Foka P. Biochem. J... 2002; 365: 561-575Google Scholar, 19Flodby P. Barlow C. Kylefjord H. Ahrlund-Richter L. Xanthopoulos K.G. J. Biol. Chem... 1996; 271: 24753-24760Google Scholar, 20Freytag S.O. Paielli D.L. Gilbert J.D. Genes Dev... 1994; 8: 1654-1663Google Scholar, 21Radomska H.S. Huettner C.S. Zhang P. Cheng T. Scadden D.T. Tenen D.G. Mol. Cell Biol... 1998; 18: 4301-4314Google Scholar). It was suggested that C/EBPα could be considered a potential tumor suppression gene in breast cancer (22Gery S. Tanosaki S. Bose S. Bose N. Vadgama J. Koeffler H.P. Clin. Cancer Res... 2005; 11: 3184-3190Google Scholar). In our previous studies we found that C/EBPβ cooperates with VDR in 1,25(OH)2D3-induced transcription (14Dhawan P. Peng X. Sutton A.L. MacDonald P.N. Croniger C.M. Trautwein C. Centrella M. McCarthy T.L. Christakos S. Mol. Cell Biol... 2005; 25: 472-487Google Scholar). In the present study we extend our initial observations in kidney and osteoblastic cells to cooperative effects between 1,25(OH)2D3 and the C/EBP family of transcription factors in breast cancer cells. We report for the first time that C/EBPα is induced by 1,25(OH)2D3 in MCF-7 breast cancer cells and that C/EBPα cooperates with Brm, an ATPase that is a component of the SWI/SNF chromatin-remodeling complex, and is a potent enhancer of VDR transcription. The induction of C/EBPα is accompanied by induction of p21WAF1/Cip1 and p27Kip1. Because the levels of VDR correlate with the antiproliferative effects of 1,25(OH)2D3 and because 1,25(OH)2D3 and C/EBPα up-regulate p21, these findings provide important mechanisms whereby 1,25(OH)2D3 may act to inhibit growth of breast cancer cells. These findings also identify C/EBPα as a 1,25(OH)2D3 target in breast cancer cells and provide evidence for C/EBPα as a candidate for breast cancer treatment. Materials—[γ-32P]ATP (3000 Ci (111 TBq)/mmol) was purchased from PerkinElmer Life Sciences. Polyvinylidene difluoride membranes and pre-stained molecular weight markers were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). T4 poly nucleotide kinase for labeling double stranded oligonucleotides for electrophoretic mobility shift assay was purchased from Invitrogen. VDR, β-actin, Brm, Brg-1, and C/EBPα antiserum and secondary antibodies against mouse and rabbit antisera were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). 1,25(OH)2D3 was a generous gift from Milan Uskokovic (Hoffmann-LaRoche, Nutley, NJ). Cell Culture—Fetal bovine serum (FBS) and charcoal-stripped FBS were from Gemini Biological Products (Calabasas, CA). Cell culture media, 0.25% trypsin-EDTA and penicillin, streptomycin, and neomycin mixture were purchased from Invitrogen. MCF-7 and MBA-MD-231 breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA.) and were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin, streptomycin, and neomycin antibiotic mixture. Cells were grown in a humidified incubator with an atmosphere of 95% air-5% CO2 at 37 °C. For treatments, cells were grown to desired confluency, and their medium was changed to DMEM supplemented with 2% charcoal-dextran-treated FBS. Treatments with vehicle or 1,25(OH)2D3 were done for the durations and with concentrations mentioned in the figure legends. Plasmids, Transfections, and Assay of Luciferase Activity—The luciferase reporter construct of human VDR promoter (-1500 to +60) was generated as previously described (23Huening M. Yehia G. Molina C.A. Christakos S. Mol. Endocrinol... 2002; 16: 2052-2064Google Scholar). The deletion construct was prepared using KpnI and NotI and re-ligating the remaining promoter construct containing (-646/+60). The C/EBP site at -919/-911 was mutated by site-directed mutagenesis using a site-directed mutagenesis kit from Stratagene. The oligonucleotides used to generate the C/EBPβ mutated site (shown in lowercase underlined letters; C/EBP site shown in bold) were as follows: 5′-CCA GAA GAT Tat gtt acg atT ACT ATT TAT TTA TAC-3′ and 5′-GTA TAA ATA AAT AGT Aat cgt aac aTA ATC TTC TGG. All these promoter constructs were used for transcription assays in MCF-7 cells. The C/EBPα promoter construct was kindly gifted by (A. Peres-Castillo, Madrid Spain) (24Menendez-Hurtado A. Santos A. Perez-Castillo A. Endocrinology.. 