Centre de Recherche en Reproduction et Fertilité, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 rue Sicotte, St-Hyacinthe, QC, J2S 2M2, Canada.
Cell Commun Signal. 2024 Aug 14;22(1):396. doi: 10.1186/s12964-024-01736-1.
Secreted frizzled-related proteins (SFRPs) comprise a family of WNT signaling antagonists whose roles in the ovary are poorly understood. Sfrp4-null mice were previously found to be hyperfertile due to an enhanced granulosa cell response to gonadotropins, leading to decreased antral follicle atresia and enhanced ovulation rates. The present study aimed to elucidate the mechanisms whereby SFRP4 antagonizes FSH action.
Primary cultures of granulosa cells from wild-type mice were treated with FSH and/or SFRP4, and effects of treatment on gene expression were evaluated by RT-qPCR and RNAseq. Bioinformatic analyses were conducted to analyse the effects of SFRP4 on the transcriptome, and compare them to those of FSH or a constitutively active mutant of FOXO1. Additional granulosa cell cultures from wild-type or Sfrp4-null mice, some pretreated with pharmacologic inhibitors of specific signaling effectors, were used to examine the effects of FSH and/or SFRP4 on signaling pathways, autophagy and apoptosis by western blotting and TUNEL.
Treatment of cultured granulosa cells with recombinant SFRP4 was found to decrease basal and FSH-stimulated mRNA levels of FSH target genes. Unexpectedly, this effect was found to occur neither via a canonical (CTNNB1-dependent) nor non-canonical WNT signaling mechanism, but was found to be GSK3β-dependent. Rather, SFRP4 was found to antognize AKT activity via a mechanism involving AMPK. This lead to the hypophosphorylation of FOXO1 and a decrease in the expression of a portion of the FSH and FOXO1 transcriptomes. Conversely, FSH-stimulated AMPK, AKT and FOXO1 phosphorylation levels were found to be increased in the granulosa cells of Sfrp4-null mice relative to wild-type controls. SFRP4 treatement of granulosa cells also induced autophagy by signaling via AKT-mTORC1-ULK1, as well as apoptosis.
This study identifies a novel GSK3β-AMPK-AKT signaling mechanism through which SFPR4 antagonizes FSH action, and further identifies SFRP4 as a novel regulator of granulosa cell autophagy. These findings provide a mechanistic basis for the phenotypic changes previously observed in Sfrp4-null mice, and broaden our understanding of the physiological roles of WNT signaling processes in the ovary.
分泌卷曲相关蛋白（SFRPs）构成 WNT 信号拮抗剂家族，其在卵巢中的作用尚不清楚。先前发现 Sfrp4 基因敲除小鼠由于对促性腺激素的颗粒细胞反应增强而具有超生育能力，导致窦卵泡闭锁减少和排卵率增加。本研究旨在阐明 SFRP4 拮抗 FSH 作用的机制。
用 FSH 和/或 SFRP4 处理来自野生型小鼠的原代颗粒细胞培养物，并通过 RT-qPCR 和 RNAseq 评估处理对基因表达的影响。生物信息学分析用于分析 SFRP4 对转录组的影响，并将其与 FSH 或 FOXO1 的组成型激活突变体的影响进行比较。来自野生型或 Sfrp4 基因敲除小鼠的其他颗粒细胞培养物，一些用特定信号转导效应物的药理学抑制剂预处理，用于通过 Western blot 和 TUNEL 检测 FSH 和/或 SFRP4 对信号通路、自噬和细胞凋亡的影响。
发现用重组 SFRP4 处理培养的颗粒细胞可降低基础和 FSH 刺激的 FSH 靶基因的 mRNA 水平。出乎意料的是，这种作用既不是通过经典（CTNNB1 依赖性）也不是非经典 WNT 信号机制发生的，而是发现它是 GSK3β 依赖性的。相反，SFRP4 被发现通过涉及 AMPK 的机制拮抗 AKT 活性。这导致 FOXO1 的低磷酸化和一部分 FSH 和 FOXO1 转录组的表达减少。相反，与野生型对照相比，在 Sfrp4 基因敲除小鼠的颗粒细胞中发现 FSH 刺激的 AMPK、AKT 和 FOXO1 磷酸化水平增加。SFRP4 处理颗粒细胞还通过 AKT-mTORC1-ULK1 信号诱导自噬以及细胞凋亡。
本研究确定了一种通过 SFRP4 拮抗 FSH 作用的新型 GSK3β-AMPK-AKT 信号机制，并进一步确定 SFRP4 是颗粒细胞自噬的新型调节剂。这些发现为先前在 Sfrp4 基因敲除小鼠中观察到的表型变化提供了机制基础，并拓宽了我们对 WNT 信号过程在卵巢中的生理作用的理解。
