Impact of the transcription factor c-Jun/AP-1 and related signaling pathways on the progression from non-alcoholic fatty liver disease to steatohepatitis and hepatocellular carcinoma
Final Report Abstract
The AP-1 transcription factor c-Jun is a major regulator of hepatocyte function in acute and chronic liver diseases. However, its role during non-alcoholic steatohepatitis (NASH) remains poorly defined. In the first part of this project, we therefore examined the expression of c-Jun in liver biopsies of patients diagnosed with steatosis and NASH. The contribution of c-Jun to the pathogenesis of NASH and subsequent fibrosis was further analyzed in hepatocyte-specific (c-Jun∆li) as well as inducible knock-out mice lacking c-Jun in both, hepatocytes and non-parenchymal liver cells (NPLCs; c-Jun∆li*) fed a methionine- and choline-deficient diet (MCDD). Disease progression from steatosis to NASH and NASH- related fibrosis strongly correlated with increased hepatocellular c-Jun expression in patients, while c-Jun expression in NPLCs strongly correlated with fibrosis stage. Analysis of MCDD- treated c-Jun∆li and control mice further revealed that c-Jun expression in hepatocytes promotes hepatocyte survival and protects against an exacerbated compensatory response called ductular reaction (DR), expression of pro-fibrogenic genes and subsequent fibrosis. The DR correlated with increased numbers of non-parenchymal Sox9 and Osteopontin (Opn) co-expressing cells as well as expression of the Opn receptor CD44, which have all been implicated in fibrogenesis. Interestingly, c-Jun and Opn expression co-localized in both, human and murine hepatocytes as well as NPLCs, raising the question whether the increased fibrosis observed in c-Jun∆li mice could be rescued by additional deletion of c-Jun in NPLCs (Jun∆li*). Indeed, NASH in c-Jun∆li* mice was characterized by reduced liver damage, impaired Opn expression, impaired DR and subsequently less fibrosis. A comparable phenotype was observed in Opn-/- mice, indicating that the phenotype observed in c-Jun∆li* mice was indeed very likely Opn-dependent. These results suggest that c-Jun exerts cell-type specific functions during NASH pathogenesis: c-Jun expression in hepatocytes promotes hepatocyte survival and protects against the NASH-related DR and fibrosis. In contrast, c-Jun expression in NPLCs rather promotes the DR and subsequent fibrosis by regulating Opn expression. In the second part of this funding period, we analyzed the functions of extracellular ATP and purinergic receptor signaling during liver tumorigenesis. Inflammation and tissue damage result in release of ATP to the extracellular space, where it orchestrates many proinflammatory responses by activation of purinergic P2 receptors. We and others have previously shown that ATP and P2Y2R control hepatocyte survival and neutrophil infiltration during acute hepatitis, as well as hepatocyte proliferation in vitro as well as following partial hepatectomy in a c-Jun dependent manner. Moreover, liver tumorigenesis was increased in CD39-/- mice, in which concentrations of extracellular ATP are increased. We therefore addressed the question how extracellular ATP and P2Y2R signalling potentially influences liver tumorigenesis. The impact of extracellular ATP on cell proliferation was analysed in vitro using hepatoma cell lines and primary hepatocytes (PMH) isolated from P2Y2R+/+ and P2Y2R-/- mice. Hepatocarcinogenesis was induced by xenotransplantation of hepatoma cells into immunodeficient Rag2-/-Il2gc-/- mice treated with P2 receptor inhibitors, as well as by diethylnitrosamine (DEN) injection of 2-week old P2Y2R+/+ and P2Y2R-/- mice, in which tumorigenesis was analysed after 8-10 months. Proliferation of HepG2 and HuH-7 hepatoma cells was strikingly reduced upon co-incubation with the purinergic receptor inhibitors Suramin and PPADS, respectively. Moreover, Suramin treatment strongly reduced tumorigenesis in mice xenotransplanted with HuH-7 cells. In keeping with this notion, liver tumorigenesis was significantly reduced in DEN-treated P2Y2R-/- mice, in which activation of pro-inflammatory pathways, such as STAT3 signalling, also appeared to be reduced at early stages of tumour initiation. These results indicate that extracellular ATP and subsequent activation of P2 receptors (in particular of P2Y2R) has pro-tumorigenic functions in two independent mouse models of HCC, which provides a rationale for further assessing the functions of this pathway for pathogenesis and therapy of HCC.
Publications
- The transcription factor c-JUN/AP-1 promotes HBV-related liver tumorigenesis in mice. Cell Death Differ 2016;23:576-582
Trierweiler C, Hockenjos B, Zatloukal K, Thimme R, Blum HE, Wagner EF, Hasselblatt P
(See online at https://doi.org/10.1038/cdd.2015.121)
