背景:间隙连接蛋白,连接蛋白43(Cx43)在人颗粒细胞-叶黄素(hGL)细胞中高度表达。已证明Cx43蛋白中某些氨基酸残基的磷酸化与间隙连接细胞间通讯(GJIC)的下降有关,随后影响卵母细胞减数分裂的恢复。作为表皮生长因子(EGF)家族的一员,β细胞素(BTC)介导哺乳动物卵泡中黄体生成素(LH)诱导的卵母细胞成熟和卵丘细胞扩增。BTC是否能调节Cx43磷酸化,其进一步降低hGL细胞中Cx43-偶联的GJIC活性仍有待确定。
方法:将从学术研究中心接受体外受精的女性中获得的永生化人颗粒细胞(SVOG细胞)和原代人颗粒细胞用作研究模型。在不同时间点与BTC孵育后,检查Cx43和磷酸化Cx43的表达水平。几种激酶抑制剂(sotrastaurin,AG1478和U0126)和靶向EGF受体(EGFR)和受体酪氨酸蛋白激酶4(ErbB4)的小干扰RNA用于验证效应的特异性并研究分子机制。实时定量PCR和蛋白质印迹分析用于检测特异性mRNA和蛋白水平,分别。使用刮擦加载和染料转移测定来评估SVOG细胞之间的GJIC。结果采用单因素方差分析。
结果:结果表明,BTC在serine368处诱导Cx43的快速磷酸化,而不改变Cx43在原代和永生化hGL细胞中的表达。此外,使用双重抑制方法(激酶抑制剂和基于siRNA的表达敲低),我们证明这种效应主要由EGFR介导,而不是ErbB4受体介导.此外,使用蛋白激酶C(PKC)激酶测定法和刮擦装载和染料转移测定法,我们发现,PKC信号传导是介导HGL细胞中Cx43磷酸化增加和随后GJIC活性降低的下游信号传导途径。
结论:BTC迅速诱导Ser368连接蛋白43的磷酸化,导致hGL细胞中GJIC活性降低。BTC诱导的细胞活性最有可能由EGFR介导的PKC依赖性信号通路驱动。我们的发现揭示了BTC调节卵母细胞减数分裂恢复过程的详细分子机制。
BACKGROUND: The gap junction protein, connexin 43 (Cx43) is highly expressed in human granulosa-lutein (hGL) cells. The phosphorylation of certain amino acid residues in the Cx43 protein has been shown to be related to a decline in gap junction intercellular communication (GJIC), which subsequently affects oocyte meiotic resumption. As a member of the epidermal growth factor (EGF) family, betacellulin (BTC) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus cell expansion in mammalian follicles. Whether BTC can regulate Cx43 phosphorylation, which further reduces Cx43-coupled GJIC activity in hGL cells remains to be determined.
METHODS: Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression levels of Cx43 and phosphorylated Cx43 were examined following cell incubation with BTC at different time points. Several kinase inhibitors (sotrastaurin, AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) and receptor tyrosine-protein kinase 4 (ErbB4) were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance.
RESULTS: The results showed that BTC induced the rapid phosphorylation of Cx43 at serine368 without altering the expression of Cx43 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR but not the ErbB4 receptor. Furthermore, using a protein kinase C (PKC) kinase assay and a scrape-loading and dye transfer assay, we revealed that PKC signaling is the downstream signaling pathway that mediates the increase in Cx43 phosphorylation and subsequent decrease in GJIC activity in response to BTC treatment in hGL cells.
CONCLUSIONS: BTC promptly induced the phosphorylation of connexin 43 at Ser368, leading to decreased GJIC activity in hGL cells. The BTC-induced cellular activities were most likely driven by the EGFR-mediated PKC-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which BTC regulates the process of oocyte meiotic resumption.