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, 126 (8), 2941-54

p53 Coordinates Decidual Sestrin 2/AMPK/mTORC1 Signaling to Govern Parturition Timing

p53 Coordinates Decidual Sestrin 2/AMPK/mTORC1 Signaling to Govern Parturition Timing

Wenbo Deng et al. J Clin Invest.

Abstract

Inflammation and oxidative stress are known risk factors for preterm birth (PTB); however, the mechanisms and pathways that influence this condition are not fully described. Previously, we showed that mTORC1 signaling is increased in mice harboring a uterine-specific deletion of transformation-related protein 53 (p53d/d mice), which exhibit premature decidual senescence that triggers spontaneous and inflammation-induced PTB. Treatment with the mTORC1 inhibitor rapamycin reduced the incidence of PTB in the p53d/d mice. Decidual senescence with heightened mTORC1 signaling is also a signature of human PTB. Here, we have identified an underlying mechanism for PTB and a potential therapeutic strategy for treating the condition. Treatment of pregnant p53d/d mice with either the antidiabetic drug metformin or the antioxidant resveratrol activated AMPK signaling and inhibited mTORC1 signaling in decidual cells. Both metformin and resveratrol protected against spontaneous and inflammation-induced PTB in p53d/d females. Using multiple approaches, we determined that p53 interacts with sestrins to coordinate an inverse relationship between AMPK and mTORC1 signaling that determines parturition timing. This signature was also observed in human decidual cells. Together, these results reveal that p53-dependent coordination of AMPK and mTORC1 signaling controls parturition timing and suggest that metformin and resveratrol have therapeutic potential to prevent PTB.

