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Am J Physiol Endocrinol Metab 286: E598-E608, 2004. First published December 9, 2003; doi:10.1152/ajpendo.00214.2003
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Integration of hormone signaling in the regulation of human 25(OH)D3 24-hydroxylase transcription

Frank Barletta, Puneet Dhawan, and Sylvia Christakos

Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103

Submitted 13 May 2003 ; accepted in final form 4 December 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The current study sought to define the molecular mechanisms involved in the cross talk between 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and activators of PKC in the regulation of 25(OH)D3 24-hydroxlyase [24(OH)ase]. Transfection of the h24(OH)ase promoter construct [-5,500/-22 luciferase; vitamin D response elements at -294/-274 and -174/-151; AP-1 site at -1,167/-1,160] in vitamin D receptor (VDR)-transfected COS-7 cells resulted in strong activation by 1,25(OH)2D3. In these cells, cotreatment with the PKC activator TPA and 1,25(OH)2D3 yielded a 27-fold increase in luciferase activity, which was 2- to 3-fold greater than activation obtained with 1,25(OH)2D3 alone (P < 0.05). Similar results were observed using LLCPK-1 kidney cells, suggesting that the previously observed enhancement of 1,25(OH)2D3-induced renal 24(OH)ase mRNA and activity by PKC activation occurs at the level of transcription. The functional cooperation between PKC activation and VDR was not found to be mediated by the AP-1 site in the h24(OH)ase promoter or by enhanced binding of GRIP or DRIP205 to VDR and was also not due to PKC-mediated phosphorylation of VDR on Ser51. Our study demonstrates that, in LLCPK-1 kidney cells, the PKC enhancement of 1,25(OH)2D3-stimulated transcription may be due, in part, to an increase in VDR concentration. In addition, inhibitors of the MAPK pathway were found to decrease the TPA enhancement (P < 0.05). Because activation of MAPK has been reported to result in the phosphorylation of SRC-1 and in functional cooperation between SRC-1 and CREB binding protein, we propose that the potentiation of VDR-mediated transcription may also be mediated through changes in the phosphorylation of specific VDR coregulators.

1,25-dihydroxyvitamin D3; vitamin D receptor; vitamin D regulation; phosphorylation; protein kinase C

A PRINCIPAL FUNCTION of the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is to maintain normal calcium homeostasis, resulting in normal bone mineralization and remodeling. 1,25(OH)2D3 has also been found to affect other physiological systems, including cell growth and differentiation, secretion of hormones, T cell proliferation, and cytokine production (11). The actions of 1,25(OH)2D3 are dependent on regulation of gene expression and are mediated through the vitamin D receptor (VDR) (27, 38). The liganded VDR heterodimerizes with the retinoid X receptor (RXR) and interacts with vitamin D response elements (VDREs) in the promoter of target genes. Transactivation is achieved through proteins of the preinitiation complex, p160 coactivators [steroid receptor coactivator 1 (SRC-1/NcoA1), glucocorticoid receptor interacting protein 1 (GRIP-1/TIF2), and activator of thyroid and retinoic acid receptors (ACTR/pCIP)] that have histone acetylase activity as well as through vitamin D receptor (VDR)-interacting proteins (the coactivator DRIP complex) that act through the recruitment of RNA polymerase (44).

The expression of vitamin D-regulated genes depends not only on VDR and associated coactivators but also on a balance between synthesis and degradation of 1,25(OH)2D3 (25,40). 1,25(OH)2D3 is produced by two sequential hydroxylations of vitamin D by 25-hydroxylase in the liver and by 25-hydroxyvitamin D3 1{alpha}-hydroxylase (1{alpha}-hydroxylase) in the kidney (40). The metabolic inactivation of 1,25(OH)2D3 is catalyzed by the C24 oxidation pathway. The 25-hydroxyvitamin D3 24-hydroxylase enzyme [24(OH)ase], which is strongly induced by 1,25(OH)2D3 in kidney and many other tissues, plays a critical function in the regulation of 1,25(OH)2D3 levels. It catalyzes the hydroxylation of 25(OH)D3 and 1,25(OH)2D3 on carbon 24, leading to the production of 24,25(OH)2D3 and 1,24,25(OH)3D3, respectively (40). The production of 24,25(OH)2D3 reduces the amount of 25(OH)D3 available to be converted to 1,25(OH)2D3, and the production of 1,24,25(OH)3D3 is believed to be the initial step in the catabolism of 1,25(OH)2D3 to calcitroic acid (46). Recent studies using the 24(OH)ase-null mutant mouse provided the first direct in vivo evidence that the C-24 pathway, initiated by the 24(OH)ase enzyme, is the major catabolic process that functions to regulate the physiological levels of 1,25(OH)2D3, thereby preventing the accumulation of toxic levels of the hormone (53). Thus 1,25(OH)2D3, by inducing the 24(OH)ase enzyme, stimulates its own deactivation.

