Targeting AXL signaling - A new strategy to reduce HSC activation and liver fibrosis

Abstract

[Background]: Activation of hepatic stellate cells is responsible for liver fibrosis associated to different hepatic pathologies and medically considered a pre-stage in the development of cirrhosis and liver cancer. GAS6 is a vitamin-K dependent protein that activates several receptor tyrosin kinases such as Axl, Mer and Tyro3. Axl activation has been related to processes leading to cell differentiation and carcinogenesis. [Methods]: HSCs were isolated from livers of wild type and GAS6 knockout mice after collagenase digestion and cultured in plastic for HSC activation. HSCs and LX2 human activated stellate cells were exposed to recombinant GAS6, Axl inhibitor (BGB324, BerGenBio) or siRNA silencing. Protein detection for p-AXL, p-AKT, alpha-SMA, beta-actin, among others, was evaluated by western blotting to analyze HSC phenotypic changes, in addition to qPCR detection of related genes, particularly Collagen-1A1, and histological markers of liver damage and fibrosis. [Results]: Since spontaneous liver fibrosis was detected in aged GAS6 deficient animals, HSCs from Gas6 knockout mice were analyzed and a significant increase in HSC activation and proliferation was detected. When analyzing Gas6-related signaling, we observed Axl overexpression and consequently AKT and MAPK activation in HSCs from the GAS6-KO mice. In fact, in HSCs from wild type animals, recombinant GAS6 phosphorylated Axl and activated AKT, inducing HSC phenotypic changes. To evaluate if the activation of the Axl/AKT system was responsible for HSC activation, an Axl inhibitor (BGB324) was used. Axl inhibition reduced murine HSC activation and proliferation. Similar results were obtained using LX2 cells and after siRNA-mediated reduction of Axl. Moreover, RNA silencing or BGB324 pre-incubation (1 microM, 1 hour) was able to repress AKT phosphorylation after GAS6 exposure (30 min, 200 ng/ml), and overnight treatment of LX2 cells was able to dosedependently decrease basal AKT levels and inhibit GAS6-dependent AKT activation. [Conclusions]: Our results point to Axl/AKT axis as a relevant mechanism in HSC activation and suggest that Axl targeting may be an interesting therapeutic strategy to reduce liver fibrosis.