title: Effect of phorbol 12-myristate 13-acetate activated signaling pathways on 1α, 25 dihydroxyvitamin D3 regulated human 25-hydroxyvitamin D3 24-hydroxylase gene expression in differentiated Caco-2 cells.
abstract: Phorbol-12-myristate-13-acetate (PMA), a protein kinase C(PKC) activator, can modulate 1α, 25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-induced expression of the 24-hydroxylase (CYP24A1) gene but this has not been studied in differentiated enterocytes, a primary 1,25(OH)(2) D(3) target cell. We found that in differentiated Caco-2 cells, an established model of the mature absorptive epithelial cell, PMA significantly enhanced 1,25(OH)(2)D(3)-induced human CYP24A1 (hCYP24A1) mRNA accumulation and hCYP24A1 promoter-luciferase reporter gene activation by 150%. Reporter gene studies further identified the region between -298 and +74 bp in the hCYP24A1 promoter as critical for the PMA enhancing effect and chromatin immunoprecipitation (ChIP) analysis showed that PMA enhanced 1,25(OH)(2)D(3)-induced binding of vitamin D receptor to this region. PMA can activate PKC, ERK1/2, and p38 MAP kinases and inhibition of these signaling pathways reduced both 1,25(OH)(2)D(3)-induced hCYP24A1 gene transcription and the enhancing effect of PMA. The PMA enhancing effect on 1,25(OH)(2)D(3) action was evident in a minimal promoter with three osteocalcin VDREs and was reduced after mutation of a putative vitamin D stimulatory site in the hCYP24A1 promoter. In contrast, mutation of a Ets binding site (EBS) in the hCYP24A1 promoter had no impact on 1,25(OH)(2)D(3) action or the PMA enhancing effect. These data suggest that in the differentiated enterocyte PMA-induced activation of several signaling pathways contribute to 1,25(OH)(2)D(3)-induced hCYP24A1 gene expression through multiple regulatory motifs within the proximal hCYP24A1 promoter.
authors.affiliation: Department of Nutrition Science, Purdue University, West Lafayette, Indiana 47907-2059, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Jiang
authors.forename: James C
authors.initials: JC
authors.lastname: Fleet
mesh_terms: D001483:Base Sequence; D018938:Caco-2 Cells; D002117:Calcitriol / Q000378:metabolism* / Q000494:pharmacology; D002454:Cell Differentiation; D020895:Enterocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013250:Steroid Hydroxylases / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D065668:Vitamin D3 24-Hydroxylase
chemical_list: D012333:RNA, Messenger; D013250:Steroid Hydroxylases; C585549:CYP24A1 protein, human; D065668:Vitamin D3 24-Hydroxylase; D011493:Protein Kinase C; D002117:Calcitriol; D013755:Tetradecanoylphorbol Acetate