Calcitonin, a Regulator of the 25-Hydroxyvitamin D3 1α-Hydroxylase Gene*

Although parathyroid hormone (PTH) induces 25-hydroxyvitamin D3 (25(OH)D3) 1α-hydroxylase (1α(OH)ase) under hypocalcemic conditions, previous studies showed that calcitonin, not PTH, has an important role in the maintenance of serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) under normocalcemic conditions. In this study we report that 1α(OH)ase transcription is strongly induced by calcitonin in kidney cells and indicate mechanisms that underlie this regulation. The transcription factor C/EBPβ is up-regulated by calcitonin in kidney cells and results in a significant enhancement of calcitonin induction of 1α(OH)ase transcription and protein expression. Mutation constructs of the 1α(OH)ase promoter demonstrate the importance of the C/EBPβ binding site at –79/–73 for activation of the 1α(OH)ase promoter by calcitonin. The SWI/SNF chromatin remodeling complex was found to cooperate with calcitonin in the regulation of 1α(OH)ase. Chromatin immunoprecipitation analysis showed that calcitonin recruits C/EBPβ to the 1α(OH)ase promoter, and Re-chromatin immunoprecipitation analysis (sequential chromatin immunoprecipitations using different antibodies) showed that C/EBPβ and BRG1, an ATPase that is a component of the SWI/SNF complex, bind simultaneously to the 1α(OH)ase promoter. These findings are the first to address the dynamics between calcitonin, C/EBPβ, and SWI/SNF in the regulation of 1α(OH)ase and provide a mechanism, for the first time, for calcitonin induction of 1α(OH)ase. Because plasma calcitonin as well as 1,25(OH)2D3 have been reported to be increased during pregnancy and lactation and in early development, these findings suggest a mechanism that may account, at least in part, for the increase in plasma 1,25(OH)2D3 during these times of increased calcium requirement.

Vitamin D is a principal factor required for maintaining normal calcium homeostasis (1). The active form of vitamin D, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) 3 is generated by two successive hydroxylations; 25-hydroxylation in the liver and 1␣-hydroxylation in the kidney (1)(2)(3). Inactivating mutations in the 25-hydroxvitamin D 3 1␣-hydroxylase (1␣(OH)ase) gene result in vitamin D dependent rickets type I despite normal intake of vitamin D, indicating the importance of the 1␣(OH)ase enzyme (4,5). Elevated parathyroid hormone (PTH) resulting from hypocalcemia is a primary signal mediating the renal synthesis of 1,25(OH) 2 D 3 (6). PTH stimulates 1␣(OH)ase transcription, resulting in increased 1,25(OH) 2 D 3 synthesis (7)(8)(9). However, under normocalcemic conditions, PTH fails to stimulate 1␣(OH)ase expression (10). Previous studies showed that calcitonin can enhance renal conversion of 25(OH)D 3 to 1,25(OH) 2 D 3 and that calcitonin, not PTH, is the major regulator of 1␣(OH)ase in the normocalcemic state (10 -13). In early development and during pregnancy and lactation calcitonin levels are increased under normocalcemic conditions and correlated to an increase in serum 1,25(OH) 2 D 3 levels (14 -17). The stimulation of 1,25(OH) 2 D 3 under normocalcemic conditions by calcitonin may have biological significance to control calcium homeostasis during the perinatal period and during pregnancy and lactation when the need for calcium is increased. However, the mechanisms involved in the stimulation of 1␣(OH)ase by calcitonin have not been examined.
Calcitonin, a 32-amino acid peptide hormone generated from the thyroid C cells, has been reported to have diverse physiological actions that include inhibition of osteoclastic bone resorption (18), inhibition of prolactin secretion (19), effects on the growth of prostate and breast cancer cells (20 -22), and effects on maternal-fetal calcium exchange (23). The calcitonin receptor, a G-protein coupled receptor, is widely expressed in numerous tissues including osteoclasts (18), kidney (12), placenta (23), and breast and prostate cancer cells (20 -22). Thus, calcitonin has both skeletal and extra-skeletal effects. Although it had been thought that a major function of calcitonin is to lower serum calcium, it has been shown that patients with medullary thyroid carcinoma, a neoplasm of C cells, have high calcitonin levels but normal serum calcium (24). In addition, in the absence of calcitonin, serum calcium is unaffected (25). The elevation in plasma 1,25(OH) 2 D 3 in rats treated with calcitonin (11) and the increased serum 1,25(OH) 2 D 3 lev-* This work was supported, in whole or in part, by National Institutes of Health Grant DK-38961 (to S. C.). This manuscript is dedicated to the memory of Iain MacIntyre (1924 -2008), a leader in the field of bone and calcium metabolism, who first discovered the effect of calcitonin on 1␣(OH)ase (13) and encouraged us in this investigation. 1  els in patients with medullary thyroid carcinoma as well as the decrease in 1,25(OH) 2 D 3 levels after surgical cure of medullary thyroid carcinoma and hypercalcitoninemia (24,26) further suggest multiple roles of calcitonin and specifically a direct effect of calcitonin on renal 1␣(OH)ase. Although 1␣(OH)ase, a mitochondrial P450 enzyme, is regulated by many factors including PTH, calcitonin, and 1,25(OH) 2 D 3 , the mechanisms involved in the regulation of 1␣(OH)ase expression are only now beginning to be defined. In the human 1␣(OH)ase promoter, an SP1 site involved in basal expression and a negative vitamin D response element, which associates with VDR/retinoid X receptor as well as histone deacetylase complex in the presence of 1,25(OH) 2 D 3 , have been reported (27,28). Regions of the mouse 1␣(OH)ase promoter that contribute to its basal activity have also been characterized (29). Differences between the mouse and human promoter have been noted, suggesting different regulation of the two genes (27,29,30). Although responsiveness to PTH has been noted for both the mouse and human promoter (7-9, 30), a direct inhibition of the activity of the mouse 1␣(OH)ase promoter by 1,25(OH) 2 D 3 has not been observed (7).
