Up-regulation of transporters and enzymes by the vitamin D receptor ligands, 1alpha,25-dihydroxyvitamin D3 and vitamin D analogs, in the Caco-2 cell monolayer.
The effects of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on gene expression and function were studied in Caco-2 cells. Microarray analyses, real-time quantitative polymerase chain reactions, and Western blotting were used to determine the mRNA and protein expression of transporters and enzymes after 1,25(OH)(2)D(3) or vehicle (0.1% ethanol) treatment for 1, 3, 6, and 10 days. The mRNA and protein expressions of the apical sodium-dependent bile acid transporter, oligopeptide transporter 1, multidrug resistance-associated protein (MRP) 3, and sulfotransferase 1E1 remained unchanged with 1,25(OH)(2)D(3) treatment, whereas those for CYP3A4, multidrug resistance protein 1, and MRP2 were significantly increased (P < 0.05). 1,25(OH)(2)D(3) treatment significantly enhanced MRP4 protein expression by increasing protein stability without affecting mRNA expression, as confirmed in cycloheximide experiments. Marked increase in 6beta-hydroxylation of testosterone by CYP3A4 was also observed in the 6-day 1,25(OH)(2)D(3)-treated (100 nM) cell lysate. The transport of [(3)H]digoxin, the P-glycoprotein (P-gp) substrate, after treatment with 100 nM 1,25(OH)(2)D(3) for 3 days revealed a higher apparent permeability (P(app)) value in the basal (B)-to-apical (A) direction over that of vehicle treatment (15.1 +/- 0.53 x 10(-6) versus 11.8 +/- 0.58 x 10(-6) cm/s; P < 0.05), whereas the P(app) in the A-to-B direction was unchanged; the efflux ratio was increased (from 5.8 to 8.0). Reduced cellular retention of 5-(and-6)-carboxy-2',7'-dichlorofluorescein, suggestive of higher MRP2 activity, was observed in the 3-day 100 nM 1,25(OH)(2)D(3)-treated cells over controls. Higher protein expression of CYP3A4, MRP2, P-gp, and MRP4 was also observed after a 6-day treatment with other vitamin D analogs (100 nM 1alpha-hydroxyvitamin D(3),1alpha-hydroxyvitamin D(2) or Hectorol, and 25-hydroxyvitamin D(3)) in Caco-2 cells, suggesting a role of 1,25(OH)(2)D(3) and analogs in the activation of enzymes and transporters via the vitamin D receptor.
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.
Jianghong
J
Fan
Shanjun
S
Liu
Yimin
Y
Du
Jodi
J
Morrison
Robert
R
Shipman
K Sandy
KS
Pang
D018938:Caco-2 Cells; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis* / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D008024:Ligands; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D018167:Receptors, Calcitriol / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000378:metabolism*
D008024:Ligands; D026901:Membrane Transport Proteins; D018167:Receptors, Calcitriol; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human