Transcriptome-based repurposing of apigenin as a potential anti-fibrotic agent targeting hepatic stellate cells.

We have used a computational approach to identify anti-fibrotic therapies by querying a transcriptome. A transcriptome signature of activated hepatic stellate cells (HSCs), the primary collagen-secreting cell in liver, and queried against a transcriptomic database that quantifies changes in gene expression in response to 1,309 FDA-approved drugs and bioactives (CMap). The flavonoid apigenin was among 9 top-ranked compounds predicted to have anti-fibrotic activity; indeed, apigenin dose-dependently reduced collagen I in the human HSC line, TWNT-4. To identify proteins mediating apigenin's effect, we next overlapped a 122-gene signature unique to HSCs with a list of 160 genes encoding proteins that are known to interact with apigenin, which identified C1QTNF2, encoding for Complement C1q tumor necrosis factor-related protein 2, a secreted adipocytokine with metabolic effects in liver. To validate its disease relevance, C1QTNF2 expression is reduced during hepatic stellate cell activation in culture and in a mouse model of alcoholic liver injury in vivo, and its expression correlates with better clinical outcomes in patients with hepatitis C cirrhosis (n = 216), suggesting it may have a protective role in cirrhosis progression.These findings reinforce the value of computational approaches to drug discovery for hepatic fibrosis, and identify C1QTNF2 as a potential mediator of apigenin's anti-fibrotic activity.

Epithelial Xbp1 is required for cellular proliferation and differentiation during mammary gland development.

Xbp1, a key mediator of the unfolded protein response (UPR), is activated by IRE1α-mediated splicing, which results in a frameshift to encode a protein with transcriptional activity. However, the direct function of Xbp1 in epithelial cells during mammary gland development is unknown. Here we report that the loss of Xbp1 in the mammary epithelium through targeted deletion leads to poor branching morphogenesis, impaired terminal end bud formation, and spontaneous stromal fibrosis during the adult virgin period. Additionally, epithelial Xbp1 deletion induces endoplasmic reticulum (ER) stress in the epithelium and dramatically inhibits epithelial proliferation and differentiation during lactation. The synthesis of milk and its major components, α/ß-casein and whey acidic protein (WAP), is significantly reduced due to decreased prolactin receptor (Prlr) and ErbB4 expression in Xbp1-deficient mammary epithelium. Reduction of Prlr and ErbB4 expression and their diminished availability at the cell surface lead to reduced phosphorylated Stat5, an essential regulator of cell proliferation and differentiation during lactation. As a result, lactating mammary glands in these mice produce less milk protein, leading to poor pup growth and postnatal death. These findings suggest that the loss of Xbp1 induces a terminal UPR which blocks proliferation and differentiation during mammary gland development.

ß-PDGF receptor expressed by hepatic stellate cells regulates fibrosis in murine liver injury, but not carcinogenesis.

BACKGROUND & AIMS: Rapid induction of ß-PDGF receptor (ß-PDGFR) is a core feature of hepatic stellate cell activation, but its cellular impact in vivo is not well characterized. We explored the contribution of ß-PDGFR-mediated pathway activation to hepatic stellate cell responses in liver injury, fibrogenesis, and carcinogenesis in vivo using genetic models with divergent ß-PDGFR activity, and assessed its prognostic implications in human cirrhosis. METHODS: The impact of either loss or constitutive activation of ß-PDGFR in stellate cells on fibrosis was assessed following carbon tetrachloride (CCl4) or bile duct ligation. Hepatocarcinogenesis in fibrotic liver was tracked after a single dose of diethylnitrosamine (DEN) followed by repeated injections of CCl4. Genome-wide expression profiling was performed from isolated stellate cells that expressed or lacked ß-PDGFR to determine deregulated pathways and evaluate their association with prognostic gene signatures in human cirrhosis. RESULTS: Depletion of ß-PDGFR in hepatic stellate cells decreased injury and fibrosis in vivo, while its auto-activation accelerated fibrosis. However, there was no difference in development of DEN-induced pre-neoplastic foci. Genomic profiling revealed ERK, AKT, and NF-κB pathways and a subset of a previously identified 186-gene prognostic signature in hepatitis C virus (HCV)-related cirrhosis as downstream of ß-PDGFR in stellate cells. In the human cohort, the ß-PDGFR signature was not associated with HCC development, but was significantly associated with a poorer outcome in HCV cirrhosis. CONCLUSIONS: ß-PDGFR is a key mediator of hepatic injury and fibrogenesis in vivo and contributes to the poor prognosis of human cirrhosis, but not by increasing HCC development.