The results of this investigation indicate that the mouse promoter, similar to the human 1␣(OH)ase promoter (9), confers positive responsiveness to calcitonin. Maximal calcitonin activation is observed using the minimal promoter region (Ϫ85/ ϩ22). Mutation of a C/EBP␤ binding site at Ϫ79/Ϫ73 resulted in marked attenuation of activation of 1␣(OH)ase transcription by calcitonin. SWI/SNF, which remodels chromatin using the energy of ATP hydrolysis, is recruited by C/EBP␤ to the 1␣(OH)ase promoter and also mediates calcitonin regulation of 1␣(OH)ase transcription. Chromatin immunoprecipitation (ChIP) analysis also showed an increase in acetylated histone 4 in response to calcitonin, suggesting cooperation between acetylation and chromatin remodeling. These findings are the first to address the dynamics between calcitonin, C/EBP␤, and SWI/SNF in the regulation of 1␣(OH)ase and provide a mechanism for the first time for calcitonin induction of 1␣(OH)ase.

EXPERIMENTAL PROCEDURES
Materials-Deoxy-[␥-32 P]ATP (3000 Ci/mmol) was obtained from PerkinElmer Life Sciences. A Random Primers DNA labeling kit was purchased from Invitrogen. The 1,25(OH) 2 D 3 RIA kit was purchased from Immunodiagnostics Systems Inc. (Fountain Hills, AZ). Prestained protein molecular weight markers and an electrochemiluminescent detection system were obtained from PerkinElmer Life Sciences. Salmon calcitonin was obtained from Sigma. C/EBP␤, BRG1, Brm, and ␤-actin antisera were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell Culture-AOK-B50 porcine renal proximal tubular cells (LLCPK1 cells that express PTH/PTHrP type I receptors as well as calcitonin receptors (31,32)) and MCT mouse renal proximal tubular cells (33) that also express both PTH and calcitonin receptors (9) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 7% heat-inactivated fetal bovine serum (Gemini Biological Products, Calabasas, CA) in a humidified atmosphere of 95% air, 5%CO 2 at 37°C. Cells were grown to 70 -80% confluence and changed to medium supplemented with 2% charcoal-dextran-treated fetal bovine serum before treatment. C33A cells (from ATCC) that lack Brm and BRG1 were used in some studies and were similarly maintained. Cells were treated with the vehicle or the compounds noted at the indicated times and concentrations.
