Human PXR-mediated induction of intestinal CYP3A4 attenuates 1α,25-dihydroxyvitamin D₃ function in human colon adenocarcinoma LS180 cells

Abstract

Oxidative catabolism of 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)] is mediated by either CYP24A1 or CYP3A4. In this paper, we tested whether induction of CYP3A4 in the LS180 intestinal cell model enhances clearance of 1α,25(OH)(2)D(3) and blunts its hormonal effect on expression of the apical membrane calcium transport protein, TRPV6. Treatment with the hPXR agonist rifampin significantly increased CYP3A4 mRNA content and catalytic activity, but had no effect on CYP24A1 or TRPV6 mRNA content. Pre-treating cells with rifampin for 48h, prior to a 24h 1α,25(OH)(2)D(3) treatment phase, was associated with a subsequent 48% increase in the elimination of 1α,25(OH)(2)D(3) and a 35% reduction of peak TRPV6 mRNA. Introduction of the CYP3A4 inhibitor, 6',7'-dihydroxybergamottin, an active inhibitor in grapefruit juice, reversed the effects of rifampin on 1α,25(OH)(2)D(3) clearance and TRPV6 expression. Over-expression of hPXR in LS180 cells greatly enhanced the CYP3A4 responsiveness to rifampin pretreatment, and elicited a greater relative suppression of TRPV6 expression and an increase in 1α,25(OH)(2)D(3) disappearance rate, compared to vector expressed cells, following hormone administration. Together, these results suggest that induction of CYP3A4 in the intestinal epithelium by hPXR agonists can result in a greater metabolic clearance of 1α,25(OH)(2)D(3) and reduced effects of the hormone on the intestinal calcium absorption, which may contribute to an increased risk of drug-induced osteomalacia/osteoporosis in patients receiving chronic therapy with potent hPXR agonists. Moreover, ingestion of grapefruit juice in the at-risk patients could potentially prevent this adverse drug effect.

Figure 1. Concentration- and time-dependent effects of rifampin and 1α,25(OH)₂D₃ on CYP3A4, CYP24A1 and TRPV6 expression in LS180 cells

Plated LS180 cells were treated with: A) 0, 0.1, 0.5 or 1 nM 1α,25(OH)₂D₃ for 12 hrs; B) 0, 10, 20 or 50 μM rifampin for 48 hrs; C) 0.5 nM 1α,25(OH)₂D₃ for 0 to 48 hrs; or D) 50 μM rifampin for 0 to 48 hrs. Levels of CYP3A4, CYP24A1 and TRPV6 mRNA were quantified by qRT-PCR analysis and normalized to the GAPDH mRNA content. Data in A) and B) are presented as a ratio of the measurements obtained with drug/hormone treatment compared to the vehicle (0.1% DMSO in A or 0.1% ethanol in B) treatment; mean of three replicate incubations ± SD are shown. Data in C) and D) are presented as a ratio of the measurements obtained at various time points to that obtained at t = 0 hrs; for each time point, the average of duplicate incubations is shown.

Figure 2. CYP3A4, CYP24A1 and TRPV6 expression and activity following sequential treatment with rifampin and 1α,25(OH)₂D₃

Plated cells were pretreated with 50 μM rifampin or 0.1% DMSO (vehicle) for 48 hrs. At the end of the pretreatment period, cells were washed with cold PBS to remove rifampin and then treated with 0.5 nM 1α,25(OH)₂D₃ or 0.1% ethanol (vehicle) for the indicated period of time (0, 4, 8, 12, 16 and 24 hrs). qRT-PCR analysis of mRNA was performed for measurement of A) CYP3A4, C) TRPV6 and D) CYP24A1 mRNA content in the cell lysate. These data were normalized to the level of GAPDH mRNA and are presented as the ratio of the indicated drug treatment to sequential vehicle control (0.1% DMSO or 0.1% ethanol). B) At the indicated times, treated cells were also incubated for 30 min with 8 μM MDZ for measurement of a 1′-OH MDZ formation rate. Observed rates for the different drug treatment conditions were normalized for the measured sequential vehicle control rates. For all time-points, the mean ± SD of 4 replicate experimental results are shown.

Figure 3. Effect of rifampin pretreatment on the elimination of 1α,25(OH)₂D₃ from the LS180 cell culture media and cell lysate

Plated cells were pretreated with 50 μM rifampin or 0.1% DMSO (vehicle) for 48 hrs. At the end of the pretreatment period, cells were washed with cold PBS to remove rifampin and then treated with 0.5 nM 1α,25(OH)₂D₃ or 0.1% ethanol (vehicle) for the indicated period of time (0, 4, 8, 12, 16 and 24 hrs). A) 1α,25(OH)₂D₃ in cell media. Data are presented as a percentage of the amount detected at t = 0; mean ± SD of 5 replicate experiments are shown. B) 1α,25(OH)₂D₃ in cell lysate. Data are presented as a mean of two independent experiments. The concentration of 1α,25(OH)₂D₃ was normalized to total protein content per well as indicated.

Figure 4. Effect of DHB treatment on CYP3A4 activity and TRPV6 expression in LS180 cells

A) Plated LS180 cells were treated with 50 μM RIF or 0.1% DMSO for 48 h, washed, and then treated with 20 μM DHB or 0.1% ethanol for 4 hrs. Following that, cells were incubated with 0.5 nM 1α,25(OH)₂D₃ + 20 μM DHB or 0.5 nM 1α,25(OH)₂D₃ alone for the indicated time, washed and then incubated with 8 μM MDZ for 30 min; formation of 1′-OH MDZ was then measured. B) Cells were treated as described for part A), except that TRPV6 mRNA content in the cell lysate was measured by qRT-PCR at the indicated time. Data are presented as a fold-change in CYP3A4 activity or TRPV6 content, relative to baseline (vehicle control pretreatment); * indicates p < 0.05 for comparison of cells pretreated with rifampin + DHB vs. rifampin alone.

Figure 5. Effects of rifampin and 1α,25(OH)₂D₃ in hPXR.1-LS180 and MOCK-LS180 cells

A) Plated hPXR.1-LS180 and MOCK-LS180 cells were treated with 50 μM rifampin or 0.1% DMSO for 48 hrs. CYP3A4 mRNA and MDZ 1′-hydroxylation activity was measured at the end of the pretreatment period. B) Plated hPXR.1-LS180 and MOCK-LS180 cells were treated with 50 μM rifampin or 0.1% DMSO for 48 hrs, washed and treated with 0.5 nM 1α,25(OH)₂D₃ for 12 hrs. TRPV6 mRNA content in the cell lysate was measured at the end of the pretreatment period (for “control” and “rifampin”) or at the end of the treatment period (for “1α,25(OH)₂D₃” or “rifampin/1α,25(OH)₂D₃”). C) Plated hPXR.1-LS180 cells and MOCK-LS180 cells were treated with 50 μM RIF or 0.1% DMSO for 48 hrs, washed, and then treated with 20 μM DHB or 0.1% ethanol for 4 hrs. Following that, cells were incubated with 0.5 nM 1α,25(OH)₂D₃ + 20 μM DHB or 0.5 nM 1α,25(OH)₂D₃ alone for 12 hrs. TRPV6 mRNA content was compared. Data are presented as the fold-change with the drug treatment relative to vehicle control; mean ± SD of 3 replicate experiments are shown. *, p < 0.05 for indicated comparison in (B) and (C).

Figure 6

A proposed mechanism for the regulation of 1α,25(OH)₂D₃-mediated TRPV6 expression by CYP3A4 induced by hPXR agonists.