The cloning of the rat and human 24(OH)ase gene promoters has made possible, for the first time, studies related to the regulation of this major target of 1,25(OH)2D3 action (7, 30, 39, 59). The 24(OH)ase gene is the most transcriptionally responsive 1,25(OH)2D3-inducible gene identified to date. It is the first vitamin D-responsive gene to be controlled by two independent VDREs (7, 30, 59). Although a number of studies by us and others (4, 13, 14, 22, 45) have defined factors that affect rat 24(OH)ase transcription, very little work has been done to examine molecular mechanisms by which 1,25(OH)2D3 and other factors affect the regulation of human 24(OH)ase transcription. In previous studies, two VDREs have been defined and a putative activating protein-1 (AP-1) site has been identified by sequence homology in the human 24(OH)ase promoter (7, 59). Signal transduction pathways that are responsive to peptide hormones and growth factors, including protein kinase C (PKC) as well as AP-1 transcription factors, have been reported to result in inhibitory or stimulatory effects on cell responsiveness to steroid hormones, including 1,25(OH)2D3 (10, 29, 35-37, 41, 42, 51, 52). Although compounds that increase PKC activity, including 12-o-tetradecanoylphorbol 13-acetate (TPA), have been shown to enhance 1,25(OH)2D3-induced 24(OH)ase mRNA and activity levels (1, 2, 8, 20, 34), the exact mechanism by which PKC affects 1,25(OH)2D3 action is not well understood. The mechanism may involve phosphorylation of VDR, since PKC has been shown to phosphorylate VDR on Ser51 (21). It is also possible that the enhanced response in the presence of TPA is due to an effect on the regulation of the VDR and/or an effect on other transcription factors. Although the human 24(OH)ase promoter has been shown to contain a putative AP-1 sequence (7), whether it is responsible for the actions of TPA is not yet known. In this study, therefore, we sought to define the molecular mechanisms involved in the cross talk between 1,25(OH)2D3 and PKC activation in the regulation of human 24(OH)ase transcription.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. [14C]chloramphenicol (50 mCi/mmol), [{gamma}-32P]ATP [3,000 Ci (111 TBq)/mmol], nylon membranes, and enhanced chemiluminescence (ECL) detection system were purchased from NEN Life Science Products (Boston, MA). Acetyl-coenzyme A, formamide, and TPA were obtained from Sigma Chemical (St. Louis, MO). Dulbecco's Modified Eagle's Medium (DMEM), Medium-199, and T4 polynucleotide kinase were purchased from Life Technologies (Gaithersburg, MD). Rat monoclonal anti-VDR antibody was purchased from Affinity BioReagents (Neshanic Station, NJ). PKC and PKA inhibitors (Gö-6983 and KT-5720) were purchased from Calbiochem (San Diego, CA). MAPK/ERK kinase (MEK) inhibitors U-0126 and PD-98059 were obtained from Cell Signaling Technology (Beverly, MA). Human (h)24(OH)ase promoter constructs (promoter regions -5,500/-22 and -316/-22 in luciferase) were a gift from Elizabeth Allegretto and Ligand Pharmaceutical (San Diego, CA) (59). 1,25(OH)2D3 and 1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor vitamin D3 (RO 26-2198) were generous gifts from M. Uskokovic (Hoffmann-LaRoche, Nutley, NJ).

Cell culture. COS-7 African Green monkey kidney cells, LLCPK-1 porcine kidney cells, and UMR-106 rat osteoblastic cells were obtained from American Type Culture Collection and were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) from Gemini (Calabassas, CA), Medium-199 supplemented with 3% FBS, and DMEM-F-12 supplemented with 5% FBS, respectively. All cell lines were grown in a humidified atmosphere of 95% air-5% CO2 at 37°C. Cells were grown to 60-70% confluence in 100-mm tissue culture dishes, and 24 h before the start of experiments the medium was replaced with medium supplemented with 2% charcoal-stripped serum. Cells were treated with vehicle or the compounds noted at the concentrations and times indicated.

Cell transfections, chloramphenicol acetyltransferase and luciferase assays. Promoter constructs containing the h24(OH)ase promoter region (-5,500/-22 or -316/-22) linked to the luciferase reporter gene were used (see Fig. 2A and Ref. 59). A tk-CAT reporter plasmid containing multiple copies of the 24(OH)ase VDRE (-174/-151) was also used (see Fig. 2A). To construct the proximal h24(OH)ase VDRE-tk CAT reporter plasmid, two complementary oligonucleotides containing the proximal VDRE of the h24(OH)ase promoter were synthesized with XbaI half-site overhangs at their 5' end. After annealing (100°C for 5 min) and cooling to room temperature, the annealed double-stranded DNA was excised from the gel and purified. The purified DNA fragment was phosphorylated by T4 polynucleotide kinase. Phosphorylated double-stranded DNA fragments were ligated into the XbaI site of the tk promoter CAT reporter gene (pBLCAT2; from J. W. Pike, University of Cincinnati, Cincinnati, OH). The number of inserts was checked by PstI digestion, and the orientation was confirmed by DNA sequencing. The VDRE multimers were inserted 105 bp upstream of the tk transcription start site. Mutations in an AP-1-like site of the h24(OH)ase promoter (TGACTCC at -151/-142 mutated to AAACAAC) were performed with a Quick Change site-directed mutagenesis kit from Stratagene (Cedar Creek, TX), as instructed in the user manual. The mutated constructs were sequenced to confirm the desired mutation. In selected experiments, cells were cotransfected with pCMV c-fos and pCMV c-jun expression vectors (provided by R. Tjian, University of California, Berkeley, CA). PKC expression vector (pCDM8-PKC{alpha}) was from E. N. Olson (University of Texas Southwestern Medical Center, Dallas, TX) (24). The HTLV-1 LTR promoter construct containing AP-1 sites, used as a positive control, was a gift from F. Kashanchi (George Washington University School of Medicine, Washington, DC). The rat osteocalcin (OC) VDRE-tk CAT construct was made by R. Gill in our laboratory. The human VDR promoter luciferase construct (-1,500/+60) was made as previously described (22). All cells were cotransfected with the appropriate reporter plasmid and a {beta}-galactosidase expression vector (pCH110; Pharmacia, Piscataway, NJ) as an internal control for transfection efficiency. Cells were transiently transfected using the calcium phosphate DNA precipitation method (3). In addition to the reporter plasmid and a {beta}-galactosidase expression vector, COS-7 cells were also transiently transfected with the hVDR expression vector pAVhVDR (from J. W. Pike) or a mutated VDR expression vector construct [S51A, also a gift from J. W. Pike (18)]. Empty vectors were used to keep the total DNA concentration the same. After transfection, cells were shocked for 1 min with phosphate-buffered saline (PBS) containing 10% dimethyl sulfoxide, washed with PBS, and treated as described [1,25(OH)2D3 (10-8 M) or TPA (100 nM)] in the appropriate medium supplemented with 2% of charcoal-dextran-treated FBS. Initial studies using different concentrations of TPA indicated that a plateau in enhanced responsiveness of VDR-mediated h24(OH)ase transcripiton was observed between 25 and 100 nM TPA. Thus the maximally effective concentration of TPA (100 nM) was used. After treatment, cells were harvested by trypsinization, pelleted, washed with PBS, and resuspended in 0.25 M Tris·HCl, pH 8.0. The cell pellets were then lysed by freezing and thawing five times. The cellular extract was collected and used for {beta}-galactosidase or protein analysis. A CAT assay was then performed by standard protocols on the cell extract normalized to {beta}-galactosidase activity and/or total protein (3, 6, 17). Autoradiograms were analyzed by densitometric scanning using the Shimadzu CS9000U Dual-Wavelength Flying Spot Scanner (Shimadzu Scientific Instruments, Princeton, NJ). For some experiments, several autoradiographic exposure times were needed for densitometric analysis. CAT activity was also quantitated by scanning TLC plates by use of the PACKARD Constant Imager System (Packard Instrument, Meriden, CT). Luciferase activity was determined by using Promega's Luciferase Assay System (Promega, Madison, WI), with minor modifications. The assay was performed using aliquots of extracts that contained equal {beta}-galactosidase activity. Cells were harvested and cellular extracts prepared as described above for the CAT analysis. Luciferase activity was quantitated using the LumiCount luminometer from Packard BioScience.



