P38α MAPK Is A Gatekeeper Of Uterine Progesterone Responsiveness At Peri-implantation Via Ube3c-mediated PGR Degradation

The success rate of normal pregnancy in a menstrual cycle is only 30%,and most pregnancy failures are caused by abnormal embryo implantation.Embryonic implantation is the process in which the embryo makes the first contact with the uterus,then establishes a physiological connection.Numbers of molecular and signal exchange between different cell types are involved in this process.Successful implantation determines subsequent embryonic development and the final pregnancy outcome.The simultaneous establishment of blastocyst activation and uterine receptivity is a prerequisite for successful embryo implantation,which is tightly regulated by ovarian secretion hormones,estrogen and progesterone.Estrogen and progesterone specify the establishment of uterine receptivity mainly through their respective nuclear receptors,ER and PR.PR is transcriptionally induced by estrogen–ER signaling in the endometrium,but how the protein homeostasis of PR in the endometrium is regulated remains elusive.The P38 MAPK family is an evolutionarily conserved serine/threonine phosphorylated kinase,which can phosphorylation their intracellular substrates and then involved in the cellular stress and inflammation response.Here,our data found that P38αwas dynamically expressed in the peri-implantation uterus in a spatiotemporal manner,showing significant activation during implantation and decidualization.In contrast,other members of the P38 MAPK family（P38β,P38γ,and P38δ）are expressed at lower levels in the uterus.Previous studies have shown that due to abnormal placental development,mice with P38αsystemic knockout are embryologically lethal.Therefore,to explore the role of P38αMAPK in early pregnancy,we constructed a P38α^f/f/Pgr-Cre(P38α^d/d)mouse model with the uterine-specific knockout of P38α.By examining the time points of major events in early pregnancy,we found that uterine P38αdeficiency mice were compromised fertility while showing normal ovulation and fertilization.RNA-seq results show the abundant expression of uterine receptivity-related genes in P38α-deficient mice.In addition,the proliferative activity of stromal cells was not effectively induced,while epithelial cells continued to proliferate in P38α^d/d D4 uterus,indicating that the absence of P38αgene in the uterus led to reduced progesterone responsiveness of stromal cells and abnormal differentiation of epithelial cells,leading to the failure establishment of the uterine receptivity.Notably,exogenous progesterone injection could not rescue the implantation defects caused by uterine P38αdeletion.Further studies found that ovarian-derived estrogen and progesterone levels show no difference between P38α^f/f and P38α^d/d mice.While the protein level of PR in the D4uterus of P38α^d/d mice was significantly decreased.Data from the endometrial organoid model and the expression of epithelial progesterone response genes,Ihh and Areg,showed that P38αdeletion did not affect the level of PR protein and progesterone response in epithelial cells.However,when the P38αMAPK signal was inhibited in uterine stromal cells,the degradation rate of PR protein by the ubiquitin-proteasome pathway increased,indicating the importance of P38αfor the stability of PR protein in stromal cells.Ubiquitination is a common protein degradation pathway in cells that attaches ubiquitin molecules to lysine residues of substrate proteins through a cascade of E1ubiquitin-activating enzyme,E2 ubiquitin-binding enzyme,and E3 ubiquitin ligase.To explore the specific mechanism involved in the regulation of PR protein ubiquitination in the uterus,we used Co-IP-MS to find that E3 ubiquitin ligase Ube3c specifically binds to PR protein in the uterus of P38α^d/d mice.Through the co-expressing of HA-PRA and Myc-Ube3c plasmid vector,we found that Ube3c can recognize and promote the ubiquitination of the Lys416,Lys441,and Lys445 sites in the DBD domain of PR protein.Additionally,when P38αwas overexpressed with Ube3c and PR,the stability of PR protein increased,while the addition of P38αMAPK signal inhibitor SB203580 could effectively restore the ubiquitin degradation of PR protein induced by Ube3c,indicating that the phosphokinase activity of P38αis the key to inhibit Ube3c inducing PR degradation.To reveal whether Ube3c is a phosphorylation substrate for P38α,we performed point mutations in the sites in Ube3c that predicted be modified by P38αand found that phosphorylation at Ser741 significantly inhibited Ube3c-induced degradation of PR protein.In summary,this study found that P38αis involved in the maintenance of the stability of PR protein in mouse uterine through phosphorylation of E3 ubiquitin ligase Ube3c,thus regulating the progesterone signaling response in stromal cells,revealing the importance of P38αMAPK signaling for the establishment of uterine receptivity.Our findings are not only conducive to further understanding the physiological process of embryo implantation but also have important guiding significance for the application of assisted reproductive technology in clinical practice.