Regulation of bovine CYP3A28 promoter by pregnane X receptor (PXR) and constitutive androstane receptor (CAR).

INTRODUCTION The regulation of cytochrome P450 3A (CYP3A) has been extensively studied in human liver. In cattle, some information on the expression and modulation of CYP3As are now available, but molecular mechanisms involved in basal and xenobiotic- mediated gene regulation are still unknown. In this study, selected transcription factor binding sites were studied for their role in the basal and nuclear receptor-driven trans-activation of CYP3A28 promoter. MATERIALS AND METHODS Putative binding sites for transcription factors were predicted within the CYP3A28 promoter region (~10 kbp) through specific software. Fragments of the CYP3A28 promoter were amplified from bovine liver gDNA and cloned into pGL4.10 vector. Bovine nuclear receptor (bPXR, bCAR and bRXRa) fulllength cDNAs were amplified and inserted into pCI-neo expression vector. Deletion of ER6, HNF-1 and HNF-4 cis-elements in the proximal promoter (PP) construct, and mutagenesis of both ER6 and HNF-4 binding elements within the PP region, and of DR5 within the Fragment 3 (F3) were performed through inverse PCR and according to Fang et al. (2012), respectively. To identify the most important regions for gene activation, cotransfection studies in HepG2 cells were performed using all the promoter constructs, bPXR and its agonist SR12813, or constitutively active bCAR. To assay the role of specific binding sites on CYP3A28 regulation, C3A cells were then co-transfected with deleted or mutated plasmids, bPXR and SR12813 or rifampicin (RIF), and bCAR. Human 3A4-XREM- luc and PBREM-tk-luc were used as positive controls. RESULTS AND CONCLUSIONS CYP3A28 promoter screening revealed PP and PP-F3 as the most important elements for bPXR and bCAR activation. 3A4-XREMluc, the PP and PP-F3 constructs were significantly activated by bPXR and SR12813 (P < 0.05), but not by RIF. Deletion of ER6, HNF-1 and HNF-4 lead to a significant decrease of PP activation by bPXR and SR12813 (P < 0.01), while the mutated single elements ER6, HNF-4 and DR5 did not. Conversely, only PP-F3 was significantly activated by bCAR (P < 0.05). Thus, CYP3A28 PP contains interaction sites for bPXR trans-activation. ER6 and HNF-1 elements seem to contribute to SR12813 response. F3 fragment is involved in bPXR- and bCAR-mediated CYP3A28 regulation, but additional binding elements, apart from DR5, need to be investigated. ACKNOWLEDGEMENTS Project supported by University of Padova (CPDA109434, 60A08-4783/14).