Effects of MAPK signaling on 1,25‐dihydroxyvitamin D‐mediated CYP24 gene expression in the enterocyte‐like cell line, Caco‐2

Abstract

We examined the role of the Extracellular signal Regulated Kinases (ERK) in 1,25 dihydroxyvitamin D (1,25(OH) 2 D 3 )-induced gene expression in the differentiated Caco-2 cells. 1,25(OH) 2 D 3 -regulated expression of the 25-hydroxyvitamin D, 24 hydroxylase (CYP24) gene (both natural gene and promoter construct) was strongly modulated by altering ERK activity (i.e. reduced by MEK inhibitors and dominant negative (dn) ERK1 and ERK2, activated by Epidermal Growth Factor) but ERK inhibition had no effect on 1,25(OH) 2 D 3 -regulated expression of the transient receptor potential cation channel, subfamily V, member 6 (TRPV6). ERK5-mediated phosphorylation of the transcription factor Ets-1 enhanced 1,25(OH) 2 D 3 -mediated CYP24 gene transcription in proliferating but not differentiated Caco-2 cells due to reduced levels of ERK5 and Ets-1 (total and phospho-protein levels) in differentiated cells. MEK inhibition reduced 1,25(OH) 2 D 3 -induced 3X-VDRE promoter activity but had no impact on the association of vitamin D receptor (VDR) with chromatin suggesting a role for co-activator recruitment in ERKmodulation of vitamin D-regulated CYP24 gene activation. Chromatin immunoprecipitation assays revealed that the ERK1/2 target, mediator 1 (MED1), is recruited to the CYP24, but not the TRPV6, promoter following 1,25(OH) 2 D 3 treatment. MED1 phosphorylation was sensitive to activators and inhibitors of the ERK1/2 signaling and MED1 siRNA reduced 1,25(OH) 2 D 3 -regulated human CYP24 promoter activity. This suggests ERK1/2 signaling enhances 1,25(OH) 2 D 3 effects on the CYP24 promoter by MED1-mediated events. Our data show that there are both promoter-specific and cell stage-specific roles for the ERK signaling pathway on 1,25(OH) 2 D 3 -mediated gene induction in enterocyte-like Caco-2 cells.

Molecular Mechanisms for Regulation of Intestinal Calcium and Phosphate Absorption by Vitamin D

“…TRPV6 mRNA level is reduced by more than 90% in the duodenum of VDR knockout mice and the TRPV6 gene is strongly regulated by 1,25(OH) 2 D in the duodenum of mice [45,58] and in Caco-2 cells [59,60]. In addition, induction of TRPV6 mRNA precedes the increase in duodenal calcium absorption that occurs following a single 1,25 (OH) 2 D injection [45].…”

CYP24A1 Regulation in Health and Disease

“…Involvement of specific MAPK-ERK signaling pathways in 1,25(OH) 2 D 3 -mediated activation of the CYP24A1 promoter may also be specific to the stage of cell differentiation. Cui et al reported that ERK1/2 signaling, but not ERK5, primarily regulates 1,25(OH) 2 D 3 -induced CYP24A1 mRNA expression in differentiated Caco-2 cells [115]. This contrasts with proliferating Caco-2 cells in which ERK5-mediated phosphorylation of Ets-1/EBS mainly potentiates 1,25(OH) 2 D 3 -mediated CYP24A1 gene transcription [115].…”

“…Cui et al reported that ERK1/2 signaling, but not ERK5, primarily regulates 1,25(OH) 2 D 3 -induced CYP24A1 mRNA expression in differentiated Caco-2 cells [115]. This contrasts with proliferating Caco-2 cells in which ERK5-mediated phosphorylation of Ets-1/EBS mainly potentiates 1,25(OH) 2 D 3 -mediated CYP24A1 gene transcription [115]. It will be interesting to establish the signaling pathways that utilize the interaction between the VDR and Ets-1 binding to modify vitamin D metabolism in disease states.…”

Vitamin D Receptor Is Not Essential for Extracellular Signal-Related Kinase Phosphorylation by Vitamin D3in Human Caco-2/TC7 Cells

“…Cui et al, 16) reported that the accumulation of CYP24 mRNA induced by 1,25-(OH) 2 D 3 was significantly reduced in the presence of MEK inhibitor, but the impact of 1,25-(OH) 2 D 3 treatment on TRPV6, another VDR targeted gene, mRNA accumulation was not 15) or VDR (Silencer Select siRNA; Ambion, Grand Island, NY, USA). Knockdown efficiency was determined by Western blotting with the indicated antibodies.…”

“…Hence Cui et al, claimed that vitamin D-dependent CYP24 but not TRPV6 gene expression requires ERK activity. 16) This suggests that the downstream effect of ERK on vitamin D is complex.…”