2000; 141: 4164-4170Google Scholar). The C/EBPα,-β, and -δ expression vectors were a gift of Simon Williams, Texas Tech University (Lubbock, TX). The dominant negative (DN) C/EBP construct was a gift from Dr. C. Vinson, NCI, National Institutes of Health. pCMV-Brm and pCMV-mutant Brm (with the ATPase site mutated, which acts as a dominant negative inhibitor) were obtained from M. Yaniv (25Muchardt C. Reyes J.C. Bourachot B. Leguoy E. Yaniv M. EMBO J.. 1996; 15: 3394-3402Google Scholar). pCMV-AML-1/ETO expression vector was from S. W. Hiebert (Vanderbilt University, Nashville, TN) Empty vectors were transfected to keep the total DNA concentration equal. Cells were transfected using Lipofectamine 2000 (Invitrogen) treated as described under "Results" in the appropriate medium supplemented with 2% charcoal-dextran-treated FBS. After treatment with vehicle or the compounds noted at the concentrations and times indicated under "Results," cells were harvested and dual luciferase assay was performed according to the Dual Luciferase assay kit manufacturer's protocol (Promega, Madison, WI). For experiments shown in Fig. 5, MDA-MB-231 cells were co-transfected with C/EBPα expression plasmid and green fluorescent protein expression plasmid with G418 resistance using Lipofectamine 2000 and incubated at 37 °C for 20 h. Control cells were transfected with vector alone. Normal growth medium (DMEM as indicated in "cell culture" above) was added the next day. Three days after transfection, selection began with increasing amounts of G418 to a final concentration of 600 μg/ml. After 2 weeks of G418 selection, cells were used for Western blot analysis, VDR transcription assays, and assessment of cell proliferation in the presence or absence of 1,25(OH)2D3. Electrophoretic Mobility Shift Assay—Complementary oligonucleotides were synthesized based on the region of the hVDR promoter containing the wild-type (-919/-911) or the mutated C/EBP site (prepared by the University of Medicine and Dentistry Molecular Resource Facility, Newark, NJ). The complementary oligonucleotides were annealed, end-radiolabeled, and purified as described before (14Dhawan P. Peng X. Sutton A.L. MacDonald P.N. Croniger C.M. Trautwein C. Centrella M. McCarthy T.L. Christakos S. Mol. Cell Biol... 2005; 25: 472-487Google Scholar) and were used for the electrophoretic mobility shift assay. The sequences of the oligonucleotides used were 5′-CCA GAA GAT TAT GTT GTA ATT ACT ATT TAT TTA TAC-3′ and 5′-TAA ATA AAT AGT AAT TAC AAC ATA ATC TTC TGG-3′ for the wild-type and 5′-CCA GAA GAT TAT GTT ACG ATT ACT ATT TAT TTA TAC-3′ and 5′-GTA TAA ATA AAT AGT AAT CGT AAC ATA ATC TTC TGG-3′ for the mutant construct. Briefly, 5 μg of the nuclear preparations from C/EBPα-transfected MCF-7 cells were incubated for 20 min at 25 °C with 2 μg of poly(dI/dC) with or without unlabeled specific or nonspecific DNA competitor or C/EBPα antibody in binding buffer (4 mm Tris-HCl (pH 7.9), 1 mm EDTA (pH 8.0), 60 mm KCl, 12% glycerol, 12 mm HEPES, 1 mm dithiothreitol). This was further incubated with 0.5 ng of the labeled oligonucleotide probe (∼100,000 cpm) and incubation for 30 min at 25 °C. The samples were separated by electrophoresis on a 6% nondenaturing polyacrylamide gel that had been pre-electrophoresed for 30 min at 100 V/cm at 4 °C in 45 mm Tris-45 mm boric acid-1 mm EDTA. Electrophoresis was conducted for 2.5 h under identical conditions. The gel was dried and exposed to x-ray film at -80 °C with intensifying screens. siRNA Transfections—The following are the sequences of the oligonucleotides used to knockdown C/EBPα expression. C/EBPα siRNA sequences are as follows: sense, 5′-GUCGGCCAGGAACUCGUCGUU-3′ and antisense, 3′-UUCAGCCGGUCCUUGAGCAGC-3′. Scrambled siRNA sequences are as follows: sense, 5′-GUAGUCCAUGGACCCGUAGUU and antisense, 3′-UUCAUCAGGUACCUGGGCAUC-3′. siRNA transfections were done using Oligofectamine™ (Invitrogen). Cells were transfected with C/EBPα siRNA following the manufacturer's instructions. One day prior to transfection, 5 × 104 cells/well were