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  		Fig. 2. A: h24(OH)ase promoter gene constructs used in transfection experiments. The putative activating protein (AP)-1 site identified by Chen and DeLuca (7) and the two vitamin D response elements (VDREs: p, proximal; d, distal) are indicated. B: enhancement of 1,25(OH)2D3-induced h24(OH)ase transcription by TPA is not mediated by the AP-1 site at -1,166/-1,160. COS-7 cells were transfected with a VDR expression vector and luciferase constructs of either the -5,500/-22 (left) or the -316/-22 (right) h24(OH)ase promoter. Cells were treated as previously described with vehicle (Basal), +D, TPA, or TPA + D for 24 h. Transfection of the h24(OH)ase promoter construct and treatment with 10-8 M 1,25(OH)2D3 resulted in an 11.7 ± 1.3-fold increase in luciferase activity over Basal in VDR-transfected COS-7 cells (n = 3). Transfection of the -316/-22 h24(OH)ase promoter construct and treatment with 10-8 M 1,25(OH)2D3 resulted in a 10.6 ± 1.3-fold increase in luciferase activity in VDR-transfected COS-7 cells over Basal (n = 3). A promoter construct containing the HTLV-1 LTR that contains 2 functional AP-1 sites was used as a positive control (C). Cells were treated with TPA (100 nM, 24 h) or transfected with empty vector (Basal) or c-fos or c-jun expression vectors (0.5 µg each). TPA treatment resulted in a 9.6 ± 1.4-fold induction in CAT activity (n = 4).

 

VDR Western blot analysis. Cells were treated and harvested by trypsinization, and nuclear extracts were prepared following the method of Dignam et al. (12). Protein content was measured by the method of Bradford (6), and equal amounts of protein from each sample (50 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane from Bio-Rad (Hercules, CA). The membrane was blocked with PBST (0.5% Tween-20 in PBS) containing 5% nonfat milk at 4°C for 16 h. After blocking, the blot was incubated with rat monoclonal anti-VDR antibody (Affinity Bioreagents) for 2 h at room temperature, washed with PBST, and incubated for an additional hour with a goat anti-rat IgG conjugated to horseradish peroxidase (Sigma). After a subsequent washing with PBST, the ECL Western blotting detection system (NEN Life Sciences) was used to detect the antigen/antibody complex. As a control, VDR Western blots were also analyzed for {alpha}-tubulin (antibody from Sigma). Western blots were quantitated by densitometric scanning. The relative optical density obtained using VDR antibody was divided by the relative optical density obtained after incubation with {alpha}-tubulin antibody to normalize for sample variation.

Electrophoretic mobility shift assay. Nuclear extracts were prepared by the method of Dignam et al. (12) from LLCPK-1 cells incubated with vehicle, 1,25(OH)2D3 (10-8 M), TPA (100 nM), or 1,25(OH)2D3 (10-8 M) plus TPA (100 nM) for 24 h. Immunoblots were used to measure VDR content of cell extracts. A DNA fragment containing the proximal VDRE of the h24(OH)ase promoter (-174/-151; prepared by the UMD Molecular Resource Facility Newark, NJ) was used as a probe. Overlapping and reverse strands were heat denatured and annealed overnight. Fifty nanograms of duplex oligo were end-labeled with [{gamma}-32P]ATP by use of T4 polynucleotide kinase (Life Technologies) and purified using a micro biospin p-30 column (Bio-Rad Laboratories). The eluted probe was used for electrophoretic mobility shift assay (EMSA) as described (4). The samples were separated by electrophoresis on a 6% nondenaturing polyacrylamide gel. Gels were dried and exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY).

Glutathione S-transferase fusion protein pull-down assay. Nuclear proteins were prepared from COS-7 cells that were transiently transfected with a VDR expression vector and incubated with either vehicle, 1,25(OH)2D3 (10-8 M), 1,25(OH)2D3 (10-8 M) plus TPA (100 nM), or RO 26-2198 (10-8 M) for 24 h. VDR protein levels in each sample were assessed by Western blot analysis using monoclonal anti-VDR antibody (Affinity Bioreagents). The glutathione S-transferase (GST)-DRIP205 (527-970) or GST-GRIP-1 fusion proteins (56), immobilized on Sepharose beads, were incubated with GST binding buffer (20 mM Tris·HCl, pH 7.9, 180 mM KCl, 0.2 mM EDTA, pH 8.0, 0.05% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) that contained 1 mg/ml BSA for 2 h. The GST binding buffer was replaced with nuclear extract aliquots, containing equal amounts of VDR protein, and incubated with 15 µg of the appropriate fusion protein for 16 h at 4°C. Interacting proteins were washed four times with GST binding buffer containing 0.1% Nonidet P-40 and then incubated with elution buffer [GST binding buffer containing 0.1% Nonidet P-40 and 0.15% Sarkosyl (Sigma)] at 4°C for 20 min. The eluted proteins were analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blotting with VDR antibody.

Statistical analysis. Significance was determined by t-test or Dunnett's multiple comparison t-statistic.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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TPA enhances 1,25(OH)2D3-induced transcription of the h24(OH)ase gene. To study the effect of the PKC activator TPA on vitamin D-induced 24(OH)ase transcription, hVDR-transfected COS-7 cells were transfected with a luciferase construct containing the region -5,500/-22 of the h24(OH)ase promoter. Cells were treated with vehicle (ethanol), 1,25(OH)2D3, (10-8 M), TPA (100 nM), or cotreated with 1,25(OH)2D3 (10-8 M) and TPA (100 nM) for 24 h (Fig. 1A). Treatment with TPA alone resulted in an increase in basal activity in the absence of 1,25(OH)2D3 (2.3-fold, P < 0.05). When COS-7 cells were cotreated, TPA and 1,25(OH)2D3 yielded a 27-fold increase in luciferase activity, which was twofold higher (P < 0.05) than the activation obtained with 1,25(OH)2D3 alone. Similar results were observed using LLCPK-1 cells (Fig. 1B).



