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.