seeded in 6-well plates (corresponding to a density of 40% at the time of transfection) without antibiotics. The transfection mixture (containing 20 μm siRNA with Oligofectamine reagent) was added to the 6-well plate 20 min after mixture preparation. After 24–48 h of transfection the medium was changed to DMEM with 2% charcoal-stripped FBS. Transfected cells were treated with vehicle or 1,25(OH)2D3 for 24–72 h as described above. Cells were then harvested, and cell viability was determined by trypan blue exclusion. Similarly transfected and treated cells were also used for preparation of nuclear extracts for Western blotting. Nuclear Extracts—For nuclear extract preparation from MCF-7 cells, cells were rinsed twice with phosphate-buffered saline at 4 °C, harvested by scraping, gently pelleted, washed, and lysed in hypotonic buffer containing 10 mm HEPES (pH 7.4), 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, phosphatase inhibitors (1 mm sodium orthovanadate, 10 mm sodium fluoride), protease inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 1 mg of pepstatin A per ml, 2 mg of leupeptin per ml, 2 mg of aprotinin per ml), and 1% Triton X-100. Nuclei were pelleted at 3,500 × g for 5 min, and cytoplasmic supernatants were separated. Nuclei were resuspended in hypertonic buffer containing 0.42 m NaCl, 0.2 mm EDTA, 25% glycerol, and the phosphatase and protease inhibitors indicated above. Soluble nuclear proteins were released by 60 min of incubation at 4 °C, and insoluble material was separated by centrifugation at 12,000 × g for 5 min. The protein concentration of the supernatant was measured by using Bradford's method (26Bradford M.M. Anal. Biochem... 1976; 72: 248-254Google Scholar), and aliquots were stored at -80 °C. Co-immunoprecipitation Assay—Co-immunoprecipitation assay was performed as described previously (14Dhawan P. Peng X. Sutton A.L. MacDonald P.N. Croniger C.M. Trautwein C. Centrella M. McCarthy T.L. Christakos S. Mol. Cell Biol... 2005; 25: 472-487Google Scholar). Briefly, nuclear extracts were isolated using the NEPER nuclear extraction reagents kit from Pierce. 1 μg of primary antibody (C/EBPα, or Brm) was added to the nuclear extract in 500 μl of immunoprecipitation buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, and 1% Triton X 100) and incubated for 4 h to overnight with rotation at 4 °C. 25 μl of protein A-Sepharose 4 Fast Flow Beads (Amersham Biosciences) were added to the nuclear extract-antibody mix, and it was further incubated with rotation at 4 °C for 1 h. After centrifugation at 3000 rpm at 4 °C for 5 min, the supernatant was discarded and the immunoprecipitated complex was washed with immunoprecipitation buffer three times, eluted and separated on 4–20% SDS-PAGE gel (Bio-Rad). The Western blot was probed with Brm or C/EBPα antibody, respectively. Chromatin Immunoprecipitation Assay—MCF-7 cells were cultured in DMEM supplemented with 10% FBS to 95% confluence and then treated with vehicle or 1,25(OH)2D3 in medium supplemented with 2% charcoal-stripped serum under the conditions and for the times indicated. Treated cells were used for the ChIP assay performed as described earlier (27Liu Y. Shen Q. Malloy P.J. Soliman E. Peng X. Kim S. Pike J.W. Feldman D. Christakos S. J. Bone Miner Res... 2005; 20: 1680-1691Google Scholar, 28Shen Q. Christakos S. J. Biol. Chem... 2005; 280: 40589-40598Google Scholar). In brief, treated cells were washed with phosphate-buffered saline and cross-link using 1% formaldehyde for 15 min. Glycine was added to a final concentration of 0.125 m to stop cross-linking. The cells were washed twice with ice-cold phosphate-buffered saline and collected by scraping. The cells were lysed for 20 min each first in buffer 1 (5 mm Pipes, pH 8.0, 85 mm KCl, 0.5% Nonidet P-40) and then in buffer 2 (1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.1). The resulting chromatin pellet was sonicated to an average DNA size of 500-bp DNA (evaluated by 1% agarose gel electrophoresis) using a Fisher model 100 sonic dismembranator at a power setting of 1. The sonicated extract was centrifuged for 10 min at 13,000 rpm at 4 °C and then