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  		Fig. 1. Effect of 12-o-tetradecanoylphorbol 13 acetate (TPA) on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced human 25-hydroxyvitamin D3 24-hydroxylase [h24(OH)ase] transcription. Vitamin D receptor (VDR)-transfected COS-7 cells (A) or LLCPK-1 kidney cells (B) were transfected with the h24(OH)ase (-5,500/-22) luciferase construct. Transfected cells were treated with vehicle (Basal), 10-8 M 1,25(OH)2D3 (+D), 100 nM TPA (TPA), or 1,25(OH)2D3 + 100 nM TPA (TPA + D) for 24 h. After treatments, cells were harvested and lysed, and luciferase activity was measured as described in MATERIALS AND METHODS. Results of 3-5 separate experiments are expressed as percent maximal response ± SE. *P < 0.05, TPA + D vs. +D. Transfection of the -5,500/-22 h24(OH)ase promoter construct and treatment with 10-8 M 1,25(OH)2D3 resulted in a 12.5 ± 1.0-fold increase in luciferase activity in COS-7 cells and a 6.7 ± 0.8-fold increase in luciferase activity in LLCPK-1 cells over Basal.

 

Enhancement of 1,25(OH)2D3-induced h24(OH)ase transcription by TPA is not mediated by the AP-1 site at -1,166/ -1,160. To define the molecular mechanism involved in the cross talk between 1,25(OH)2D3 and activation of PKC in the regulation of human 24(OH)ase transcription, studies were done using COS-7 cells. COS-7 cells were transiently cotransfected with hVDR and an h24(OH)ase promoter construct that contains the -5,500/-22 promoter region in the presence or absence of pCMV c-fos and pCMV c-jun expression vectors. After transfection, the cells were treated with vehicle (ethanol), 1,25(OH)2D3 (10-8 M), TPA (100 nM), or a cotreatment of 1,25(OH)2D3 (10-8 M) and TPA (100 nM) for 24 h. The 24(OH)ase promoter construct containing region -5,500/-22 contains a consensus AP-1 site at -1,166/-1,160 (7) (Fig. 2A). Expression of c-fos and c-jun (0.5 µg each) did not stimulate the 1,25(OH)2D3 or TPA and 1,25(OH)2D3-induced transcription of the -5,500/-22 h24(OH)ase promoter construct but rather inhibited the induction (Fig. 2B). The inhibition may be due to squelching of transcription factors necessary for VDR-mediated transcription such as CBP and/or SRC-1 (28, 52). As a positive control, to ensure that the c-fos and c-jun expression vectors are able to mediate transcription through an AP-1 site, a human T-cell leukemia virus type 1 long terminal repeat (HTLV-1 LTR) promoter construct, containing functional AP-1 sites, was cotransfected with c-fos and c-jun expression vectors (0.5 µg each) in COS-7 cells (Fig. 2C). Transcription of the AP-1-responsive promoter construct was increased by treatment with TPA or by transfection of c-fos and c-jun (Fig. 2C). The h24(OH)ase promoter construct -316/-22 lacks the putative AP-1 site at -1,166/-1,160 (Fig. 2A). TPA and 1,25(OH)2D3 cotreatment of COS-7 cells transfected with the promoter construct containing the -316/-22 region of the human 24(OH)ase promoter still resulted in an enhancement over the 1,25(OH)2D3-induced transcription (3.9-fold, P < 0.05; Fig. 2B). These results indicate that the consensus AP-1 sequence at -1,166/-1,160 in the h24(OH)ase promoter is not involved in mediating the enhancement of 1,25(OH)2D3-induced h24(OH)ase transcription by TPA. Expression of c-fos and c-jun also did not stimulate but rather inhibited the 1,25(OH)2D3- or TPA- and 1,25(OH)2D3-induced transcription of the -316/-22 construct. Using the -316/-22 promoter construct, mutation of an AP-1-like sequence (TGACTCC to AAACAAC) at -151/-142 adjacent to the proximal VDRE (-172/-153) also did not alter the TPA responsiveness (data not shown). A nonfunctional AP-1-like sequence was also noted adjacent to the rat proximal 24(OH)ase (14).

TPA enhances 1,25(OH)2D3-induced transcription of the h24OHase VDRE (-174/-151)-tk CAT in COS-7 cells. To investigate whether the enhancement of 1,25(OH)2D3-induced transcription by TPA occurs through the VDRE and not via other sequences in the promoter, COS-7 cells were cotransfected with the h24(OH)ase proximal (-174/-151) VDRE-tk CAT promoter construct and a pAVhVDR expression vector. After transfection, the cells were cultured in the presence of vehicle (ethanol), 1,25(OH)2D3 (10-8 M), or a cotreatment of 1,25(OH)2D3 (10-8 M) and TPA (100 nM) for an additional 24 h (0-24 h; Fig. 3A). Similar to the -5,500/-22 and the -316/-22 h24(OH)ase promoter constructs, cotreatment of TPA plus 1,25(OH)2D3 resulted in a 3.7-fold (P < 0.05) enhancement of the 1,25(OH)2D3-induced transcription of the 24(OH)ase VDRE construct, indicating that the proximal VDRE in the h24(OH)ase promoter is sufficient to confer transcriptional activation by TPA. Similar results were observed using LLCPK-1 cells (not shown). To determine the effects of TPA at shorter incubation times, COS-7 cells were cotransfected with an hVDR expression vector and the 24(OH)ase VDRE-tk CAT promoter construct and pretreated with TPA for the times indicated, followed by the addition of 1,25(OH)2D3 for 24 h in the absence of TPA (0.5-4 h; Fig. 3A). At all time points, TPA treatment resulted in an increase in the 1,25(OH)2D3-induced transcription. Pretreatment for 4 h with TPA resulted in an enhancement in VDR-mediated transcription that was not significantly different from the enhancement observed with cotreatment of TPA and 1,25(OH)2D3 for 24 h (P > 0.5). To determine whether the enhancement observed using the h24(OH)ase VDRE by TPA was mediated by the action of PKC, COS-7 cells were pretreated with TPA for 4 h in the presence or absence of the specific PKC inhibitor Gö-6983 (500 nM). In the presence of Gö-6983, the TPA enhancement was not observed [P > 0.5 vs. treatment with 1,25(OH)2D3; Fig. 3B]. Pretreatment with the PKC inhibitor for 4 h in the absence of TPA did not affect 1,25(OH)2D3-induced transcription (not shown). In addition, transfection of 1 or 3 µg of PKC expression vector (pCDM8-PKC{alpha}) containing a cDNA encoding bovine PKC{alpha} (24) followed by treatment with 1,25(OH)2D3 (10-8 M for 24 h) resulted in a two- to threefold enhancement in transcription, respectively, over the levels of transcription observed with 1,25(OH)2D3 alone (data not shown). PKC expression vector alone had no effect on transcription. Treatment with 500 nM KT-5720 (a PKA inhibitor) for 4 h in the presence of TPA followed by treatment with 1,25(OH)2D3 alone for 24 h did not affect TPA-mediated enhancement or 1,25(OH)2D3-induced transcription (data not shown).