Transient Transfection and Luciferase Assay-For transfection studies, the mouse 1␣(OH)ase promoter Ϫ1651/ϩ22 placed upstream of a luciferase reporter gene in the pGL2b vector and the deletion constructs Ϫ144/ϩ22, Ϫ85/ϩ22, and Ϫ74/ϩ22 were kindly provided by Dr. H. F. DeLuca (University of Wisconsin at Madison, Madison, WI). pMex-C/EBP␤ was a gift of Dr. Simon Willimas, Texas Tech University (Lubbock, TX). A-C/EBP, a dominant negative that heterodimerizes with C/EBP family members and blocks DNA binding, was provided by Dr. Charles Vinson (NIH, Bethesda, MD) (34). The C/EBP␤ promoter luciferase construct (Ϫ1400/ϩ16) was provided by Dr. Christian Trautwein (35). pCMV-mutant Brm (with the ATPase site mutated that acts as a dominant negative inhibitor) was obtained from M. Yaniv (Institut Pasteur, Paris). PBJ5 BRG1 (K785R) with the ATPase site mutated was from J. DiRenzo and M. Brown (36). Mutant Brm and BRG1 (with the ATPase sites mutated) have previously been characterized and shown to be incorporated into the SWI/SNF complex resulting in interference in transcriptional activation (37). LIP, the dominant negative isoform of C/EBP␤, originally described by Descombes and Schibler (38), was provided by A. Dusso (Washington University School of Medicine, St. Louis, MO). Cells were seeded in a 24-well culture dish 24 h before transfection at 70% confluence. Empty vectors were transfected to keep the total DNA concentration equal. Cells in each well were transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Efficiency of transfection, as assessed by green fluorescent protein cotransfection and subsequent visualization, was estimated at 75-85% for AOK-B50 cells. Efficiency of transfection of MCT cells, similarly analyzed, was 25-35%. Maximal induction of 1␣(OH)ase transcription (using transfected promoter constructs) by calcitonin in MCT cells was 2.4 Ϯ 0.3-fold (compared with 9 -10-fold for AOK-B50 cells). Thus, for most studies examining transcriptional regulation of 1␣(OH)ase using transfected cells, AOK-B50 cells were used. Cells were treated 24-h post-transfection for another 24 h. Time course studies with calcitonin indicated a peak of activation at 6 h (1 nM calcitonin resulted in a 20 -21fold induction in transcription using either the Ϫ1651/ϩ22 or the Ϫ85/ϩ22 promoter construct). Studies were done at 24 h (suboptimal conditions) similar to previous studies with calcitonin (100 nM) (9). Cells were washed twice with phosphatebuffered saline and harvested by incubating with 1ϫ passive lysis buffer, supplied by the dual-luciferase reporter assay kit (Promega, Madison, Wisconsin). The luciferase activity assay was performed according to the protocol of the manufacturer and normalized based on protein content of the cell lysates. Protein levels were determined by the Bradford assay (39).
Site-directed Mutagenesis-The C/EBP␤ binding site at Ϫ79/ Ϫ73 was mutated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The oligonucleotides used to generate the mutated site at Ϫ79/Ϫ73 were as follows: 5Ј-GGA GTC TGG GAG ACT CTG AAG AGC-3Ј (top strand) and 5Ј-GCT CTT CAG AGT CTC CCA GAC TCC-3Ј (lower strand). The mutated constructs were confirmed by DNA sequencing.
Western Blot Analysis-For Western blot analysis of 1␣(OH)ase, 50 g of protein from total cell extracts was loaded onto a 15% SDS-polyacrylamide gel, separated by electrophoresis, and transferred onto a polyvinylidene difluoride membrane (Bio-Rad). Membranes were blocked with 5% milk, Tris-buffered saline for 30 min and incubated at room temperature with 1␣(OH)ase antibody (from Armbrecht et al. laboratory (40); rabbit antiserum was raised against a synthetic peptide consisting of the last 12 amino acids of the mouse/pig 1␣(OH)ase sequence) at a dilution of 1:1000 in 1% milk, Tris-buffered saline for 60 min. Membranes were then rinsed with Tris-buffered saline (TBS) and incubated at room temperature with goat anti-rabbit IgG-horseradish peroxidase antibody (sc-2004; Santa Cruz Biotechnology) at 1:10,000 in 1% milk, TBS for 30 min. The antigen-antibody complex was detected by the electrochemiluminescent detection system. For analysis of C/EBP␤ protein, nuclear extracts were prepared, and Western blot analysis was performed as previously described (41).
ChIP Assay-Cells were cultured to 95% confluence before the experiment and then treated with calcitonin for the different times to perform the ChIP assay as previously described (42). Briefly, cells were first washed with phosphate-buffered saline and subjected to a cross-link reaction with 1% formaldehyde for 15 min. The cross-link reaction was stopped by adding glycine to a final concentration of 0.125 M. Cells were washed with phosphate-buffered saline twice, collected by scraping, and lysed sequentially in 5 mM Pipes, pH 8.0, 85 mM KCl, 0.5% Nonidet P-40, and then in 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, for 20 min individually. The chromatin pellets were sonicated to an average DNA size of 500 bp using a Fisher model 100 sonic dismembrator at a power setting of 2. The sonicated extract was centrifuged for 10 min at maximum speed 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, and 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 TE buffer (10 mM Tris, 1 mM EDTA) twice. The protein-DNA was eluted by using 1% SDS and 0.1 M NaHCO 3 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 (Valencia, CA) and subjected to PCR using the primers designed to amplify the fragment containing the C/EBP binding site (upper, 5Ј-CTA CAC AGA CCA CTT GCA AA-3Ј; lower, 5Ј-TCA TGT CTG TGT TTG GGG AG-Ј). PCR products were resolved in 1% agarose gel and visualized using ethidium bromide staining. PCR was carried out in the linear range of DNA amplification. DNA acquired before precipitation was collected and used as the input. 10% of input was used for PCR evaluation. PCR using the primers designed to amplify the upstream region of 1␣(OH)ase promoter (Ϫ1300/Ϫ980) were used as a negative control. DNA acquired from immunoprecipitates performed with IgG was subjected to PCR using the primers designed to amplify the fragment containing the C/EBP binding site to exclude nonspecific binding.
