Protein kinase A-dependent pSer(675) -β-catenin, a novel signaling defect in a mouse model of congenital hepatic fibrosis

Abstract

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 addressed the relationships between increased cAMP and β-catenin. In cholangiocytes isolated and cultured from Pkhd1(del4/del4) mice, stimulation of cAMP/PKA signaling (forskolin 10 μM) stimulated Ser(675) -phosphorylation of β-catenin, its nuclear localization, and its transcriptional activity (western blot and TOP flash assay, respectively) along with a down-regulation of E-cadherin expression (immunocytochemistry and western blot); these changes were inhibited by the PKA blocker, PKI (1 μM). The Rho-GTPase, Rac-1, was also significantly activated by cAMP in Pkhd1(del4/del4) cholangiocytes. Rac-1 inhibition blocked cAMP-dependent nuclear translocation and transcriptional activity of pSer(675) -β-catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1(del4/del4) and was inhibited by: (1) PKI, (2) silencing β-catenin (siRNA), and (3) the Rac-1 inhibitor NSC 23766.

Conclusion: These data show that in fibrocystin-defective cholangiocytes, cAMP/PKA signaling stimulates pSer(675) -phosphorylation of β-catenin and Rac-1 activity. In the presence of activated Rac-1, pSer(675) -β-catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1(del4/del4) cholangiocytes. β-Catenin-dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target.

Figure 1. Expression and transcriptional activity of p⁶⁷⁵-β-catenin in Pkhd1^del4/del4 cholangiocytes

A) Representative Western blot and quantitative analysis showing a significantly higher expression of p⁶⁷⁵-β-catenin in Pkhd1^del4/del4 compared WT and PC-KO cholangiocytes both at baseline (C) and after treatment with forskolin (Forsk). The PKA inhibitor (PKI) significantly reduced the expression of p⁶⁷⁵-β-catenin (n=4). B) WT and cystic cholangiocytes were transfected with a TCF/LEF Top Flash reporter or its mutant, Fop Flash. Luciferase activity was normalized to Renilla luciferase activity. Transcriptional activity of β-catenin was higher in Pkhd1^del4/del4 compared to WT and PC-KO cholangiocytes, was increased by forskolin, and was inhibited by PKI and by quercetin (Querc), (n=6). (*p<0.05 vs WT and PC-KO untreated cells; ^{^}p<0.01 vs controls; ^●p<0.01 vs Forsk; ^op<0.05 vs controls).

Figure 2. E-cadherin is down-regulated in Pkhd1^del4/del4 cholangiocytes

A) WT and Pkhd1^del4/del4 cholangiocytes were cultured over membrane inserts and labelled with a monoclonal rabbit anti E-cadherin antibody (green) and the nuclei were stained with DAPI (blue). A clear reduction and mislocalization of E-cadherin is present in Pkhd1^del4/del4 cholangiocytes (magnification 400x). B) Representative Western blot and quantitative analysis illustrating a reduction of E-cadherin in Pkhd1^del4/del4 compared to WT cholangiocytes. Forskolin further decreased the expression of E-cadherin in Pkhd1^del4/del4 but not in WT cholangiocytes (n=4; *p<0.05 vs WT; ^op<0.05 vs controls). C) Gene expression of Zeb-1, a negative regulator of E-cadherin , was significantly reduced in Pkhd1^del4/del4 cholangiocytes after treatment with the β-catenin inhibitor, quercetin (Q), or with the Rac-1 inhibitor, NSC 23766 (NSC). Data are normalized to untreated control (C) cells. (n=7; *p<0.05 vs controls).

Figure 3. Cell motility is increased in Pkhd1^del4/del4 cholangiocytes

Cell motility was examined by the Boyden chamber assay. Cells that had migrated to the underside of the porous polycarbonate membrane were quantified under a phase-contrast microscope. Representative images are shown in (A) (Original magnification 40X). B) Cell motility was significantly higher in Pkhd1^del4/del4 with respect to WT and PC-KO cholangiocytes, was cAMP/PKA-dependent, due to the boosting effect of forskolin (F) and the inhibitory effect of PKI, and was inhibited by the Rac-1 inhibitor, NSC 23766, by the β-catenin inhibitor, quercetin (Querc), and by β-catenin silencing (siRNA). (n=7; *p<0.05 vs WT and PC-KO untreated cells; ^{^}p<0.01 vs controls; ^●p<0.01 vs Forsk; ^○p<0.05 vs controls).

Figure 4. Rac-1 activity is cAMP/PKA-dependent in Pkhd1^del4/del4 cholangiocytes

Rac-1 (A), Cdc42 (B) and RhoA (C) activities were measured by an ELISA that recognized the activated forms. All the three Rho GTPases were activated by forskolin (F) and inhibited by the PKA inhibitor, PKI, in Pkhd1^del4/del4 cholangiocytes but not in WT. However, the effects on Rac-1 were significantly higher and with a different kinetic with respect to Cdc42 and RhoA. (n=8; *p<0.05 vs untreated cells (C); ^○p<0.05 vs forskolin).

Figure 5. In Pkhd1^del4/del4 cholangiocytes, nuclear translocation of p⁶⁷⁵-β-catenin is Rac-1-dependent

Representative Western blots and quantitative analyses on cytosolic (A) and on nuclear (B) cell lysates. In Pkhd1^del4/del4 cholangiocytes, but not in WT or PC-KO, forskolin (F) augmented the expression of p⁶⁷⁵-β-catenin. Treatment with the Rac-1 inhibitor, NSC 23766 (NSC), significantly blocked the expression of p⁶⁷⁵-β-catenin in the nuclear fraction. (n=4, *p<0.05 vs untreated cells (C); ^○p<0.05 vs forskolin)

Figure 6. Transcriptional activity of p⁶⁷⁵-β-catenin in Pkhd1^del4/del4 cholangiocytes is Rac-1 dependent

WT and Pkhd1^del4/del4 cholangiocytes were transfected with a TCF/LEF Top Flash reporter or its mutant, Fop Flash. Luciferase activity was normalized to Renilla luciferase activity. Treatment with the Rac-1 inhibitor, NSC 23766, significantly inhibited the transcriptional activity of β-catenin. (n=6; *p<0.05 vs untreated cells (C); ^○p<0.05 vs forskolin (F).