Goals: To analyze the role of C/EBPbeta phosphorylation on hepatic stellate cell survival/cell death.
Background: Activation and survival of stellate cells is critical for the development of liver fibrosis. C/EBPbeta phosphorylation regulates stellate cell survival by affecting caspase 8 activation. The mechanisms responsible for these effects are unknown.
Study: We study the effects of caspase 8 activators signaling through death receptors. In addition, we assess the role of C/EBPbeta phosphorylation on the susceptibility of stellate cells to apoptotic stimuli. Finally, we investigated whether C/EBPbeta is associated with the caspase 8 complex protein FLIP, a critical inhibitor of caspase 8.
Results: Primary mouse stellate cells from C/EBPbeta wild type and the phosphorylation mimic C/EBPbetaGlu transgenic mice were treated with lipopolysaccharide [an inducer of tumor necrosis factor-alpha (TNF-alpha)], FAS, or TNF-alpha. Stellate cell apoptosis was determined by assessing the binding of annexin-V to exposed phosphatidylserine of plasma membranes. TNF-alpha and FAS, but not lipopolysaccharide, induced annexin-V binding at 6 hours in C/EBPbeta wild type stellate cell. However, the stimulation of apoptosis by TNF-alpha and FAS was markedly blocked in C/EBPbetaGlu stellate cells (P<0.001). Stellate cells activated on a collagen type 1 matrix expressed both C/EBPbeta and FLIPL. Treatment of stellate cells with a MAP kinase kinase1 (MEK1) inhibitor blocked FLIPL cellular localization, suggesting that MEK1 signaling through C/EBPbeta modulates FLIP activity. The colocalization of C/EBPbeta and FLIPL was disrupted by activation of the FAS receptor, by blocking the association of C/EBPbeta with the long form of FLIP, FLIPL.
Conclusions: The MAPK-RSK-C/EBPbeta signaling may modulate stellate cell survival through caspase 8-associated protein FLIPL. This step is critical for liver fibrosis and if blocked with competitor peptides may prevent fibrogenesis.