Up-Regulation of Transporters and Enzymes by the Vitamin D Receptor Ligands, 1α,25-Dihydroxyvitamin D₃and Vitamin D Analogs, in the Caco-2 Cell Monolayer

Abstract

The effects of 1␣,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 Ͻ 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 6␤-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 Ϯ 0.53 ϫ 10 Ϫ6 versus 11.8 Ϯ 0.58 ϫ 10 Ϫ6 cm/s; P Ͻ 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-2Ј,7Ј-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 1␣-hydroxyvitamin D 3 , 1␣-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.The intestine plays an important role in the absorption of orally administered drugs. The expression and proximity of metabolic enzymes and efflux transporters in the enterocyte contribute to intestinal first-pass removal and delimit the tissue accumulation of endo-and xenobiotics. In the small intestine, cytochrome P450 3A4 (CYP3A4) accounts for approximately 70% of total cytochrome P450 content and is responsible for the metabolism of approximately 50% of drugs currently in use (Pelkonen et al., 2008). The ATPdependent drug efflux P-glycoprotein (P-gp), encoded by the multidrug resistance 1 gene (MDR1) located in the apical membrane of the enterocyte, is involved in the active excretion of a variety of lipophilic, cationic drugs from the intestine ). The multidrug resistance-associated proteins (MRPs), another important subfamily of Article, publication date, and citation information can be found at

Seasonal Variation in Blood Drug Concentrations and a Potential Relationship to Vitamin D

“…Although all three drugs are primarily metabolized by CYP3A (Lampen et al, 1998;Iwasaki, 2007;Staatz et al, 2010), they are also substrates of the drug efflux pump P-glycoprotein (P-gp), which limits oral bioavailability by pumping absorbed drug molecules back to the intestinal lumen (Lo and Burckart, 1999). Similar to CYP3A4, P-gp is induced by vitamin D, but at the vitamin D concentration investigated (1,25(OH) 2 vitamin D 3 100 nM) the level of P-gp induction is much lower than that of CYP3A4 (Fan et al, 2009). If the relative influence of CYP3A4 and P-gp on the overall bioavailability varies among cyclosporine, sirolimus, and tacrolimus, this could affect their sensitivity to vitamin D-associated enzyme induction.…”

“…In vitro studies have indicated that vitamin D may induce the expression of CYP3A4 by a vitamin D receptor-mediated increase in gene transcription (Schmiedlin-Ren et al, 1997;Fan et al, 2009), and this activity would result in increased metabolism of CYP3A4 drug substrates. Indeed, these molecular findings suggest a potential influence of vitamin D on the turnover of many drugs, but the relevance for drug exposure in patients has not been explored.…”

Vitamin D and colon cancer

“…In addition, 1,25(OH) 2 D 3 regulates many other genes related to proliferation, including MYC, FOS and JUN (Pálmer et al 2001, 2003, Meyer et al 2012. The c-MYC oncogene is deregulated and over-expressed in most cancers, and its down-regulation by 1,25(OH) 2 Fan et al (2009) in vitro model developed to evaluate the cross talk between tumour-associated macrophages and CRC cells, 1,25(OH) 2 D 3 restores the sensitivity of CRC cells to TRAIL-induced apoptosis by interfering with the release of IL1b by macrophages (Kaler et al 2010).…”

Temporal changes in tissue 1α,25-dihydroxyvitamin D3, vitamin D receptor target genes, and calcium and PTH levels after 1,25(OH)2D3treatment in mice

“…VDR-responsive drug-metabolizing enzymes include the cytochrome P-450s [human CYP3A4, CYP2B6, and CYP2C9 (17,50), and rodent Cyp3a1, Cyp3a9, and Cyp24a1 (12,14)] and sulfotransferase-2A1 (SULT2A1) (20). VDRresponsive transporters include the rat apical sodium-dependent bile acid transporter (Asbt) (10), human organic aniontransporting polypeptide (OATP1A2) (21), multidrug resistance protein-1 or P-glycoprotein (MDR1/P-gp) (44), and the human and rodent multidrug resistance-associated proteins (MRP2/Mrp2, Mrp3, MRP4/Mrp4) both in vitro and in vivo (14,22). Our laboratory has shown that VDR transactivates P-gp in brain microvessel endothelia (18) in vitro and P-gp in murine kidney and brain but not ileum and liver in vivo, leading to hastened efflux of digoxin in the brain and kidney (11).…”