Re-ChIP experiments were also done using sequential chromatin immunoprecipitations and two different antibodies (␣-C/EBP␤ and ␣-BRG1) to assay for the simultaneous presence of these two factors at the same site in the 1␣(OH)ase promoter. In Re-ChIP experiments, on the second day of the ChIP experiment complexes were eluted in 60 l of elution buffer containing 10 mM dithiothreitol for 30 min at 37°C. The eluted samples were diluted 50 times with ChIP dilution buffer and subjected again to the ChIP procedure using the second antibody (␣-BRG1) (42).
Electrophoretic Mobility Shift Assay-22-Mer complementary oligonucleotides spanning the C/EBP binding site at Ϫ79/ Ϫ73 of the mouse 1␣(OH)ase promoter or mutated C/EBP binding site were used for the gel shift assays. The sequences of the oligo nucleotides were 5Ј-CTT CAG CCA ATC CCA GAC GCG-3Ј and 5Ј-CGC GTC TGG GAT TGG CTG AAG-3Ј for the wild type and 5Ј-CTT CAG AGT CTC CCA GAC GCG-3Ј and 5Ј-CGC GTC TGG GAG ACT CTG AAG-3Ј for the mutant construct. Overlapping oligonucleotide strands were heat-denatured and annealed overnight. Fifty nanograms of duplex oligonucleotides were 5Ј-end-labeled with [␥-32 P]ATP and T4 polynucleotide kinase (Invitrogen) and purified using a Micro Bio-Spin P-30 column (Bio-Rad). Aliquots of nuclear preparations from calcitonin-treated AOK-B50 cells (5 g of protein) were incubated for 20 min at 27°C with 2 g of poly(dI-dC) with or without unlabeled wild type or mutant DNA competitor or C/EBP␤ 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) followed by the addition of 0.3-0.5 ng of the labeled oligonucleotide probe (ϳ100,000 cpm) and incubation for 30 min at 27°C. The samples were separated by electrophoresis on a 6% nondenaturing polyacrylamide which 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 for 2.5 h under identical conditions. The dried gels were exposed to x-ray film at Ϫ80°C with an intensifying screen.
Immunoprecipitation-Immunoprecipitation experiments were done to examine the interaction of C/EBP␤ and BRG1. Nuclear extracts were prepared from MCT cells, and protein concentration was determined by the Bradford method (39). 500 g of each preparation was used for immunoprecipitation with the addition of 4 g of C/EBP␤ antiserum or 4 g of BRG1 antiserum for 24 h at 4°C. 30 l of protein A-Sepharose 4 Fast Flow Beads (Amersham Biosciences) were added to each sample and, after further incubation by rotating at 4°C for 3 h, the immunoprecipitated complex was collected by centrifuging at 3000 rpm for 5 min. The complex was separated by 7.5 or 15% SDS-PAGE and probed with BRG1 antibody or C/EBP␤ antibody (42).

1,25(OH) 2 D 3
Production-To determine that 1␣(OH)ase protein (examined by Western blot) was catalytically active, 1,25(OH) 2 D 3 production in AOK-B50 cells in response to calcitonin was measured. Cells were cultured 60 -70% confluent in T25 flasks before serum deprivation overnight and then treated with vehicle or calcitonin for different doses for 12 h. After treatment, cells were incubated with 100 nM 25(OH)D 3 for 4 h. Cellular 1,25(OH) 2 D 3 production was determined by radioimmunoassay using the 1,25(OH) 2 D 3 radioimmunoassay kit according to the protocol of the manufacturer (IDS, Inc., Fountain Hills, AZ). Samples of medium containing 100 nM 25(OH)D 3 were incubated without cells to control for crossreactivity of 25(OH)D 3 in the assay. Cell pellets were lysed, delipidated, and immunoextracted using an immobilized monoclonal antibody to the 1␣-hydroxyl group. The extracted samples were incubated with primary antibody overnight at 4°C, and then 125 I-1,25(OH) 2 D 3 was added and incubated for another 2 h at room temperature. Primary antibody-bound or free 1,25(OH) 2 D 3 was separated using an immobilized second antibody. Bound radioactivity, quantified with a gamma counter, was inversely proportional to 1,25(OH) 2 D 3 production in the samples (40).
Statistical Analysis-Results are expressed as the mean Ϯ S.E., and significance was determined by analysis with Students' t test for two-group comparison or analysis of variance for multiple group comparisons.

