Jiangsu Province Key Laboratory of Reproductive Medicine, Nanjing Medical University, PR China.
Mol Cell Endocrinol. 2010 May 5;319(1-2):39-46. doi: 10.1016/j.mce.2010.01.014. Epub 2010 Jan 18.
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.
孤儿核受体 NR4A1 是核受体超家族的成员，广泛表达于不同的细胞类型，并介导多种生物学过程。最近出现的证据表明，NR4A1 参与了性腺和肾上腺中几种类固醇生成酶基因的转录调控。然而，其在卵巢间质细胞中的功能仍有待确定。本研究通过免疫组织化学染色，发现 NR4A1 存在于卵巢间质细胞和颗粒细胞中。为了探索 NR4A1 在调节卵巢间质细胞类固醇生成的类固醇生成酶基因转录中的作用，我们构建了增强 NR4A1 表达的重组腺病毒 AdCMV-NR4A1 和敲低 NR4A1 表达的重组腺病毒 AdH1-NR4A1，分别用于增强和敲低卵巢间质细胞中 NR4A1 的表达。随后通过实时 RT-PCR 分析 StAR、CYP11A1、CYP17 和 HSD3B2 的表达模式。此外，通过放射免疫法测量培养上清液中睾酮的浓度。结果表明，在卵巢间质细胞中过表达 NR4A1 可刺激 StAR、CYP11A1、CYP17 和 HSD3B2 的表达，从而增加睾酮的产生。相反，内源性 NR4A1 的敲低导致 StAR、CYP11A1、CYP17 和 HSD3B2 的表达和睾酮的产生显著减少。由于已知内分泌器官中 NR4A1 的表达受 cAMP/PKA 介导的激素（如 ACTH 和 LH）的调节，因此本研究应用 forskolin（FSK），即 cAMP/PKA 通路的激活剂，处理培养的卵泡。FSK 可快速增加 NR4A1 的 mRNA 水平，随后增加 StAR、CYP11A1、CYP17 和 HSD3B2 的水平。总之，本研究结果表明，NR4A1 在卵巢间质细胞中 StAR、CYP11A1、CYP17 和 HSD3B2 的转录调控中发挥了先前未知的作用。NR4A1 对这些类固醇生成酶的调节可能影响卵巢间质细胞产生雄激素的能力。
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