Abstract

The effects of 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] 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)₂D₃ 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)₂D₃ treatment, whereas those for CYP3A4, multidrug resistance protein 1, and MRP2 were significantly increased (P < 0.05). 1,25(OH)₂D₃ treatment significantly enhanced MRP4 protein expression by increasing protein stability without affecting mRNA expression, as confirmed in cycloheximide experiments. Marked increase in 6β-hydroxylation of testosterone by CYP3A4 was also observed in the 6-day 1,25(OH)₂D₃-treated (100 nM) cell lysate. The transport of [³H]digoxin, the P-glycoprotein (P-gp) substrate, after treatment with 100 nM 1,25(OH)₂D₃ 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 × 10^-6 versus 11.8 ± 0.58 × 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)₂D₃-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 1α-hydroxyvitamin D₃,1α-hydroxyvitamin D₂ or Hectorol, and 25-hydroxyvitamin D₃) in Caco-2 cells, suggesting a role of 1,25(OH)₂D₃ and analogs in the activation of enzymes and transporters via the vitamin D receptor.