Estrogen and Aging effects on Transvaginal Transpoet

描述(申请人提供)：我们的长期目标是了解调节女性生殖道上皮细胞通透性的机制。我们发现雌激素可调节人正常宫颈外-阴道上皮细胞(ECVE)的细胞外间隙阻力(R-Lis)，并降低细胞旁阻力。外侧细胞间隙(和R-Lis)的几何形状由定义该间隙的上皮细胞的形状决定，并取决于细胞骨架的刚性。基于新的初步结果，我们提出了我们的Maior假说，即雌激素通过重塑皮质肌球蛋白框架来降低细胞骨架的刚性。这项研究有四个具体目标：(1)了解雌激素如何重塑细胞骨架皮质肌球蛋白框架。我们的假设-A是，刚性皮质框架的形成依赖于非肌肉肌球蛋白IIB与皮质肌动蛋白的相互作用。我们认为，雌激素通过诱导非肌球蛋白Lib从皮质肌动蛋白环上解离，将细胞骨架重塑为一种灵活的结构。(2)了解上皮细胞皮质肌球蛋白骨架环的结构基础。我们的假设B是，在上皮细胞中，皮质肌球蛋白-肌动蛋白环的稳定性取决于肌动蛋白与同源二聚化的非肌肉肌球蛋白IIB细丝的相互作用。我们认为，非肌肉肌球蛋白IIB重链的解聚抑制了肌球蛋白镁ATPase的活性，并导致非肌肉肌球蛋白IIB从皮质肌球蛋白框架中解离。(3)了解非肌肉肌球蛋白IIB重链的磷酸化调节镁ATPase活性的机制。我们的假设-C是，非肌肉肌球蛋白IIB重链的磷酸化抑制了肌球蛋白细丝的同质二聚，并阻断了肌球蛋白镁ATPase。另一种假设是，通过直接在运动区对非肌肉肌球蛋白Lib重链进行磷酸化，可以独立于二聚体来调节镁ATPase。(4)了解雌激素诱导非肌球蛋白IIB重链磷酸化的信号通路。我们的假设D是，雌激素的作用是通过激活EGFR-MAPK通路开始的，它涉及酪蛋白激酶II(CKII)作为末端激酶。扩展的假说假设，包括Rho-Kinase和一种未知的磷酸酶在内的多分子复合体参与了CKII诱导的非肌肉肌球蛋白IIB重链的磷酸化的调节器。实验将利用从接受手术的妇女身上获得的人类宫颈和阴道组织，以及在过滤器上生长的人类ECVE细胞的培养。项目的健康相关性：研究结果可能提供有关雌激素调节女性生殖道上皮通透性的新数据，并提高我们对生殖生理学的理解：生殖道炎症和传染病的病理生理学；以及改善妇女的健康。

DESCRIPTION (provided by applicant): 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.