NF-κB regulates the expression of Nucling, a novel apoptosis regulator, with involvement of proteasome and caspase for its degradation

Abstract

Nucling is identified as a novel regulator of apoptosis. In this study, we investigated the nuclear factor-κB (NF-κB)-dependent mechanism of Nucling expression, as well as the degradation pathways mediated by proteasome system and caspase activation. Using chromatin immunoprecipitation assay in wild-type mouse embryonic fibroblasts (WT MEFs), we found that NF-κB p65 could bind to the κB motifs in the promoter regions of Nucling gene at four putative-binding sites. By real-time PCR and immunoblot, we confirmed that Nucling expression was up-regulated by tumour necrosis factor-α (TNF-α) stimulation in WT MEFs, but not in NF-κB p50 knockout MEFs. On the other hand, we investigated the degradation of Nucling in connection with proteasome and caspase by using cycloheximide chase. The results showed that Nucling is a short-lived protein, and its degradation was recovered by proteasome inhibitor MG132. Moreover, under TNF-α stimulation, degradation of Nucling was recovered by pan-caspase inhibitor zVAD-fmk. Taken together, we propose a mechanism of Nucling intracellular metabolism. Nucling expression is induced by canonical NF-κB signalling pathway, whereas Nucling is undergoing proteasome degradation, as well as being cleaved by caspase system under stress conditions. This opens a new perspective for studying the NF-κB dependent regulation mechanism of cell death and survival.