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

We have previously shown that oval cells harboring a genetically inactivated Met tyrosine kinase (Met.sup.-/- oval cells) are more sensitive to TGF-[beta]-induced apoptosis than cells expressing a functional Met (Met.sup.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-[beta] induced a mitochondria-dependent apoptotic cell death in Met.sup.flx/flx and Met.sup.-/- oval cells, associated with a marked increase in levels of the BH3-only proteins Bim and Bmf. Bmf plays a key role during TGF-[beta]-mediated apoptosis since knocking down of BMF significantly diminished the apoptotic response in Met.sup.-/- oval cells. TGF-[beta] also induced oxidative stress accompanied by NADPH oxidase 4 (Nox4) mRNA up-regulation and decreased protein levels of antioxidant enzymes. Antioxidants inhibit both TGF-[beta]-induced caspase 3 activity and Bmf up-regulation, revealing an oxidative stress-dependent Bmf regulation by TGF-[beta]. Notably, oxidative stress-related events were strongly amplified in Met.sup.-/- oval cells, emphasizing the critical role of Met in promoting survival. Pharmacological inhibition of PI3K did impair HGF-driven protection from TGF-[beta]-induced apoptosis and increased sensitivity of Met.sup.flx/flx oval cells to TGF-ß by enhancing oxidative stress, reaching apoptotic indices similar to those obtained in Met.sup.-/- oval cells. Interestingly, both PI3K inhibition and/or knockdown itself resulted in caspase-3 activation and loss of viability in Met.sup.flx/flx oval cells, whereas no effect was observed in Met.sup.-/- 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-[beta]-induced oxidative stress and apoptosis.