Luigi Locatelli

Altered store operated calcium entry increases cyclic 3′,5′‐adenosine monophosphate production and extracellular signal‐regulated kinases 1 and 2 phosphorylation in polycystin‐2‐defective cholangiocytes

Mutations in polycystins (PC1 or PC2/TRPP2) cause progressive polycystic liver disease (PLD). In PC2‐defective mice, cyclic 3′,5′‐adenosine monophosphate/ protein kinase A (cAMP/PKA)‐dependent activation of extracellular signal‐regulated kinase/ mammalian target of rapamycin (ERK‐mTOR) signaling stimulates cyst growth. We investigated the mechanisms connecting PC2 dysfunction to altered Ca2+ and cAMP production and inappropriate ERK signaling in PC2‐defective cholangiocytes. Cystic cholangiocytes were isolated from PC2 conditional‐KO (knockout) mice (Pkd2flox/−:pCxCreER™; hence, called Pkd2KO) and compared to cholangiocytes from wild‐type mice (WT). Our results showed that, compared to WT cells, in PC2‐defective cholangiocytes (Pkd2KO), cytoplasmic and ER‐Ca2+ (measured with Fura‐2 and Mag‐Fluo4) levels are decreased and store‐operated Ca2+ entry (SOCE) is inhibited, whereas the expression of Ca2+‐sensor stromal interaction molecule 1 (STIM1) and store‐operated Ca2+ channels (e.g., the Orai1 channel) are unchanged. In Pkd2KO cells, ER‐Ca2+ depletion increases cAMP and PKA‐dependent ERK1/2 activation and both are inhibited by STIM1 inhibitors or by silencing of adenylyl cyclase type 6 (AC6). Conclusion: These data suggest that PC2 plays a key role in SOCE activation and inhibits the STIM‐dependent activation of AC6 by ER Ca2+ depletion. In PC2‐defective cells, the interaction of STIM‐1 with Orai channels is uncoupled, whereas coupling to AC6 is maximized. The resulting overproduction of cAMP, in turn, potently activates the PKA/ERK pathway. PLD, because of PC2 deficiency, represents the first example of human disease linked to the inappropriate activation of store‐operated cAMP production. (HEPATOLOGY 2012)

Cyclic AMP/PKA-dependent paradoxical activation of Raf/MEK/ERK signaling in polycystin-2 defective mice treated with sorafenib.

Mutations in polycystins are a cause of polycystic liver disease. In polycystin‐2 (PC2)‐defective mice, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)‐dependent activation of the Rat Sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen signal‐regulated kinase–extracellular signal‐regulated kinase (ERK) 1/2 pathway stimulates the growth of liver cysts. To test the hypothesis that sorafenib, a Raf inhibitor used for the treatment of liver and kidney cancers, inhibits liver cyst growth in PC2‐defective mice, we treated PC2 (i.e., Pkd2flox/−:pCxCreERTM [Pkd2cKO]) mice with sorafenib‐tosylate for 8 weeks (20‐60 mg/kg/day). Sorafenib caused an unexpected increase in liver cyst area, cell proliferation (Ki67), and expression of phosphorylated ERK (pERK) compared with Pkd2cKO mice treated with vehicle. When given to epithelial cells isolated from liver cysts of Pkd2cKO mice (Pkd2cKO‐cells), sorafenib progressively stimulated pERK1/2 and cell proliferation [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium and bromodeoxyuridine assay (MTS)] at doses between 0.001 and 1 μM; however, both pERK1/2 and cell proliferation significantly decreased at the dose of 10 μM. Raf kinase activity assay showed that whereas B‐Raf is inhibited by sorafenib in both wild‐type (WT) and Pkd2cKO cells, Raf‐1 is inhibited in WT cells but is significantly stimulated in Pkd2cKO cells. In Pkd2cKO cells pretreated with the PKA inhibitor 14‐22 amide, myristolated (1 μM) and in mice treated with octreotide in combination with sorafenib, the paradoxical activation of Raf/ERK1/2 was abolished, and cyst growth was inhibited. Conclusion: In PC2‐defective cells, sorafenib inhibits B‐Raf but paradoxically activates Raf‐1, resulting in increased ERK1/2 phosphorylation, cell proliferation, and cyst growth in vivo. These effects are consistent with the ability of Raf inhibitors to transactivate Raf‐1 when a PKA‐activated Ras promotes Raf‐1/B‐Raf heterodimerization, and are inhibited by interfering with cAMP/PKA signaling both in vitro and in vivo, as shown by the reduction of liver cysts in mice treated with combined octreotide and sorafenib. (HEPATOLOGY 2012)