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  		Fig. 3. h24(OH)ase proximal VDRE is sufficient to mediate enhancement of 1,25(OH)2D3-induced h24(OH)ase transcription by activation of PKC, and the effect of TPA is cell type specific and not due to a general increase in transcription. A: COS-7 cells transfected with hVDR expression vector and the h24(OH)ase (-174/-151) VDRE-tk CAT construct were treated with TPA (100 nM) and 1,25(OH)2D3 simultaneously for 24 h or were pretreated with 100 nM TPA for indicated times (0.5, 1, 2, and 4 h) to determine the effect of TPA at shorter incubation times. After TPA pretreatment, cells were incubated with 1,25(OH)2D3 alone for 24 h. Shown is a representative autoradiogram; similar results were observed in several different experiments. B: PKC inhibitor Gö-6983 blocks TPA-induced enhancement of transcription. COS-7 cells transfected with hVDR and the 24(OH)ase VDRE-tk CAT construct were pretreated for 4 h with TPA (100 nM) in the absence or presence of the specific PKC inhibitor Gö-6983 (500 nM), followed by incubation with 1,25(OH)2D3 alone (10-8 M) for 24 h (TPA + D and TPA + D + Gö-6983, respectively). Results of 3-6 separate experiments are expressed as percent maximal response ± SE after normalization for {beta}-galactosidase activity. Transfection of the h24(OH)ase VDRE tk CAT construct and treatment with 10-8 M 1,25(OH)2D3 resulted in a 10.5 ± 1.8-fold induction in CAT activity in VDR-transfected COS-7 cells. *P < 0.05, TPA + D vs. +D. TPA + D + Gö-6983 vs. +D, P > 0.5. C: UMR-106 cells were transfected with the 24(OH)ase VDRE-tk CAT construct and treated as previously described with vehicle (-D/Basal), +D, or TPA + D for 24 h. Representative autoradiogram is shown. Results of 3 separate experiments are expressed as percent maximal response ± SE after normalization for {beta}-galactosidase activity. TPA treatment did not significantly affect 1,25(OH)2D3-induced transcription (P > 0.5, n = 3). Similar results were observed using the h24(OH)ase -5,500/-22 promoter construct (not shown). D: COS-7 cells were cotransfected with hVDR expression vector, and 4 µg of a control reporter plasmid pRSV-CAT, which places CAT under the control of Rous sarcoma virus long-terminal repeat or pBL2-CAT, which uses the minimal promoter of the thymidine kinase gene. Cells were treated as previously described with vehicle (Basal), TPA, or TPA + D for 24 h. Results of 3 separate experiments are expressed as percent maximal response ± SE after normalization for {beta}-galactosidase activity. P > 0.5 Basal vs. TPA or TPA + D.

 

Activation of PKC does not enhance 1,25(OH)2D3-dependent 24(OH)ase transcription in UMR-106 cells. UMR-106 cells, expressing endogenous VDR and transfected with the 24(OH)ase VDRE (-174/-151)-tk CAT construct, were treated with vehicle (ethanol), 1,25(OH)2D3, (10-8 M), TPA (100 nM), or 1,25(OH)2D3 plus TPA. Unlike the results obtained in LLCPK-1 and VDR-transfected COS-7 cells, treatment of UMR-106 cells with 1,25(OH)2D3 plus TPA did not affect 1,25(OH)2D3-induced 24(OH)ase transcription (+D vs. TPA + D, P > 0.5; Fig. 3C).

TPA does not affect transcription of control reporter plasmids. COS-7 cells were cotransfected with a minimal promoter construct [pBL(tk)CAT2 or pRSV CAT] along with the pAVhVDR expression vector to ensure that the TPA enhancement of 1,25(OH)2D3-induced transcription is not due to a general increase in transcription. After transfection, the cells were treated with vehicle (ethanol), TPA (100 nM), or a cotreatment of 1,25(OH)2D3 (10-8 M) plus TPA (100 nM) for 24 h (Fig. 3D). TPA alone or in the presence of 1,25(OH)2D3 did not significantly affect the activity of either minimal promoter (TPA or TPA + D vs. Basal, P > 0.5). These results indicate that the enhancement of 1,25(OH)2D3-induced transcription by TPA is not due to a general increase in transcription.

Mechanism of the effect of activation of PKC on VDR-mediated transcription. TPA has been shown to induce PKC-mediated phosphorylation of VDR on Ser51 (21). To determine whether the TPA-induced enhancement occurs by stimulating phosphorylation of VDR, COS-7 cells were cotransfected with the h24(OH)ase VDRE (-174/-151) promoter construct and an hVDR expression plasmid where Ser51 was mutated to alanine (Fig. 4). In the presence of the mutated VDR, there was a 1,25(OH)2D3-dependent increase in transactivation, and this induction was significantly enhanced 2.6-fold by the cotreatment with TPA (+D vs. TPA + D, P < 0.05; Fig. 4). These results indicate that the TPA-induced enhancement of vitamin D-dependent transcription does not require phosphorylation of VDR on Ser51.



