PKA dependent p-Ser-675\u3b2-catenin, a novel signaling defect in a mouse model of Congenital Hepatic Fibrosis

Background and Aims: Genetically-determined loss of fibrocystin function causes Congenital Hepatic Fibrosis (CHF), Caroli Disease (CD) and Autosomal Recessive Polycystic Kidney Disease (ARPKD). Cystic dysplasia of the intrahepatic bile ducts and progressive portal fibrosis characterize liver pathology in CHF/CD. At a cellular level, several functional morphological and signaling changes have been reported including increased levels of 3'-5'-cyclic adenosine monophosphate (cAMP). In this study, we have addressed the relationships between increased cAMP and \u3b2-catenin. Methods and Results: In cholangiocytes isolated ad cultured from Pkhd1del4/del4 mice, stimulation of cAMP/PKA signaling (forskolin 10 \u3bcM) stimulated Ser-675-phosphorylation of \u3b2-catenin, its nuclear localization and its transcriptional activity (Western blot and TOP Flash assay, respectively) along with a downregulation of E-cadherin expression (Immunocytochemistry and Western blot); these changes were inhibited by the PKA blocker, PKI (1 \u3bcM). The Rho-GTPase, Rac-1, was also significantly activated by cAMP in Pkhd1del4/del4 cholangiocytes. Rac-1 inhibition blocked cAMP-dependent nuclear translocation and transcriptional activity of pSer-675\u3b2-catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1del4/del4 and was inhibited by: 1) PKI, 2) silencing \u3b2-catenin (siRNA) and 3) the Rac-1 inhibitor, NSC 23766. Conclusions: These data show that in fibrocystin-defective cholangiocytes, cAMP stimulates pSer-675phosphorylation of \u3b2-catenin and Rac-1 activity. In the presence of activated Rac-1, pSer-675-\u3b2-catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1del4/del4 cholangiocytes. \u3b2-catenin dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target