The fully active dihydroxylated metabolite of vitamin D 3 induces the expression of CYP3A4 and, to a lesser extent, CYP2B6 and CYP2C9 genes in normal differentiated primary human hepatocytes. Electrophoretic mobility shift assays and cotransfection in HepG2 cells using wild-type and mutated oligonucleotides revealed that the vitamin D receptor (VDR) binds and transactivates those xenobiotic-responsive elements (ER6, DR3, and DR4) previously identified in CYP3A4, CYP2B6, and CYP2C9 promoters and shown to be targeted by the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR). Full VDR response of various CYP3A4 heterologous/homologous promoter-reporter constructs requires both the proximal ER6 and the distal DR3 motifs, as observed previously with rifampicinactivated PXR. Cotransfection of a CYP3A4 homologous promoter-reporter construct (including distal and proximal PXR-binding motifs) and of PXR or CAR expression vectors in HepG2 cells revealed the ability of these receptors to compete with VDR for transcriptional regulation of CYP3A4. In conclusion, this work suggests that VDR, PXR, and CAR control the basal and inducible expression of several CYP genes through competitive interaction with the same battery of responsive elements.Cytochrome P450 (CYP) 1 enzymes are mainly expressed in the liver and catalyze the metabolic conversion of xenobiotics, including environmental pollutants and drugs, to more polar and easily disposable derivatives (2, 3). CYP genes from the CYP2 and CYP3 families are inducible by many xenobiotics, notably including barbiturates and rifampicin. Two nuclear receptors, the pregnane X receptor (PXR; NR1I2) and the constitutive androstane receptor (CAR; NR1I3), have recently been shown to mediate CYP2 and CYP3 gene induction in animals and man (4 -6). Both PXR and CAR form heterodimers with the retinoid X receptor (RXR; NR2B1). PXR is activated by a wide spectrum of xenobiotics and steroids (4, 7, 8) and controls CYP3A4 and CYP3A7 induction by targeting two specific responsive elements present in the regulatory region of these genes (4, 7-12). The first of these is the proximal PXR-responsive element, located at -160. It consists of an everted repeat of the nuclear receptor half-site AGGTCA separated by 6 nucleotides (ER6); this element is necessary but not sufficient for full transactivation of the CYP3A4 promoter. Indeed, full PXRmediated induction requires the presence of a second distal xenobiotic-responsive element (dPXRE), located between -7800 and Ϫ7200 (9). This element is composite and consists of two direct repeats separated by 3 nucleotides (DR3), encompassing an ER6 motif. In contrast to PXR, CAR is sequestered in the cytoplasm and translocates into the nucleus upon activation, notably in response to phenobarbital (6, 13). Several groups have identified a complex phenobarbital-responsive element module that consists of two nuclear receptor-binding sites (termed NR1 and NR2) and one nuclear factor 1 binding site (12,14). Both NR1 and NR2 are imperfect DR4...

We have investigated the effect of cytokines, including interleukin-6 (Il-6), interleukin-la (Il-la), and tumor necrosis factor-alpha ("Fa), on the inducible expression of cytochrome P450s (CYP) CYPlA1, CYPlA2, and CYP3A4 in human hepatocytes in primary culture. The ability of these cultures to mimic the acute phase response when stimulated with cytokines was evaluated using immunoblotting to measure the production of albumin, ferritin, fibrinogen, and ceruloplasmin. The cytokines exhibited specific patterns of action on the production of these proteins. Albumin was depressed by all the cytokines. In contrast to 11-6 and Il-la, T " -a reduced the production of fibrinogen and ceruloplasmin but stimulated the production of ferritin. When cells were treated with the CYP inducer alone, large increases in the expression of CYPlAl and CYPlA2 by P-naphthoflavone and of CYP3A4 by rifampicin were observed at messenger RNA (mRNA) and protein levels, by ribonuclease protection and immunoblotting, respectively. When the cells were treated with the inducer plus cytokines, the induction of mRNA was greatly reduced. Again, specific patterns of action were revealed: 11-6 had the most potent effect on CYP3A4, whereas TNF-a was the most potent with CYPlA genes. In all cases, changes at the protein levels paralleled changes at the mRNA levels. In cells preinduced with P-naphthoflavone or rifampicin, the decay with time of the levels of the CYPlA2 or CYP3A4 proteins, after the removal of the inducer, was not affected by cytokines. We conclude that cytokines strongly repress the inducibility of CYPlAs and CYP3A4 genes at a transcriptional or a posttranscriptional level, but affect neither the rate of translation of CYP mFWAs nor the rate of degradation of the CYP proteins in these cultures. (HEPATOLOGY 1995; 221 143-1 153.) Abbreviations: CYP, cytochrome P450s; TNF-a, tumor necrosis factoralpha; 11-10, interleukin-1 alpha; mRNA, messenger RNA, cDNA, complementary DNA, NO, nitric oxide.From the 'INSERM U 128,