Abstract
Simple SummaryTNF-α is considered to be a potential therapeutic drug for cancer, but the systemic toxicity problem still exists in clinical use. MCPIP1 plays a crucial role in anti-inflammatory and anti-viral contexts, as well as in the inhibition of cell growth. This study aims to investigate the roles of MCPIP1 in TNF-α-treated cells and their underlying molecular mechanisms. MCPIP1 was found to enhance TNF-α-induced apoptosis by activating the caspase cascade, and pan-caspase inhibitor and the caspase-1/-4 inhibitor could reverse TNF-α/MCPIP1-mediated apoptosis. MCPIP1 was first identified with cleavage site of caspase-1/-4 and could be cleaved during apoptosis. Downregulation of NF-κB activation and a caspase-8 inhibitor, cFLIP, were associated with TNF-α/MCPIP1-mediated apoptosis.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-α-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-α/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 234~238. As expected, the WT MCPIP1 but not the MCPIP1-D141N mutant suppressed NF-κB activation, as evidenced by inhibition of IκB kinase (IKK) phosphorylation and IκB degradation using Western blotting, IKK activity using in vitro kinase activity, and NF-κB translocation to nuclei using an immunofluorescence assay. Interestingly, MCPIP1 also significantly inhibited importin α3 and importin α4 expressions, which are major nuclear transporter receptors for NF-κB. Inhibition of NF-κB activation further downregulated expression of the caspase-8 inhibitor, cFLIP. In summary, the results suggest that MCPIP1 could enhance the TNF-α-induced apoptotic pathway through decreasing NF-κB activation and cFLIP expression.
Highlights
Tumor necrosis factor (TNF)-α is a multifunctional cytokine that binds to the TNF receptor (TNFR) and transduces cell survival, apoptosis and necroptosis signals
MG132 treatment did not block the cleavage of Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) induced by TNF-α with overexpression of MCPIP1 (Figure S2). These results suggested that MCPIP1 overexpression enhanced TNF-α-induced apoptosis through a caspase cascade, and MCPIP1 cleavage might be mediated through a caspase-1/-4 pathway
It is well known that importin α family proteins are involved in the nuclear transloInterestingly, we found for the first time that MCPIP1 was cleaved during apoptosis, cation of many transcription factors
Summary
Tumor necrosis factor (TNF)-α is a multifunctional cytokine that binds to the TNF receptor (TNFR) and transduces cell survival, apoptosis and necroptosis signals. After TNF-α binds to its receptor, TNFR1, two protein complexes can subsequently be formed. The first is called the TNFR1 signaling complex (TNF-RSC, known as complex-I), which is related to NF-κB’s transcriptional activity and cell survival; the second complex is called complex-II and is related to apoptosis [2]. Members of complex-I include TNFR1-associated death domain protein (TRADD), receptor-interacting protein kinase (RIP1), TNFR-associated factor 2 (TRAF2) or TRAF5, and the cellular inhibitor of apoptosis protein 1 and 2 (cIAP1 and cIAP2). The cIAP is a ubiquitin E3 ligase, which can catalyze K63-linked polyubiquitination of RIP1 [3,4]
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