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  		Fig. 4. TPA-induced enhancement of 1,25(OH)2D3-induced transcription is not due to PKC-mediated phosphorylation of VDR on Ser51. COS-7 cells were cotransfected with 500 ng of S51A mutant VDR expression vector and the h24(OH)ase VDRE (-174/-151) promoter construct. Cells were treated as previously described with vehicle (Basal), +D, or TPA + D for 24 h. Representative autoradiogram is shown (A). Results of 3 separate experiments are expressed as percent maximal response ± SE after normalization for {beta}-galactosidase activity (B). Transfection of the 24(OH)ase VDRE (-174/-151)-tk CAT construct in COS-7 cells cotransfected with the S51A mutant VDR and treatment with 10-8 M 1,25(OH)2D3 resulted in a 14.6 ± 1.0-fold induction in CAT activity. Cotreatment with TPA and 1,25(OH)2D3 significantly enhanced 1,25(OH)2D3-induced transcription using the mutant VDR with the PKC phosphorylation site (Ser51) mutated (P < 0.05 +D vs. TPA + D).

 

GST pull-down assays were performed to assess any effect that TPA may have on the binding of VDR to GRIP-1 and DRIP205 (Fig. 5, A and B). The interaction of VDR with each cofactor was increased when nuclear extracts were used from 1,25(OH)2D3-treated VDR-transfected COS-7 cells, consistent with previous results indicating that VDR cofactor interactions are 1,25(OH)2D3 dependent (44, 56). The cotreatment of 1,25(OH)2D3 plus TPA was unable to increase the interaction of VDR with either coactivator protein over that observed with 1,25(OH)2D3 alone (Fig. 5, A and B). These results suggest that the enhancement of VDR-mediated transcription induced by TPA does not involve an enhanced binding of these coactivator proteins to the VDR. As a positive control, cells were treated with a potent analog of 1,25(OH)2D3, RO 26-2198. The hexafluoro substitution in the analog has been reported to increase metabolic stability against inactivating hydroxylases (54). Treatment of COS-7 cells with the analog resulted in increased DRIP-VDR and GRIP-VDR interaction compared with treatment with 1,25(OH)2D3, indicating that the effect was not saturated by treatment with 1,25(OH)2D3.



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  		Fig. 5. Enhancement of VDR-mediated transcription by PKC activation does not correlate with increased interaction of VDR with DRIP205 or with GRIP. A: glutathione S-transferase (GST) pull-down assay was performed as previously described (4), using 15 µg of GST-DRIP205 (527-970) fusion protein and equal amounts of VDR protein from nuclear extracts of VDR expression vector-transfected COS-7 cells treated, as described (see MATERIALS AND METHODS), with vehicle (Basal), +D, or TPA + D or with a superagonist, analog RO 26-2198 (A1), for 24 h. Western blot analysis with a monoclonal anti-VDR antibody was performed to visualize VDR binding (left). Results of 4 separate experiments are expressed as relative signal intensity (100 = maximal intensity) ± SE after normalization to VDR input (right) [+D vs. TPA + D (P > 0.5); positive control, +D vs. A1 (*P < 0.05)]. B: GST pull-down assays were performed as in A, except that GST-GRIP1 fusion protein was used instead of GST-DRIP205. GST pull-down assay shown in B represents a typical example of experiments that were replicated 2-4 times.

 

To further study the mechanism by which TPA functions to enhance VDR-mediated transcription, we examined the effect of TPA on the expression of VDR. VDR protein levels were analyzed by Western blot analysis. In COS-7 cells transiently transfected with hVDR expression vector, treatment with 1,25(OH)2D3 (10-8 M; +D) or 1,25(OH)2D3 + TPA (100 nM; TPA + D) did not alter the levels of VDR (fold induction over basal, as analyzed by Western blot: 1.1 ± 0.3, +D; 1.36 ± 0.2, TPA + D; P > 0.1 vs. basal). Unlike COS-7 cells overexpressing VDR, treatment of LLCPK-1 cells with 1,25(OH)2D3 resulted in an increase in endogenous VDR protein levels. TPA alone also resulted in an increase in VDR levels compared with those obtained in the presence of 1,25(OH)2D3. The VDR protein levels were further increased (2.4-fold; +D vs. TPA + D, P < 0.05) when LLCPK-1 cells were cotreated with 1,25(OH)2D3 plus TPA (Fig. 6A). A similar enhancement of VDR levels was observed after treatment of LLCPK-1 cells with TPA for 4 h followed by 1,25(OH)2D3 treatment for 24 h (TPA + D vs. +D, 1.7 ± 0.2-fold enhancement, P < 0.05). Treatment for 4 h with TPA in the presence of Gö-6983 (500 nM) resulted in an inhibition of the enhancement in VDR levels (P > 0.5, TPA + D + Gö-6983 vs. +D), suggesting that the enhancement by TPA in VDR protein levels is mediated by the action of PKC. When the rat OC VDRE-tk CAT construct was transfected in LLCPK-1 cells, TPA enhancement of VDR-mediated transcription was still observed (data not shown), indicating that the TPA enhancement is not h24(OH)ase promoter specific. These results suggest that the increase in VDR-mediated transcription by TPA in LLCPK-1 cells may be due, at least in part, to an increase in VDR protein levels.



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  		Fig. 6. TPA + D treatment results in an increase in VDR content in LLCPK-1 cells. A: Western blot analysis was performed using 50 µg of nuclear protein prepared from LLCPK-1 cells treated with vehicle (Basal), 1,25(OH)2D3 (+D), TPA, or 1,25(OH)2D3 + TPA for 24 h. Detection was by immunoblotting with a monoclonal VDR antibody. The membrane was reprobed using {alpha}-tubulin antibody. Graphic representation of the data is normalized on the basis of results obtained after immunoblotting with {alpha}-tubulin antibody. Results of 3 separate experiments are expressed as percent maximal response ± SE (+D vs. TPA + D, *P < 0.05). B: gel mobility shift assays were done using equal amounts of nuclear protein (5 µg) prepared from LLCPK-1 cells treated as described in A and 32P-labeled oligonucleotide containing the h24(OH)ase VDRE (-174/-151). Preincubation with cold VDRE (cold VDRE + D; lane 5) or monoclonal VDR antibody (not shown) depleted the binding to the labeled probe. Data are representative of 3 experiments.

 

Gel shift assays were also performed. Binding reactions were done using equal amounts of nuclear protein prepared from LLCPK-1 cells and an oligonucleotide containing the proximal h24(OH)ase VDRE (Fig. 6B). The observed protein-DNA interaction was induced using nuclear extracts from 1,25(OH)2D3-treated LLCPK-1 cells. When nuclear extracts were prepared from cells treated with TPA alone, the protein-DNA interaction was similar to that obtained under basal conditions. Cotreatment with 1,25(OH)2D3 and TPA (increased VDR levels are observed under these conditions; Fig. 6A) resulted in a protein-DNA interaction that was enhanced over the binding observed in the presence of 1,25(OH)2D3 alone, supporting the results of the Western blot.

