Mouse Hepatic Oval Cells Require Met-Dependent PI3K to Impair TGF-β-Induced Oxidative Stress and Apoptosis

Abstract

We have previously shown that oval cells harboring a genetically inactivated Met tyrosine kinase (Met ^−/− oval cells) are more sensitive to TGF-β-induced apoptosis than cells expressing a functional Met (Met ^flx/flx), demonstrating that the HGF/Met axis plays a pivotal role in oval cell survival. Here, we have examined the mechanism behind this effect and have found that TGF-β induced a mitochondria-dependent apoptotic cell death in Met ^flx/flx and Met ^−/− oval cells, associated with a marked increase in levels of the BH3-only proteins Bim and Bmf. Bmf plays a key role during TGF-β-mediated apoptosis since knocking down of BMF significantly diminished the apoptotic response in Met ^−/− oval cells. TGF-β also induced oxidative stress accompanied by NADPH oxidase 4 (Nox4) mRNA up-regulation and decreased protein levels of antioxidant enzymes. Antioxidants inhibit both TGF-β-induced caspase 3 activity and Bmf up-regulation, revealing an oxidative stress-dependent Bmf regulation by TGF-β. Notably, oxidative stress-related events were strongly amplified in Met ^−/− oval cells, emphasizing the critical role of Met in promoting survival. Pharmacological inhibition of PI3K did impair HGF-driven protection from TGF-β-induced apoptosis and increased sensitivity of Met ^flx/flx oval cells to TGF-ß by enhancing oxidative stress, reaching apoptotic indices similar to those obtained in Met ^−/− oval cells. Interestingly, both PI3K inhibition and/or knockdown itself resulted in caspase-3 activation and loss of viability in Met ^flx/flx oval cells, whereas no effect was observed in Met ^−/− oval cells. Altogether, results presented here provide solid evidences that both paracrine and autocrine HGF/Met signaling requires PI3K to promote mouse hepatic oval cell survival against TGF-β-induced oxidative stress and apoptosis.