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。在第一项建议中，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（绒毛蛋白-CD97）小鼠的肠道活检中，受刺激的氯化物分泌增加，而在稳定的CD97 siRNA结肠直肠细胞克隆中，氯化物分泌减少。这些和其他数据表明CD97调节囊性纤维化跨膜传导调节剂（CFTR）。CFTR基因的突变导致囊性纤维化，这是最常见的单基因疾病，可将寿命缩短至40年。本研究的目的是验证cd97在多个层面调控CFTR-的假设，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）。我们建立了用于福斯克林诱导（FIS）和辐射诱导肿胀（RIS）的小鼠肠道干细胞的三维培养。因此，在新的双Cd97/ CFTR转基因小鼠的类器官中，除了用这些动物进行经典实验外，我们将验证Cd97是否调节CFTR。CD97与野生型和突变型CFTR直接相互作用的电生理分析策略是在非洲爪蟾卵母细胞中进行两个电极电压钳和膜片钳实验，这两个受体共同表达。我们将阐明CD97是否调控和挽救突变的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（RIGS）的发展。我们的研究结果可能为通过CD97调节囊性纤维化患者CFTR突变的表达和/或功能开辟新的治疗途径。哺乳动物出生后肠道的生长分为两个阶段，这两个阶段可能是由不同的分子模式或调节机制引起的。在正常显微镜解剖下，肠长和直径呈圆柱形增长，在出生后断奶前占主导地位。有趣的是，Tg（绒毛蛋白- cd97）小鼠在出生后发育出具有正常显微解剖结构的巨肠。因此，这些小鼠是鉴定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的相互作用参与肠道生长的调节，CD97调节出生后小肠隐窝分裂，CD97调节肠上皮中限时营养（乳）因子的受体。有了这些结果，我们也可以增加我们对短肠综合征患者治疗过程中缺失的有利的圆柱形肠道生长模式的认识。

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.