1␣(OH)ase Expression and Transcription Are Induced by
Calcitonin in Kidney Cells-Previous studies have shown increased 1␣(OH)ase mRNA and enzymatic activity upon calcitonin administration in normocalcemic animals (10). AOK-B50 porcine renal proximal tubule cells (LLCPK1 cells that express PTH/PTH-related protein (PTHrP) type I receptors as well as calcitonin receptors (31,32)) have previously been used to study the regulation of 1␣(OH)ase by 1,25(OH) 2 D 3 and PTH (7,8) and represent a good in vitro model of hormonal regulation of renal 1␣(OH)ase in proximal tubules. Therefore, these cells were used to investigate the mechanisms involved in the calcitonin-mediated 1␣(OH)ase activation. We found by Western blot that calcitonin induced 1␣(OH)ase protein levels (Fig.  1A). The 1␣(OH)ase protein stimulated by calcitonin in AOK-B50 cells effectively converted 25(OH)D 3 to 1,25(OH) 2 D 3 (Fig.  1B). Fig. 1C indicates the correlation between 1␣(OH)ase protein and 1,25(OH) 2 D 3 production in these cells in response to calcitonin (r ϭ 0.97, p Ͻ 0.01). To examine the mechanism of activation of 1␣(OH)ase by calcitonin, AOK-B50 cells were transfected with the mouse 1␣(OH)ase promoter (Ϫ1651/ϩ22) as well as different deletion constructs (Fig. 2). The enhancement of luciferase activity using the Ϫ1651/ϩ22 construct by calcitonin was concentration-dependent ( Fig. 2A). A 9.3-10.4fold induction in response to calcitonin (100 nM) was observed using the Ϫ1651/ϩ22, Ϫ144/ϩ22, and Ϫ85/ϩ22 constructs, with a significant decrease in calcitonin-induced transcription observed using the Ϫ74/ϩ22 construct (4.9 Ϯ 0.4-fold induction; p Ͻ 0.05 compared with other constructs) (Fig. 2B). The enhancement of luciferase activity of the Ϫ85/ϩ22 construct by calcitonin was also concentration-dependent and similar to the response observed using the Ϫ1651/ϩ22 1␣(OH)ase promoter; Fig. 2A (data not  shown). Thus, although the Ϫ74/ ϩ22 region contributes to the calcitonin responsiveness, the decrease in transcription observed using the Ϫ74/ϩ22 construct suggests the presence of a calcitoninresponsive region between Ϫ74 and Ϫ85 that is required for maximal activation of the 1␣(OH)ase promoter by calcitonin.
C/EBP␤ Can Enhance the Calcitonin-mediated Induction of 1␣(OH)ase and Calcitonin Induces C/EBP␤ in Kidney Cells-Because examination of the mouse 1␣(OH)ase promoter indicated by sequence homology a putative C/EBP binding site at Ϫ79/ Ϫ73, we investigated the possibility that C/EBP␤ may be involved in the regulation of 1␣(OH)ase by calcitonin. C/EBP␤ (0.025, 0.05 g) significantly enhanced calcitonin induction of 1␣(OH)ase transcription 2.0 -4.2-fold (p Ͻ 0.05 compared with calcitonin alone; maximal stimulation of transcription in the presence of C/EBP␤ (0.05 g) and calcitonin was 42.0-fold; Fig. 3A). Using the Ϫ1651/ ϩ22 construct as well as the Ϫ144/ϩ22 construct, C/EBP␤ (0.025 and 0.05 g) similarly significantly enhanced calcitonin induced transcription (1.7-4.0-fold; p Ͻ 0.05 compared with calcitonin alone (1, 10, and 100 nM)). Using the Ϫ74/ϩ22 promoter construct, significant enhancement of calcitonin induction of transcription by C/EBP␤ (0.01-0.05 g) was not observed (p Ͼ 0.1 compared with calcitonin alone). However, using the Ϫ1651/ϩ22 and the Ϫ144/ϩ22 constructs and 0.2 g of C/EBP␤, a significant inhibition of calcitonin induced transcription was observed (10.0 Ϯ 1-fold versus 4.0 Ϯ 0.5-fold and 9.1 Ϯ 0.2-fold versus 4.9 Ϯ 0.3-fold induction in 1␣(OH)ase transcription, calcitonin (100 nM) versus calcitonin ϩ 0.2 g of C/EBP␤ (p Ͻ 0.05 compared with calcitonin alone) with each promoter construct, respectively). This inhibition of calcitonin induced transcription was not observed using the Ϫ85/ϩ22 promoter construct and higher concentrations of C/EBP␤ (0.1-0.5 g), suggesting an upstream regulatory region of inhibition of 1␣(OH)ase transcription in the presence of high concentrations of C/EBP␤. A-C/EBP, which functions as a dominant negative inhibitor for C/EBPs, however, inhibited calcitonin induction of 1␣(OH)ase transcription dose-dependently (Fig. 3B). This finding suggests that endogenous C/EBP is required for calcitonin induction of transcription, further supporting a predominant positive, cooperative role of C/EBP␤ with calcitonin in the induction of 1␣(OH)ase transcription. Note there was no effect of A-C/EBP on basal levels of 1␣(OH)ase transcription even at high concentrations (1 g; open bar A-C/EBP; Fig. 3B). LIP also decreased calcitonin induction of 1␣(OH)ase transcription; however, basal levels of 1␣(OH)ase transcription were similarly decreased (not shown).  APRIL 24, 2009 • VOLUME 284 • NUMBER 17

