The Nur77 (Nr4a1) gene, encoding the orphan nuclear receptor NUR77 (NR4A1), is an immediate early response gene whose expression is rapidly induced by a variety of physiologic stimuli. Nur77 is expressed in several organs, including the classic steroidogenic tissues: gonads and adrenal. In MA-10 Leydig cells, NUR77 has been shown to regulate expression of several genes involved in steroidogenesis and male sex differentiation. In Leydig cells, androgen biosynthesis is controlled primarily by the pituitary gonadotropin luteinizing hormone (LH) acting via its receptor (LH-R), which in turn activates the adenylate cyclase/cyclic adenosine monophosphate (cAMP) signaling pathway. Even though Nur77 expression is induced both at the mRNA and protein levels in response to LH/forskolin/cAMP in Leydig cells, the mechanisms involved remain largely unknown. Here, we report that cAMP-mediated induction of Nur77 expression at the protein, mRNA, and promoter levels in MA-10 cells involves different mechanisms. We found that increased NUR77 protein requires transcription and translation, whereas increased Nur77 mRNA does not require de novo protein synthesis, and would therefore rely on transcription factors already present in the cell. In addition, our detailed analysis of the Nur77 promoter in MA-10 cells revealed that distinct regulatory elements are involved in basal and cAMP-induced Nur77 transcription. Finally, we found that maximal cAMP-mediated increase in Nur77 promoter activity involves a Ca
Expression of steroidogenic enzyme-encoding genes in testicular Leydig cells is complex and involves several transcription factors including the orphan nuclear receptor NUR77 (NR4A1) and the bZIP factor CCAAT/enhancer binding protein b (EBPb). How these transcription factors are integrated into a functional network, however, remains to be fully understood. Here, we report that the transcription factor C/EBPb can activate the Nur77 promoter as revealed by transient transfections in MA-10 Leydig cells. Through 5 0 progressive deletions and site-directed mutagenesis, the C/EBPb-mediated activation of the Nur77 promoter was found to be dependent on a novel species-conserved C/EBP element located at K110 bp. We also demonstrate using electromobility shift assay that C/EBPb specifically binds to this element. Furthermore, we report a functional cooperation between C/EBPb and the p50 subunit of NF-kB that involves a previously uncharacterized kB element located at K18 bp. Promoter analysis revealed that either the C/EBP or the kB element was sufficient to sustain the C/EBPb-p50 cooperation thus suggesting that both factors physically interact. Altogether, our results provide new data regarding Nur77 transcription in testicular Leydig cells in addition to providing new insights into the interplay between transcription factors involved in Leydig cell gene expression and function.
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