Short Communication C-JUN N-TERMINAL KINASE MODULATES 1,25-DIHYDROXYVITAMIN D3-INDUCED CYTOCHROME P450 3A4 GENE EXPRESSION

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is known to induce the expression of cytochrome P450 3A4 (CYP3A4) in human colon carcinoma Caco-2 cells. Recently, it was demonstrated that the vitamin D receptor (VDR) regulates 1,25(OH)2D3-induced CYP3A4 gene expression through the xenobiotic-responsive element and the vitamin D-responsive element located on the 5 -flanking region of the CYP3A4 gene. On the other hand, we previously reported that protein kinases such as protein kinase C and tyrosine kinases contribute to the induction of CYP3A4 mRNA by 1,25(OH)2D3. In the present study, we examined the involvement of mitogen-activated protein kinases (MAPKs) in the 1,25(OH)2D3-induced CYP3A4 gene expression using MAPK inhibitors. Curcumin, a c-Jun N-terminal kinase (JNK) pathway inhibitor, and anthra[1,9-cd]pyrazole-6(2H)-one (SP600125), a JNK inhibitor, suppressed the induction of CYP3A4 mRNA by 1,25(OH)2D3, but not 2 -amino-3 -methoxyflavone (PD098059), a mitogen-activated protein kinase kinase-extracellular signalregulated kinase (ERK) pathway inhibitor, or 4-(4-fluorophenyl)2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a p38 inhibitor. In addition, we demonstrated that SP600125 dose-dependently inhibited the CYP3A4 promoter activity induced by 1,25(OH)2D3 using the reporter plasmid of the CYP3A4 promoter. However, SP600125 did not affect 1,25(OH)2D3-induced transactivation of the DR3 via VDR. These results indicate that JNK, but not ERK or p38, is required for the optimal activation of the CYP3A4 gene induced by 1,25(OH)2D3. Cytochrome P450 (P450) plays an important role in the oxidative metabolism of numerous endogenous and exogenous compounds. In humans, cytochrome P450 3A4 (CYP3A4) is the predominant P450 isoform in the liver and small intestinal epithelial cells (Watkins et al., 1987; Paine et al., 1997) and is responsible for the metabolism of more than 60% of therapeutic drugs. Intestinal CYP3A4 is thought to contribute to the first-pass metabolism of orally administered drugs (Paine et al., 1997). The CYP3A4 gene is inducible by many xenobiotics, including rifampicin, dexamethasone, and phenobarbital (Pichard et al., 1990). The nuclear receptor, pregnane X receptor, is known to contribute to the CYP3A4 gene induction by these drugs (Kliewer et al., 1998; Lehmann et al., 1998; Goodwin et al., 1999). It has recently been demonstrated that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a potent inducer of the CYP3A4 gene in the human colon carcinoma cell line Caco-2, which has been extensively used as an experimental model of small intestinal cells (Schmiedlin-Ren et al., 1997; Hara et al., 2000). 1,25(OH)2D3, the most active metabolite of vitamin D, functions to regulate cellular proliferation and differentiation and calcium homeostasis in the intestine, bone, and kidney (Christakos et al., 1996). Most of these physiological activations are mediated by the vitamin D receptor (VDR), which belongs to the nuclear hormone receptor superfamily. The VDR acts as a ligand-inducible transcription factor through heterodimerization with the retinoid X receptor and binding to the vitamin D response element (VDRE) within the vitamin Dinducible genes (Christakos et al., 1996). Recently, a direct repeat separated by three nucleotides (DR3) and an everted repeat separated by six nucleotides (ER6) within the 5 -flanking region of the CYP3A4 gene were identified as VDRE (Thummel et al., 2001; Drocourt et al., 2002). We and others demonstrated that the VDR is an essential factor for 1,25(OH)2D3-induced CYP3A4 expression (Thummel et al., 2001; Drocourt et al., 2002; Hara et al., 2002). On the other hand, several reports have demonstrated that the phosphorylation step is critical for the expression of xenobiotic-induced P450 genes such as CYP1A1 and CYP3A (Chen and Tukey, 1996; Galisteo et al., 1999). We also showed that alteration of the cellular phosphorylation state mediated by protein kinase C (PKC) and protein tyrosine kinases affects the 1,25(OH)2D3-mediated induction of CYP3A4 mRNA in Caco-2 cells (Hara et al., 2002). In addition, the inhibition of the phosphorylation reduced the VDRmediated enhancement of osteocalcin gene transcription (Desai et al., 1995). These results suggest that the phosphorylation step is critical for the complete CYP3A4 induction by 1,25(OH)2D3 via VDR. In the present study, we examined whether MAPKs are involved in the 1,25(OH)2D3-induced expression of the CYP3A4 gene via VDR. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of