Ethanol sensitizes hepatocytes for TGF-ß-triggered apoptosis.

Alcohol abuse is a global health problem causing a substantial fraction of chronic liver diseases. Abundant TGF-ß-a potent pro-fibrogenic cytokine-leads to disease progression. Our aim was to elucidate the crosstalk of TGF-ß and alcohol on hepatocytes. Primary murine hepatocytes were challenged with ethanol and TGF-ß and cell fate was determined. Fluidigm RNA analyses revealed transcriptional effects that regulate survival and apoptosis. Mechanistic insights were derived from enzyme/pathway inhibition experiments and modulation of oxidative stress levels. To substantiate findings, animal model specimens and human liver tissue cultures were investigated. RESULTS: On its own, ethanol had no effect on hepatocyte apoptosis, whereas TGF-ß increased cell death. Combined treatment led to massive hepatocyte apoptosis, which could also be recapitulated in human HCC liver tissue treated ex vivo. Alcohol boosted the TGF-ß pro-apoptotic gene signature. The underlying mechanism of pathway crosstalk involves SMAD and non-SMAD/AKT signaling. Blunting CYP2E1 and ADH activities did not prevent this effect, implying that it was not a consequence of alcohol metabolism. In line with this, the ethanol metabolite acetaldehyde did not mimic the effect and glutathione supplementation did not prevent the super-induction of cell death. In contrast, blocking GSK-3ß activity, a downstream mediator of AKT signaling, rescued the strong apoptotic response triggered by ethanol and TGF-ß. This study provides novel information on the crosstalk between ethanol and TGF-ß. We give evidence that ethanol directly leads to a boost of TGF-ß's pro-apoptotic function in hepatocytes, which may have implications for patients with chronic alcoholic liver disease.

Hepatocyte fate upon TGF-ß challenge is determined by the matrix environment.

Primary hepatocytes are a versatile tool to investigate all aspects of liver function, and frequently used in drug development and testing. Upon TGF-ß challenge, hepatocytes either undergo an epithelial to mesenchymal transition (EMT) or apoptosis: culture on stiff collagen (monolayer) results in EMT whereas hepatocytes in a soft collagen matrix (sandwich) undergo programmed cell death. In this study, we analyzed the transcriptional programs triggered by TGF-ß under different culture conditions. Our results indicate that TGF-ß initiates an identical transcription profile in hepatocytes irrespective of the cellular environment. The fact that we nevertheless observe two vastly different phenotypes indicates that the matrix environment rather than the TGF-ß induced expression signature is the major determinant of the hepatocellular response. To confirm the impact of the surrounding matrix environment on the hepatocytes׳ phenotype in response to TGF-ß signaling, we studied the effect of Snail overexpression and knockout in both culture conditions. Neither genetic manipulation showed an impact on hepatocytes' fate upon TGF-ß treatment, confirming the crucial role of the surrounding matrix. Our findings provide novel insights into the hepatocellular basis of the fate decision between EMT and apoptotic cell death, and might explain why liver cells can react in very different manners to identical stimuli when tissue remodeling has changed the matrix environment, as occurs in a fibrotic liver.