JOURNAL OF BIOLOGICAL CHEMISTRY 11063
A similar dose-dependent inhibition of calcitonin-induced 1␣(OH)ase transcription by A-C/EBP was observed using the Ϫ1651/ϩ22 promoter construct (not shown). Western blot analysis also indicated enhancement of calcitonin induction of 1␣(OH)ase protein levels by C/EBP␤ (Fig. 3C). In addition, calcitonin was found to induce the transcription of C/EBP␤ as well as C/EBP␤ protein expression (Fig. 3D). Note in Fig. 3D, right panel, that the induction of C/EBP␤ protein by calcitonin precedes the induction of 1␣(OH)ase protein by calcitonin (see Fig.  1A), consistent with a role for C/EBP␤ in calcitonin induction of 1␣(OH)ase.

C/EBP␤ Binding Site (Ϫ79/Ϫ73) on the Mouse 1␣(OH)ase Promoter Is Detected by Electrophoretic Mobility Shift
Assay-Gel shift analysis was performed to determine whether calcitonin can modify the binding of transcription factors involved in the 1␣(OH)ase promoter activation. Calcitonin treatment resulted in increased binding of nuclear extracts from AOK-B50 cells to the site at Ϫ79/Ϫ73. No binding was observed using the mutated sequence or preincubation with the unlabeled wild type oligonucleotide (Fig. 5B). Preincubation with C/EBP␤ antibody depleted the binding (Fig. 5C), indicating the ability of C/EBP␤ to bind to the element. Gel shift analysis using COS-7 cell extracts transfected with C/EBP␤ expression vector also showed binding of C/EBP␤ to the site (Ϫ79/ Ϫ73). No binding was observed using COS-7 cell nuclear extracts transfected with vector alone (data not shown).

Mutation of the C/EBP␤ Binding Site (Ϫ79/Ϫ73) Inhibits the Activation of 1␣(OH)ase Transcription Mediated by Calcitonin-
Mutation of the C/EBP binding site at Ϫ79/Ϫ73 within the Ϫ85/ϩ22 construct inhibited the response to calcitonin (Fig.  6). Mutation of this site within the Ϫ1651/ϩ22 promoter construct also markedly reduced the response to calcitonin (Fig. 6). These findings indicate that the C/EBP␤ binding site at Ϫ79/ Ϫ73 plays an important role in the calcitonin effect on 1␣(OH)ase transcription.
Calcitonin Modulates Binding of Transcription Factors to the Mouse 1␣(OH)ase Promoter-To understand mechanisms involved in the calcitonin induction of mouse 1␣(OH)ase in vivo, we first determined whether C/EBP␤ and SWI/SNF interact within the nuclei of kidney cells, and then we examined the  recruitment of C/EBP␤ and the binding of SWI/SNF as well as acetylated histone H4 to the 1␣(OH)ase promoter using the ChIP assay. Calcitonin was reported to induce 1␣(OH)ase mRNA expression in MCT cells (mouse proximal tubular cell line) (9), and our studies using MCT cells showed an enhance-ment by C/EBP␤ of calcitonin induced 1␣(OH)ase transcription (2.1 Ϯ 0.2-fold; p Ͻ 0.05 compared with calcitonin alone). We used this cell line in our ChIP assays because the mouse 1␣(OH)ase promoter sequence, unlike the porcine sequence, is well defined. Using nuclear extracts prepared from MCT cells and immunoprecipitation using BRG1 antibody and Western blot with C/EBP␤ or using C/EBP␤ antibody for immunoprecipitation and BRG1 antibody for Western blot, C/EBP␤ and BRG1 were found to be components of the same nuclear complex in MCT cells (Fig. 7A). ChIP analysis shows that calcitonin recruits C/EBP␤ to the 1␣(OH)ase promoter in vivo (Fig. 7, B  and C). Re-ChIP analysis shows that C/EBP␤ and BRG1 bind simultaneously to the 1␣(OH)ase promoter (Fig. 7, B and C). These factors do not interact with an upstream sequence of the 1␣(OH)ase promoter (Ϫ1300/Ϫ980), indicating specificity of the C/EBP␤/BRG1 interaction within the context of the proximal promoter. ChIP analysis also indicated an increase in acetylated histone 4 in response to calcitonin (Fig. 7, B and C).