Figures

Figure 1
Figure 1. Met and Rsv attenuate preterm birth and dystocia.
(A and B) Treatment schedules for Met and Rsv treatment. (C and D) Percent preterm birth and live pups under Met or Rsv treatment in p53fl/fl and p53d/d females. Three of six p53d/d females that had preterm birth also showed dystocia. §One of ten p53d/d females showed both preterm birth and dystocia. *P < 0.05, χ2 test. (E and F) Percent preterm birth and live pups under Met or Rsv in combination with P4 4 hours before an LPS (10 μg) injection in p53fl/fl and p53d/d mice. One of two p53d/d females that had preterm birth also showed dystocia. §One of four p53d/d females showing preterm birth also had dystocia. *P < 0.05, χ2 test.
Figure 2
Figure 2. Met treatment improves decidual health.
(A) Met reduced the number of γH2AX-positive cells in p53d/d deciduae. Scale bar: 250 μm. (B) Met attenuated decidual senescence in p53d/d females. Scale bar: 500 μm. (C) Met upregulated Prlr expression in p53d/d deciduae. Scale bar: 500 μm. (D) pAMPK localization is low in p53d/d deciduae and increased after Met treatment. Scale bar: 250 μm. bv, blood vessel; Dec, decidua; Lb, labyrinth; Sp, spongiotrophoblast. (E and F) pAMPK, pRAPTOR, and pS6 expression and quantitation in day 16 deciduae. pAMPK and pRAPTOR levels were lower in p53d/d females with increased pS6 levels. Scale bar: 500 μm. *P < 0.05, mean ± SEM, Student’s t test.
Figure 3
Figure 3. Met and Rsv regulate COX2-derived PG synthesis.
(A) Upregulated Cox2 in p53d/d deciduae was inhibited by Met. Dec, decidua; Lb, labyrinth; Sp, spongiotrophoblast. Scale bar: 500 μm. (B and C) Decidual PG levels on day 17 after Met treatment. PGF levels were higher in p53d/d than in p53fl/fl mice after LPS injection, and levels were suppressed by combined treatment with Met and P4 (n = 6, *P < 0.05, mean ± SEM, Student’s t test). (D) PG levels in decidualized stromal cells after treatment with Met or Rsv. Met reduced PGF levels in both p53fl/fl and p53d/d decidualized stromal cells, while Rsv had little effect. Met or Rsv inhibited PGE2 levels in p53fl/fl and p53d/d decidualized stromal cells (n = 6, *P < 0.05, mean ± SEM, Student’s t test). (E and F) Met and Rsv attenuated Ptgs2 (encoding COX2) mRNA levels in decidualized mouse stromal cells (n = 3, *P < 0.05, mean ± SEM, Student’s t test).
Figure 4
Figure 4. Met, Rsv, and AICAR affect pAMPK and pS6 levels in a concentration-dependent manner.
(A and B) pAMPK levels and quantitation were significantly upregulated by Met (10 mM) with reduced pS6 levels. (C and D) Levels of pAMPK and pS6 and quantitation were altered at 50, 100, and 500 μM by Rsv. (E and F) Levels of pAMPK and pS6 and quantitation were significantly altered at 500 μM AICAR. *P < 0.05, mean ± SEM, ANOVA.
Figure 5
Figure 5. p53 inversely regulates pAMPK and mTORC1 signaling.
(A and B) Met or rapamycin (Rapa) inhibited pS6 in decidualized mouse stromal cells; Met alone stimulated pAMPK signaling. (CF) Met (10 mM) or Rsv (50 μM) treatment of p53fl/fl and p53d/d decidualized mouse stromal cells increased pAMPK levels in p53d/d decidual cells with concomitant decreased pS6 levels. (G and H) pS6 levels significantly increased in p53–/– MEFs compared with control cells. pAMPK and pS6 levels in WT and p53–/– MEFs were inversely regulated by Met. *P < 0.05, mean ± SEM, ANOVA.
Figure 6
Figure 6. p53 regulates SESN2 expression in deciduae.
(A and B) Sesn2 expression is downregulated in p53d/d deciduae on day 16 with no significant changes in Sesn1 expression compared with p53fl/fl deciduae (n = 4, *P < 0.05, mean ± SEM, Student’s t test). (C and D) Sesn2 expression is downregulated in p53–/– MEFs with no significant changes in Sesn1 expression (n = 3, *P < 0.05, mean ± SEM, Student’s t test). (E and F) SESN2 protein levels were downregulated in p53d/d deciduae and upregulated by Met on day 16 (n = 3, *P < 0.05, mean ± SEM, ANOVA). (G) SESN2 is downregulated in p53d/d deciduae, and Met pretreatment upregulated SESN2 expression in p53d/d deciduae. Dec, decidua; RBC, red blood cells. Scale bar: 250 μm. (H and I) Putative p53 binding sites for Sesn2 in mouse and human. (J) Increases in luciferase activity of Sesn2 after treatment with Nutlin or Dox in WT MEFs, but not in p53–/– MEFs (n = 6, *P < 0.05, mean ± SEM, ANOVA). TSS, transcription start site.
Figure 7
Figure 7. p53 induces SESN2 expression to modulate AMPK and mTORC1 signaling.
(AE) Nutlin and Doxo upregulated the levels of p53, SESN2, and pAMPK with downregulation of pS6 levels in decidualized stromal cells (n = 3, *P < 0.05, mean ± SEM, ANOVA). (FI) SESN2 overexpression inversely regulated pAMPK and pS6 levels (n = 3, *P < 0.05, mean ± SEM, Student’s t test).
Figure 8
Figure 8. p53-sestrin interactions coordinate AMPK and mTORC1 signaling in human decidual cells.
(AD) Met or Rsv inversely regulated pAMPK and pS6 levels in HuF cells (n = 3, *P < 0.05, mean ± SEM, Student’s t test). (EJ) pAMPK and pS6 levels were inversely regulated by Met or Rsv in human primary decidual cells isolated from term placentas. Each sample was isolated from a discrete decidua from a single patient. pAMPK and pS6 levels were inversely regulated by Met or Rsv (n = 4, *P < 0.05, mean ± SEM, ANOVA). (K and L) SESN1 and SESN2 mRNA was upregulated by P4 in term decidual cells, but not in preterm decidual cells (n = 3, *P < 0.05, mean ± SEM, Student’s t test).
Figure 9
Figure 9. A schematic representation of the role of decidual p53-sestrin interactions to integrate AMPK and mTORC1 signaling in determining parturition timing.
Increased AMPK signaling by SESNs in mouse and human decidual cells with downregulation of mTORC1 signaling by p53 and further enhanced by Met or Rsv provides protection against preterm birth (PTB) This downregulation of mTORC1 signaling with increased pAMPK activity is reflected in attenuated protein translation involving pS6K and/or p4EBP1. This perhaps helps rescue PTB.

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