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Title: mTOR Signaling and MMP expression in response to Myometrial Stretch and in Preterm Labor
Authors: Heather Burkin, Ph.D., Carolina Wandscheer, Ph.D., Craig Ulrich, Ph.D.,, Kyle VonSchimmelmann, Patricia Cachila, Constanza Spelius, B.S., and Iain Buxton, Pharm.D.
Institutional Affiliation: Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada, United States, 89557
Key Words:Pregnancy, preterm labor, myometrium, smooth muscle, parturition
Objectives:Recent literature has implicated mTOR signaling in regulating birth timing and TIMP2 expression. We hypothesized alterations in mTOR and TIMP2 expression or activity might be associated with some cases of preterm labor. We performed experiments to determine if human preterm labor is associated with increased myometrial Akt/mTOR signaling or altered expression/activity of Matrix Metalloproteinases (MMP-2 and MMP-9) and their endogenous inhibitor TIMP-2. An estimated 10% of preterm births are attributed to abnormal uterine distension, so we determined if mTOR signaling increases in response to stretch in pregnant human myometrial tissue.
Setting: Experiments were performed in the Burkin laboratory at the University of Nevada School of Medicine. Uterine biopsies were obtained with IRB approval and written informed consent from mothers undergoing Cesarean section and transported to the laboratory in physiological buffer for immediate processing.
Methods:Freshly isolated term non-laboring myometrial tissue from six patients was dissectedand four uterine strips from each patient were mounted vertically in tissue baths and tested for the ability to contract in response to 50 mM KCl. Strips were left unstretchedor stretched to 10g tension for 0, 10, 20, or 30 min and snap frozen.Protein was extracted and analyzed by western blot.MMP levels were determined by gelatin zymography.
Results:We observed increased mTOR expression in myometrial samples from all pregnant groups (term in labor, term not in labor, preterm in labor, and preterm not in labor) compared to nonpregnant, but did not observe significant changes in phospho-mTOR or phospho-Akt between groups. TIMP2 protein levels were lower in all pregnant states, but were not significantly different between pregnant groups. We observed higher pro-MMP-2 and MMP-2 enzymatic activity in preterm laboring myometrial tissue (p<0.05). Pro-MMP-9 enzymatic activity was increased in the preterm laboring (P<0.01) groupand MMP-9 activity was higher in term nonlaboring(P<0.01) and preterm laboring (P<0.01) myometrium. Results for MMP-2 and pro-MMP-9 levels were reproducible by western blot.
Interpretation: We did not observe significant changes in phospho-mTOR or phospho-Akt between groups, likely due to the large variation between patient samples. We are assessing the impact of myometrial stretch on this signaling pathway.We have shown that MMP-2 and MMP-9 are present in pregnant human myometrial tissue and that levels of MMP-2 and MMP-9 are highest in preterm laboring samples. Myometrial TIMP-2 was low in all pregnant groups. Our results suggest a possible disinhibition of MMP activity in pregnant myometrium. Because MMP-2 and MMP-9 inhibitors can rapidly affectthe oxytocin-induced contractile response in rat myometrium, our future studies will examine effects of MMP-2 and MMP-9 on human myometrial contractility.