Abstract
Activation of NF-κB transcription factors is critical for innate immune cells to induce inflammation and fight against microbial pathogens. On the other hand, the excessive and prolonged activation of NF-κB causes massive inflammatory damage to the host, suggesting that regulatory mechanisms to promptly terminate NF-κB activation are important to prevent immunopathology. We have previously reported that PDLIM2, a PDZ-LIM domain-containing protein, is a nuclear ubiquitin E3 ligase that targets the p65 subunit of NF-κB for degradation, thereby suppressing NF-κB activation. Here we show that PDLIM7, another member of LIM protein family, is also a ubiquitin E3 ligase that inhibits NF-κB-mediated inflammatory responses. PDLIM7 directly polyubiquitinates p65 and promotes its proteasomal degradation. Moreover, PDLIM7 heterodimerizes with PDLIM2 to promote synergistic PDLIM2-mediated degradation of p65. Mechanistically, PDLIM7 promotes K63-linked ubiquitination of PDLIM2 and then the proteasome/autophagosome cargo protein p62/Sqstm1 binds to both polyubiquitinated PDLIM2 and the proteasome, thereby facilitating the delivery of the NF-κB-PDLIM2 complex to the proteasome and subsequent p65 degradation. Consistently, double knockdown of PDLIM7 and either PDLIM2 or p62/Sqstm1 results in augmented proinflammatory cytokine production compared to control cells or single knockdown cells. These data delineate a new role for PDLIM7 and p62/Sqstm1 in the regulation of NF-κB signaling by bridging a ubiquitin E3 ligase and the proteasome.
Highlights
Innate immune cells, such as macrophages and dendritic cells, recognize invading microbial pathogens by pattern recognition molecules, such as Toll-like receptors (TLRs)
These data suggested that, among PDZ-LIM protein family members, only PDLIM2 and PDLIM7 are ubiquitin E3 ligases that mediate polyubiquitination of p65 in dendritic cells. These observations prompted us to select PDLIM7 for further analysis. Since both PDZ and LIM domains function as a protein-protein interaction domain, previous studies of PDLIM7 have been focused on the identification of its binding partners [10, 12] and have shown that PDLIM7 can associate with the insulin receptor [18], the receptor tyrosine kinase RET [19], and protein kinase C (PKC) [20] via its LIM domain, and β-tropomyosin [21] through its PDZ domain, the biological significance of these interactions has not been elucidated
We examined the effect of PDLIM7 on TLR-induced, nuclear factor κB (NF-κB)-dependent gene activation in a reporter assay
Summary
Innate immune cells, such as macrophages and dendritic cells, recognize invading microbial pathogens by pattern recognition molecules, such as Toll-like receptors (TLRs). The transcription factor nuclear factor κB (NF-κB) is critically involved in TLR-mediated activation of these innate cells. The NF-κB p65/p50 heterodimer is sequestrated in the cytoplasm by interacting with IκBα, an inhibitor of NF-κB. Signaling through TLR causes the proteasomal degradation of IκBα. Heterodimers of p65 and p50 translocate to the nucleus, bind to NF-κB sites in the enhancers of multiple inflammation-related genes, PDLIM7 Regulates NF-κB p65 Degradation including those encoding proinflammatory cytokines, and induce their transcription [1, 2]. Inflammatory responses must eventually be terminated, otherwise, excessive and prolonged activation of NF-κB signaling will cause massive damage to the host and could lead to autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, multiple sclerosis, and inflammatory bowel disease [3, 4]. A system to negatively regulate inflammatory signaling is crucial for preventing immunopathology [5]
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