EXPRESSION AND FUNCTION OF THE GUANYLIN LIGAND FAMILY

大肠杆菌热稳定肠毒素（STa）与其 肠受体对启动毒素诱导的 分泌物和牙周病。然而，STa的作用可能是 分子模拟的结果; STa受体的主要作用可能 介导鸟苷素家族的一个或多个成员的作用 哺乳动物的肽配体。此应用程序的目标是 阐明鸟苷素的体内功能。该应用程序将解决 三个具体目标：1）我们将测试假设，顺式活性 鸟苷素基因的元件赋予细胞特异性表达， 肠上皮细胞我们将使用体外转录检测 用鸟苷酶-荧光素酶报告基因构建体和体内转基因 用鸟苷肽-人生长激素构建体的小鼠来研究元素 其调节鸟苷素基因表达。鸟苷素的鉴别 启动子区域指导表达到绒毛肠上皮细胞和杯状细胞 细胞可以增强鸟苷素在肠中表达的策略 在粘蛋白附近，鸟苷素受体和囊性纤维化 跨膜调节因子（CFTR）。2)我们将用 肠特异性启动子-鸟苷素基因构建体，以测试 假设鸟苷素在肠内过表达将导致 增加基础肠Cl分泌。我们将确定 在基础和鸟苷素/STa刺激的细胞中， 肠分泌和鸟苷酸-STa受体（G-STaR）表达（G-STaR）。 星星mRNA、鸟苷酸环化酶活性和配体结合）。转基因 线不仅可用于表征体内效应 但它们也可能用作分泌性腹泻的模型 或在治疗胎粪性肠梗阻中， 校正CF小鼠模型。3)我们将使用有针对性的技术 鸟苷素基因的破坏，以测试这一假设，即损失的鸟苷素基因， 鸟苷素在肠中的表达将减少净肠液 体内分泌。我们将描述这种损失的肠分泌 类似于目标2中所述的研究， 互补过表达模型。这种“敲除”动物模型可以 也可用于研究其他鸟苷素相关农药的作用， 例如，在一个实施例中，uroguanylin或异常合成鸟苷素和用于未来研究 包括与G-STaR基因已经被 针对性地总之，我们将使用分子遗传学方法来定义 鸟苷素的体内作用和细胞定位。我们将开发 动物模型，这是有用的，以解决这一具体目标， 的建议，并阐明发病机制，以及 通过CFTR介导的基础肠Cl分泌。

Binding of Escherichia coli heat-stable enterotoxin (STa) to its intestinal receptor is critical to the initiation of toxin-induced secretion and diarrheal disease. However, the action of STa may be a result of molecular mimicry; the primary role for the STa receptor may be to mediate the action of one or more members of the guanylin family of mammalian peptide ligands. The goal of this application is to elucidate the in vivo function of guanylin. This application will address three specific aims: 1) We will test the hypothesis that cis-active elements of the guanylin gene confer cell-specific expression to intestinal epithelial cells. We will use in vitro transcriptional assays with guanylin-luciferase reporter gene constructs and in vivo transgenic mice with guanylin-human growth hormone constructs to study elements which regulate guanylin gene expression. Identification of guanylin promoter regions that direct expression to villus enterocytes and goblet cells may enhance strategies for expression of guanylin in the intestine in proximity to mucin, the guanylin receptor and the cystic fibrosis transmembrane regulator (CFTR). 2) We will make transgenic mice with intestinal specific promoter-guanylin gene constructs to test the hypothesis that intestinal overexpression of guanylin will result in increased basal intestinal Cl secretion. We will determine the effect of overexpression of guanylin on basal and guanylin/STa stimulated intestinal secretion, and guanylin-STa receptor (G-STaR) expression (G- STaR mRNA, guanylate cyclase activity and ligand binding). Transgenic lines will not only be useful for characterization of the in vivo effects of guanylin but they may also be useful as a model of secretory diarrhea or in treating the meconium ileus equivalent seen in the partially corrected CF mouse model. 3) We will use the technique of targeted disruption of the guanylin gene, to test the hypothesis that loss of guanylin expression in the intestine will decrease net intestinal fluid secretion in vivo. We will characterize intestinal secretion in this loss of function model akin to the studies described in Aim 2 for the complementary overexpression model. This "knock-out" animal model may also be useful to study the actions of other guanylin-related pesticides, e.g., uroguanylin or aberrant synthetic guanylins and for future studies involving crossbreeding to animals in which the G-STaR gene has been targeted. In summary, we will use molecular genetic approaches to define the in vivo role and cellular localization of guanylin. We will develop animal models which be useful to address the specific aims of this proposal and to elucidate the mechanisms of diarrheal disease as well as basal intestinal Cl secretion mediated through the CFTR.