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

项目概要 肠液分泌是肠上皮细胞的主要功能，由囊性纤维化介导 跨膜电导调节阴离子通道，CFTR。压力和 CFTR 与常见的 腹泻病，例如影响 20% 美国人的肠易激综合症 (IBS-D)。 CFTR 是 还与遗传、炎症、常见大肠杆菌感染和其他病因引起的腹泻有关。但如何 压力会加剧或导致腹泻尚不清楚。该提案研究了新的机制 调节肠道内的 CFTR，旨在增进我们对肠液分泌和发育的了解 新疗法。我们对确定治疗腹泻的新型药物靶点特别感兴趣 由遗传、传染性和压力相关病因引起的疾病。 CFTR 受直接监管 肠上皮细胞中 cAMP(PKA) 和 cGMP(PKG) 依赖性磷酸化和囊泡运输。这个提议 重点关注糖皮质激素 (GC) 和血清糖皮质激素激酶 1 的关键但之前未被认识的作用 (SGK1) 调节肠道 CFTR 以及针对该途径治疗腹泻的潜力。 SGK1 是 受糖皮质激素 (GC)、盐皮质激素、细胞应激和其他因素的转录调节，以及 通过转录、翻译和运输有效调节离子运输。然而，目前尚不清楚如何 GC 和 SGK1 调节肠道内的 CFTR。这里要测试的假设是 GC、SGK1 和激酶 信号通路调节肠道中的 CFTR，并可能加剧腹泻疾病。该假设将 通过以下目标进行测试：（1）确定GC和激酶信号传导在调节CFTR中的作用 肠道功能、mRNA 和蛋白质表达。 (2)考察GC和激酶在调节中的作用 肠道内的 CFTR 膜运输。 (3) 检查 GC-SGK1 通路在 cGMP 引起的腹泻中的作用 疾病。这些目标将通过采用两种培养的动物模型（SGK1 KO 和 Nedd4-2KO）来实现 表达内源性 CFTR 的肠道细胞，以及包括使用腔室在内的科学方法 电生理学、体内运输分析、shRNA 沉默、激酶信号分析、质谱分析、 qPCR、免疫印迹和免疫荧光染色。总的来说，这些研究将开辟新天地 扩大我们对肠道 CFTR 调节机制的理解，并有可能发现新的 治疗由多种病因引起的 CFTR 介导的腹泻的生理学目标。 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