Intestinal fluid secretion is a principal function of enterocytes and is mediated by the cystic fibrosis transmembrane conductance regulator anion channel, CFTR. Enterotoxins increase CFTR activity and result in diarrhea, the second leading cause of mortality globally in children under age five. CFTR is also implicated in diarrhea associated with Irritable Bowel Syndrome (IBS-D), which affects 20% of the US population. In contrast, impaired fluid secretion resulting from loss of CFTR function is the cardinal feature of cystic fibrosis (CF). 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 a 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 expression, function, and traffic in the intestine and are therefore important to 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 SGK1 pathways in diarrheal disease.
These aims will be achieved by employing three animal models (rat, SGK1 KO, and Nedd4-2KO), human jejunum enteroids (HJE's), cultured cells 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
The secretion of fluid is a principal function of the intestine that is altered under conditions of stress, infections to result in diarrhea. This proposal explores the cellular mechanisms responsible for fluid secretion in the intestine under the condition of stress and infections. This work will increase our understanding of how stress leads to diarrhea and identify targets for treatment. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page
