Nucleo-cytoplasmic cycling of the vitamin D receptor in the enterocyte-like cell line, Caco-2.
We examined the effects of 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on the distribution and mobility of the vitamin D receptor (VDR) in the enterocyte-like Caco-2 cell. Confocal microscopy showed that a green fluorescent protein-vitamin D receptor (GFP-VDR) fusion protein is predominantly nuclear (58%) and it does not associate with the apical or basolateral membrane of proliferating or polarized, differentiated cells. In contrast to the previously studied cell types, neither endogenous VDR nor GFP-VDR levels accumulate in the nucleus following 1,25(OH)(2)D(3) treatment (100 nM, 30 min). However, in nuclear photobleaching experiments nuclear GFP-VDR import was significantly increased by 1,25(OH)(2)D(3) during both an early (0-5 min) and later (30-35 min) period (20% per 5 min). Compared to the natural ligand, nuclear import of GFP-VDR was 60% lower in cells treated with the 1,25(OH)(2)D(3) analog, 1-alpha-fluoro-16-ene-20-epi-23-ene-26,27-bishomo-25-hydroxyvitamin D(3) (Ro-26-9228, 5 min, 100 nM). Downstream events like ligand-induced association of VDR with chromatin at 1 h and the accumulation of CYP24 mRNA were significantly lower in Ro-26-9228 treated cells compared to 1,25(OH)(2)D(3) (60 and 95% lower, respectively). Collectively our data are consistent with a role for ligand-induced nuclear VDR import in receptor activation. In addition, ligand-dependent VDR nuclear import appears to be balanced by export, thus accounting for the lack of nuclear VDR accumulation even when VDR import is significantly elevated.
Klopot
Anna
A
Department of Foods and Nutrition and the Interdepartmental Nutrition Program, Purdue University, West Lafayette, IN 47907-2059, USA.
Hance
Kenneth W
KW
Peleg
Sara
S
Barsony
Julia
J
Fleet
James C
JC
D001483:Base Sequence; D018938:Caco-2 Cells; D002117:Calcitriol / Q000494:pharmacology; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017931:DNA Primers; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D021381:Protein Transport; D018167:Receptors, Calcitriol / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D018167:Receptors, Calcitriol; D049452:Green Fluorescent Proteins; D002117:Calcitriol