Role of A20(TNFAIP3) in mediating TNFα-induced pathways activation in mouse hepatoma cell lines and primary hepatocytes

Backround/Aims:

The tumor necrosis factor-α (TNFα)/NF-kB pathway is crucial for the regulation of apoptosis and proliferation during chronic liver disease, regeneration, as well as carcinogenesis. Recently, a first mathematical model of TNFα/NF-kB signaling provided an important basis to quantitatively disentangle the complex interplay of pathway components in hepatocytes (Pinna et al., 2012 Front Syst Physiol). However, it is not understood how TNFα activates or inhibits e.g., apoptosis depending on specific environmental conditions. In this work we therefore aim to analyze the impact of signalosome-associated TNFα-induced protein 3 (TNFAIP3, synonym: A20) as a putative factor able to switch from a survival to an apoptotic phenotype in mouse hepatocytes and hepatoma cell lines.

Methods:

The time-resolved response of NF-kB pathway constituents and target genes (e.g., p65, p-p65, IkB-α, p-IkB-α, A20) were quantitatively defined using western immunoblotting and real-time PCR after TNFα stimulation (10 ng/ml) of primary mouse hepatocytes and hepatoma cells lines (e.g., Hepa1 – 6). Pathway activity was also analyzed after gene-specific inhibition of A20 in HCC cells. Moreover, the cross talk with other important pathways was quantitatively examined (e.g., MAKP/JNK/p38).

Results:

Comparison of the accurate NF-κB signaling dynamics between HCC cells and primary mouse hepatocyte revealed no significant differences after TNFα treatment suggesting that mechanisms involved in phenotypic changes are mainly located at the signalosome level in both cell types. The TNFα/NF-kB target genes IkB-α and TNFAIP3/A20 were induced in all cells in a comparable manner. Importantly, TNFα administration and simultaneous siRNA-mediated inhibition of A20 in hepatoma cells showed increased phosphorylation of c-Jun N-terminal kinase (JNK) but no significant effects on p38 MAPK phosphorylation.

Conclusion/Outlook:

Both, hepatocytes and HCC cells respond to TNFα-induced activation of the NF-kB pathway in a comparable manner. However, we present A20 as a critical component regulating the cross-talk of specific downstream pathways after TNFαstimulation in cancer cells. Currently, we analyze if this cross-talk may affect the biological outcome of liver-derived cells (e.g., apoptosis).