Contributions of metabolic dysregulation and inflammation to nonalcoholic steatohepatitis, hepatic fibrosis, and cancer.

PURPOSE OF REVIEW: We review accumulating evidence that nonalcoholic steatohepatitis (NASH), a more advanced form of nonalcoholic fatty liver disease (NAFLD), predisposes patients to the risk of developing hepatocellular carcinoma (HCC), and we summarize recent advances in the elucidation of cancer-promoting pathways in NASH. We highlight the potential role of progenitor cells and hepatic stellate cells (HSCs) in promoting the early events that could culminate in cancer, as well as the emerging contribution of the gut-liver axis in promoting inflammation, senescence, and tumor growth in NASH and HCC. Finally, we review the role of bile acid receptors, vitamin D, and protective cellular pathways such as autophagy. RECENT FINDINGS: Studies have recently uncovered roles for gut microbiota, bile acid receptors and vitamin D in regulating the progression from NAFLD to HCC. Intriguing findings linking senescence and autophagy in hepatic stellate cells to HCC have also been discovered, as well as a link between dysregulated progenitor cell regulation and HCC. SUMMARY: NAFLD is the most common cause of chronic liver disease in the United States and Western Europe. The lack of definitive mechanisms underlying development of NASH among patients with NAFLD and its progression to HCC limit diagnosis and management, but new findings are paving the way for better biomarkers and therapies.

Calpain induces N-terminal truncation of ß-catenin in normal murine liver development: diagnostic implications in hepatoblastomas.

Hepatic competence, specification, and liver bud expansion during development depend on precise temporal modulation of the Wnt/ß-catenin signaling. Also, loss- and gain-of-function studies have revealed pleiotropic roles of ß-catenin in proliferation and hepatocyte and biliary epithelial cell differentiation, but precise mechanisms remain unknown. Here we utilize livers from different stages of murine development to determine ß-catenin signaling and downstream targets. Although during early liver development full-length ß-catenin is the predominant form, at late stages, where full-length ß-catenin localizes to developing biliary epithelial cells only, a 75-kDa truncated ß-catenin species is the principal form localizing at the membrane and in the nucleus of differentiating hepatocytes. The truncated species lacks 95 N-terminal amino acids and is transcriptionally active. Our evidence points to proteolytic cleavage of ß-catenin by calpain as the mechanism of truncation in cell-free and cell-based assays. Intraperitoneal injection of a short term calpain inhibitor to timed pregnant female mice abrogated ß-catenin truncation in the embryonic livers. RNA-seq revealed a unique set of targets transcribed in cells expressing truncated versus full-length ß-catenin, consistent with different functionalities. A further investigation using N- and C-terminal-specific ß-catenin antibodies on human hepatoblastomas revealed a correlation between full-length versus truncated ß-catenin and differentiation status, with embryonal hepatoblastomas expressing full-length ß-catenin and fetal hepatoblastomas expressing ß-catenin lacking its N terminus. Thus we conclude that calpain-mediated cleavage of ß-catenin plays a role in regulating hepatoblast differentiation in mouse and human liver, and the presence of the ß-catenin N terminus correlates with differentiation status in hepatoblastomas.

Beta-catenin signaling in hepatic development and progenitors: which way does the WNT blow?

The Wnt/ß-catenin pathway is an evolutionarily conserved signaling cascade that plays key roles in development and adult tissue homeostasis and is aberrantly activated in many tumors. Over a decade of work in mouse, chick, xenopus, and zebrafish models has uncovered multiple functions of this pathway in hepatic pathophysiology. Specifically, beta-catenin, the central component of the canonical Wnt pathway, is implicated in the regulation of liver regeneration, development, and carcinogenesis. Wnt-independent activation of beta-catenin by receptor tyrosine kinases has also been observed in the liver. In liver development across various species, through regulation of cell proliferation, differentiation, and maturation, beta-catenin directs foregut endoderm specification, hepatic specification of the foregut, and hepatic morphogenesis. Its role has also been defined in adult hepatic progenitors or oval cells especially in their expansion and differentiation. Thus, beta-catenin undergoes tight temporal regulation to exhibit pleiotropic effects during hepatic development and in hepatic progenitor biology.