Hepatocyte growth factor activator and its inhibitors in mouse uterus

描述（由申请人提供）：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、癌等病理状况中起着重要作用。在细胞外环境中，单链HGF蛋白水解转化为活性异二聚体HGF是HGF介导的信号转导的关键限制步骤，然而，子宫中控制HGF激活的机制尚不清楚。本研究的长期目标是确定子宫内HGF激活的相关因素，并阐明其潜在的调控机制。HGF激活因子（HGF activator， HGFA）是一种丝氨酸蛋白酶，在体内对HGF的激活最有效，但关于其在子宫中的表达尚无相关信息。本应用的具体目的是研究HGF/c-Met调控系统（包括HGFA及其抑制剂HAI-1和HAI-2）在子宫中的表达和类固醇激素调控，并表征HGFA在HGF/c-Met信号激活中的作用。为了实现这一目标，本研究将采用体内和体外模型检测小鼠子宫中HGFA、HAI-1、HAI-2、HGF和c-Met的表达以及雌激素和黄体酮对它们的调节作用。HGFA在HGF激活中的作用将通过HGFA表达与HGF激活、c-Met磷酸化和子宫内膜上皮细胞和基质细胞增殖的相关性来研究。在体外研究中，我们将利用重组HGFA、HGFA和HGF的中和抗体来证实HGFA通过激活HGF的特异性作用。本文采用的方法包括子宫内膜基质和上皮细胞培养、Western分析、免疫组织化学、实时荧光定量pcr、原位杂交、HGF激活试验和细胞增殖试验。这些实验数据将为HGFA在子宫生理中的细胞定位、调控和功能提供新的信息。此外，本研究将为深入研究子宫中HGF激活的分子机制奠定基础，甚至可能为治疗以HGF表达和功能异常为特征的子宫疾病（如子宫内膜异位症）提供新的策略。这些研究结果将为子宫HGF系统的调控提供新的信息，并可能导致针对子宫内膜病理生理（如子宫内膜异位症）的新疗法的发展。

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.