DISCUSSION
Although the synthesis of 1,25(OH) 2 D 3 is induced in a hypocalcemic state by secondary hyperparathyroidism, under normocalcemic conditions, calcitonin, not PTH, has been reported to play a major role in 1,25(OH) 2 D 3 synthesis (10). The data here provide a mechanism, for the first time that accounts at least in part for the calcitonin induction of 1␣(OH)ase. C/EBP␤ and SWI/SNF were found to mediate the calcitonin induction of 1␣(OH)ase transcription. A positive C/EBP␤ site at Ϫ79/Ϫ73 on the 1␣(OH)ase promoter was identified.
C/EBPs, and in particular C/EBP␤, have been implicated in numerous different processes including hormonal control of nutrient metabolism, differentiation, and regulation of cell type-specific gene expression (45). The levels of C/EBP proteins have previously been reported to differentially modulate gene transcription (46,47). With regard to vitamin D metabolism, C/EBP␤ has previously been shown to be induced by 1,25(OH) 2 D 3 in kidney and osteoblasts and to act as an enhancer of negative vitamin D-mediated transcription of 24(OH)ase (41). ␥-interferon induction of C/EBP␤ expression has been shown to contribute to ␥-interferon transcriptional control of 1␣(OH)ase expression in monocytes/macrophages (48,49). It was concluded that C/EBP␤ is the essential transcription factor controlling immune-mediated 1␣(OH)ase transcription (48,49).
Recent studies examining mechanisms involved in the PTH regulation of mouse 1␣(OH)ase transcription in kidney cells noted that the orphan receptor nuclear receptor 4A2 (NR4A2 or Nurr 1) and not C/EBP␤ is a key factor involved in the induction of 1␣(OH)ase transcription by PTH (8). NR4A2 was previously shown to have an important role in brain in normal dopamine cell functions (50) in the regulation of osteopontin in osteoblasts (51) and in the regulation of key cytokines in T cells (52). In AOK-B50 cells PTH induces NF4A2, and C/EBP␤ was found to inhibit the NF4A2 induction of 1␣(OH)ase transcription (8). We found that the 1␣(OH)ase promoter was more sensitive to calcitonin stimulation than to PTH (maximal induction by PTH of 1␣(OH)ase transcription in AOK-B50  (OH)ase (Ϫ85/ϩ22) promoter (wild type (WT) or mutant (MT)) or 1␣(OH)ase (Ϫ1651/ϩ22) promoter (wild type or mutant). After 24 h, cells were treated with vehicle or 100 nM calcitonin (CT) for another 24 h. 1␣(OH)ase promoter activity was measured by firefly luciferase activity/protein concentration and represented as -fold induction (mean Ϯ S.E.) by comparison to basal levels (3-5 observations/group). *, p Ͻ 0.05 compared with wild type, calcitonin-treated. cells is ϳ2-fold (8) 4 , further suggesting that different factors are involved in the regulation of 1␣(OH)ase by CT and PTH. Our study is the first demonstration of the induction of C/EPB␤ by calcitonin in kidney cells. It is of interest that PTH, which induces C/EBP␤ in osteoblastic cells, does not induce C/EBP␤ in AOK-B50 cells or other kidney cells (unlike calcitonin) (41). Thus, different mechanisms are involved in the calcitonin induction and PTH induction of 1␣(OH)ase transcription.
SWI/SNF chromatin remodeling complex has been shown to participate in cell cycle control, gene regulation, development, and differentiation (53). Although SWI/SNF has been found to associate with several transcription activators including steroid receptors, erythroid Kruppel-like factors, and heat shock factor 1 (54), only a few transcription factors have the capacity to recruit SWI/SNF to the promoter region. Among them is C/EBP␤, which has been reported to cooperate with SWI/ SNF to regulate the expression of myeloid genes (43), the osteocalcin gene in osteoblastic cells (44), and mammary-specific casein genes (55). Similar to our study of 1␣(OH)ase gene transcription in kidney cells, BRG1-DN inhibited osteocalcin gene transcription in osteoblastic cells and ␤ and ␥ casein transcription in EpH4 cells (epithelial cells derived from normal mouse mammary gland) (44,55), and ChIP/Re-ChIP analysis indicated that BRG1 and C/EBP␤ interact within the context of the osteocalcin promoter (44). Also, extracellular matrix protein was found to cooperate with prolactin to induce the recruitment of BRG1 and C/EBP␤ to the ␤ and ␥ casein promoters (55). ChIP analysis also demonstrated enhanced histone acetylation after activation in the ␤ casein promoter (55). In the regulation of osteocalcin it has been suggested that SWI/SNF and histone acetylation cooperate in mediating changes in chromatin structure that facilitate osteocalcin transcription (44). In our studies ChIP analysis indicated an increase in acetylated histone H4 in response to calcitonin, similarly suggesting cooperation between acetylation and chromatin remodeling. It is possible that acetylation may allow SWI/SNF to remain stably bound to the promoter, thus facilitating remodeling of nucleosomes. It has previously been reported that acetylation of histone H4 results in firm association of SWI/SNF through BRG1 (56). Thus, increased calcitonin levels would result in enhanced C/EBP␤ binding to the 1␣(OH)ase promoter, recruitment of SWI/SNF, and cooperation between acetylation and chromatin remodeling, allowing for efficient initiation of transcription. In future studies it will be of interest to examine additional coactivators that may be involved in the C/EBP␤-SWI/SNF mediated calcitonin induction of transcription. Because CBP, a histone acetyltransferase, has been reported to interact with C/EBP␤ and is involved in C/EBP activation of transcription (41,57), it is possible that CBP may be involved in the calcitonin regulation of 1␣(OH)ase and in the cooperation between acetylation and chromatin remodeling.
