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.
Cui
Min
M
Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana, USA.
Klopot
Anna
A
Jiang
Yan
Y
Fleet
James C
JC
D000818:Animals; D001483:Base Sequence; D000075686:COP9 Signalosome Complex; D018938:Caco-2 Cells; D002117:Calcitriol / Q000494:pharmacology*; D015220:Calcium Channels / Q000235:genetics; D002454:Cell Differentiation; D049109:Cell Proliferation; D042002:Chromatin Assembly and Disassembly; D017931:DNA Primers / Q000235:genetics; D020895:Enterocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D018167:Receptors, Calcitriol / Q000378:metabolism; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D013250:Steroid Hydroxylases / Q000235:genetics; D050916:TRPV Cation Channels / Q000235:genetics; D065668:Vitamin D3 24-Hydroxylase
C402762:COPS2 protein, human; D015220:Calcium Channels; D017931:DNA Primers; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018167:Receptors, Calcitriol; D012097:Repressor Proteins; D050916:TRPV Cation Channels; C422827:TRPV6 protein, human; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D000075686:COP9 Signalosome Complex; D002117:Calcitriol