The effect of differentiation on 1,25 dihydroxyvitamin D‐mediated gene expression in the enterocyte‐like cell line, Caco‐2

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.