Robert Nicholas M, Martin Luc J, Tremblay Jacques J
Ontogeny-Reproduction Research Unit, CHUL Research Centre, Ste-Foy, Québec, Canada G1V 4G2.
Biol Reprod. 2006 Feb;74(2):322-30. doi: 10.1095/biolreprod.105.044560. Epub 2005 Oct 19.
Insulin-like 3 (INSL3) is a hormone produced by fetal and adult Leydig cells of the testis and by theca and luteal cells of the adult ovary. In males, INSL3 regulates testicular descent during fetal life, whereas in adults, it acts as a germ cell survival factor. In the ovary, INSL3 regulates oocyte maturation. Despite its importance for male sex differentiation and reproductive function in both sexes, very little is known regarding the molecular mechanisms that regulate Insl3 expression. So far, the nuclear receptor NR5A1 is the only transcription factor known to regulate the mouse Insl3 promoter in Leydig cells. NR5A1 by itself, however, cannot explain the spatiotemporal expression pattern of the Insl3 gene. In the present study, we have identified the orphan nuclear receptor NR4A1 as a novel regulator of INSL3 transcription in Leydig cells. Using RT-PCR, we found that Nr4a1 is coexpressed with Insl3 in purified Leydig cells and in several Leydig cell lines. Through detailed analyses of the mouse and human INSL3 promoter in Leydig cells, we have mapped a novel regulatory element located at -100 bp that is essential and sufficient to confer NR4A1 responsiveness. Consistent with a role for NR4A1 in Insl3 transcription, chromatin immunoprecipitation assays revealed that endogenous NR4A1 binds to the proximal Insl3 promoter in vivo. Finally, we found that NR4A1 is also implicated in cAMP-induced Insl3 transcription in Leydig cells. Taken together, our identification of NR4A1 as an important regulator of mouse and human INSL3 promoter activity helps us to better define the tissue-specific regulation of the INSL3 gene in gonadal cells.
胰岛素样3（INSL3）是一种由睾丸的胎儿和成年睾丸间质细胞以及成年卵巢的卵泡膜细胞和黄体细胞产生的激素。在男性中，INSL3在胎儿期调节睾丸下降，而在成年人中，它作为生殖细胞存活因子发挥作用。在卵巢中，INSL3调节卵母细胞成熟。尽管其对两性的男性性别分化和生殖功能很重要，但关于调节Insl3表达的分子机制却知之甚少。到目前为止，核受体NR5A1是已知的唯一能调节睾丸间质细胞中小鼠Insl3启动子的转录因子。然而，NR5A1本身并不能解释Insl3基因的时空表达模式。在本研究中，我们已确定孤儿核受体NR4A1是睾丸间质细胞中INSL3转录的新型调节因子。使用逆转录聚合酶链反应（RT-PCR），我们发现Nr4a1与Insl3在纯化的睾丸间质细胞和几种睾丸间质细胞系中共同表达。通过对睾丸间质细胞中小鼠和人类INSL3启动子的详细分析，我们绘制了一个位于-100 bp处的新型调控元件，该元件对于赋予NR4A1反应性是必不可少且足够的。与NR4A1在Insl3转录中的作用一致，染色质免疫沉淀分析表明内源性NR4A1在体内与近端Insl3启动子结合。最后，我们发现NR4A1也参与了cAMP诱导的睾丸间质细胞中Insl3的转录。综上所述，我们将NR4A1鉴定为小鼠和人类INSL3启动子活性的重要调节因子，这有助于我们更好地定义性腺细胞中INSL3基因的组织特异性调控。