Macrophage recruitment by fibrocystin‐defective biliary epithelial cells promotes portal fibrosis in congenital hepatic fibrosis

Congenital hepatic fibrosis (CHF) is a disease of the biliary epithelium characterized by bile duct changes resembling ductal plate malformations and by progressive peribiliary fibrosis, in the absence of overt necroinflammation. Progressive liver fibrosis leads to portal hypertension and liver failure; however, the mechanisms leading to fibrosis in CHF remain elusive. CHF is caused by mutations in PKHD1, a gene encoding for fibrocystin, a ciliary protein expressed in cholangiocytes. Using a fibrocystin‐defective (Pkhd1del4/del4) mouse, which is orthologous of CHF, we show that Pkhd1del4/del4 cholangiocytes are characterized by a β‐catenin‐dependent secretion of a range of chemokines, including chemokine (C‐X‐C motif) ligands 1, 10, and 12, which stimulate bone marrow‐derived macrophage recruitment. We also show that Pkhd1del4/del4 cholangiocytes, in turn, respond to proinflammatory cytokines released by macrophages by up‐regulating αvβ6 integrin, an activator of latent local transforming growth factor‐β1. While the macrophage infiltrate is initially dominated by the M1 phenotype, the profibrogenic M2 phenotype increases with disease progression, along with the number of portal myofibroblasts. Consistent with these findings, clodronate‐induced macrophage depletion results in a significant reduction of portal fibrosis and portal hypertension as well as of liver cysts. Conclusion: Fibrosis can be initiated by an epithelial cell dysfunction, leading to low‐grade inflammation, macrophage recruitment, and collagen deposition; these findings establish a new paradigm for biliary fibrosis and represent a model to understand the relationship between cell dysfunction, parainflammation, liver fibrosis, and macrophage polarization over time. (Hepatology 2016;63:965–982)

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

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 Pkhd1del4/del4 mice, stimulation of cAMP/PKA signaling (forskolin 10 μM) stimulated Ser675‐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 Pkhd1del4/del4 cholangiocytes. Rac‐1 inhibition blocked cAMP‐dependent nuclear translocation and transcriptional activity of pSer675‐β‐catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1del4/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 pSer675‐phosphorylation of β‐catenin and Rac‐1 activity. In the presence of activated Rac‐1, pSer675‐β‐catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1del4/del4 cholangiocytes. β‐Catenin‐dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target. (Hepatology 2013;58:1713–1723)

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

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 Pkhd1del4/del4 mice, stimulation of cAMP/PKA signaling (forskolin 10 μM) stimulated Ser675‐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 Pkhd1del4/del4 cholangiocytes. Rac‐1 inhibition blocked cAMP‐dependent nuclear translocation and transcriptional activity of pSer675‐β‐catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1del4/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 pSer675‐phosphorylation of β‐catenin and Rac‐1 activity. In the presence of activated Rac‐1, pSer675‐β‐catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1del4/del4 cholangiocytes. β‐Catenin‐dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target. (Hepatology 2013;58:1713–1723)

Protein kinase a-dependent pSer675-β-catenin, a novel signaling defect in a mouse model of congenital hepatic fibrosis: Hepatology, Vol. 00, No. X, 2013

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 Pkhd1del4/del4 mice, stimulation of cAMP/PKA signaling (forskolin 10 μM) stimulated Ser675‐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 Pkhd1del4/del4 cholangiocytes. Rac‐1 inhibition blocked cAMP‐dependent nuclear translocation and transcriptional activity of pSer675‐β‐catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1del4/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 pSer675‐phosphorylation of β‐catenin and Rac‐1 activity. In the presence of activated Rac‐1, pSer675‐β‐catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1del4/del4 cholangiocytes. β‐Catenin‐dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target. (Hepatology 2013;58:1713–1723)