In this study, we demonstrate that Itch forms a ubiquitin-editing complex with Cyld via
the interaction through WW-PPXY motifs. Itch-Cyld complex promotes the transition of K63-linked to K48-linked ubiquitination by sequentially cleaving K63-linked ubiquitin chain and by catalyzing K48-linked ubiquitination on Tak1. During the early phase of TNF- or TLR-induced inflammatory response RING type E3 ligases TRAF2 and TRAF6 promote K63-linked ubiquitination within the kinase domain29
, promoting phosphorylation of Tak1 that leads to IKK-NF-κB activation and transcription of proinflammatory cytokine gene expression42
. We propose that the combined activity of Itch and Cyld shifts Tak1 ubiquitination from K63-linked toward K48-linked chains that triggers the proteasomal degradation of Tak1, thus terminating the inflammatory response. Itch-Cyld association was detected only following stimulation of BMDMs with TNF suggesting the requirement of extracellular signals. At present the molecular mechanisms that regulate Itch-Cyld complex formation remain unclear. One possibility is that Cyld expression is upregulated by TNF and IL-1β, via
IKK-NF-κB pathway16, 24, 25
triggers its association with Itch. Alternatively, Itch-Cyld association could be regulated by NF-κB-dependent phosphorylation mediated mechanisms. Such a phenomenon has recently been demonstrated for the recruitment of Itch to A20 complex by TAXIBP134, 43
The molecular basis for the chronic multi-organ inflammatory disorder observed in Itch−/−
mice has been attributed to biased TH
2 differentiation due to the defect in the degradation of JunB13
. Our previous studies have shown that Itch also regulates T cell tolerance, as Itch−/−
T cells are resistant to anergy induction44
and TGF-β mediated suppression11
. Our present results indicate a critical role for Itch in the regulation innate immune cells via
targeting Tak1 for proteasomal degradation. These findings along with recent reports that Itch is a component of A20, TAXIBP1 and RNF11 complex34, 43
raise the possibility that Itch has more prominent role as a negative regulator of inflammatory signaling in the innate immune cells.
Itch seems to be a specific ligase that targets Tak1 for K48-linked ubiquitination as Cbl-b a functionally related E3 ligase8
which also regulates NF-κB pathway45
but failed to ubiquitinate Tak1. Similarly, several other DUBs including A20, Cezanne, USP4, and USP21 are shown to negatively regulate NF-κB pathway39
. In our assay system we did not detect DUB activity of A20 or Cezanne towards Tak1, suggesting the specificity of Cyld. However, we do not completely rule out the involvement of additional DUBs or E3 ligases in the regulation of Tak1 and downstream signaling.
An additional observation from our studies is that Itch−/−
TAMs produced elevated tumor promoting inflammatory cytokines. Consistently, Itch−/−
mice exhibited aggressive growth and metastasis of transplanted LLC tumors. However, it is not clear whether the enhanced tumor growth observed is exclusively due to the chronic production of tumor promoting cytokines by TAMs. Aberrant T cell response in Itch−/−
mice8, 10, 11, 13
may also contribute to tumor growth. Further detailed analysis is essential for a clear and better understanding of these mechanisms that could lead to innovative therapeutic targeting.
Consistent with our findings, cytokines produced by activated innate immune cells, particularly TAMs, have been associated with inflammation and cancer6
. Clinical studies have shown that increased numbers of TAMs are correlated with angiogenesis, metastasis and poor prognosis6
. Tumor-promoting role of TAMs is linked to NF-κB-mediated production of pro-inflammatory cytokines, including TNF and IL-67
supporting the notion that excessive, chronically produced pro-inflammatory cytokines contribute to tumor progression. Our findings have identified an unanticipated co-ordination between Itch and Cyld that restricts prolonged inflammatory signaling and secretion of tumor promoting inflammatory cytokines by the macrophages.
Our studies also show that Tak1 selective inhibitor, (5Z)-7-Oxozeaenol inhibits tumor promoting cytokine production by BMDMs. Since NF-κB has emerged as a key internal factor that promotes inflammation and cancer1, 2
, several approaches have been designed to block NF-κB, including inhibitors of IKK and proteasome inhibitors to inhibit IκB degradation46
. Although proteasome inhibitors like bortezomib block NF-κB in a variety of cells and are being used in clinical trials against multiple myeloma, the results of the phase I/II clinical trials indicate the drug has limited activity against advanced NSCLC47, 48
. In addition, complications have been reported after global inhibition of NF-κB using genetic and pharmacological approaches49
. This highlights the requirement for more specific inhibitors to dampen inflammation and tumor progression. Our results provide a mechanistic insight into regulation of Tak1 for termination of inflammatory cytokine production which can be exploited therapeutically to target inflammatory cells in the tumor microenvironment. By targeting inflammatory cells, which are genetically normal and stable compared to genetically unstable carcinoma cells, development of drug resistance could be minimized. In addition, a combination of conventional cytotoxic cancer therapy along with inhibitors that block inflammatory responses could potentially enhance current therapeutic strategies.