P38α MAPK is a gatekeeper of uterine progesterone responsiveness at peri-implantation via Ube3c-mediated PGR degradation

Abstract

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. Here, we demonstrated that the uterine-selective depletion of P38α derails normal uterine receptivity ascribed to the dramatic down-regulation of PR protein and disordered progesterone responsiveness in the uterine stromal compartment, leading to defective implantation and female infertility. Specifically, Ube3c, an HECT family E3 ubiquitin ligase, targets PR for polyubiquitination and thus proteasome degradation in the absence of P38α. Moreover, we discovered that P38α restrains the polyubiquitination activity of Ube3c toward PR by phosphorylating the Ube3c at serine741 . In summary, we provided genetic evidence for the regulation of PR protein stability in the endometrium by P38α and identified Ube3c, whose activity was modulated by P38α-mediated phosphorylation, as an E3 ubiquitin ligase for PR in the uterus.

Keywords: P38α; Ube3c; progesterone receptor; uterine receptivity.

Fig. 1.

P38α is indispensable for normal implantation. (A) ISH of Mapk14 in D1, D4, D5, D6, and D8 uteri. The pink site indicates the location of Mapk14. Positive signals were not detected in the uterus labeled with the Sense probe. Le, luminal epithelium; S, stroma; Bl, blastocyst; Em, embryo (Scale bars, 100 μm). (B) qRT-PCR analysis to reveal the knockout efficiency of Mapk14 in P38α^f/f and P38α^d/d uteri on D4. Data shown represent the means ± SEM; ***P < 0.001. (C and D) IHC and Western blot analysis revealed the efficient ablation of P38α and p-P38 at protein level (Scale bars, 100 μm). (E) Average litter sizes in P38α^f/f and P38α^d/d mice. Number within the bar indicates the number of mice tested. (F) Gross morphological implantation sites in P38α^f/f mice compared with P38α^d/d mice as determined by blue dye injection on D5. The unimplanted embryos were recovered from P38α^d/d uteri (Scale bars, 100 μm). (G) Average number of implantation sites in P38α^f/f and P38α^d/d mice on D5 morning of pregnancy. Mice that failed to recover any embryos were excluded in statistical analysis. Number within the bar indicates the number of mice tested. (H) Morphological implantation sites in P38α^f/f mice compared with P38α^d/d mice as determined by blue dye injection on D6. (I) Average number of implantation sites in P38α^f/f and P38α^d/d mice on D6 morning of pregnancy. Number within the bar indicates the number of mice tested. **P < 0.01. (J) Representative hematoxylin and eosin staining of cross-sections of P38α^f/f and P38α^d/d implantation sites on D5 (Scale bars, 100 μm). (K) IHC staining of COX2 in P38α^f/f and P38α^d/d uteri on D5 of pregnancy (Scale bars, 100 μm).

Fig. 2.

P38α depletion derails uterine receptivity accompanying decreased expression of P4-responsive genes. (A) Volcano plots showing the differentially expressed genes in P38α^f/f and P38α^d/d uteri on D4 of pregnancy. (B) Heat map showing the expression level of differentially expressed genes in P38α^f/f and P38α^d/d uteri on D4 of pregnancy. (C and D) qRT-PCR analysis of implantation-related marker gene expression in P38α^f/f and P38α^d/d uteri on D4 of pregnancy. Data shown represent the means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. (E–G) IHC (E and G) and ISH (F) staining of implantation-related marker gene expression in P38α^f/f and P38α^d/d uteri on D4 of pregnancy (Scale bars, 100 μm). (H) The proliferation of uterine cells was compared in P38α^f/f and P38α^d/d mice by IHC staining for Ki67 (Scale bars, 100 μm).

Fig. 3.

Loss of P38α compromises PR protein level rather than mRNA level in uterine stromal cells. (A and B) qRT-PCR analysis of Esr1 and Pgr expression in P38α^f/f and P38α^d/d uteri on D4. NS, not significant. (C) ISH of Pgr in P38α^f/f and P38α^d/d uteri on D4 of pregnancy (Scale bars, 100 μm). (D and E) Western blot and IHC staining show the protein level of ERα and PR in P38α^f/f and P38α^d/d uteri on D4 of pregnancy (Scale bars, 100 μm). (F) qRT-PCR analysis of Pgr expression in P38α^f/f and P38α^d/d ovariectomized mouse uteri treated with E2 for the indicated times. NS, not significant. (G and H) Western blot and IHC staining were performed to compare the protein level of PR in P38α^f/f and P38α^d/d ovariectomized mouse uteri when treated with E2 for the indicated times (Scale bars, 100 μm).

Fig. 4.

Inhibition of P38α MAPK reduces the half-life of PR protein. (A) Western blot analysis of PR in CHX (50 μg/mL)-treated P38α^f/f and P38α^d/d uterine stromal cells with or without SB203580/PH797804 treatment. (B and C) The half-life of PR in isolated uterine stromal cells when treated with the vehicle/SB203580/PH797804 for 12 h, followed by treatment with cycloheximide (50 μg/mL) for the indicated times. Veh, vehicle. (D) Western blot analysis of PR in the isolated uterine stromal cells in the presence of proteasome inhibitor MG-132 in the indicated treatment group.

Fig. 5.

Ube3c promotes PR protein degradation via polyubiquitination. (A) Venn diagrams showing the number of proteins that interact with PR in P38α^f/f and P38α^d/d uteri on D4 of pregnancy. (B and C) qRT-PCR and Western blot analysis showing the expression pattern of Ube3c in P38α^f/f and P38α^d/d uteri on D4 of pregnancy. NS, not significant. (D and E) Western blot analysis revealed the stability (D) and ubiquitination (E) of HA-PRA with or without overexpression of Myc-Ube3c in 293T cells. IP, immunoprecipitation; Ub, ubiquitin.

Fig. 6.

Ube3c mediates PR ubiquitination at K416, K441 and K445. (A) Domain architecture of PR protein. (B) Western blot analysis showing the protein level of HA-P1, HA-P2, and HA-PRA with or without Myc-Ube3c in 293T cells. (C) Lysine (red K) sites in DBD domain of PR. (D and E) Western blot and immunoprecipitation revealed the stability (D) and polyubiquitination (E) of different point mutation forms of HA-PRA.

Fig. 7.

P38α regulates the catalytic activity of Ube3c. (A) Western blot analysis showing the weakened catalytic activity of Myc-Ube3c result from the overexpression of P38α in 293T cells. (B) Immunoprecipitation and Western blot assay to analyze the p-Ser in Ube3c. (C) Potential serine sites in Ube3c that can be phosphorylated by P38α. (D and E) Western blot analysis revealed the p-Ser level (D) and catalytic activity (E) of different point mutation forms of Myc-Ube3c.

Fig. 8.

Illustrative model of uterine P38α function during uterine receptivity establishment. In the presence of P38α, the E3 ubiquitin ligase Ube3c was phosphorylated at Ser741, which down-regulated its polyubiquitination activity toward PR and thus maintained the proper PR level in the stroma cell. The deficiency of P38α led to reduced Ube3c phosphorylation, which displayed aberrant high activity to induce the PR polyubiquitination and proteasome-mediated protein degradation.