ISG15 deficiency in hepatic stellate cells promotes TGFβ2-induced liver fibrosis by counteracting CREB1 ISGylation

BACKGROUND: Interferon (IFN)-stimulated gene 15 (ISG15) is a downstream molecule of the IFN pathways central to many cellular processes. ISG15 mainly exerts its function through a post-translational modification process known as ISGylation. OBJECTIVE: In this study, the role of ISG15 in the activation of hepatic stellate cells (HSCs) and liver fibrosis was examined. DESIGN: Liver fibrosis was established by carbon tetrachloride (CCl4), bile duct ligation (BDL) surgery and metabolic dysfunction-associated steatohepatitis (MASH) diet between HSC-specific deletion of ISG15 (ISG15cKO) and wild type mice. Using genetic strategies in vitro, the role of ISG15 in HSCs was established. Immunoprecipitation, luciferase reporter assays and chromatin-immunoprecipitation assays (ChIP) in combination with proteomics sequencing in HSCs were used to study the associated downstream mechanisms. RESULTS: ISG15 was underexpressed in activated HSCs and fibrotic livers, showing an inverse correlation with α-smooth muscle actin in patients with liver fibrosis. ISG15cKO mice developed spontaneous hepatic fibrosis and showed exacerbated CCl4/BDL-induced fibrogenesis. In vitro, ISG15 modulated HSC activation, proliferation and excessive extracellular matrix production. ISG15 deficiency in HSCs promoted transforming growth factorβ2 (TGFβ2) transcription by enhancing phosphorylated cAMP responsive element binding protein 1 (CREB1) activity, thereby inducing CREB1 binding on TGFβ2 promoter regions to activate TGFβ2/SMAD2 signalling. ISGylation directly binds CREB1 on Lys-304 and Lys-305 to inhibit p-CREB1 activity. Overexpression of ISG15 in HSCs or pharmacological inhibition of CREB1 by 666-15 could abolish ISG15 deficiency-induced liver fibrosis in CCl4-treated mice. CONCLUSIONS: ISG15 regulated HSC activation and liver fibrosis in part via the CREB1/TGFβ2/SMAD2 regulatory pathway. Utilisation of ISG15-CREB1 signalling may be a potential therapeutic target for liver fibrosis.