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 D₃ [1α,25(OH)₂D₃] 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)₂D₃ 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 48 h, prior to a 24 h 1α,25(OH)₂D₃ treatment phase, was associated with a subsequent 48% increase in the elimination of 1α,25(OH)₂D₃ 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)₂D₃ 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)₂D₃ 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)₂D₃ 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.

Introduction

Osteomalacia, characterized by defective bone mineralization, most commonly occurs in adults with vitamin D deficiency. It is often associated with a reduced dietary intake or synthesis of vitamin D, however osteomalacia has also been reported in patients receiving long-term therapy with certain drugs including rifampin, an antimicrobial agent [1], and the anti-epileptic drugs, carbamazepine, phenytoin and phenobarbital [2], [3]. Although the clinical manifestation and histology of drug-induced osteomalacia resembles that found in other adults with vitamin D deficiency [4], the molecular mechanism of this adverse drug effect is still unclear.

A primary function of vitamin D in maintaining calcium homeostasis and bone health is accomplished by stimulation of transcellular and paracellular calcium transport in the small intestine through vitamin D receptor (VDR)-dependent pathways [5]. One protein thought to regulate the rate and extent of intestinal calcium absorption is TRPV6. TRPV6 is a calcium channel protein expressed in the intestinal luminal epithelium and its synthesis and function is regulated in part by 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] through a VDR signaling pathway [5], [6], [7], [8]. The basal duodenal TRPV6 expression is correlated with serum 1α,25(OH)₂D₃ levels in men, as well as with VDR expression in both men and women, although TRPV6 expression in women is influenced predominantly by an age effect, not by vitamin D level [8]. In addition, TRPV6 transcription responds rapidly to 1α,25(OH)₂D₃ in the human duodenum [9]. Moreover, studies with TRPV6 knockout mice demonstrate that transporting calcium by TRPV6 may be the rate-limiting step and gatekeeper of transcellular calcium flux [10]. Interestingly, 1α,25(OH)₂D₃ might also mediate paracellular transport of calcium by a non-TRPV6 mediated mechanism [11].

Metabolic inactivation of 1α,25(OH)₂D₃ constitutes a negative feedback mechanism for regulating its effect on calcium absorption in the small intestine [12]. Human cytochrome P450 24A1 (CYP24A1) and P450 3A4 (CYP3A4) contribute to the inactivation of 1α,25(OH)₂D₃ [13], [14], [15], [16]. However, the relative contributions of these two enzymes to vitamin D catabolism may be tissue-dependent. For example, in the healthy kidney, CYP24A1 activity likely dominates 1α,25(OH)₂D₃ catabolism, because the expression of CYP3A4 is very low in that organ [17], [18], [19]. In contrast, in tissues such as the liver and small intestine, CYP3A4 could play a more important role in 1α,25(OH)₂D₃ catabolism, because constitutive levels of CYP3A4 are high, whereas CYP24A1 expression is very low [17]. In addition, local production and hormonal effects of 1α,25(OH)₂D₃ in the duodenum were indicated in previous studies [9]. Thus, 1α,25(OH)₂D₃-mediated calcium absorption could be disrupted locally by drugs that alter CYP3A4 expression in the intestinal epithelium, independent of any change in the systemic vitamin D level [20].

In fact, it has been reported that treatment of healthy volunteers with the pregnane X receptor (hPXR) agonist rifampin causes preferential induction of human duodenal CYP3A4, but not CYP24A1, mRNA content [17]. Thus, acceleration of intestinal 1α,25(OH)₂D₃ catabolism by stimulation of CYP3A4 synthesis may result in down-regulation of calcium transporters, such as TRPV6, and an impairment of calcium absorption. Long-term impairment of intestinal calcium absorption by chronic drug administration and CYP3A4 induction could result in osteomalacia [17]. Those drugs associated with osteomalacia share the capacity to activate the hPXR and constitutive androstane receptor (CAR) and thus up-regulate the expression of multiple cytochrome P450 enzymes, including CYP3A4 [21], [22], [23].

In this investigation, we tested the hypothesis that induction of CYP3A4 by hPXR agonists alters the intestinal cellular clearance of 1α,25(OH)₂D₃, and thereby affects the transcription of calcium transporters such as TRPV6, LS180 cell line was used as a model for intestinal enterocytes due to the robust expression of both VDR and hPXR [24], [25]. We report that hPXR agonists induce CYP3A4, but not CYP24A1 in LS180 cells, and under most conditions enhance 1α,25(OH)₂D₃ catabolism and decrease the transcriptional response of TRPV6 to 1α,25(OH)₂D₃. The findings presented here provide a viable molecular mechanism of drug-induced osteomalacia by hPXR activators.