Our long-term objective is to understand the mechanisms that regulate the permeability of epithelia of the female reproductive tract. We have discovered that estrogen modulates the Resistance of the Lateral Intercellular Space (R-LIS) of human normal ectocervical-vaginal epithelial cells (ECVE), and decreases the paracellular resistance. The geometry of the lateral intercellular space (and the R-LIS) is determined by the shape of epithelial cells that define this space, and depends on the rigidity of the cytoskeleton. Based on novel preliminary results we advance our Maior Hypothesis that estrogen decreases the rigidity of the cytoskeleton by remodeling the cortical acto-myosin frame. The study has four Specific Aims: (1) To understand how estrogen remodels the cytoskeletal cortical acto-myosin frame. Our Hypothesis-A is that formation of a rigid cortical frame depends on the interaction of nonmuscle myosin IIB with cortical actin. We propose that estrogen remodels the cytoskeleton into a flexible structure by inducing disassembly of nonmusele myosin lIB from the cortical actin ring. (2) To understand the structural basis of the cortical acto-myosin cytoskeletal ring in epithelial cells. Our Hypothesis-B is that in epithelial cells the stability of the cortical myosin-actin ring depends on the interaction of actin with homodimerized nonmuscle myosin IIB filaments. We propose that dedimerization of nonmuscle myosin IIB heavy chains inhibits myosin MgATPase activity, and leads to disassociation of nonmuscle myosin IIB from the cortical acto-myosin frame. (3) To understand the mechanism by which phosphorylation of nonmuscle myosin IIB heavy chains regulates MgATPase activity. Our Hypothesis-C is that that phosphorylation of nonmuscle myosin IIB heavy chains inhibits homodimerization of myosin filaments and blocks myosin MgATPase. The alternative hypothesis is that MgATPase can be regulated independent of dimerization, by phosphorylation of nonmuscle myosin lIB heavy chains directly at the motor domain. (4) To understand the signaling pathway of the estrogen-induced phosphorylation of nonmuscle myosin IIB heavy chains. Our Hypothesis-D is that the effect of estrogen is initiated by activation of the EGFR-MAPK pathway, and it involves casein kinase II (CKII) as the terminal kinase. The extended hypothesis postulates involvement of multimolecular complexes, including Rho-kinase and an unidentified phosphatase, as modulators of CKII-induced phosphorylation of nonmuscle myosin IIB heavy chains. Experiments will utilize tissues of human ectocervix and vagina obtained from women undergoing surgery, and cultures of human ECVE cells grown on filters. Health relatedness of the project: The results of the study may provide novel data about estrogen regulation of the permeability of the female reproductive tract epithelia, and improve our understanding of the physiology of reproduction: the pathophysiology of inflammatory and infectious disease in the genital tract; and for improving woman's health. ? ? ?
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