Influence of USP2-Isoforms on TNF-a dependent NFkB activation in primary hepatocytes versus hepatoma cells

Backgrounds and aims:

Ubiquitination and deubiquitination of proteins have diverse functions within the cell. Among others, both are involved in regulation of signalling pathways. Within the Tumour necrosis factor (TNF)-a dependent NFkB signalling pathway multiple ubiquitination and deubiquitination steps occur to induce cell proliferation and survival but TNF-a also triggers apoptosis. The deubiquitinating enzyme Ubiquitin-specific protease 2 (USP2) seems to play an important role within this pathway. Two different USP2 isoforms exists, a shorter USP2–41kDa and a longer USP2–69kD isoform. We analysed both USP2 isoforms within the NFkB pathway in primary hepatocytes versus hepatoma cells.

Methods:

We performed NFkB promoter assays and Real-time PCR analysis. Apoptosis was triggered by ActinomycinD/TNF-a (ActD/TNFa) treatment and measured by Western Blot using caspase-8 and caspase-3 antibodies.

Results:

Western Blot analysis showed that USP2–41kD was predominantly expressed in primary mouse hepatocytes whereas USP2–69kD is expressed in mouse hepatoma cells. Downregulation of USP2 (USP2–69kD) in Hepa1–6 cells followed by ActD/TNF-a treatment led to a stronger induction of apoptosis compared to control siRNA transfected cells. In contrast, primary hepatocytes showed a decreased rate of apoptosis in the absence of USP2 (USP2–41kD). Furthermore, downmodulation of USP2 in primary hepatocytes led to an increased NFkB promoter activity.

Conclusions:

USP2 seems to be differently regulated in primary hepatocytes versus hepatoma cells, whereas USP2–69kD showed anti-apoptotic activity we observed pro-apoptotic activity for USP2–41kD. Our results showed that USP2–69kD could be a possible candidate to target tumour cells for induction of cell death.