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