Glucocorticoid and SGK1 regulation of CFTR in the intestine: role in diarrhea

糖皮质激素和 SGK1 对肠道 CFTR 的调节：在腹泻中的作用

• 批准号: 10681492
• 负责人: Nadia A. Ameen
• 金额: $ 53.32万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681492
• 关键词: Affect; Animal Model; Anions; Biological Assay; Biotinylation; Cell membrane; Cells; Cellular Stress; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic Nucleotides; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Development; Diarrhea; Electrophysiology (science); Endocrine; Enterocytes; Enterotoxins; Escherichia coli; Escherichia coli Infections; Etiology; Fluids and Secretions; Genetic; Genetic Transcription; Glucocorticoids; Human; Immunofluorescence Immunologic; Infection; Inflammatory; Intestines; Ion Transport; Irritable Bowel Syndrome; Knockout Mice; Knowledge; Link; Mass Spectrum Analysis; Mediating; Mediator; Membrane Protein Traffic; Messenger RNA; Mineralocorticoids; Mus; Neurosecretory Systems; Pathway interactions; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Physiologic pulse; Physiological; Population; Rattus; Regulation; Role; Serum; Sgk protein; Signal Pathway; Signal Transduction; Stains; Stress; Surface; Testing; Translations; Western Blotting; Work; airway epithelium; diarrheal disease; in vivo; interest; knock-down; mRNA Expression; mouse model; mutant; nerve stem cell; new therapeutic target; novel; novel therapeutics; protein expression; small hairpin RNA; stem; trafficking; ubiquitin ligase

PROJECT SUMMARY Intestinal fluid secretion is a principal function of enterocytes and is mediated by the cystic fibrosis transmembrane conductance regulator anion channel, CFTR. Stress and CFTR are linked to common diarrheal diseases such as Irritable Bowel Syndrome (IBS-D) that affects 20% of the US population. CFTR is also linked to diarrhea due to genetic, inflammatory, common E. Coli infections, and other etiologies. But how stress exacerbates or leads to diarrhea is not understood. This proposal examines novel mechanisms that regulate CFTR in the intestine with the aim of advancing our knowledge of gut fluid secretion and developing new therapeutics. We have a special interest in identifying new and novel druggable targets to treat diarrheal diseases stemming from genetic, infectious, and stress-related etiologies. CFTR is regulated by direct cAMP(PKA) and cGMP(PKG)-dependent phosphorylation and vesicular traffic in enterocytes. This proposal centers on a key but previously unrecognized role for glucocorticoids (GCs) and serum glucocorticoid kinase 1 (SGK1) in regulating intestinal CFTR and the potential for targeting this pathway to treat diarrhea. SGK1 is transcriptionally regulated by glucocorticoids (GCs), mineralocorticoids, cell stress, and other factors, and potently regulates ion transport by transcription, translation, and traffic. However, nothing is known about how GCs and SGK1 regulate CFTR in the intestine. The hypothesis to be tested here is that GC, SGK1, and kinase signaling pathways regulate CFTR in the intestine and can exacerbate diarrheal diseases. The hypothesis will be tested through the following aims: (1) Determine the role of GC and kinase signaling in regulating CFTR function, mRNA, and protein expression in the intestine. (2) Examine the role of GC and kinases in regulating CFTR membrane traffic in the intestine. (3) Examine the role of GC-SGK1 pathways in cGMP-elicited diarrheal diseases. These aims will be achieved by employing two animal models (SGK1 KO, and Nedd4-2KO), cultured intestinal cells expressing endogenous CFTR, and scientific approaches including Ussing chamber electrophysiology, in vivo trafficking assays, shRNA silencing, kinase signaling assays, mass spectrometry, qPCR, immunoblotting and immunofluorescence staining. Collectively, these studies will break new ground and expand our understanding of mechanisms regulating CFTR in the intestine and potentially identify novel physiologic targets to treat CFTR-mediated diarrhea stemming from diverse etiologies. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page

项目摘要 肠液分泌是肠上皮细胞的主要功能，由囊性纤维化介导 跨膜电导调节器阴离子通道，CFTR。压力和CFTR与常见有关 腹泻疾病（例如肠易激综合征（IBS-D））影响了20％的美国人群。 CFTR是 由于遗传，炎症，常见的大肠杆菌感染和其他病因，还与腹泻有关。但是如何 不了解压力加剧或导致腹泻。该建议研究了新的机制 调节肠内CFTR，目的是促进我们对肠道分泌的了解并发展 新治疗学。我们对识别新的和新颖的可药物治疗腹泻有特别的兴趣 源于遗传，传染性和与压力相关的病因引起的疾病。 CFTR受直接监管 CAMP（PKA）和CGMP（PKG）依赖性磷酸化和肠肠道流量。这个建议 以糖皮质激素（GCS）和血清糖皮质激素激酶1的关键但以前无法识别的作用为中心1 （SGK1）在调节肠道CFTR和靶向这种治疗腹泻的途径的潜力中。 SGK1是 由糖皮质激素（GCS），盐皮质激素，细胞应激和其他因素和其他因素和其他因素和其他因素调节的转录。 通过转录，翻译和流量来有效调节离子传输。但是，什么都不知道 GCS和SGK1在肠中调节CFTR。这里要检验的假设是GC，SGK1和激酶 信号通路调节肠中的CFTR，并会加剧腹泻疾病。该假设将 通过以下目的进行测试：（1）确定GC和激酶信号在调节CFTR中的作用 肠中的功能，mRNA和蛋白质表达。 （2）检查GC和激酶在调节中的作用 肠道中的CFTR膜流量。 （3）检查GC-SGK1途径在CGMP引诱的腹泻中的作用 疾病。这些目标将通过使用两种动物模型（SGK1 KO和NEDD4-2KO）来实现。 表达内源性CFTR的肠细胞以及包括使用室内的科学方法 电生理学，体内运输测定，shRNA沉默，激酶信号测定，质谱法， QPCR，免疫印迹和免疫荧光染色。总的来说，这些研究将破坏新的基础 并扩展我们对调节CFTR的机制的理解，并有潜在地识别新颖的 生理靶标，以治疗来自不同病因的CFTR介导的腹泻。 PHS 398/2590（修订版06/09）页面延续格式页面

项目成果

Activation of AMPK inhibits cholera toxin stimulated chloride secretion in human and murine intestine.

• DOI: 10.1371/journal.pone.0069050
• 发表时间: 2013
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Rogers AC;Huetter L;Hoekstra N;Collins D;Collaco A;Baird AW;Winter DC;Ameen N;Geibel JP;Kopic S
• 通讯作者: Kopic S