ATF6 knockdown decreases apoptosis, arrests the S phase of the cell cycle, and increases steroid hormone production in mouse granulosa cells
ATF6 knockdown decreases apoptosis, arrests the S phase of the cell cycle, and increases steroid hormone production in mouse granulosa cells
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ATF6 敲低可减少小鼠颗粒细胞的细胞凋亡、阻止细胞周期的 S 期并增加类固醇激素的产生
DOI：
10.1152/ajpcell.00222.2016
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Jin Yaping
中科院分区：
文献类型：
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作者：
Xiong Yongjie;Chen Huatao;Lin Pengfei;Wang Aihua;Wang Lei;Jin Yaping
转录激活因子6（Activating Transcription Factor 6，ATF 6）是内质网（Endoplasmic Reticulum，ER）膜上的一种感受蛋白，是内质网应激信号通路中的一个重要因子。ER应激参与卵泡发生、卵泡生长和排卵;然而，ATF 6在小鼠颗粒细胞中的生理功能在很大程度上仍然未知。本研究的目的是通过RNA干扰、免疫组化染色、实时定量PCR、Western印迹、流式细胞术、末端脱氧核苷酸转移酶介导的脱氧UTP缺口末端标记（TUNEL）测定和ELISA，评估ATF 6在小鼠颗粒细胞凋亡、细胞周期和类固醇激素产生方面的作用，以及与卵泡发育相关的几个关键基因。免疫组织化学染色显示，ATF 6广泛分布于成年雌性小鼠各种卵泡的颗粒细胞和卵母细胞中。FSH或LH处理显著增加小鼠颗粒细胞中的ATF 6蛋白水平。同时，利用短发夹RNA介导的干扰技术，成功构建了ATF 6基因的缺失质粒，并在mRNA和蛋白水平上进行了验证。流式细胞术和TUNEL分析表明，在小鼠颗粒细胞中，ATF 6耗竭减少凋亡并阻滞细胞周期的S期。与这些结果一致，p53、caspase-3、B细胞淋巴瘤2（Bcl-2）相关X蛋白、CCAAT增强子结合蛋白同源蛋白、细胞周期蛋白A1、细胞周期蛋白B1和细胞周期蛋白D2 mRNA表达减少，而Bcl-2和葡萄糖调节蛋白78 kDa mRNA表达增加。有趣的是，ATF 6敲低明显增加小鼠颗粒细胞中孕酮和雌二醇的产生。细胞色素P450 1b 1（Cyp 1b 1）mRNA水平下调，而Cyp 11 a1，类固醇急性调节，Cyp 19 a1 mRNA水平上调，保持与类固醇激素的变化。此外，在小鼠颗粒细胞中，ATF 6破坏显著增加了胰岛素样生长因子结合蛋白4（Igfbp 4）的表达，并降低了透明质酸合酶2（Has 2）、胰高血糖素内过氧化物合酶2（Ptgs 2）和前列腺素F受体（Ptgfr）的表达，这些蛋白质对卵泡发育至关重要。但经衣霉素处理后，Has 2、Ptgs 2和Ptgfr的表达水平相对升高，而Igfbp 4的表达降低。总的来说，这些结果表明，ATF 6，作为一个关键的球员在ER应激信号，可能会调节细胞凋亡，细胞周期，类固醇激素的合成，以及其他相关的卵泡在小鼠颗粒细胞，这可能间接参与的发展，排卵，和闭锁的卵泡通过影响颗粒细胞的生理功能。目前的研究扩展了我们的理解，并提供了新的见解ATF 6的生理意义，ER应激的关键信号转导，在卵巢颗粒细胞。
Activating transcription factor 6 (ATF6), a sensor protein located in the endoplasmic reticulum (ER) membrane, is an important factor in the ER stress signaling pathway. ER stress is known to be involved in folliculogenesis, follicular growth, and ovulation; however, the physiological function of ATF6 in mouse granulosa cells remains largely unknown. The aim of this study was to assess the role of ATF6 in mouse granulosa cells with respect to apoptosis, the cell cycle, and steroid hormone production, as well as several key genes related to follicular development, via RNA interference, immunohistochemical staining, real-time quantitative PCR, Western blotting, flow cytometry, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay, and ELISA. Immunohistochemical staining revealed that ATF6 was extensively distributed in the granulosa cells of various ovarian follicles and oocytes in adult female mice. FSH or LH treatment significantly increased ATF6 protein levels in mouse granulosa cells. In the meantime, a recombinant plasmid was used to deplete ATF6 successfully using short hairpin RNA-mediated interference technology, which was verified at both the mRNA and protein levels. Flow cytometry and TUNEL assay analysis indicated that ATF6 depletion decreased apoptosis and arrested the S phase of the cell cycle in mouse granulosa cells. Consistent with these results, p53, caspase-3, B cell lymphoma 2 (Bcl-2)-associated X protein, CCAAT-enhancer-binding protein homologous protein, cyclin A1, cyclin B1, and cyclin D2 mRNA expression decreased, whereas Bcl-2 and glucose-regulated protein 78 kDa mRNA expression increased. Interestingly, ATF6 knockdown obviously increased progesterone and estradiol production in mouse granulosa cells. Cytochrome P450 1b1 (Cyp1b1) mRNA levels were downregulated, whereas Cyp11a1, steroidogenic acute regulatory, and Cyp19a1 mRNA levels were upregulated, in keeping with the changes in steroid hormones. Furthermore, ATF6 disruption remarkably increased insulin-like growth factor binding protein 4 (Igfbp4) expression and decreased hyaluronan synthase 2 (Has2), prostaglandin-endoperoxide synthase 2 (Ptgs2), and prostaglandin F receptor (Ptgfr) expression in mouse granulosa cells, which are proteins crucial for follicular development. But, after treating with tunicamycin, the levels of Has2, Ptgs2, and Ptgfr increased relatively, whereas Igfbp4 expression decreased. Collectively, these results imply that ATF6, as a key player in ER stress signaling, may regulate apoptosis, the cell cycle, steroid hormone synthesis, and other modulators related to folliculogenesis in mouse granulosa cells, which may indirectly be involved in the development, ovulation, and atresia of ovarian follicles by affecting the physiological function of granulosa cells. The present study extends our understanding and provides new insights into the physiological significance of ATF6, a key signal transducer of ER stress, in ovarian granulosa cells.