Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â ABSTRACT
PRINCIPAL INVESTIGATOR: Heather Burkin
PROJECT TITLE: MyometrialmTOR Regulation of Preterm Labor
PROGRESS TOWARDS GOAL: The long-term goals of this research are to provide new methods to identify preterm labor risk, to better understand the fundamental nature of labor and parturition, and prevention of preterm labor and delivery through basic research on the human myometrium. Specific Aim 1: Is human preterm labor associated with increased myometrialmTOR signaling? Increased mTOR signaling appears to play a pivotal role in regulating birth timing in mice. We tested the hypothesis that human myometrialmTOR expression and/or activity are altered in patients experiencing preterm labor. We used Western blotting to determine if total mTOR protein is increased in preterm and term laboring myometrium compared to preterm and term non-laboring myometria. We observed an increase in total mTOR expression in myometrial samples from all pregnant groups compared to nonpregnant myometrium. However, we did not observe significant changes in phospho-mTOR between groups, likely due to the amount of variation observed in the nonpregnant samples. We also examined the activity of the upstream kinase Akt and the downstream effector S6K1. We did not observe significant differences in Akt or S6K1 phosphorylation between groups. Specific Aim 2: Is Akt/mTOR signaling increased in response to mechanical stretch in pregnant human myometrial tissue? We have previously shown that mTOR phosphorylation in response to mechanical stretch is isolated, cultured human uterine cells. In this Aim, we are using Western blotting and phosphorylation assays to confirm activation of the Akt/mTOR pathway (phosphorylation of Akt, mTOR, and S6K1) occurs in response to mechanical stretch in human myometrial tissues. Freshly isolated term non-laboring myometrial tissue from six patients was dissected and four uterine strips from each patient were mounted vertically in tissue baths and tested for the ability to contract in response to 50 mMKCl. Strips were left unstretched or stretched to 10g tension for 0, 10, 20, or 30 min and snap frozen. Protein extracts have been prepared and are currently being subjected to western analyses. Specific Aim 3: Is TIMP2 expression or activity decreased in preterm laboring human myometrial tissue? We used Western blotting to determine if TIMP2 protein levels are lower in preterm and term laboring than in non-laboring myometria. While TIMP2 protein levels were lower in all pregnant states than in nonpregnant myometrium, TIMP2 levels were not significantly different between pregnant groups. We have ordered an enzyme assay kit to determine if TIMP2 activity is lower in preterm and term laboring. The required kit has just arrived and these experiments are underway. In our first CTR application, one of reviewer suggested we also examine related enzymes (MMPs and other TIMPs). We wanted to look at TIMP2 first because we had observed significant differences in its transcript levels between preterm laboring and nonlaboring patient myometrium and because it was associated with PPROM, but I replied that if time and budget allowed, we would follow up with experiments to look at related enzymes, particularly MMP2 and MMP9. We actually did this and 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) group and 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 confirmed by western blot. Conclusions: 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 currently 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 affect the oxytocin-induced contractile response in rat myometrium, our future studies will examine effects of MMP-2 and MMP-9 on human myometrial contractility.