Key Laboratory of Fertility Preservation and Maintenance, Ministry of Education, Key Laboratory of Reproduction and Genetics in Ningxia, Department of Histology and Embryology of Basic Medical College, Ningxia Medical University, Yinchuan 750004, China.
College of Life Science, Capital Normal University, Beijing 100048, China.
Int J Mol Sci. 2023 Feb 1;24(3):2749. doi: 10.3390/ijms24032749.
Female infertility is caused by premature ovarian failure (POF), which is triggered by the endoplasmic reticulum (ER) stress-mediated apoptosis of granulosa cells. The ER unfolded protein response (UPR) is initiated to promote cell survival by alleviating excessive ER stress, but cellular apoptosis is induced by persistent or strong ER stress. Recent studies have reported that reticulophagy is initiated by ER stress. Whether reticulophagy is activated in the ER stress-mediated apoptosis of granulosa cells and which pathway is initiated to activate reticulophagy during the apoptosis of granulosa cells are unknown. Therefore, the role of reticulophagy in granulosa cell death and the relationship between ER stress and reticulophagy were investigated in this work. Our results suggest that the ER stress inducer tunicamycin causes POF in mice, which is attributed to the apoptosis of granulosa cells and is accompanied by the activation of UPR and reticulophagy. Furthermore, granulosa cells were treated with tunicamycin, and granulosa cell apoptosis was triggered and increased the expression of UPR and reticulophagy molecules. The expression of ATF4 was then downregulated by RNAi, which decreased the levels of autophagy and the reticulophagy receptor CCGP1. Furthermore, ATF4 targets MAP1LC3A, as revealed by the ChIP sequencing results, and co-IP results demonstrated that MAP1LC3A interacts with CCPG1. Therefore, reticulophagy was activated by ER stress through the ATF4-MAP1LC3A-CCPG1 pathway to mitigate ER stress. Additionally, the role of reticulophagy in granulosa cells was investigated by the knockdown of CCPG1 with RNAi. Interestingly, only a small number of granulosa cells died by apoptosis, whereas the death of most granulosa cells occurred by necroptosis triggered by STAT1 and STAT3 to impair ER proteostasis and the ER protein quality control system UPR. Taken together, the results indicate that the necroptosis of granulosa cells is triggered by up- and downregulating the reticulophagy receptor CCPG1 through STAT1/STAT3-(p)RIPK1-(p)RIPK3-(p)MLKL and that reticulophagy is activated by ER stress through the ATF4-MAP1LC3A-CCPG1 pathway.
女性不孕是由卵巢早衰（POF）引起的，这是由内质网（ER）应激介导的颗粒细胞凋亡引起的。内质网未折叠蛋白反应（UPR）的启动是为了通过缓解过度的 ER 应激来促进细胞存活，但持续或强烈的 ER 应激会诱导细胞凋亡。最近的研究表明，内质网应激会引发网质体自噬。在颗粒细胞 ER 应激介导的凋亡中是否激活了网质体自噬，以及在颗粒细胞凋亡过程中启动了哪个途径来激活网质体自噬，目前尚不清楚。因此，本工作研究了网质体自噬在颗粒细胞死亡中的作用以及 ER 应激与网质体自噬之间的关系。我们的结果表明，内质网应激诱导剂衣霉素导致小鼠发生 POF，这归因于颗粒细胞的凋亡，并伴有 UPR 和网质体自噬的激活。此外，用衣霉素处理颗粒细胞，触发颗粒细胞凋亡并增加 UPR 和网质体自噬分子的表达。然后通过 RNAi 下调 ATF4 的表达，降低自噬和网质体自噬受体 CCGP1 的水平。此外，ChIP 测序结果显示 ATF4 靶向 MAP1LC3A，共免疫沉淀结果表明 MAP1LC3A 与 CCGP1 相互作用。因此，内质网应激通过 ATF4-MAP1LC3A-CCPG1 途径激活网质体自噬，以减轻 ER 应激。此外，通过 RNAi 敲低 CCGP1 研究了网质体自噬在颗粒细胞中的作用。有趣的是，只有少数颗粒细胞通过凋亡死亡，而大多数颗粒细胞通过 STAT1 和 STAT3 触发的坏死性凋亡死亡，从而损害 ER 蛋白稳态和 ER 蛋白质量控制系统 UPR。总之，结果表明，通过 STAT1/STAT3-(p)RIPK1-(p)RIPK3-(p)MLKL 上调和下调网质体自噬受体 CCGP1 触发颗粒细胞的坏死性凋亡，并且内质网应激通过 ATF4-MAP1LC3A-CCPG1 途径激活网质体自噬。
