Hepatic NFAT signaling regulates the expression of inflammatory cytokines in cholestasis.

Abstract Background & Aims The Nuclear Factor of Activated T-cells (NFAT) plays an important role in immune response by regulating the expression of inflammatory genes. However, it is not known whether it takes part in bile acid (BA)-stimulated expression of proinflammatory cytokines in hepatocytes in cholestatic livers. Methods Gene and protein expression and cellular localization were assessed in primary hepatocyte cultures (mouse and human) and cholestatic liver tissues (murine models and patients with PBC and PSC) by Q-PCR, Western blot and immunohistochemistry. Specific NFAT inhibitors were used in vivo and in vitro. Gene reporter assay and ChIP-PCR were used to determine promoter activity. Results NFAT isoform c1 and c3 were expressed in human and mouse hepatocytes. When treated with cholestatic levels of BA, both human and mouse hepatocytes increased NFATc3 nuclear translocation which was associated with elevated mRNA levels of IL-8, Cxcl2, and Cxcl10 in these cells. Blocking NFAT activation with pathway-specific inhibitors or knocking down Nfatc3 expression significantly decreased BA-induction of these cytokines in mouse hepatocytes. Nuclear expression of NFATc3/Nfatc3 protein was increased in cholestatic livers, both in mouse models (bile duct ligation or Abcb4-/- mice) and in patients with PBC and PSC in association with tissue elevations of Cxcl2 or IL-8, respectively. Gene reporter assays and ChIP-PCR demonstrated that NFAT response element in its promoter played a key role in BA-induced human IL-8 expression. Finally, blocking NFAT activation in vivo in Abcb4-/- mice reduced cholestatic liver injury. Conclusions NFAT plays an important role in BA-stimulation of hepatic cytokines in cholestasis. Blocking hepatic NFAT activation may reduce cholestatic liver injury in humans.