Functional characterization of CD97 in intestinal epithelial cells

肠上皮细胞中 CD97 的功能特征

• 批准号: 147293537
• 负责人: Professor Dr. Salah Amasheh
• 金额: --
• 依托单位: Institut für Veterinär-Physiologie (WE02)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/147293537?language=en
• 关键词: Functional; characterization; CD97; intestinal; epithelial

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.

CD 97是一种粘附-GPCR，在正常人肠的上皮粘附连接中表达。在第一个提议中，我们使用Cd 97转基因小鼠评估了这些细胞中CD 97的功能。在本提案中，将检查两个新的临床相关方面：A在Tg（villin-CD 97）小鼠的肠活检中，刺激的氯化物分泌增加，而在稳定的CD 97 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，CD 97调节囊性纤维化跨膜传导调节因子（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，其寿命仅缩短至40年。本提案的目的是验证假设-CD 97调节CFTR-在不同水平，包括表达（转录，翻译，运输，定位，稳定性）和功能（折叠，打开/关闭）的CFTR。我们建立了小鼠肠道干细胞的三维培养，用于毛喉素诱导（FIS）和辐射诱导肿胀（RIS）。因此，在新的双Cd 97/Cftr转基因小鼠的类器官中，除了用这些动物进行经典实验之外，我们还将验证CD 97是否调节CFTR。CD 97与野生型和突变的CFTR的直接相互作用的电生理学分析的策略是用共表达两种受体的非洲爪蟾卵母细胞进行的双电极电压钳和膜片钳实验。我们将阐明CD 97是否调节和拯救突变的CFTR，以及CFTR的激活或增加是否参与了辐射诱导的胃肠道综合征（RIGS）的发展。我们的研究结果可能开辟新的治疗方法，通过CD 97调节囊性纤维化患者突变CFTR的表达和/或功能。B哺乳动物出生后肠道的生长分为两个阶段，这可能是由不同的分子模式或调节机制引起的。圆柱形肠生长，即在正常显微解剖结构下长度和直径的增加，在出生后断奶前占主导地位。有趣的是，Tg（villin-CD 97）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生长的未知分子模式的独特模型。我们将验证以下假设：CD 97与CFTR的相互作用参与肠道生长的调节，CD 97调节出生后小肠中的隐窝分裂，CD 97调节肠上皮中时间限制性营养（乳）因子的受体。通过这些结果，我们还可以增加我们对短肠综合征患者治疗期间缺失的有利圆柱形肠生长模式的诱导的知识。