Sayasith Khampoun, Sirois Jean
Centre de Recherche en Reproduction Animale, Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec J2S 7C6, Canada.
Centre de Recherche en Reproduction Animale, Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec J2S 7C6, Canada.
Mol Cell Endocrinol. 2015 Aug 15;411:49-57. doi: 10.1016/j.mce.2015.04.010. Epub 2015 Apr 24.
A disintegrin and metalloprotease-17 (ADAM17) is thought to play a key role in the release of soluble and active epiregulin (EREG) and amphiregulin (AREG) in ovarian follicles but its transcriptional regulation in follicular cells remains largely unknown. The objectives of this study were to characterize the regulation of ADAM17 transcripts in bovine follicles prior to ovulation and to investigate its transcriptional control in bovine granulosa cells. To study the regulation of ADAM17 transcripts, RT-PCR analyses were performed using total RNA extracted from bovine follicles collected between 0 h and 24 h post-hCG. Results showed that levels of ADAM17 mRNA were low prior to hCG (0 h), markedly and transiently increased 6-12 h post-hCG (P <0.05), and returned to low baseline levels at 24 h post-hCG in granulosa and theca interna cells of preovulatory follicles. To determine the transcriptional control of ADAM17 expression, primary cultures of bovine granulosa cells were used. Forskolin (FSK) stimulation induced a pattern of ADAM17 mRNA up-regulation in vitro similar to that observed by hCG in vivo. 5'-Deletion mutagenesis studies identified a minimal region of the bovine ADAM17 promoter containing basal and FSK-inducible activities, which were dependent on the presence of a consensus AP1 cis-element. Electrophoretic mobility shift assays revealed an interaction between AP1 and the trans-acting factor Fra2. Chromatin immunoprecipitation assays confirmed an endogenous interaction between Fra2 and the ADAM17 promoter in granulosa cell cultures. FSK-inducible ADAM17 promoter activity and mRNA expression were suppressed by PKA and ERK1/2 inhibitors but not by a p38MAPK inhibitor, pointing to the importance of PKA and ERK1/2 signaling pathways in the up-regulation of bovine ADAM17 mRNA. Collectively, these findings describe the gonadotropin/FSK-dependent up-regulation of ADAM17 transcripts in bovine preovulatory follicles and unravel for the first time some of the molecular mechanisms involved in ADAM17 gene expression in granulosa cells of a monoovulatory species.
解整合素金属蛋白酶17（ADAM17）被认为在卵巢卵泡中可溶性活性表皮调节素（EREG）和双调蛋白（AREG）的释放中起关键作用，但其在卵泡细胞中的转录调控仍 largely未知。本研究的目的是表征排卵前牛卵泡中ADAM17转录本的调控，并研究其在牛颗粒细胞中的转录控制。为了研究ADAM17转录本的调控，使用从hCG后0小时至24小时收集的牛卵泡中提取的总RNA进行RT-PCR分析。结果显示，在hCG（0小时）之前，ADAM17 mRNA水平较低，在hCG后6至12小时显著且短暂升高（P<0.05），并在排卵前卵泡的颗粒细胞和内膜细胞中于hCG后24小时恢复到低基线水平。为了确定ADAM17表达的转录控制，使用了牛颗粒细胞的原代培养物。福司柯林（FSK）刺激在体外诱导了ADAM17 mRNA上调模式，类似于体内hCG观察到的模式。5'-缺失诱变研究确定了牛ADAM17启动子的一个最小区域，其包含基础和FSK诱导活性，这取决于共有AP1顺式元件的存在。电泳迁移率变动分析揭示了AP1与反式作用因子Fra2之间的相互作用。染色质免疫沉淀分析证实了颗粒细胞培养物中Fra2与ADAM17启动子之间的内源性相互作用。FSK诱导的ADAM17启动子活性和mRNA表达被PKA和ERK1/2抑制剂抑制，但不被p38MAPK抑制剂抑制，这表明PKA和ERK1/2信号通路在牛ADAM17 mRNA上调中的重要性。总体而言，这些发现描述了牛排卵前卵泡中促性腺激素/FSK依赖性的ADAM17转录本上调，并首次揭示了单排卵物种颗粒细胞中ADAM17基因表达所涉及的一些分子机制。
