MCPIP1 Enhances TNF-α-Mediated Apoptosis through Downregulation of the NF-κB/cFLIP Axis

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) is rapidly produced under proinflammatory stimuli, thereby feeding back to downregulate excessive inflammation. In this study, we used the stable, inducible expressions of wild-type (WT) MCPIP1 and an MCPIP1-D141N mutant in T-REx-293 cells by means of a tetracycline on (Tet-on) system. We found that WT MCPIP1 but not MCPIP1-D141N mutant expression dramatically increased apoptosis, caspase-3, -7, -8, and -9 activation, and c-Jun N-terminal kinase (JNK) phosphorylation in TNF-alpha-treated cells. The pan-caspase inhibitor, z-VAD-fmk, and the caspase-1 inhibitor, z-YVAD-fmk, but not the JNK inhibitor, SP600125, significantly reversed apoptosis and caspase activation in TNF-alpha/MCPIP1-treated cells. Surprisingly, MCPIP1 itself was also cleaved, and the cleavage was suppressed by treatment with the pan-caspase inhibitor and caspase-1 inhibitor. Moreover, MCPIP1 was found to contain a caspase-1/-4 consensus recognition sequence located in residues 234 similar to 238. As expected, the WT MCPIP1 but not the MCPIP1-D141N mutant suppressed NF-kappa B activation, as evidenced by inhibition of I kappa B kinase (IKK) phosphorylation and I kappa B degradation using Western blotting, IKK activity using in vitro kinase activity, and NF-kappa B translocation to nuclei using an immunofluorescence assay. Interestingly, MCPIP1 also significantly inhibited importin alpha 3 and importin alpha 4 expressions, which are major nuclear transporter receptors for NF-kappa B. Inhibition of NF-kappa B activation further downregulated expression of the caspase-8 inhibitor, cFLIP. In summary, the results suggest that MCPIP1 could enhance the TNF-alpha-induced apoptotic pathway through decreasing NF-kappa B activation and cFLIP expression.