The phosphoproteomic analysis of human endometrium reveals the regulatory roles of GSK3 <b>β</b> thr390 phosphorylation in endometrial decidualization

Non-receptive endometrium is one major cause of female fertility defects. While transcriptomic analyses have illuminated gene expression changes, the role of dynamic phosphoproteomic modifications remains under-investigated. We profiled the proteomic and phosphoproteomic landscape of the human endometrium during the establishment of endometrial receptivity at pre-receptive LH + 5, receptive LH + 7 and LH + 9 in which thousands of proteins and phosphor sites displayed dynamic changes. Among them, GSK3β protein levels were constant but Thr390 phosphorylation in human endometrium were increased from LH + 5 to LH + 7 and LH + 9, and reduced in the endometrial stroma of patients with Recurrent Implantation Failure. Knockdown of GSK3β impaired the decidualization of primary human endometrial stromal cells (HESCs) which can be rescued by wildtype GSK3β but not T390A mutant GSK3β. Through the analysis of our endometrial phosphoproteome, siP38α knockdown in HESCs, and uterine specific P38α knockout mice, we identified GSK3β Thr390 was mainly phosphorylated by P38α, whose deficiency was also detrimental to stroma decidualization. RNA-Seq analysis of HESCs indicated FOXO1 as the key transcription factor that mediated the conserved transcriptomic changes during decidualization by the siGSK3β and siP38α treatment. Further analysis indicated GSK3β knockdown shifted FOXO1 out of the nucleus and reduced the total protein levels of FOXO1. More interestingly, FOXO1 overexpression mitigates the decidualization defects caused by GSK3β deficiency. This study describes dynamic phosphorylation events during endometrial receptivity and highlights the critical role of P38α-dependent GSK3β Thr390 phosphorylation in endometrial decidualization. These findings offer mechanistic insight into potential therapeutic targets for enhancing endometrial receptivity.