Hepatocyte growth factor activator and its inhibitors in mouse uterus

描述(申请人提供)：越来越多的证据表明，肝细胞生长因子(HGF)在子宫发育和子宫内膜异位症和癌症等病理疾病中发挥着重要作用。在细胞外环境中，单链HGF的蛋白水解性转化为活性的异二聚体HGF是HGF介导的信号转导的关键限制步骤，然而，子宫内控制HGF激活的机制尚不清楚。目前这项提议的长期目标是确定HGF在子宫中激活的相关因素，并阐明潜在的调控机制。肝细胞生长因子激活剂(HGF Actifator，HGFA)是一种丝氨酸蛋白酶，在体内对HGF的激活作用最强，但关于其在子宫中的表达尚不清楚。本应用的具体目的是检测HGF/c-Met调节系统包括HGFA及其抑制物HAI-1和HAI-2在子宫中的表达和类固醇激素调节，并表征HGFA在激活HGF/c-Met信号中的作用。为了实现这一目标，本研究将使用体内和体外模型检测HGFA、HAI-1、HAI-2、HGF和c-Met在小鼠子宫中的表达以及雌激素和孕激素对它们的调节。HGFA在HGF激活中的作用将通过HGFA表达与HGF激活、c-Met磷酸化以及子宫内膜上皮和间质细胞增殖的相关性来检验。在体外研究中，将利用重组HGFA、HGFA和HGF的中和抗体，通过HGF的激活来证实HGFA的特异性作用。方法包括子宫内膜间质和上皮细胞培养、Western分析、免疫组织化学、Real-Time RT-PCR、原位杂交、HGF活性检测和细胞增殖检测。这些实验获得的数据将为HGFA在子宫生理中的细胞定位、调节和功能提供新的信息。此外，本研究将为深入研究子宫内HGF激活的分子机制奠定基础，甚至可能为开发治疗子宫内膜异位症等以HGF表达和功能异常为特征的子宫疾病提供新的策略。这些拟议研究的结果将为子宫HGF系统的调节提供新的信息，并可能导致针对子宫内膜异位症等子宫内膜病理生理的新疗法的开发。

DESCRIPTION (provided by applicant): Increasing evidence indicates that hepatocyte growth factor (HGF) plays an important role in uterine development and pathological conditions such as endometriosis and carcinoma. Proteolytic conversion of single chain HGF to active heterodimeric HGF in the extracellular milieu is a critical limiting step in HGF- mediated signal transduction, however, the mechanisms controlling HGF activation in the uterus remain unexplored. The long-term goal of the current proposal is to determine the factors involved in the activation of HGF in the uterus, and elucidate the underlying regulatory mechanisms. HGF activator (HGFA) is a serine protease and is most potent in processing HGF activation in vivo, but no information is available regarding its expression in the uterus. The specific aim of this application is to examine the expression and steroid hormonal regulation of the HGF/c-Met regulatory system including HGFA and its inhibitors HAI-1 and HAI-2 in the uterus, and characterize the role of HGFA in the activation of HGF/c-Met signaling. To accomplish this goal, this study will use both in vivo and in vitro models to examine the expression of HGFA, HAI-1, HAI-2, HGF and c-Met in the mouse uterus as well as their regulation by estrogen and progesterone. The role of HGFA in HGF activation will be examined by correlating HGFA expression with HGF activation, c-Met phosphorylation and endometrial epithelial and stromal cell proliferation. Recombinant HGFA, neutralizing antibodies for HGFA and HGF will be utilized during in vitro study to confirm the specific effect of HGFA through HGF activation. Methods used in this proposal include endometrial stromal and epithelial cell culture, Western analysis, Immunohistochemistry, Real-time RT-PCR, In situ hybridization, HGF activation assay and Cell proliferation assay. Data obtained from these experiments will provide novel information on the cellular localization, regulation and function of HGFA in uterine physiology. Further more, this research will provide groundwork for thorough investigation on the molecular mechanisms controlling HGF activation in the uterus, and may even lead to the development of new strategies in treating uterine diseases characterized by abnormal expression and function of HGF, such as endometriosis. The findings from the proposed studies will provide novel information on the regulation of the uterine HGF system and may lead to the development of new therapies to target endometrial pathophysiologies such as endometriosis.