Because activation of PKC can activate the Ras signal transduction pathway (16) and TPA has been reported to induce MEK-dependent ERK phosphorylation, we asked whether the phorbol ester-mediated enhancement of 1,25(OH)2D3-induced transcription involved activation of ERK. When LLCPK-1 cells were pretreated with TPA for 1 h in the presence of the MEK inhibitor U-0126 followed by treatment with 1,25(OH)2D3 alone for 24 h, a significant decrease was observed in the TPA enhancement of VDR-mediated transcription (P < 0.05; TPA + D + U-0126 vs. TPA + D; Fig. 7A). Under these conditions [1 h TPA treatment followed by incubation with 1,25(OH)2D3 for 24 h], an increase in VDR protein levels in LLCPK-1 cells over the levels observed with 1,25(OH)2D3 treatment is not observed (data not shown). A similar decrease in the TPA enhancement of VDR-mediated transcription was observed with the use of VDR-transfected COS-7 cells incubated with TPA for 1 h in the presence of U-0126 (Fig. 7B). By use of LLCPK-1 cells or VDR-transfected COS-7 cells, pretreatment with the MEK inhibitor for <=4 h in the absence of TPA, followed by treatment with 1,25(OH)2D3 alone for 24 h, did not affect 1,25(OH)2D3-induced transcription (not shown). Similar results were obtained using PD-98059, another MAPK pathway inhibitor. Thus activation of MAPK is also involved in the TPA-mediated enhancement of VDR-mediated transcription. Because the MEK kinase inhibitors, unlike the PKC inhibitor, only partially blocked the effect of TPA, our findings suggest that both MAPK-dependent and -independent pathways are involved in the TPA-mediated enhancement of transcription.



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  		Fig. 7. Effect of MEK inhibitor U-0126 on TPA-induced enhancement of VDR-mediated transcription. LLCPK-1 cells (A) or VDR-transfected COS-7 cells (B) were transfected with h24(OH)ase VDRE (-174/-151)-tk CAT construct and pretreated for 1 h with TPA (100 nM) in the absence or presence of MEK inhibitor U-0126 (10 µM), followed by incubation with 1,25(OH)2D3 (10-8 M) alone for 24 h (TPA + D and TPA + D +U-0126, respectively). Results of 3 separate experiments are expressed as percent maximal response ± SE after normalization for {beta}-galactosidase activity (in both LLCPK-1 cells and COS-7 cells, TPA + D vs. TPA + D + U-0126, *P < 0.05). Transfection of h24(OH)ase VDRE into LLCPK-1 cells and in VDR-transfected COS-7 cells and treatment with 1,25(OH)2D3 resulted in a 5.1 ± 0.8- and a 10.0 ± 1.5-fold increase in CAT activity, respectively, compared with Basal.

 


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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We investigated the role that PKC plays in enhancing VDR-mediated transcription of the h24(OH)ase gene. Although previous studies indicate that activators of PKC can enhance 1,25(OH)2D3-induced 24(OH)ase mRNA and activity levels (1,2, 8, 20, 34), our study shows that this enhancement of the 1,25(OH)2D3 response by TPA occurs at the transcriptional level. We show that the effect of TPA is not mediated by the AP-1 site in the h24(OH)ase promoter (-1,167/-1,160). In LLCPK-1 cells, the enhancement may be due, in part, to an increase in VDR content. Our findings also suggest that the potentiation of VDR-mediated transcription involves activation of the MAPK pathway. Because the VDR-associated cofactor SRC-1 is phosphorylated through MAPK and this phosphorylation is involved in regulation of steroid receptor activation (48, 49), the effect of TPA may also be mediated by differential cofactor recruitment mediated by phosphorylation of specific VDR coregulators.

The concentration of VDR has been shown to be a significant factor in the response of vitamin D-dependent genes (5, 9, 22, 23). Parathyroid hormone (PTH) and activation of PKA have also been reported to regulate VDR and the levels of transactivation of vitamin D-dependent genes in response to 1,25(OH)2D3 (22, 36, 58). In the kidney, in vivo studies have noted cell type specificity in the regulation of VDR in response to low calcium and subsequent elevation of PTH (23). In the proximal convoluted tubule, VDR was found to be downregulated, resulting in inhibition of 24(OH)ase expression. However, in the distal nephron, under low dietary calcium, VDR was not downregulated, and 24(OH)ase was induced (23). The effect of PKC activation on VDR regulation has also been reported to be cell type specific. Studies using rat osteosarcoma (ROS 17/2.8) and human promyelocytic leukemic (HL-60) cells, similar to our results, indicate that activation of PKC can lead to increased VDR content (20, 47). Additional reports using UMR-106 osteoblastic, rat intestinal epithelial (IEC-6 and IEC-18), and mouse fibroblast (NIH-3T3) cells indicate that PKC activation results in either no change or a decrease in VDR levels (34, 35, 55, 58). These findings suggest that the effect of PKC activity on VDR levels is cell type specific. We report that activation of PKC results in an increase in VDR content in LLCPK-1 cells. Experiments done in our laboratory using the -1,500/+60 hVDR promoter construct (22) indicate that cotransfection with PKC{alpha} expression vector (1 µg) results in a 1.9 ± 0.3-fold stimulation of transcription, which is further stimulated by TPA (100 nM, 4 h; 3.8 ± 0.5-fold vs. Basal, P < 0.05; data not shown). These results suggest that PKC activation can affect hVDR transcription. Qi et al. (43) recently identified a functional AP-1 site in the mouse VDR promoter, also suggesting transcriptional regulation of VDR by PKC activation. In future studies, it will be of interest to determine the exact mechanisms involved in the cell type-specific regulation of VDR by PKA- and PKC-signaling pathways.

