title: The orphan nuclear receptor NR4A1 regulates transcription of key steroidogenic enzymes in ovarian theca cells.
abstract: Orphan nuclear receptor NR4A1, a member of the nuclear receptor superfamily, is widely expressed in different cell types and mediates diverse biological processes. Recent emerging evidence suggests that NR4A1 is involved in the transcriptional regulation of several steroidogenic enzyme genes in gonads and adrenals. However, its function in ovarian theca cells remains to be defined. In the present study, immunohistochemical staining of NR4A1 in healthy human ovaries indicate that it is expressed in theca cells and granulosa cells. In an effort to explore the function of NR4A1 in the transcript regulation of steroidogenic enzyme genes responsible for ovarian theca cell steroidogenesis, we constructed recombinant adenovirus AdCMV-NR4A1 and AdH1-NR4A1 to enhance or knockdown the expression of NR4A1 in theca cells, respectively. The expression patterns of StAR, CYP11A1, CYP17 and HSD3B2 were subsequently analyzed by real-time RT-PCR. Moreover, concentrations of testosterone in the spent medium were measured by radioimmunoassay. Our results show that overexpression of NR4A1 in theca cells stimulates the expression of StAR, CYP11A1, CYP17 and HSD3B2, leading to increased testosterone production. Conversely, knockdown of the endogenous NR4A1 exhibits a significant decrease in StAR, CYP11A1, CYP17 and HSD3B2 expression and testosterone production. Since expression of NR4A1 in the endocrine organs is known to be regulated by both cAMP/PKA mediated hormones, ACTH and LH, forskolin (FSK), an activator of cAMP/PKA pathway, was applied to the cultured follicles. FSK rapidly increases the NR4A1 mRNA levels followed by an increase in StAR, CYP11A1, CYP17 and HSD3B2. Collectively, our results outline a previously unrecognized role for NR4A1 in the transcriptional regulation of StAR, CYP11A1, CYP17 and HSD3B2 in ovarian theca cells. Modulation of these steroidogenic enzymes by NR4A1 could influence the capacity of the ovarian theca cells to produce androgen.
authors.affiliation: Jiangsu Province Key Laboratory of Reproductive Medicine, Nanjing Medical University, PR China.
authors.forename: Mei
authors.initials: M
authors.lastname: Li
authors.forename: Kai
authors.initials: K
authors.lastname: Xue
authors.forename: Jing
authors.initials: J
authors.lastname: Ling
authors.forename: Fei-Yang
authors.initials: FY
authors.lastname: Diao
authors.forename: Yu-Gui
authors.initials: YG
authors.lastname: Cui
authors.forename: Jia-Yin
authors.initials: JY
authors.lastname: Liu
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1 / Q000235:genetics* / Q000378:metabolism; D010053:Ovary / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011375:Progesterone Reductase / Q000235:genetics / Q000378:metabolism; D011863:Radioimmunoassay; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism; D013739:Testosterone / Q000096:biosynthesis; D013799:Theca Cells / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: C539821:NR4A1 protein, human; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D010750:Phosphoproteins; D000098682:Steroidogenic Acute Regulatory Protein; D013739:Testosterone; C533068:3 beta-hydroxysteroid dehydrogenase type II; D011375:Progesterone Reductase; D013254:Steroid 17-alpha-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme