In the presence of ethanol TGF-β effects switch from pro-survival to apoptotic in hepatocytes

Signalling of the pro-fibrogenic cytokine TGF-β is activated in liver disease including alcoholic liver disease (ALD). Since TGF-β was described as both, survival promoting or apoptosis inducing factor depending on the cellular context we analyzed its effects in combination with ethanol. The mild cytotoxic effects of ethanol or TGF-β on monolayer-cultured primary mouse hepatocytes is strongly enhanced when both substances were added simultaneously, resulting in strong apoptosis with cytochrome-C release from mitochondria and activation of Caspase 3. Ethanol (or the combination of ethanol and TGF-β) reduced basal activity of p-Akt accompanied by increased TGF-β receptor type II (TβRII) expression. Blunting PI3K with small molecule inhibitor LY294002 (40µM) similarly led to increased TβRII expression, indicating a functional link between loss of activated Akt and TβRII expression. TGF-β alone had contrary effects towards hepatocytes that is increasing pAkt levels and reducing TβRII expression. Furthermore, the pro-apoptotic effect of TGF-β plus ethanol was defined as Smad-dependent and was strongly enhanced when selectively the Smad-1 pathway was inhibited (by overexpressed Smad-6) leaving the Smad-2/-3 pathway (even more) active. In summary, ethanol decreased phospho-Akt levels in hepatocytes leading to increased TβRII expression which allowed strongly enhanced TGF-β dependent apoptosis. The results also support previous data that showed that the TGF-β/Smad-1 pathway regulates many survival- and regeneration-associated genes while the TGF-β/Smad-2/-3 route induces apoptotic and anti-proliferative genes. Ongoing animal experiments will clarify the role of the Smad-1 pathway for HSC activation (which was inhibited by blocking Smad-1 in HSC in vitro) and liver regeneration after acute CCl4 intoxication in vivo. We expect that the Smad-1 pathway is essential for both processes in vivo.