Nur77 Transcriptional Regulation Of Target Genes And Identification Of The Physiological Function Of A Preliminary Study

Nur77 is one member of nuclear receptor family.As a transcription factor,Nur77 participates in a variety of biological processes including T cell development, inflammatory response,steroid hormone synthesis,hepatic glucose metabolism and etc.Our previous report showed that Nur77 was upregulated during the activation-induced cell death(AICD) in T lymphocytes,however,no target gene transactivated by Nur77 is found in this process.In this study,we aim to further understand the functions and mechanisms of Nur77 via searching its novel target genes.Since cis-element,NBRE,found in almost all known promoters of Nur77-targeted genes is highly conservative we searched the novel Nur77 targeted genes by Genebank data mining for the promoters with NBRE and then validated by various wet-lab experiments.We produced firstly a program NBREscan in Perl,with which we scanned the genome-wide promoter sequences[—1000 bp to +200 bp relative to the TSS] downloaded from DBTSS database for the genes with NBREs in their promoters. We found 483 candidate genes,from which we selected 14 function-associated genes to construct the promoter-Luciferase report plasmids.Cotransfection of HEK 293T cells with Nur77 expression vector and the report plasmids demonstrated that three genes with NBREs in their promoters were transactivated by Nur77,i.e.,one known target gene of Nur77,HSD3B2,and two novel ones,ESR1 and SerpinA3.We then identified the transcriptional regulation of the two novel genes by Nur77 in proper cell lines.As the result,SerpinA3 expression,but not ESR1,was upregulated by Nur77 in HepG2 cells,indicating that ESR1 might not be a Nur77 regultated gene.We thereafter focused our attention on the transactivation of SerpinA3 by Nur77 in detail.After the transactivation of SerpinA3 promoter by Nur77 was confirmed in several cell lines,EMSA(electrophoretic mobility shift assay) demonstrated that Nur77 was bound specifically with NBRE(—182 to—175) in the SerpinA3 promoter,suggesting that the NBRE in SerpinA3 promoter is a Nur77-dependent functional motif.In addition,a NBRE-like element(—93 to—88) was found to be contributed to the transactivation by Nur77.ChIP(chromatin immunoprecipitation) assay revealed that both endogenous and exogenous expressed Nur77 were bound with the SerpinA3 promoter in HepG2 cells.These data allowed us draw a conclusion that Nur77 transactivates SerpinA3 expression.We further investigated the transcription and regulation of SperinA3 in HepG2 cells.It was found that Nur77 regulated basal expression of SerpinA3 in HepG2 cells.Over-expression of both wild-type Nur77 and EGFP-Nur77 fusion proteins augmented the basal expression of SerpinA3,while knockdown of Nur77 via RNA interference supressed its expression in HepG2 cells.These data indicated that Nur77 is involved in the regulation of SerpinA3 basal expression in the HepG2 human liver cancer cells.Studies on IL-6-induced SerpinA3 expression demonstrated that Nur77 enhanced IL-6-induced SerpinA3 expression.Truncation of the NBRE(—182 to—175) impaired the expression of SerpinA3 induced by IL-6.Stimulation of HepG2 cells with IL-6,SerpinA3,total Nur77 and its nuclear content were all increased, while overexpression of the dominant negative Nur77 decreased SerpinA3 expression,suggesting that SerpinA3 expression is kept the pace with the concentration of the nuclear transcription factor Nur77 in cell nuclear.In order to optimize the expression of SerpinA3 regulated by Nur77,several cell lines were selected and treated with various chemicals.It was found that both Nur77 and SerpinA3 expression in PMA plus Ionomycin-treated Jurkat T lymphocytes were upregulated and their expression change seemed functional-related.ChIP assay confirmed that Nur77 bound with the SerpinA3 promoter in the cells after stimulation,suggesting that Nur77 transactivates SerpinA3 during T cell activation.However,molecular mechanism of this phenomenon remains to be further explored.In summary,we provided the evidences for the first time that SerpinA3 is a novel target gene of Nur77,and both SerpinA3 and Nur77 may play important roles in physiological and pathological conditions,such as inflammatory response and T cell activation.