Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell‐matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvβ6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvβ6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter‐driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvβ6 during liver injury induced by 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen‐inducible Cre‐loxP system down‐regulated integrin αvβ6 in DDC‐damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvβ6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis‐related genes, smaller areas of alpha‐smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvβ6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)‐β1 activation in vitro. Conclusions: CTGF and integrin αvβ6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF‐β1. CTGF and integrin αvβ6 are potential therapeutic targets to control DRs and fibrosis in related liver disease. (Hepatology 2015;61:678‐691)

The liver possesses an extraordinary ability to regenerate after injury. Hepatocyte-driven liver regeneration is the default pathway in response to mild-to-moderate acute liver damage. When replication of mature hepatocytes is blocked, facultative hepatic progenitor cells (HPCs), also referred to as oval cells (OCs) in rodents, are activated. HPC/OCs have the ability to proliferate clonogenically and differentiate into several lineages including hepatocytes and bile ductal epithelia. This is a conserved liver injury response that has been studied in many species ranging from mammals (rat, mouse, and human) to fish. In addition, improper HPC/OC activation is closely associated with fibrotic responses, characterized by myofibroblast activation and extracellular matrix production, in many chronic liver diseases. Matrix remodeling and metalloprotease activities play an important role in the regulation of HPC/OC proliferation and fibrosis progression. Thus, understanding molecular mechanisms underlying HPC/OC activation has therapeutic implications for rational design of anti-fibrotic therapies.

Liver fibrosis is a common outcome of most chronic liver insults/injuries that can develop into an irreversible process of cirrhosis and, eventually, liver cancer. In recent years, there has been significant progress in basic and clinical research on liver cancer, leading to the identification of various signaling pathways involved in tumorigenesis and disease progression. Slit glycoprotein (SLIT)1, SLIT2, and SLIT3 are secreted members of a protein family that accelerate positional interactions between cells and their environment during development. These proteins signal through Roundabout receptor (ROBO) receptors (ROBO1, ROBO2, ROBO3, and ROBO4) to achieve their cellular effects. The SLIT and ROBO signaling pathway acts as a neural targeting factor regulating axon guidance, neuronal migration, and axonal remnants in the nervous system. Recent findings suggest that various tumor cells differ in SLIT/ROBO signaling levels and show varying degrees of expression patterns during tumor angiogenesis, cell invasion, metastasis, and infiltration. Emerging roles of the SLIT and ROBO axon-guidance molecules have been discovered in liver fibrosis and cancer development. Herein, we examined the expression patterns of SLIT and ROBO proteins in normal adult livers and two types of liver cancers: hepatocellular carcinoma and cholangiocarcinoma. This review also summarizes the potential therapeutics of this pathway for anti-fibrosis and anti-cancer drug development.

Liver fibrosis is the common outcome of many chronic liver diseases, resulting from altered cell-cell and cell-matrix interactions that promote hepatic stellate cell (HSC) activation and excessive matrix production. This study aimed to investigate functions of cellular communication network factor 2 (CCN2)/Connective tissue growth factor (CTGF), an extracellular signaling modulator of the CYR61/CTGF/Nov (CCN) family, in liver fibrosis. Tamoxifen-inducible conditional knockouts in mice and hepatocyte-specific deletion of this gene in rats were generated using the Cre-lox system. These animals were subjected to peri-central hepatocyte damage caused by carbon tetrachloride. Potential crosstalk of this molecule with a new profibrotic pathway mediated by the Slit2 ligand and Roundabout (Robo) receptors was also examined. We found that Ccn2/Ctgf was highly upregulated in periportal hepatocytes during carbon tetrachloride-induced hepatocyte damage, liver fibrosis and cirrhosis in mice and rats. Overexpression of this molecule was observed in human hepatocellular carcinoma (HCC) that were surrounded with fibrotic cords. Deletion of the Ccn2/Ctgf gene significantly reduced expression of fibrosis-related genes including Slit2, a smooth muscle actin (SMA) and Collagen type I during carbon tetrachloride-induced liver fibrosis in mice and rats. In addition, Ccn2/Ctgf and its truncated mutant carrying the first three domains were able to interact with the 7th -9th epidermal growth factor (EGF) repeats and the C-terminal cysteine knot (CT) motif of Slit2 protein in cultured HSC and fibrotic murine livers. Ectopic expression of Ccn2/Ctgf protein upregulated Slit2, promoted HSC activation, and potentiated fibrotic responses following chronic intoxication by carbon tetrachloride. Moreover, Ccn2/Ctgf and Slit2 synergistically enhanced activation of phosphatidylinositol 3-kinase (PI3K) and AKT in primary HSC, whereas soluble Robo1-Fc chimera protein could inhibit these activities. These observations demonstrate conserved cross-species functions of Ccn2/Ctgf protein in rodent livers. This protein can be induced in hepatocytes and contribute to liver fibrosis. Its novel connection with the Slit2/Robo signaling may have therapeutic implications against fibrosis in chronic liver disease.

Yes‐associated protein (YAP), a central effector in the Hippo pathway, is involved in the regulation of organ size, stem cell self‐renewal, and tissue regeneration. In this study, we observed YAP activation in patients with alcoholic steatosis, hepatitis, and cirrhosis. Accumulation of this protein in the nucleus was also observed in murine livers that were damaged after chronic‐plus‐single binge or moderate ethanol ingestion combined with carbon tetrachloride intoxication (ethanol/CCl ₄ ). To understand the role of this transcriptional coactivator in alcohol‐related liver injury, we knocked out the Yap1 gene in hepatocytes of floxed homozygotes through adeno‐associated virus (AAV8)‐mediated deletion utilizing Cre recombinase. Yap1 hepatocyte‐specific knockouts (KO) exhibited hemorrhage, massive hepatic necrosis, enhanced oxidative stress, elevated hypoxia, and extensive infiltration of CD11b ⁺ inflammatory cells into hepatic microenvironments rich for connective tissue growth factor (Ctgf) during ethanol/CCl ₄ ‐induced liver damage. Analysis of whole‐genome transcriptomics indicated upregulation of genes involved in hypoxia and extracellular matrix (ECM) remodeling, whereas genes related to hepatocyte proliferation, progenitor cell activation, and ethanol detoxification were downregulated in the damaged livers of Yap1 KO. Acetaldehyde dehydrogenase (Aldh)1a1 , a gene that encodes a detoxification enzyme for aldehyde substrates, was identified as a potential YAP target because this gene could be transcriptionally activated by a hyperactive YAP mutant. The ectopic expression of the human ALDH1A1 gene caused increase in hepatocyte proliferation and decrease in hepatic necrosis, oxidative stress, ECM remodeling, and inflammation during ethanol/CCl ₄ ‐induced liver damage. Taken together, these observations indicated that YAP was crucial for liver repair during alcohol‐associated injury. Its regulation of ALDH1A1 represents a new link in liver regeneration and detoxification.