Essential role of Sharpin in TNFα dependent NFκB activation in primary hepatocytes

Backgrounds&aims: Tumour necrosis factor (TNF)-α triggers cell survival and apoptosis in hepatocytes. The cell survival pathway depends on post-translational modifications (PTM) leading to activation of NFκB. An essential PTM is ubiquitination that regulates NFκB activation at different steps. One important ubiquitination step depends on the linear ubiquitin chain assembly complex (LUBAC) that is responsible for ubiquitination of IKKγ (NEMO). As recently shown, LUBAC consists of HOIP, HOIL–1L and Sharpin. We analysed TNFα dependent NFκB activation in Sharpin-deficient primary hepatocytes (PH).

Methods: Using Sharpin-deficient PH we performed NFκB promoter assays and Real-time PCR analysis. Apoptosis was detected by FACS analysis and caspase–3 activity was measured using a colorimetric assay. Phosphorylation and degradation of IκBα were analysed by Western Blot. We performed LPS treatment of Sharpin-deficient mice and examined liver damage by measuring serum ALT activities.

Results: TNFα-induced NFκB activation as well as expression of NFκB target genes is strongly reduced in PH of Sharpin-deficient mice. This is due to a reduced and delayed phosphorylation and degradation of IκBα. TNFα treatment of Sharpin-deficient PH led to the activation of caspase–8 and caspase–3 resulting in apoptosis. Furthermore, in vivo experiments showed that Sharpin-deficient mice were more susceptible to LPS treatment than control mice. While LPS-treated control mice survived and showed no liver damage, 43% of Sharpin-deficient mice died within 8h following LPS administration and showed significant liver damage.

Conclusions: TNFα is continuously produced in the liver at low level, because of the fact that the liver is exposed to bacterial toxins and food antigens from the gut via the portal vein. Sharpin is part of LUBAC that is active in TNFα dependent NFκB activation. Our results revealed that Sharpin is one part of the hepatocellular defence favouring the cell survival pathway.

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LUBAC - NFκB pathway - Sharpin - TNFα - ubiquitination