Although we identified a positive C/EBP␤ binding site within the Ϫ85/ϩ22 region of the mouse 1␣(OH)ase promoter, it has been noted that C/EBP␤ has both activation and repression functions, depending on the promoter context and co-factor interaction (46). C/EBP␤ has been reported to activate genes by recruiting chromatin remodeling complexes and by cooperat-4 Y. Zhong and S. Christakos, unpublished observation. FIGURE 7. C/EBP␤ and BRG1 are components of the same nuclear complex and calcitonin modulates C/EBP␤ and BRG1 recruitment to the 1␣(OH)ase promoter. A, nuclear extracts were prepared from MCT cells and used for immunoprecipitation (IP) with C/EBP␤ antibody, BRG1 antibody, or control rabbit IgG. WB, Western blot. B, ChIP analysis of CEBP␤, acetylated histone H4 (AcH4), and Re-ChIP analysis of BRG1 binding to the 1␣(OH)ase promoter. MCT cells were treated with vehicle or calcitonin for 1 and 4 h and cross-linked by 1% formaldehyde for 15 min. Cross-linked cell lysates were subjected to immunoprecipitation first with C/EBP␤ antibody (␣-C/EBP␤) and then with BRG1 antibody (␣-BRG1). DNA precipitates were isolated and then subjected to PCR using specific primers designed according to the C/EBP␤ site on the mouse 1␣(OH)ase promoter (see "Experimental Procedures"). Analysis of input DNA (0.2%) was taken before precipitation (Input). Recruitment of Brm to the 1␣(OH)ase promoter was not observed (it should be noted that, although Brm and BRG1 were detected in AOK-B50 cell nuclear extracts, BRG1 but not Brm was detected by Western blot analysis of nuclear extracts of MCT cells). Using a distal 1␣(OH)ase promoter region (Ϫ1300/Ϫ980) binding of C/EBP␤ and BRG1 was not observed. C, quantitation of ChIP analyses (ϮS.E.).
ing with transcription factors such as Myb and CBP/P300 (43,44,57,58). C/EBP␤ also possesses the capacity to suppress gene expression directly through its repression domains or indirectly through other co-factors. Studies from the Wahli laboratory (59) have shown inhibition of peroxisome proliferator-activated receptor ␤expression by C/EBP␤ and its association with histone deacetylase in the control of differentiation and proliferation of keratinocytes. It can also bind directly to the C/EBP element on the osteoblast-specific Runx2 promoter independent of deacetylase activity to repress gene expression, resulting in an inhibition of retinoic acid-induced osteoblast differentiation (60). In our studies, C/EBP␤ at low concentrations enhances calcitonin-induced 1␣(OH)ase transcription, but at high concentrations repression of 1␣(OH)ase transcription was observed. Thus, although the predominant effect of C/EPB␤ is enhancement, as indicated by increased expression of 1␣(OH)ase protein in C/EBP␤-transfected cells and inhibition of calcitonin-induced 1␣(OH)ase transcription by A-C/EBP as well as by LIP, it is possible that C/EBP␤ may have a dual role depending on the level of C/EBP␤, the hormonal context, the specific intracellular environment, and the level of 1␣(OH)ase expression.
In summary, calcitonin is a hormone that has diverse physiological actions, including a role in the maintenance of 1,25(OH) 2 D 3 levels. Previous evidence indicated that the stimulation of 1,25(OH) 2 D 3 under normocalcemic conditions by calcitonin has physiological importance during pregnancy, lactation, and early development (14 -16). Our findings provide a mechanism for the first time for calcitonin induction of 1␣(OH)ase and identify key regulators involved in the maintenance of 1,25(OH) 2 D 3 levels.