背景:分泌型卷曲相关蛋白(SFRP)包含WNT信号拮抗剂家族,其在卵巢中的作用知之甚少。以前发现Sfrp4-null小鼠由于颗粒细胞对促性腺激素的反应增强而肥沃,导致窦卵泡闭锁减少和排卵率提高。本研究旨在阐明SFRP4拮抗FSH作用的机制。
方法:用FSH和/或SFRP4处理来自野生型小鼠的颗粒细胞的原代培养物,并且通过RT-qPCR和RNAseq评价处理对基因表达的影响。进行生物信息学分析以分析SFRP4对转录组的影响,并将它们与FSH或FOXO1的组成型活性突变体进行比较。来自野生型或Sfrp4-null小鼠的其他颗粒细胞培养物,一些用特定信号效应物的药物抑制剂预处理,用于检查FSH和/或SFRP4对信号通路的影响,蛋白质印迹和TUNEL的自噬和凋亡。
结果:发现用重组SFRP4处理培养的颗粒细胞可降低FSH靶基因的基础和FSH刺激的mRNA水平。出乎意料的是,发现这种效应既不通过规范(CTNNB1依赖性)也不通过规范的WNT信号机制发生,但被发现是GSK3β依赖性的。相反,发现SFRP4通过涉及AMPK的机制使AKT活性前变性。这导致FOXO1的低磷酸化和FSH和FOXO1转录组的一部分的表达减少。相反,FSH刺激的AMPK,发现相对于野生型对照,Sfrp4缺失小鼠的颗粒细胞中的AKT和FOXO1磷酸化水平增加。SFRP4处理颗粒细胞还通过经由AKT-mTORC1-ULK1的信号传导诱导自噬以及细胞凋亡。
结论:本研究确定了一种新的GSK3β-AMPK-AKT信号机制,SFPR4通过该机制拮抗FSH作用,并进一步鉴定SFRP4为颗粒细胞自噬的新型调节因子。这些发现为先前在Sfrp4-null小鼠中观察到的表型变化提供了机制基础,并拓宽了我们对卵巢中WNT信号传导过程的生理作用的理解。
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.