In human and non-human primates, trophoblasts invade the uterus and uterine vasculature. Endovascular trophoblasts migrate against the flow of blood, eventually reaching uterine spiral arteries which they remodel. Trophoblast-endothelial interaction is essential for placental development and establishing a blood supply to the fetus. Despite its importance, the factors that regulate trophoblast migration and function within the uterine vasculature remain poorly understood. Impaired trophoblast invasion and incomplete vascular remodeling occur in preeclampsia and intrauterine growth restriction. Identification of regulatory mechanisms controlling trophoblast invasion will provide a better understanding of placental development and insights into the etiology of these disorders. ?1 Integrins are important in trophoblast adhesion and migration on endothelium and are downregulated in preeclampsia. Endovascular trophoblasts are exposed to blood flow-derived shear stress (FSS) but little is known about how FSS regulates trophoblast function. We showed that trophoblast migration and ?1 integrin expression were stimulated by FSS. Preliminary results show that FSS-mediated upregulation of ?1 integrin is dependent on the mucin, MUC1. This competitive renewal asks, how does FSS induce ?1 integrin expression, and how do trophoblasts sense FSS? We propose that MUC1 forms part of a trophoblast flow sensing network that includes membrane-type matrix metalloproteases (MT-MMPs) and ErbB1. We also hypothesize that FSS-induced, MUC1-mediated signaling events activate estrogen receptors (ERs) and Sp1 transcription factors, causing upregulation of ?1 integrin. This application continues to use early gestation rhesus monkey trophoblasts as well as a novel rhesus trophoblast stem cell line as models to test these hypotheses.
The first aim characterizes the role of MUC1 and estrogen receptors in FSS-induced upregulation of ?1 integrin expression. FSS-dependent association of the MUC1 cytoplasmic domain and ERs will be studied using co-immunoprecipitation and confocal microscopy. We will examine the effects of FSS and MUC1 siRNAs on Sp1 and ER-dependent transcriptional regulation of ?1 integrin using promoter-reporter assays.
The second aim uses siRNAs and inhibitors to test the idea that FSS-induced phosphorylation of MUC1 results from association of MUC1 and ErbB1. We will study the effect of these interventions on trophoblast migration on endothelial cells. In the third aim we will apply an optical trap force directly to individual MUC1 molecules on the trophoblast surface and determine whether or not relevant downstream signaling events occur.
The fourth aim tests the idea that trophoblast migration on endothelium under FSS conditions is dependent on MT-MMPmediated MUC1 ectodomain shedding. The last aim tests the idea that MUC1 cytoplasmic domain internalization is required for FSS-induced trophoblast migration. Internalization will be studied under static and flow conditions and correlated with MUC1 phosphorylation and MUC1 association with ErbB1 and ERs. FSSinduced trophoblast migration will be quantified in cells in which MUC1 internalization has been blocked.
