Regulation Mechanism Of Human Cytochrome P4501A1

T-2 toxin is one of the type A trichothecenes produced mainly by the Fusarium genus.Due to its broad distribution and highly toxic nature,it causes great concern as a threat to the animal breeding and human health.T-2 toxin strongly inhibits eukaryotic protein,DNA,RNA synthesis,DNA damage and eventually cell apoptosis.Furthermore,chronic low-dose exposure triggers immunosuppression,cardiovascular lesions and various malignant tumors.According to the transcriptome data and previous results shown that T-2 toxin can up-regulate the CYP1A1 m RNA level.The expression of CYP1A1 is related to the occurrence of prostate cancer,and overexpression of CYP1A1 results in various malignancies.Therefore,it is necessary to clarify the basal regulatory mechanism and T-2 toxin-induced expression of CYP1A1,which can provide theoretical basis for the CYP1A1-related diseases and the toxicological mechanism of T-2 toxin.In this study,we first studied the basal expression of CYP1A1.The dual-luciferase reporter assay system shown that the promote activity of CYP1A1 was regulated by the proximal(-126/-35)and distal promoter(-1024/-936).Then,we predicted the cis-acting elements XRE and GC box in the proximal and distal promoter region.Site-specific mutagenesis and deletion of the core cis-acting elements,and the results showed that the XRE and GC box determined the promoter activity in the human CYP1A1 proximal and distal promoter regions.AhR and Sp1 respectively bound to the XRE and GC box.Besides,knockdowned AhR and Sp1 significantly inhibited the protein expression and promoter activity of CYP1A1.In order to further verify the relationship between AhR and Sp1,we found that AhR and Sp1,possibly via a DNA-mediated interactio by immunoprecipitation(IP).In the(m GC box)-Luc-transfected cells,Sp1 overexpression minimally restored promoter activity,while AhR overexpression significantly increased the promoter activity.This result implied that a high level of AhR overcame the deficiency in the binding of Sp1 to the proximal CYP1A1 promoter.However,XRE mutation sharply decreased the promoter activity,compared to that allowed by the-126/-1 construct,and neither Sp1 nor AhR overexpression enhanced the promoter activity.These results indicated that XRE site in the proximal promoter region likely determined the transactivation of CYP1A1 expression.DNA Affinity Purification Assay(DAPAs)shown that AhR bound to the proximal cis elements and determined the proximal transactivation of basal CYP1A1 expression.Then,and Ch IP were carried out to validate AhR bound to the proximal cis elements and determined the proximal transactivation of basal CYP1A1 expression.Based on the above results,AhR bound to the XRE/GC box and recruited Sp1 to activate the basal transcription of CYP1A1.T-2 toxin significantly increased the protein and m RNA levels of CYP1A1 but markedly inhibited AhR protein expression by RT-PCR and western blot,which indicate that T-2 toxin upregulated the expression of CYP1A1 in a manner independent of AhR.Next,we treated the Hep G2 cells with the actinomycin D(Act D),a transcription inhibitor,either short or long periods of Act D pretreatment markedly diminished the upregulation of CYP1A1 m RNA levels when cells were treated with T-2 toxin for 15 h,which implied that T-2 toxin upregulated CYP1A1 expression through a mechanism coupling to transcription.Then,the dual-luciferase reporter assay system confirmed that the overlapping cis elements of Sp1/NRF1 binding sites in the CYP1A1 promoter region(from-1172 to-1141)regulated the induction of its expression by T-2 toxin.Deletion or mutation of the Sp1/NRF1 binding sites suppressed T-2 toxin-induced expression.Furthermore,DAPA and Ch IP indicated that T-2 toxin increased the enrichment of NRF1 and Sp1 bound to the promoter,suggested that NRF1 and Sp1 probably involved in the T-2 toxins induced-expression of CYP1A1.To elucidate the role of NRF1 and Sp1 in the induced expression of CYP1A1 in T-2 toxin-treated cells,we found that knockdown NRF1 or Sp1 significantly inhibited the protein expression of CYP1A1 and reduced promoter activity by 66.5% or 74.2%,respectively.Therefore,we confirmed that NRF1 and Sp1 regulated the induced expression of CYP1A1.T-2 toxin upregulated the protein levels of Sp1 but did not change those of NRF1,how NRF1 and Sp1 regulated the inducible expression of CYP1A1 remained to be clarified.IP results indicated that T-2 toxin promoted the physical interaction of NRF1 protein with Sp1 protein.To prove whether NRF1 and Sp1 coordinately regulated the induction of CYP1A1 expression,we performed knockdown experiments and found that knockdown of either NRF1 or Sp1 strongly suppressed CYP1A1,suggesting that CYP1A1 regulation requires the joint participation of NRF1 and Sp1.IF and nuclear and cytoplasmic separation indicated that T-2 toxin promoted the nuclear enrichment of NRF1.Upon further elucidating the relationship between Sp1 and the nuclear enrichment of NRF1,we confirmed that T-2 toxin strongly enhanced the interaction between NRF1 and Sp1,which implied that nuclear translocation of NRF1 facilitated its interaction with Sp1.Knockdown of Sp1 abolished NRF1 nuclear enrichment.Therefore,we concluded that T-2 toxin facilitated interaction between NRF1 and Sp1,and then promoted T-2 toxin induced the expression of CYP1A1.Our results elaborated the basal expression and T-2 toxin-induced the expression of CYP1A1 in Hep G2 cells.We first identified AhR regulates the basal expression of CYP1A1 by recruiting Sp1.Besides,T-2 toxin upregulates the expression of CYP1A1 by enhancing NRF1 and Sp1 interaction.These results advance the current understanding of the regulation mechanism of CYP1A1 in human,provides new insights to understand the tumorigenic features of T-2 toxin.