diluted into ChIP dilution buffer (16.7 mm Tris-HCl, pH 8.1, 150 mm NaCl, 0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA). Immunoprecipitations were performed at 4 °C overnight with the indicated antibody. After a 1-h incubation with salmon sperm DNA and bovine serum albumin-pretreated Zysorbin (Zymed Laboratories Inc., San Francisco, CA), the precipitates were collected by centrifugation. Precipitates were washed sequentially in buffer I (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 150 mm NaCl), buffer II (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1, 500 mm NaCl), buffer III (0.25 m LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mm EDTA, 10 mm Tris-HCl, pH 8.1), and twice in TE buffer (10 mm Tris, 1 mm EDTA). The protein-DNA was then eluted by using 1% SDS and 0.1 m NaHCO3 for 15 min twice. Cross-links were reversed by incubating at 65 °C overnight in elution buffer with 0.2 m NaCl. DNA fragments were purified using Qiagen QIAquick PCR purification kits, and PCR was performed using the primers designed to amplify fragments of human VDR promoter C/EBP motif (upper, 5′-GAGGCGAATAGCAATATCTTCC-3′; lower, 5′-GAGACCTGGAATTGTGGATGG-3′). PCR analysis was performed in the linear range of DNA amplification. 248-bp PCR products were resolved in 1% agarose gel and visualized using ethidium bromide staining. DNA obtained before precipitation was used as the input. 10% of input was used for PCR reaction. Statistical Analysis—Results are expressed as means ± S.E., and significance was determined by analysis by Student's t test for two-group comparison or by analysis of variance for multiple-group comparison. C/EBPα Expression Is Induced by 1,25(OH)2D3 in MCF-7 Breast Cancer Cells—Shown in Fig. 1A is the 1,25(OH)2D3-induced inhibition of MCF-7 cell proliferation. Because C/EBPα has been reported to have a growth inhibitory role in breast cancer (22Gery S. Tanosaki S. Bose S. Bose N. Vadgama J. Koeffler H.P. Clin. Cancer Res... 2005; 11: 3184-3190Google Scholar), we tested the possibility that 1,25(OH)2D3 may regulate the expression of C/EBPα in breast cancer cells. When MCF-7 breast cancer cells were treated with 1,25(OH)2D3 (10 nm for 8–24 h), Western blot analysis, using nuclear extracts from the vehicle or 1,25(OH)2D3-treated cells, showed that 1,25(OH)2D3 induced C/EBPα expression in MCF-7 breast cancer cells (Fig. 1B, left panel; maximum induction, 4.2-fold). The induction of C/EBPα by 1,25(OH)2D3 in MCF-7 cells was accompanied by an increase in the expression of VDR (maximum 3.7-fold at 24 h) (Fig. 1B, left panel). A dose response of the stimulation by 1,25(OH)2D3 (1–100 nm for 24 h), shown in Fig. 1B (right panel), similarly indicates that induction of C/EBPα by 1,25(OH)2D3 is accompanied by an increase in VDR. C/EBPβ is not induced by 1,25(OH)2D3 in MCF-7 cells (not shown). Induction of C/EBPα mRNA by 1,25(OH)2D3 in MCF-7 cells is shown in Fig. 1C. 1,25(OH)2D3 also enhanced the expression of p21WAF1/Cip1 and p27Kip1 in MCF-7 cells (Fig. 1D). To examine a possible mechanism of regulation of C/EBPα by 1,25(OH)2D3 in MCF-7 breast cancer cells, MCF-7 cells were transfected with the rat C/EBPα promoter (-1171/+23) and treated with 1,25(OH)2D3. 1,25(OH)2D3 failed to show any effect on the activity of the C/EBPα promoter (Fig. 1E). The C/EBPα promoter was also unresponsive to 1,25(OH)2D3 when VDR-transfected COS-7 cells were used (not shown). As a positive control, MCF-7 cells were transfected with C/EBPβ expression vector. C/EBPβ significantly induced C/EBPα transcription 5.5-fold (p < 0.01 compared with cells transfected with vector alone; Fig. 1E). The unresponsiveness of the C/EBPα promoter to 1,25(OH)2D3 (similar to what we observed using the C/EBPβ promoter and transfection in osteoblastic cells (14Dhawan P. Peng X. Sutton A.L. MacDonald P.N. Croniger C.M. Trautwein C. Centrella M. McCarthy T.L. Christakos S. Mol. Cell Biol... 2005; 25: 472-487Google Scholar)) suggests the possibility that C/EBPα is regulated by 1,25(OH)2D3 at other sites yet to be defined or that 1,25(OH)2D3 may be regulating C/EBPα at a post transcriptional level. Enhancement of VDR Transcription by C/EBPα in MCF-7 Cells—Because the induction by 1,25(OH)2D3 of C/EBPα is accompanied by an increase in VDR (Fig. 1B) and because sequence analysis of the hVDR promoter revealed the presence of a putative C/EBP site at -919/-911 and two CRE sites at -571/-567 and -361/-357 (Fig. 2A (29Miyamoto K. Kesterson R.A. Yamamoto H. Taketani Y. Nishiwaki E. Tatsumi S. Inoue Y. Morita K. Takeda E. Pike J.W. Mol. Endocrinol... 