Tenugu Swathi, Senthilkumaran Balasubramanian
Department of Animal Biology, School of Life Sciences, University of Hyderabad, P.O. Central University, Hyderabad, Telangana 500046, India.
Department of Animal Biology, School of Life Sciences, University of Hyderabad, P.O. Central University, Hyderabad, Telangana 500046, India.
J Steroid Biochem Mol Biol. 2025 Oct;253:106817. doi: 10.1016/j.jsbmb.2025.106817. Epub 2025 Jun 18.
Steroidogenic acute regulatory protein (Star) is a key factor that predominantly mediates the rate-limiting step of steroidogenesis which involved in the translocation of cholesterol from the outer to inner mitochondrial membrane in most of the vertebrates. To understand the transcriptional regulation of star (star1) in fish, the 5' upstream region of the star was cloned from both common carp and catfish genomic libraries and in silico promoter motif analysis predicted putative binding sites of C-jun, AP-1, Gata2, and Oct1 in common carp while C-jun, Gata1, Gata2, and Oct1 binding sites were predicted in star promoter of catfish. Luciferase reporter assay through sequential deletion constructs in TM3 cells revealed significantly high promoter activity (P < 0.001) having C-jun site in both common carp and catfish but not with other motifs largely. Increased activity of star promoter was observed upon hCG induction, in vitro, indicating gonadotropin dependency. Site-directed mutagenesis and chromatin immunoprecipitation assays further confirmed the binding of C-jun to the upstream promoter region of star in both species. Expression of c-jun was significantly (P < 0.05) higher during the spawning phase of the testicular cycle in both species. Both c-jun and star were found to be gonadotropin inducible and androgen-dependent in both species. Further, transient gene silencing through c-jun-siRNA in both the species decreased expression of c-jun, star, and other AP-1 factors such as c-fos, junB, sox family genes, steroidogenic enzyme genes, sperm-related factor, testis-related genes, along with reduced testosterone and 11-ketotestosterone levels. The protein level of C-jun was decreased upon transient gene silencing in both species. This was the first report to show that C-jun binds to the upstream promoter motif of stars in both common carp and catfish and also adds valuable new insights that define the molecular mechanisms of c-jun regulating star transcription during steroidogenesis in teleost and probably testicular function.
类固醇生成急性调节蛋白（Star）是一个关键因子，在大多数脊椎动物中，它主要介导类固醇生成的限速步骤，该步骤涉及胆固醇从线粒体外膜向内膜的转运。为了解鱼类中star（star1）的转录调控，从鲤鱼和鲶鱼的基因组文库中克隆了star的5'上游区域，通过计算机启动子基序分析预测了鲤鱼中C-jun、AP-1、Gata2和Oct1的假定结合位点，而在鲶鱼的star启动子中预测了C-jun、Gata1、Gata2和Oct1的结合位点。通过在TM3细胞中进行连续缺失构建体的荧光素酶报告基因测定，发现在鲤鱼和鲶鱼中具有C-jun位点的启动子活性显著较高（P < 0.001），但在很大程度上与其他基序无关。体外hCG诱导后观察到star启动子活性增加，表明其对促性腺激素的依赖性。定点诱变和染色质免疫沉淀试验进一步证实了两种物种中C-jun与star上游启动子区域的结合。在两种物种的睾丸周期产卵期，c-jun的表达均显著升高（P < 0.05）。在两种物种中，c-jun和star均被发现可被促性腺激素诱导且依赖雄激素。此外，通过在两种物种中使用c-jun-siRNA进行瞬时基因沉默，降低了c-jun、star以及其他AP-1因子（如c-fos、junB）、sox家族基因、类固醇生成酶基因、精子相关因子、睾丸相关基因的表达，同时降低了睾酮和11-酮睾酮水平。在两种物种中，瞬时基因沉默后C-jun的蛋白水平均降低。这是首次报道表明C-jun在鲤鱼和鲶鱼中均与star的上游启动子基序结合，并且还提供了有价值的新见解，即定义了硬骨鱼类固醇生成过程中c-jun调节star转录以及可能的睾丸功能的分子机制。
