Functional characterization of CD97 in intestinal epithelial cells

CD97是一种黏附-GPCR，在正常人肠上皮黏附连接中表达。在第一个方案中，我们使用CD97转基因小鼠评估了CD97在这些细胞中的功能。在目前的建议中，将研究两个新的临床相关方面：a在TG(Villin-CD97)小鼠的肠道活检组织中，刺激的氯离子分泌增加，而在稳定的CD97 siRNA结肠细胞克隆中，氯离子分泌减少。这些和其他数据表明，CD97调节囊性纤维化跨膜电导调节因子(CFTR)。CFTR基因的突变是囊性纤维化的原因，囊性纤维化是最常见的单基因疾病，寿命只有40岁。本研究的目的是在不同水平上验证CD97调控CFTR的假说，包括CFTR的表达(转录、翻译、运输、定位、稳定性)和功能(折叠、打开/关闭)。我们建立了小鼠小肠干细胞的3D培养，用于Forsklin诱导的(FIS)和辐射诱导的肿胀(RIS)。因此，除了通过对这些动物的经典实验，我们还将在新的双CD97/CFTR转基因小鼠的器官中验证CD97是否调控CFTR。对CD97与野生型和突变型CFTR的直接相互作用进行电生理分析的策略是两个电极电压钳和膜片钳实验-以共同表达这两个受体的非洲爪哇卵母细胞为对象。我们将阐明CD97是否调节和挽救突变的CFTR，以及CFTR的激活或增加是否参与辐射诱导的胃肠道综合征(RARS)的发生。我们的结果可能为囊性纤维化患者CD97基因突变CFTR的表达和/或功能调控开辟新的治疗途径。哺乳动物出生后肠道发育分两个阶段进行，可能是由不同的分子模式或调节机制引起的。圆柱形肠道生长，即具有正常显微解剖的长度和直径的增加，在断奶前出生后占主导地位。有趣的是，TG(Villin-CD97)小鼠出生后发育出具有正常显微解剖的巨。因此，这些小鼠是识别圆柱形肠道生长的未知分子模式的独特模型。我们将验证以下假设：CD97与CFTR的相互作用参与了肠道生长的调节，CD97调节出生后小肠中的隐窝分裂，CD97调节肠道上皮中一种适时受限的营养(牛奶)因子的受体。有了这些结果，我们还可以增加我们对短肠综合征患者在治疗过程中丢失诱导有利的圆柱形肠道生长模式的了解。

CD97, an adhesion-GPCR, is expressed in epithelial adherens junctions of the normal human intestine. In the first proposal we evaluated the function of CD97 within these cells using Cd97-transgenic mice. In the present proposal two new, clinically relevant aspects will be examined: A In intestinal biopsies of Tg(villin-CD97) mice the stimulated chloride secretion increased whereas in stable CD97 siRNA colorectal cell clones chloride secretion decreased. These and other data suggest that CD97 regulates the cystic fibrosis transmembrane conductance regulator (CFTR). Mutations in the CFTR gene are responsible for cystic fibrosis, the most frequent monogenetic disease which reduces life span to 40 years only. The aim of the present proposal is to verify the hypothesis -CD97 regulates the CFTR- at various levels, including expression (transcription, translation, transport, localization, stability) and function (folding, opening/closure) of the CFTR. We established the 3D culture of murine intestinal stem cells for its use in forskolin-induced (FIS) and radiation-induced swelling (RIS). Thus, in organoids of new double Cd97/Cftr-transgenic mice we will verify, beside by classical experiments with these animals, whether CD97 regulates the CFTR. A strategy for the electrophysiological analysis of a direct interaction of CD97 with the wildtype- and mutated CFTR are two electrode voltage clamp and patch clamp-experiments with Xenopus laevis oocytes which coexpress both receptors. We will clarify whether CD97 regulates and rescues the mutated CFTR and whether an activation or increase of the CFTR is involved in the development of a radiation-induced gastrointestinal syndrome (RIGS). Our results may open new therapeutic approaches to regulate the expression and/or function of the mutated CFTR by CD97 in patients with cystic fibrosis. B In mammals intestinal growth after birth proceeds in two phases which are caused probably by different molecular pattern or regulatory mechanisms. Cylindrical intestinal growth, that is an increase in length and diameter with normal microscopic anatomy, dominates after birth before weaning. Interestingly, Tg(villin-CD97) mice develop a megaintestine with normal microscopic anatomy after birth. Thus, these mice are a unique model to identify the unknown molecular pattern of cylindrical intestinal growth. We will verify the following hypothesis: The interaction of CD97 with the CFTR is involved in the regulation of intestinal growth, CD97 regulates crypt fissioning in the small intestine after birth and CD97 regulates a receptor for a timely-restricted trophic (milk)-factor in the intestinal epithelium. With these results we could also increase our knowledge on the missing induction of a favored cylindrical intestinal growth pattern during therapy of patients with short bowel syndrome.