title: Integration of hormone signaling in the regulation of human 25(OH)D3 24-hydroxylase transcription.
abstract: The current study sought to define the molecular mechanisms involved in the cross talk between 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and activators of PKC in the regulation of 25(OH)D(3) 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)(2)D(3). In these cells, cotreatment with the PKC activator TPA and 1,25(OH)(2)D(3) yielded a 27-fold increase in luciferase activity, which was 2- to 3-fold greater than activation obtained with 1,25(OH)(2)D(3) alone (P < 0.05). Similar results were observed using LLCPK-1 kidney cells, suggesting that the previously observed enhancement of 1,25(OH)(2)D(3)-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 Ser(51). Our study demonstrates that, in LLCPK-1 kidney cells, the PKC enhancement of 1,25(OH)(2)D(3)-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.
authors.affiliation: Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: Barletta
authors.forename: Puneet
authors.initials: P
authors.lastname: Dhawan
authors.forename: Sylvia
authors.initials: S
authors.lastname: Christakos
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D002522:Chlorocebus aethiops; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics; D006728:Hormones / Q000502:physiology*; D006801:Humans; D018374:LLC-PK1 Cells; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D020239:Receptor Cross-Talk / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D013250:Steroid Hydroxylases / Q000096:biosynthesis* / Q000235:genetics; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D065668:Vitamin D3 24-Hydroxylase
chemical_list: D006728:Hormones; D003577:Cytochrome P-450 Enzyme System; D008156:Luciferases; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate