Regulation Mechanisms Of Human And Porcine CYP3A Family Genes

Cytochrome P450s belong to a heme monooxygenase superfamily named according to their characteristic absorption at 450 nm.P450 genes are categorized into different families and subfamilies.This supeerfamily play crucial roles in the metabolism of endogenous substances,including sterols,fatty acids,and vitamins,and the biotransformation of exogenous compounds,including drugs,environmental pollutants,and toxins.In particular,more than 60% of known clinical drugs can be metabolized by the human CYP3A4 and CYP3A5.Due to their physiological and anatomical similarity to humans,pigs represent an important animal model for drug evaluations in humans.The following three CYP3 A isoforms have been identified to be expressed in porcine cells:CYP3A22,CYP3A29 and CYP3A46.The amino acid similarity analysis indicated that porcine CYP3A22,3A29 and 3A46 have the highest identity with human CYP3A4.The saimilar amino acid sequence and fuction reminder us that the porcine CYP3As and human CYP3As may share the same core mechanism for the regulation of basal transactivation.T-2 toxin,which is a secondary metabolite by Fusarium species,belongs to type A trichothecenes.T-2 toxins was proved to be one type of the most severe mycotoxins to animals and humans.T-2 toxin was also potentially dangerous for human health.Porcine CYP3 A were key enzymes in the metabolism of T-2 toxin in porcine liver tissue.Therefore,explore the mechanism of T-2 toxin on the regulation of its key metabolic enzymes,CYP3As,will help us to further understand the toxicological effects and the mechanism of metabolic transformation caused by T-2 toxin.To investigate the mechanism of the regulation of the basal transcription of CYP3 A genes in both porcine and human hepatocytes,a sequence alignment was performed to analyze the conservation of cis-acting elements,CCAAT box and GC box,in the proximal promoter of different CYP3As isoforms.And NF-Y and Sp1 binding to CCAAT box and GC box,respectively.The lucferase assays indicated that Sp1 and NF-Y play important roles in porcine and human CYP3As basal expression,but the variations in the cis-acting elements altered their individual contributions to the basal transcription levels.The electrophoretic mobility shift,DNA affinity precipitation,and chromatinimmunoprecipitation assays confirmed the directly bind in these proximal promoter of porcine and human CYP3As.The changes of the distance between the NF-Y and Sp1 binding sites resulted in significant decreases in the promoter activity of CYP3As.These results indicated that the basal transcriptional regulation of CYP3A in hepatocytes is evolutionarily conserved.Porcine CYP3As have been identified as a key enzyme in the metabolism of T-2 toxin.And the porcine CYP3As expression were induced by T-2 toxin treated.The induction effect was abolished bu the pretreated of ACTD,indicated that T-2 toxin induced the expression of porcine CYP3As by transcriptional level.The lucferase assays revealed that Sp1 and NF-Y mediated the inductive activition.Furthermore,we observed that T-2 toxin induced the nuclear translocation of Sp1.These results indicatied that T-2 toxin modified the post-translational modification of Sp1,then mediated the increased expression of porcine CYP3As.Taken together,our results show that human and porcine CYP3As are target genes for NF-Y and Sp1 transactivation,and the distance between the CCAAT box and GC box is crucial for the constitutive expression of the CYP3As.Our study reveals that the coordinated regulation by NF-Y and Sp1 is a conserved basal mechanism for the regulation of human and porcine CYP3 A genes.And revealed that the transactivation of porcine CYP3As by T-2 toxin depends on the recruitment of NF-Y and Sp1 to the CCAAT box and GC box,respectively.These research could help us to understand the molecular mechanism of the metabolic mechanism and toxicological effects of various drugs and toxic substances,and provide a theoretical basis for the development and utilization of drugs.