Activators of PKC have been shown to affect the activity of other members of the steroid receptor family of transcription factors. Activation of PKC by treatment with TPA enhances the glucocorticoid-induced expression of liver tyrosine aminotransferase (TAT) (31, 32). It was suggested that PKC may enhance glucocorticoid-induced TAT expression by facilitating the translocation of the glucocorticoid receptor (GR) to the nucleus (31). In addition, TPA has been reported to increase GR content in HL-60 cells, and this increase is correlated with HL-60 cell differentiation (19). In T47D (37) or in MCF7 breast cancer cells (10), enhanced GR- or estrogen receptor (ER)-mediated transcription, respectively, is observed in the presence of TPA. TPA did not show enhanced activation with estradiol in Chinese hamster ovary cells, indicating cell type specificity. TPA-enhanced transcription in the breast cancer cells was not associated with an increase in cellular receptor content or due to a promoter AP-1 site. The authors suggested that the effect of TPA may be due to regulation of cofactor proteins. Recently, a novel steroid-associated coactivator, GT-198, that interacts with several steroid receptors, including GR and ER, and stimulates their transcriptional activity has been identified in specific tissues as well as in MCF7 breast cancer cells and other cancer cell lines (33). GT-198 is phosphorylated by protein kinases, including PKC, that regulate its transcriptional activity. Therefore, GT-198 may be one factor involved in integrating the effects of TPA and GR or ER previously observed. Thus, similar to our findings, TPA enhancement of the activity of other steroid receptors is cell type specific, and more than one mechanism is involved in the effect of TPA.

In addition to 24(OH)ase, the expression of the vitamin D-inducible calcium-binding proteins OC and osteopontin (OPN) is also modulated by activation of PKC. PKC activation has been reported to inhibit 1,25(OH)2D3-stimulated OC production in human osteoblastic cells and to enhance the 1,25(OH)2D3 induction of OPN gene expression in UMR osteoblastic cells (15, 58). The enhancement of OPN transcription by TPA in UMR-106 osteoblastic cells has been linked to an AP-1 site in the OPN promoter (58). An AP-1 consensus sequence closely juxtaposed to the VDRE has been identified in the human OC promoter (42). It has been proposed that AP-1 proteins may function to induce or inhibit OC gene expression depending on the state of cell differentiation (41). Although the 24(OH)ase promoter contains an AP-1 site, our studies using 24(OH)ase promoter constructs lacking the AP-1 site suggest, similar to studies related to GR and ER transcription in breast cancer cells (10, 37), that this AP-1 site is not involved in the PKC-mediated enhancement of transcription. In addition, when the human 24(OH)ase promoter constructs were cotransfected with c-fos and c-jun, there was a repression of transactivation (Fig. 2). The repression may be due to sequestration of VDR coactivator proteins by c-fos and/or c-jun. The sequestration of coactivator proteins by c-fos and c-jun has been suggested as one mechanism involved in the repression by c-fos and c-jun of transcriptional activation of other nuclear receptors (28, 52).

Our investigation suggests that the ability of PKC to enhance vitamin D-dependent transactivation is independent of its capacity to phosphorylate VDR. The phosphorylation of VDR by casein kinase II at Ser208 and by PKC at Ser51, in two distinct functional domains, account for ~90% of the total phosphorylation. These sites have been suggested to be necessary for maximal transactivation (21, 26). Consensus MAPK sites are not present in VDR (13). The phosphorylation of VDR by PKC, unlike VDR phosphorylation by casein kinase II, is not 1,25(OH)2D3 dependent. However, the phosphorylation of VDR at Ser51 is increased by the PKC activator TPA (21). In our study, in the presence of the Ser51 to alanine mutant VDR, TPA, similar to what was observed using the wild-type VDR, enhanced the 1,25(OH)2D3-dependent 24(OH)ase transcriptional activity. These findings suggest that the phosphorylation of VDR on Ser51 is not required for the increased activation by TPA of the hormone-induced transactivation of 24(OH)ase transcription.

Activation of PKC did not result in enhanced transcription by increasing the interaction of VDR with DRIP205 or GRIP. Although increased interaction between VDR and DRIP205 was shown to be correlated to enhanced VDR-mediated transcription by inhibition of phosphatases (4), our findings did not indicate increased interaction of VDR and DRIP in the presence of TPA. This finding indicates that increased interaction of VDR and DRIP205 is not a general mechanism coupling extracellular signals to vitamin D action.

Our findings suggest that more than one signaling pathway is involved in the effect of TPA. Activation of PKC can activate the Ras signal transduction pathway and lead to the phosphorylation and activation of the MAPK family member ERK (16). Recently, Rowan and colleagues (48, 49) demonstrated that the p160 coactivator protein SRC-1 is a substrate for the ERK kinase (Thr1179, Ser1185, and Ser395) and that mutation of the phosphorylated residues (Thr1179 and Ser1185 to alanine) resulted in a decrease in progesterone receptor-mediated transactivation due to a loss of cooperation between SRC-1 and CREB binding protein. ERK2-mediated phosphorylation of RXR{alpha} at Ser260 has also been reported and shown to be necessary for maximal VDR-mediated 24(OH)ase transcription in COS-1 cells. (13). These results indicate that phosphorylation of coactivator proteins by ERK activation plays an important role in nuclear receptor-mediated transcription. Thus our findings showing a decreased response after pretreatment with a MEK inhibitor, although preliminary, suggest that the TPA-stimulated enhancement of 1,25(OH)2D3-induced 24(OH)ase transcription may, in part, be the result of activation of the MAPK pathway and phosphorylation of coactivator proteins.

The physiological significance of the TPA-mediated transcriptional enhancement is that PKC activation, mediated by peptides and growth factors, by enhancing 1,25(OH)2D3 induction of 24(OH)ase, contributes to the prevention of the accumulation of toxic levels of the hormone in renal cells. Our data provide evidence for transcriptional mechanisms involved in the enhancement by TPA of 1,25(OH)2D3-induced 24(OH)ase expression and suggest that the enhancement may be due in LLCPK-1 cells to an increase in VDR concentration and may also be due in part to the stimulation of the MAPK pathway that can result in the phosphorylation of VDR-associated cofactors and enhanced transcriptional activation.


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 ABSTRACT
 MATERIALS AND METHODS
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 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38961.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the assistance of Yan Liu and Xiaorong Peng in certain aspects of this investigation as well as the assistance of Demetri Merianos, a medical student who contributed to this work as part of a summer research program.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Christakos, Dept. of Biochemistry and Molecular Biology, UMDNJ New Jersey Medical School, 185 South Orange Ave., Newark, N.J. 07103 (E-mail: christak{at}umdnj.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 REFERENCES
 

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