The mechanism of 1,25(OH)2D3-mediated CYP24 gene expression in Caco-2 cells

The activity of 1,25(OH)2D3 is regulated by its synthesis and degradation. The degradation pathway of 1,25(OH) 2D3 is initiated by 25-hydroxyvitamin D3 24-hydroxylase (CYP24). CYP24 gene is tightly regulated by vitamin D, which forms a natural feedback loop to control cellular 1,25(OH)2D3 actions. Various kinase pathways have been found to modulate the genomic action of 1,25(OH)2D3 but this has not been well studied in enterocytes, a primary 1,25(OH)2D3 target cell. In this study, we found that phorbol-12-myristate-13-acetate (PMA), a traditional PKC activator significantly enhanced 1,25(OH)2D3 induced CYP24 gene expression but had no impact on another 1,25(OH)2D3 target gene TRPV6. Moreover, this enhancement effect of PMA is mediated through -298 to +74bp region of human CYP24 promoter. We found that three kinase pathways, classic PKC, ERK1/2 and p38 kinase were activated by PMA and were involved in both 1,25(OH)2D3 induced and PMA enhanced CYP24 gene and promoter activity. In the study of DNA elements on human CYP24 promoter -298 to +74bp, we also found that VSE but not EBS site were involved in both basal 1,25(OH)2D3 and its synergistic effect with PMA on CYP24 promoter activity. In addition, we found that inhibition of Sp transcription factor binding to GC-boxes on human CYP24 promoter and siRNA knockdown of Sp3 reduced both 1,25(OH)2D3 alone and PMA enhanced CYP24 promoter activity. We also found that Sp3 transcription factor could interact with VDR when phosphorylated by ERK1/2. Finally, ChIP assays showed that PMA could increase 1,25(OH)2D3-induced interaction of VDR with the human CYP24 promoter and this is associated with increased binding of Sp3 to the same promoter. Our data demonstrated that signaling pathways are important regulators in 1,25(OH)2D3 mediated CYP24 gene expression in intestinal enterocyte like Caco-2 cells. Our data also suggest that MAP kinase signaling pathway mediates 1,25(OH)2D 3 induced and PMA enhanced CYP24 gene transcription through phosphorylation of Sp3 which leads to enhanced Sp3-VDR interactions and improved VDR-RXR binding to the human CYP24 promoter