Phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, can modulate 1a, 25 dihydroxyvitamin D3 (1,25(OH)2D3)-induced expression of the 24-hydroxylase (CYP24A1) gene but this has not been studied in differentiated enterocytes, a primary 1,25(OH)2D3 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)2D3-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 +74bp in the hCYP24A1 promoter as critical for the PMA enhancing effect and chromatin immunoprecipitation (ChIP) analysis showed that PMA enhanced 1,25(OH)2D3-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)2D3-induced hCYP24A1 gene transcription and the enhancing effect of PMA. The PMA enhancing effect on 1,25(OH)2D3 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)2D3 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)2D3-induced hCYP24A1 gene expression through multiple regulatory motifs within the proximal hCYP24A1 promoter. J. Cell. Biochem. 113: 15991607, 2012. (C) 2011 Wiley Periodicals, Inc.

Phorbol 12-myristate 13-acetate (PMA) increased 1,25(OH)2D3-induced human 25 hydroxyvitamin d-24 hydroxylase (hCYP24A1) gene expression and vitamin D receptor (VDR) binding to the hCYP24A1 promoter. It did not alter transient receptor potential cation channel, subfamily V, member 6 (TRPV6) expression, VDR binding to the TRPV6 promoter, or VDR binding to a crude chromatin preparation. PMA activated Extracellular signal-Regulated Kinases (ERK) 1/2 and p38 mitogen activated protein kinases (MAPK) and inhibiting these kinases reduced 1,25(OH)2D3-induced and PMA-enhanced hCYP24A1 promoter activity. Mithramycin A inhibits Specific Protein (Sp) family member binding to DNA and reduced 1,25(OH)2D3-induced and PMA-enhanced hCYP24A1 promoter activity. Sp1 or Sp3 siRNA knockdown reduced 1,25(OH)2D3-regulated hCYP24A1 promoter activity but only Sp3 siRNA reduced PMA-enhanced hCYP24A1 promoter activity. PMA increased MAPK-dependent Sp3 phosphorylation, Sp3–VDR interactions, and Sp3 binding to the hCYP24A1 promoter. These data suggest that MAPK signaling contributes to 1,25(OH)2D3-induced and PMA-enhanced CYP24A1 gene transcription by modulating Sp3 function.

CYP24 is an enzyme critical for degradation of 1, 25 dihydroxyvitamin (1,25D) and its gene is strongly regulated by 1,25D. Multiple signaling pathways have been shown to modulate 1,25D‐mediated CYP24 gene expression. Phorbol ester (PMA) treatment activates PKC, as well as ERK1/2 and p38 MAP kinase signaling pathways. In addition, PMA treatment enhances 1,25D‐induced CYP24 gene transcription (mRNA, 180%; reporter gene 150–250%) in Caco‐2 cells. Inhibition of PKC, ERK, and p38 kinase signaling pathways each reduced PMA enhanced and 1,25D induced CYP24 mRNA and reporter genes by 20–40%. Mithramycin A, an inhibitor of Sp protein binding to DNA, dose‐dependently reduced both 1,25D‐induced and PMA‐enhanced CYP24 promoter activity. Bioinformatics revealed 11 putative Sp1/Sp3 sites in the −298 to +74 bp region of the human CYP24 promoter. siRNA knockout of Sp1 reduced 1,25D action by only 30% but had no effect on PMA‐mediated enhancement of CYP24 induction. In contrast, Sp3 siRNA decreased both 1,25D‐mediated CYP24 induction and PMA enhancement of that effect by 20%. Immunoprecipitation showed that PMA can phosphorylate a serine residue on Sp3 and that this is blocked by inhibition of MEK1 with U0126. These data show that a portion of the impact that PMA has on 1,25D‐mediated CYP24 induction is due to activation of ERK1/2 and subsequent phosphorylation/activation of the Sp3 transcription factor. Supported by NIDDK Award DK054111 to JCF.Grant Funding Source: NIDDK Award DK054111 to JCF

