br Funding and Role of Funder br Conflicts

Funding and Role of Funder
Conflicts of Interest
Author Contributions
Acknowledgements This work was supported by the Intramural Research Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. We thank Dr. Oksana Gavrilova for her excellent suggestions and assistance with AZIP animal experiments; and Sheila Smith RN for her outstanding assistance with clinical protocols, patients, and patient samples.
Introduction Chronic tissue injury leads to fibrosis in many organs, including the liver, lungs, kidneys, and heart. In chronic liver disease, the development of fibrosis is the first step toward the progression to cirrhosis and its complications (such as organ failure, esophageal variceal bleeding, and hepatocellular carcinoma), irrespective of the underlying etiology (Bataller and Brenner, 2005), and there is currently no effective therapy. Although many pathways and cytokines such as transforming growth factor (TGF)-β, platelet-derived growth factor (PDGF), Toll-like receptor (TLR) 4, sphingosine-1-phosphate, AKT, and p38 mitogen-activated protein kinase (MAPK) have been characterized as mediators of liver fibrosis, the underlying molecular mechanism is still not well defined. Wnt/β-catenin is involved in virtually every aspect of embryonic development and in the pathogenesis of many human diseases (Clevers, 2006), and is also involved in homeostatic self-renewal in adult tissues, such as liver and lung repair following an injury (Monga, 2011; Beers and Morrisey, 2011). Recently, Wnt/β-catenin has been reported to be associated with organ fibrosis (Dees and Distler, 2013; Chilosi et al., 2003) suggesting that they may be new therapeutic targets in liver fibrosis (Cheng et al., 2008). Hepatic stellate pkc inhibitors (HSCs) represent a major fibrogenic cell type in the liver (Bataller and Brenner, 2005). Following a liver injury, HSCs undergo an activation process and change their phenotype from quiescent retinoid storing HSCs to collagen-producing and contractile myofibroblast-like cells (Friedman, 2000). Wnt signaling is stimulated in activated HSCs compared to quiescent cells, and the inhibition of Wnt signaling by the transduction of the adenoviral Wnt co-receptor antagonist Dickkopf-1 restores HSC quiescence and increases apoptosis in cultured HSCs (Cheng et al., 2008). Macrophages may perform both injury-inducing and repair-promoting tasks simultaneously in an injured organ. The depletion of liver macrophages aggravates hepatocellular damage, while suppressing liver fibrosis following bile duct ligation (BDL) (Osawa et al., 2010). Moreover, the depletion of macrophages decreases myofibroblasts in a liver tumor (Osawa et al., 2013b). By contrast, macrophage depletion during the fibrosis resolution period induces the failure of matrix degradation (Duffield et al., 2005), suggesting that hepatic macrophages are involved in the regression of hepatic fibrosis (Friedman, 2005). The roles of β-catenin in macrophages have been reported. Macrophage-specific knockdown of β-catenin causes insufficient skin wound healing due to defects in migration, in the adhesion to fibroblasts, and in TGF-β production (Amini-Nik et al., 2014). Following activation by upstream signaling from Wnt, β-catenin translocates to the nucleus. Nuclear β-catenin recruits the Kat3 transcriptional co-activators, cAMP-response element-binding protein (CREB)-binding protein (CBP) (Takemaru and Moon, 2000) or EP300 (p300) (Hecht et al., 2000), to stimulate the transcription of its target genes, and distinct roles have been reported for CBP and p300. CBP/β-catenin-mediated transcription is critical for proliferation/non-differentiation, whereas p300/β-catenin-mediated transcription initiates differentiation (Teo and Kahn, 2010; Lenz and Kahn, 2014). ICG-001, a selective inhibitor of the CBP/β-catenin interaction, attenuates bleomycin-induced lung fibrosis and reverses the established fibrosis (Henderson et al., 2010). ICG-001 also ameliorates renal interstitial fibrosis induced by unilateral ureteral obstruction (Hao et al., 2011). C-82 is a second-generation specific CBP/β-catenin antagonist developed by Prism Pharma, which inhibits the binding between β-catenin and CBP and increases the binding between β-catenin and p300 similar to ICG-001. PRI-724 is phosphorylated-C-82 and is rapidly hydrolyzed to its active form C-82 in vivo, and pre-clinical studies have shown a very acceptable toxicity profile (Lenz and Kahn, 2014). A phase I safety study for hepatitis C virus (HCV)-related cirrhosis patients has been ongoing in our hospital since September 2014. To attempt to clarify the precise roles of CBP/β-catenin on liver fibrosis, in this study, we investigated the effects of PRI-724 and of its active form, C-82, on chronic liver injury mouse models and primary isolated mouse HSCs.