An incomplete understanding of the molecular mechanisms responsible for myometrial activation from the quiescent pregnant state to the active contractile state during labor has hindered the development of effective therapies for preterm labor. Myometrial stretch has been implicated clinically in the initiation of labor and the etiology of preterm labor, but the molecular mechanisms involved in the human have not been determined. We investigated the mechanisms by which gestation-dependent stretch contributes to myometrial activation, by using human uterine samples from gynecologic hysterectomies and Cesarean sections. Here we demonstrate that the Ca requirement for activation of the contractile filaments in human myometrium increases with caldesmon protein content during gestation and that an increase in caldesmon phosphorylation can reverse this inhibitory effect during labor. By using phosphotyrosine screening and mass spectrometry of stretched human myometrial samples, we identify 3 stretch-activated focal adhesion proteins, FAK, p130Cas, and alpha actinin. FAK-Y397, which signals integrin engagement, is constitutively phosphorylated in term human myometrium whereas FAK-Y925, which signals downstream ERK activation, is phosphorylated during stretch. We have recently identified smooth muscle Archvillin (SmAV) as an ERK regulator. A newly produced SmAV-specific antibody demonstrates gestation-specific increases in SmAV protein levels and stretch-specific increases in SmAV association with focal adhesion proteins. Thus, whereas increases in caldesmon levels suppress human myometrium contractility during pregnancy, stretch-dependent focal adhesion signaling, facilitated by the ERK activator SmAV, can contribute to myometrial activation. These results suggest that focal adhesion proteins may present new targets for drug discovery programs aimed at regulation of uterine contractility.
Src is a known regulator of focal adhesion turnover in migrating cells; but, in contrast, Src is generally assumed to play little role in differentiated, contractile vascular smooth muscle (dVSM). The goal of the present study was to determine if Src-family kinases regulate focal adhesion proteins and how this might affect contractility of non-proliferative vascular smooth muscle. We demonstrate here, through the use of phosphotyrosine screening, deconvolution microscopy imaging, and differential centrifugation, that the activity of Src family kinases in aorta is regulated by the alpha agonist and vasoconstrictor phenylephrine, and leads to focal adhesion protein phosphorylation and remodeling in dVSM. Furthermore, Src inhibition via morpholino knockdown of Src or by the small molecule inhibitor PP2 prevents phenylephrine-induced adhesion protein phosphorylation, markedly slows the tissues ability to contract, and decreases steady state contractile force amplitude. Significant vasoconstrictor-induced and Src-dependent phosphorylation of Cas pY-165, FAK pY-925, paxillin pY-118, and Erk1/2 were observed. However, increases in FAK 397 phosphorylation were not seen, demonstrating differences between cells in tissue versus migrating, proliferating cells. We show here that Src, in a cause and effect manner, regulates focal adhesion protein function and, consequently, modulates contractility during the action of a vasoconstrictor. These data point to the possibility that vascular focal adhesion proteins may be useful drug discovery targets for novel therapeutic approaches to cardiovascular disease.
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