The purpose of this study was to determine the impact of 1α, 25 dihydroxyvitamin D (1,25(OH)2D) on hypoxia‐inducible factor 1α (HIF‐1α) expression, an important angiogenic transcription factor, in MCF10A and oncogene (H‐ras and ErbB2) transfected MCF10A breast epithelial cells which are models of cancer progression. Treatment with 1,25(OH)2D (10 nM) increased both mRNA (64% + 19 greater than vehicle, p=0.01) and protein (370% + 41 greater than vehicle, p<0.0001) of HIF‐1α in MCF10A cells in 12 hours. After 24 hrs, HIF‐1α mRNA expression returned to baseline, but HIF‐1α protein level remained higher (590%+150 greater than vehicle, p=0.02). Treatment with a transcriptional inhibitor, actinomycin D, prevented the 1,25(OH)2D induction of HIF‐1α protein expression in MCF10A cells. In contrast, treatment with 1,25(OH)2D (24 hr) did not change HIF‐1α protein expression in H‐ras transfected or ErbB2‐transfected MCF10A cells. However, mRNA level of HIF‐1α was decreased (23%+4.6 less than vehicle, p=0.008) in the H‐ras transfected cells, but was not different with 1,25(OH)2D (24 hr) treatment in ErbB2‐transfected cells compared to vehicle. These results suggest that 1,25(OH)2D induction of HIF‐1α expression is through transcriptional regulation in MCF10A breast epithelial cells and the effect may be modulated in the presence of H‐ras or ErbB2 oncogenes. Supported by NIH DK069965

We examined 1,25 dihydroxyvitamin D (1,25(OH) 2 D 3 )‐induced expression of 25‐hydroxyvitamin D 3 24‐hydroxylase (CYP24) and apical calcium channel (TRPV6) mRNA levels in 2‐, 9‐, and 15‐day cultures Caco‐2 cells that model proliferating, post‐proliferative, and differentiated enterocytes. 1,25(OH) 2 D 3 ‐induced (10 nM, 8 h) CYP24 and TRPV6 mRNA levels were significantly greater in differentiated and post‐proliferative than proliferating Caco‐2 cells (>16X and >3X, respectively). Neither CYP24 mRNA half‐life nor induction of a −298 bp rat CYP24 promoter‐luciferase reporter construct (10 nM 1,25(OH) 2 D 3 , 24 h) were different between proliferating and post‐proliferating Caco‐2 cells. We next tested whether the blunted response of natural genes to 1,25(OH) 2 D 3 in proliferating Caco‐2 cells is due to altered chromatin remodeling. VDR and coactivator protein levels do not increase with differentiation but the level of the co‐repressor Alien falls by 50% with differentiation. Over‐expression of Alien reduced 1,25(OH) 2 D 3 ‐induced activity of a minimal VDRE containing promoter‐luciferase construct by more than 60% in differentiated Caco‐2 cells while siRNA knockdown of Alien in proliferating Caco‐2 cells increased 1,25(OH) 2 D 3 ‐induced CYP24 mRNA level by 40%. These observations suggest that Alien is a regulator of VDR‐mediated gene transcription in Caco‐2 cells. In addition, we found that 1,25(OH) 2 D 3 ‐induced association of VDR with chromatin and with the CYP24 promoter was lower in proliferating cells. This suggests that decreased recruitment of VDR to vitamin D response elements also contributes to the blunted transcriptional responsiveness to 1,25(OH) 2 D 3 in proliferating Caco‐2 cells. J. Cell. Physiol. 218: 113–121, 2009. © 2008 Wiley‐Liss, Inc.

A number of kinase pathways have been found to modulate the genomic action of 1α, 25 dihydroxyvitamin D3 (1,25 D) but this has not been well studied in enterocytes, a primary 1,25 D target cell. We examined the role of protein kinase C (PKC) on 1,25 D action in differentiated Caco-2 cells, an established model for 1,25 D-mediated intestinal calcium (Ca) absorption. Activation of PKC with phorbol ester (PMA 100 nM, 5 min pre-treatment) significantly enhanced 1,25 D (10 nM, 2 h)-induced accumulation of 24-hydroxylase (CYP24) mRNA by 160% but had no impact on induction of the mRNA for the apical Ca channel TRPV6. Inhibition of Ca-dependent PKC isoforms (2 and 10 μM Go6976) reduced basal (by 40 and 70%) and PMA-enhanced effects of 1,25 D on CYP24 mRNA accumulation. MAPK family members ERK1/2 and p38 kinase were activated by PMA treatment (100 nM, 5min). However, while MEK (10 μM U0126) and p38 kinase (8.75 μM SB202190) inhibition reduce 1,25 D-mediated CYP24 mRNA accumulation by 50–60%, only the p38 inhibitor reduced the PMA effect on 1,25 D action (by 40%). Thus, our data show that PMA enhances 1,25 D-induced CYP24 mRNA accumulation through activation of Ca-dependent PKC isoforms and p38 kinase. In addition, the induction of CYP24 mRNA by 1,25 D in enterocytes is dependent upon PKC, p38 kinase and ERK 1/2. However, the molecular targets for these interactions remain to be determined. Supported by NIDDK Award DK54111 to JCF.