Di-Luoffo M, Brousseau C, Tremblay J J
Reproduction, Mother and Child Health, Centre de recherche du centre hospitalier universitaire de Québec, Québec City, QC, Canada.
Centre de recherche en biologie de la reproduction, Department of Obstetrics, Gynecology and Reproduction, Faculty of Medicine, Université Laval, Québec City, QC, Canada.
Andrology. 2016 Mar;4(2):335-44. doi: 10.1111/andr.12150. Epub 2016 Jan 8.
Leydig cells are essential for male reproductive development and health throughout life. Production of androgens [testosterone, dihydrotestosterone (DHT)] as well as intermediate steroids [progesterone, dihydroprogesterone (DHP)] is tightly regulated. In the mouse, the 3α-hydroxysteroid dehydrogenase enzyme (3α-HSD, AKR1C14) catalyses the interconversion of DHP and DHT into less potent steroids. Despite its importance, nothing is currently known regarding the regulation of Akr1c14 expression in Leydig cells. Recently, the transcription factors MEF2 and NR2F2 were identified in the mouse testis including in Leydig cells where they were found to regulate expression of genes involved in steroidogenesis. Analyses of transcriptomic data from MEF2- or NR2F2-deficient MA-10 Leydig cells revealed a significant decrease in Akr1c14 mRNA levels. Using qPCR, we confirmed that Akr1c14 mRNA levels were decreased in MEF2- and in NR2F2-deficient conditions. Conversely, overexpression of MEF2A or/and NR2F2 in MA-10 Leydig cells led to an increase in endogenous Akr1c14 mRNA levels. Recruitment of MEF2 and NR2F2 to the Akr1c14 promoter was confirmed by ChIP while DNA precipitation assays revealed direct binding of MEF2 but not NR2F2 to this region. In functional promoter studies, NR2F2 was found to activate the Akr1c14 promoter while MEF2A on its own had no effect. Combination of both NR2F2 and MEF2A led to a cooperative activation of the Akr1c14 promoter and this required intact MEF2 and NR2F2 elements. Finally, co-immunoprecipitation experiments showed that MEF2 and NR2F2 are present in the same protein complex. In conclusion, our results identify a novel cooperation between MEF2 factors and NR2F2 in the expression of the Akr1c14 gene involved in the regulation of DHP/DHT levels.
睾丸间质细胞对于男性一生的生殖发育和健康至关重要。雄激素[睾酮、双氢睾酮（DHT）]以及中间类固醇[孕酮、双氢孕酮（DHP）]的产生受到严格调控。在小鼠中，3α-羟基类固醇脱氢酶（3α-HSD，AKR1C14）催化DHP和DHT相互转化为活性较低的类固醇。尽管其很重要，但目前对于睾丸间质细胞中Akr1c14表达的调控情况尚不清楚。最近，在小鼠睾丸中，包括在睾丸间质细胞中鉴定出了转录因子MEF2和NR2F2，发现它们可调节参与类固醇生成的基因的表达。对MEF2或NR2F2缺陷的MA-10睾丸间质细胞的转录组数据进行分析后发现，Akr1c14 mRNA水平显著降低。使用qPCR，我们证实了在MEF2和NR2F2缺陷条件下，Akr1c14 mRNA水平降低。相反，在MA-10睾丸间质细胞中过表达MEF2A或/和NR2F2会导致内源性Akr1c14 mRNA水平升高。通过染色质免疫沉淀（ChIP）证实了MEF2和NR2F2与Akr1c14启动子的结合，而DNA沉淀试验显示MEF2可直接结合到该区域，而NR2F2则不能。在功能性启动子研究中，发现NR2F2可激活Akr1c14启动子，而单独的MEF2A则无作用。NR2F2和MEF2A共同作用可协同激活Akr1c14启动子，这需要完整的MEF2和NR2F2元件。最后，免疫共沉淀实验表明MEF2和NR2F2存在于同一蛋白复合物中。总之，我们的结果确定了MEF2因子和NR2F2在参与DHP/DHT水平调节的Akr1c14基因表达中存在新型合作关系。
