p62 Negatively Regulates TLR4 Signaling via Functional Regulation of the TRAF6-ECSIT Complex

Abstract

Sequestosome 1 (SQSTM1, p62), a ubiquitin binding protein, plays a role in cell signaling, oxidative stress, and autophagy. However, its functional role in inflammatory signaling is controversial. Recent studies have shown that p62 is negatively implicated in inflammatory responses. But, the precise molecular mechanisms by which p62 regulates inflammatory responses remain unclear. In this study, we report on a new regulatory role for p62 in TLR4-mediated signaling. p62 overexpression led to the suppression of NF-κB activation and the production of pro-inflammatory cytokines, TNF-α, IL-6, and IL-1β in response to TLR4 stimulation. In contrast, p62 −/− mouse embryonic fibroblast (MEF) cells exhibited marked enhancement of NF-κB activation and production of pro-inflammatory cytokines by TLR4 stimulation, compared to p62 +/+ MEF cells. Additionally, the TLR4-induced activation of signal transduction was significantly augmented in p62 −/− MEF cells, indicating that p62 was negatively implicated in TLR4-mediated signaling. Biochemical studies revealed that p62 interacted with the internal domain of evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), which is critical for associating with the TNF receptor associated factor 6 (TRAF6)-ECSIT complex to activate NF-κB in TLR4 signaling. Interestingly, p62-ECSIT interaction inhibited the interaction between TRAF6 and ECSIT and attenuated the ubiquitination of ECSIT. Furthermore, upon LPS challenge, the mortality of p62 −/− ( p62 -knockout) mice was markedly enhanced compared to p62 +/+ ( p62 wild-type) mice. Taken together, our data demonstrate that p62 negatively regulated TLR4 signaling via functional regulation of the TRAF6-ECSIT complex.

Autophagy Promotes Duck Tembusu Virus Replication by Suppressing p62/SQSTM1-Mediated Innate Immune Responses In Vitro

“…These results indicated that p62 played a positive role in antiviral immune responses and a negative role in DTMUV replication. Recent reports show that p62 acts as a regulator of host immunity by its function as a cargo adaptor in selective autophagy process [52][53][54], whereas SQSTM1/p62 not only functions as a cargo adaptor in the autophagy pathway but also acts as a signaling hub that regulates various physiological processes such as NF-κB signal and antioxidant stress [19,55,56]. Our findings provided evidence of the function of p62 as a regulator of antiviral immune responses and indicated that p62 regulated not only the NF-κB signal but also the IRF7 signal.…”

p62 is Negatively Implicated in the TRAF6-BECN1 Signaling Axis for Autophagy Activation and Cancer Progression by Toll-Like Receptor 4 (TLR4)

“…Although the regulatory role of p62 in inflammatory responses is controversial [9,10], it is thought to be involved in the induction of inflammatory cytokine production via TNF Tumor necrosis factor) receptor-associated factor 6 (TRAF6) polyubiquitination and, thereby, NF-κB activation [11,12]. A recent report has shown that p62 is negatively implicated in Toll-like receptor 4 (TLR4)-mediated signaling through inhibition of TRAF6-evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) association and the ubiquitination of ECSIT by TRAF6 [13]. Importantly, p62 −/− KO mice exhibited a higher mortality rate following LPS challenge [13], suggesting that p62 might negatively regulate TLR4-mediated signaling for the activation of NF-κB.…”

“…A recent report has shown that p62 is negatively implicated in Toll-like receptor 4 (TLR4)-mediated signaling through inhibition of TRAF6-evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) association and the ubiquitination of ECSIT by TRAF6 [13]. Importantly, p62 −/− KO mice exhibited a higher mortality rate following LPS challenge [13], suggesting that p62 might negatively regulate TLR4-mediated signaling for the activation of NF-κB.…”

“…TLR4-mediated signaling induces the activation of NF-κB and autophagy induction via TRAF6-ECSIT signaling axis and TRAF6-BECN1 signaling axis, respectively [20][21][22][23][24][25][26]. Since p62 negatively regulates TLR4-mediated signals through the inhibition of TRAF6-ECSIT signaling [13], we therefore investigated whether p62 is functionally implicated in the TRAF6-BECN1 signaling axis, thereby regulating autophagy activation, and cancer cell migration and invasion induced by TLR4. We found that p62 interrupted the association of the TRAF6-BECN1 complex, and that inhibited BECN1 ubiquitination, leading to inhibition of autophagy activation induced by TLR4.…”

AMPKα1 Regulates Lung and Breast Cancer Progression by Regulating TLR4-Mediated TRAF6-BECN1 Signaling Axis

“…Western blotting and IP assays were performed as previously described [13,15,16,[23][24][25][26][27][28][29][30][31]. Briefly, mock vector as control vector, MYC-TRAF6, FLAG-AMPKαl, and MYC-BECN1 were transfected into HEK293T cells using Lipofectamine 2000 for 38-48 h. Cell lysates were immunoprecipitated with anti-FLAG or anti-MYC antibody.…”