1997; 11: 1165-1179Google Scholar)) (C/EBP family members have been reported to bind to a CRE or a C/EBP site (30Wilson H.L. Roesler W.J. Mol. Cell Endocrinol... 2002; 188: 15-20Google Scholar)), the possibility that C/EBPα may have a role in the 1,25(OH)2D3-mediated enhancement of VDR in MCF-7 breast cancer cells was examined. MCF-7 cells were co-transfected with hVDR promoter (-1500/+60) and C/EBPα expression vector. A dose-dependent enhancement of hVDR transcription by C/EBPα was observed (maximum enhancement of 11 ± 1.2-fold at 0.25 μg of C/EBPα (Fig. 2B)). Unlike C/EBPα, C/EBPβ or C/EBPδ had no effect on VDR transcription (Fig. 2B). To further confirm the role of C/EBPα as an enhancer of the hVDR transcription, MCF-7 cells were transfected with the hVDR promoter (-1500/+60) in the presence of C/EBPα expression vector and dominant negative C/EBP (DN C/EBP). DN C/EBP, which lacks the transactivation domain sequence but retains the dimerization and DNA binding domain, suppressed the C/EBPα mediated enhancement of hVDR transcription in MCF-7 cells in a dose-dependent manner (Fig. 2C). AML-ETO has earlier been reported to block C/EBPα DNA binding and transactivation (31Pabst T. Mueller B.U. Harakawa N. Schoch C. Haferlach T. Behre G. Hiddemann W. Zhang D.E. Tenen D.G. Nat. Med... 2001; 7: 444-451Google Scholar). Increasing concentrations of AML-ETO also suppressed the C/EBPα mediated activation of the hVDR promoter (Fig. 2D). These results indicate, for the first time, the involvement of C/EBPα in VDR transcription in MCF-7 cells. Identification of the C/EBPα Activation Domain in the VDR Promoter—Sequences in the hVDR promoter involved in transactivation by C/EBP were identified by deletion mutant analysis and site-directed mutagenesis. Deletion of the hVDR promoter to -646 (DEL C/EBP) eliminated the ability of C/EPBα to transactivate the hVDR promoter, suggesting the involvement of the site at -919/-911 in the induction by C/EBPα of hVDR transcription (Fig. 3, A and B). To investigate the specific contribution of this element to the induction of hVDR transcription by C/EBPα, a mutant hVDR promoter construct was generated with the C/EBP site at -919/-911 mutated (MT CEBP). Mutation of this site within the -1500/+60 promoter construct blocked the C/EBPα-mediated induction of transcription (Fig. 3B), suggesting that C/EBPα acts through this site to induce hVDR transcription. Gel mobility shift assays were performed using synthetic oligonucleotide corresponding to the wild-type (-919/-911) C/EBP binding sequences and nuclear extracts from control and 1,25(OH)2D3-treated MCF-7 cells transfected with C/EBPα. An interaction of MCF-7 cell nuclear extract with this element was observed and binding was enhanced in the presence of 1,25(OH)2D3 (Fig. 3C, lanes 2 and 3). A supershift was observed in the presence of C/EBPα antibody, indicating the specificity of this interaction for C/EBPα (Fig. 3C, lanes 4 and 6). Binding was competed by excess cold oligonucleotide, confirming specificity (Fig. 3C, lanes 5 and 7). C/EBPα failed to bind to mutant radiolabeled oligonucleotide, and the oligonucleotide containing the mutated C/EBP sequences could not deplete the binding of C/EBPα to the labeled WT probe (not shown). RNA Interference Knockdown of C/EBPα Reduces the Anti-proliferative Effect of 1,25(OH)2D3 in MCF-7 Cells—MCF-7 cells were transfected with C/EBPα siRNA or scrambled siRNA (Fig. 4A). C/EBPα siRNA transfection reduced the expression of C/EBPα as well as VDR protein in MCF-7 cells (Fig. 4B). Treatment of MCF-7 cells or control transfected MCF-7 cells (scrambled siRNA) with 10 nm 1,25(OH)2D3 decreased the number of the viable cell

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