مقالات پذیرفته شده در سومین کنگره بین المللی زیست پزشکی
Endometrial Cellular senescence and preterm birth
Endometrial Cellular senescence and preterm birth
Fereshteh Moheb Afzali ,1,*
1. Department of Molecular and Cellular Biology , Faculty of Advanced Sciences and Technology , Tehran Medical Sciences Branch , Islamic Azad University , Tehran, Iran.
Introduction: Aging is a contributing factor to cellular senescence.it is possible that endometrial cellular senescence due to maternal aging can increase the risk of preterm birth. Several risk factors of preterm birth, including genetics, stress and inflammation/infection have been shown to contribute to the senescence process in other systems, and we speculate that these factors pathologically push decidual senescence toward the threshold. Endometrial cellular senescence in p53 d/d females is associated with heightened signaling of Mammalian target of rapamycin complex1(mTORC1),a serine/threonine kinase, plays important roles in cell proliferation and survival. In the mouse model of preterm labor in which Trp53 is conditionally deleted in the uterus, decidual senescence begins prematurely due to activation of an pAkt-mTORC1-p21-Cox2 signaling axis and reaches the senescence threshold in a shorter gestational time frame, leading to preterm delivery.
Methods: Mice. Mice with uterine deletion of Trp53 (Trp53loxP/loxPPgrCre/+= p53d/d) were generated as described previously. Cdkn1a−/−Trp53loxP/loxPPgrCre/+(p21d/d p53d/d) mice were generated by mating p53d/d mice with Cdkn1a−/− (p21d/d) mice. For experiments, littermate p53f/f, p53d/d, p21d/dp53f/f, and p21d/dp53d/d females on the mixed background were mated with WT fertile males to induce pregnancy. All mice used in this investigation and protocols were reviewed and approved by the Cincinnati Children’s Research Foundation’s Institutional Animal Care and Use Committee. Mice were provided with autoclaved rodent LabDiet 5010 (Purina)and UV light-sterilized RO/DI constant circulation water ad libitum.
Analysis of Parturition. Preterm birth was defined as birth occurring earlier than day 19 of pregnancy. The mTORC1 inhibitor rapamycin (0.25 mg/kg body weight/day) was suspended in 5% PEG400 and 5% Tween-80 dissolved in water by constant stirring and was given as a single oral gavage on days 8, 10, and/or 12 of pregnancy. The control group received vehicle alone. LPS (25 μg/mouse i.p.; Sigma-Aldrich) was administered on day 16 of pregnancy.
Western Blot Analysis. Protein extraction and Western blot analysis were performed. Immunoblots were performed using extracts of uteri from which placenta and fetuses, including fetal membranes, were removed. Protein lysates were run on 10% SDS/PAGE gels. Antibodies for total S6 (1:1,000; Cell Signaling) and phospho-S6 (serine 235/236, 1:1,000; Cell Signaling), COX2 (1:5,000; Cayman), p21 (1:1,000; Santa Cruz Biotechnology), liver- and lung-type prostaglandin F synthase (PGFS; a gift from Kikuko Watanabe, University of East Asia, Shimonoseki, Japan),actin (1:1,000; Santa Cruz Biotechnology), and 20α-HSD (a gift from Geula Gibori, University of Illinois, Chicago, IL) were used. The same blots were used for quantitative analyses of each protein. Bands were visualized using an ECL kit (GE Healthcare). Actin served as a loading control.
Immunohistochemistry. Immunostaining of pS6 was performed in formalinfixed, paraffin-embedded sections using antibodies to pS6 (Cell Signaling). PAS staining was used to identify dNK cells .To compare intensity of immunostaining between p53f/f and p53d/d tissues, tissue sections from both genotypes were processed onto the same slide.
SA-β-gal Staining. Staining of SA-β-gal activity was performed as described Previously. SA-β-gal staining, a well-established marker of cellular senescence, is performed at pH 5.5–6.0. To compare the intensity of SA-β-gal staining, sections from different genotypes and on different days of pregnancy were processed on the same slide.
Statistics. Statistical analyses were performed using the two-tailed Student t test and Fisher’s exact probability test, as appropriate. P < 0.05 was considered statistically significant.
Results: This observation is supported by a previous in vitro study showing that loss of p53 increased mTORC1 signaling and cellular senescence in a human fibrosarcoma cell line. Taken together, these results suggest that rapamycin rescued preterm birth by attenuating the senescence process.
These results suggested that p21 deficiency would also rescue premature delivery in mice deleted of uterine p53.With these data in hand, we then asked whether the effects of rapamycin or p21 deficiency in rescuing preterm birth in p53d/d mice are mediated by cyclooxygenase-2 (COX2).
increased levels of COX2 and PGF synthase(PGFS),were associated with preterm birth in p53d/d mice, and this phenotype was rescued by oral administration of celecoxib, a selective COX2 inhibitor. These results demonstrate that inhibition of mTORC1 signaling negatively regulates the status of p21 and COX2.
Conclusion: Our results emphasize anew role of endometrial cellular senescence involving the p53-m TORC1-p21-Cox2 signaling axis in determining the timing of birth.