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Hypoxia-reoxygenation can induce inflammation by activating NF-κB. In endothelial cells this process is critical for pathogenesis of many chronic inflammatory conditions such as atherosclerosis and auto-immune disease. Recent publication from Evans’s lab shows the critical role of de-ubiquitinating enzyme Cezanne regulating its extent of NF-κB activation and expression of inflammatory genes1. In particular, they showed that poly-ubiquitination of TRAF6 is a specific anti-inflammatory mechanism control by Cezanne. In this editorial, we will briefly review the TRAF6-mediated NF-κB signaling and other post-translational modifications, which play a key role in modulating EC inflammation.
NF-κB (nuclear factor κB) transcription factor complexes consist of a heterodimer of p65 (RelA) and p50 or p522. In most non-stimulated cells, p65-containing NF-κB complexes are kept in an inactive cytoplasmic form, bound to one of a family of inhibitor proteins, the IκBs (inhibitory κBs). Two IκB kinases, IKKα and IKKβ, target phosphorylation of IκB following the hypoxia-reoxygeneration, cytokine, or UV stress stimulation. Phosphorylation of IκBs promotes their ubiquitination and degradation by the proteasome, which releases the p65 complex, allowing it to translocate to the nucleus3. An ubiquitin E2 conjugating enzyme of the Ubc4/5 family and the SCF-βTrCP E3 ligase (Skp1-Cul1-F-box ligase containing the F-box protein βTrCP) execute ubiquitination of IκB. Once IκB is phosphorylated, βTrCP1 and βTrCP2 associate with phosphorylated IκB 4, 5. The polyubiquitinated IκB is selectively degraded by the 26S proteasome, and then mature p52 and p65 subunits translocate into the nucleus to increase NF-κB activity4. It is now well established that NF-κB activation is a key event in inflammation-based cardiovascular pathogenesis including atherosclerotic plaque formation, diabetes-mediated endothelial inflammation, and ischemia/reperfusion injury6.
The regulatory subunit NF-κB essential modulator (NEMO/IKKγ) of the cytoplasmic IκB kinase (IKKα and IKKβ) complex (IKK complex) plays a central role in the upstream events of NF-κB activation7. Although IKKα and IKKβ contain serine/threonine kinase domains, NEMO does not have apparent catalytic domains, but several protein interaction motifs exist. Interestingly, NEMO is also an ubiquitin-binding protein. This suggests that not only the IκB but also IKK complex is regulated by ubiquitination4. The coiled-coil region of NEMO contains an ubiquitin-binding domain (NEMO ubiquitin binding (NUB)), and IKK complex activation is significantly inhibited by the mutation of NUB8.
Ubiquitin is a 8.5 kDa molecule protein that contains seven lysines, and is frequently ligated to proteins to form polyubiquitin chains. There is some specificity inherent in the sites of ubiquitin binding to another ubiquitin and forming polyubiquitin chains. For example, when proteins bind to lysine-48 (K48)-linked polyubiquitin chains, the protein is targeted to the proteasome for degradation. In contrast, K63-linked polyubiquitin chain (K63-Ub chain) may regulate proteins through a degradation-independent mechanism. For example, specifically IKK complex is activated when links to K63-Ub chain4, 9-11 (Figure).
Particularly interesting situation is when polyubiquitinating ligase is itself ubiquinated. For example, under Toll-like receptor (TLR) activation, IRAK1 is phosphorylated by IRAK4 and then associates with and activates the ubiquitin E3 ligase TRAF6. Working together with the ubiquitin E2 complex composed of Ubc13 and Uev1A11, 12, TRAF6 catalyzes the formation of K63-linked polyubiquitin chains on itself and other protein (Figure). TRAF6 promotes the binding of the TAB2 (TGF-β-activated kinase 1 and MAP3K7-binding protein 2) subunit of the TAK1 (TGF-β-activated kinase 1) kinase complex and NEMO via K63 Ub-chains, promoting TAK1 kinase auto-phosphorylation and activation of TAK111, 13. Next, the K63-linked polyubiquitin chains bind NEMO to recruit the IKK complex, thereby accelerating TAK1-mediated IKKβ phosphorylation and activation, which phosphorylates IκB and causes its degradation14. Thereby activating NF-κB.
It has been reported that Cezanne is a de-ubiquitinating enzyme that suppresses NF-κB activation in response to TNFα or IL-1 by removing polyubiquitin chains from signaling intermediaries such as TRAF3 and RIP1(receptor-interacting protein 1)15, 16. In this current issue, Luong et al. report that Cezanne can also inhibit NF-κB-dependent inflammatory activation in response to hypoxia-reoxygenation by reducing TRAF6 K63 polyubiquitination. They furthermore showed lacking Cezanne a critical role for the enzyme in renal inflammation and injury in mice kidneys exposed to ischemia followed by reperfusion1.
The role of TRAF6 auto-ubiquitination on NF-κB activation needs to be treated cautiously. It is suggested that K63-linked autoubiquitination of TRAF6 is essential for the formation and activation of a complex involving TAK1 kinase and its adapters, TAB1 and TAB217. However, Walsh et al. have reported that both TRAF6 autoubiquitination and the TRAF6 RING finger domain do not appear to be indispensable for the formation of the TAB1-TAB2-TAK1 complex, as well as subsequent TAK1 and NF-kB activation18. Furthermore, lysine-deficient TRAF6, which cannot be autoubiquitinated, remains competent to induce ubiquitination of IKKγ/NEMO, and subsequent TRAF6-mediated activation of NF-κB18. Therefore, although autoubiquitination can be a marker of TRAF6 activation (this report), it is unclear if this ubiquitination contributes towards TRAF6 function (Figure).
Three de-ubiquitination enzymes (DUBs), Cezanne, CYLD (the cylindromatosis tumor suppressor protein) and A20, have been reported to play an important role in inhibiting NF-κB activation upstream of the IKK complex4. It has been reported that TRAF2/5/6 and RIP1 are differentially poly-ubiquitinated and/or activated by TNF receptor, IL-1R, TLR, and TCR (T-cell receptor) activation. Therefore, the substrate specificity of DUBs determines the physiological role for each DUB. Two DUBs, CYLD (the cylindromatosis tumor suppressor protein) and A20 have been reported to play an important role in inhibiting NF-κB activation upstream of the IKK complex4 (Figure). CYLD inhibits IKK by cleaving K63-linked polyubiquitin chains on TRAF2, TRAF6 and NEMO19, 20 Interestingly, in patients with the cylindromas (a type of tumor), a mutation in the DUB domains of CYLD has been reported, suggesting its role of tumorgenesis19. A20 also cleaves K63-linked polyubiquitin chains, but it does so from RIP (in the TNFα pathway)21 and TRAF6 (in the LPS pathway)22. The uniqueness of A20 is based on its dual functions as a ubiquitin ligase through its zinc finger domains. A20 can add K48-linked polyubiquitin chains on RIP after the K63-linked chains are removed by A20 itself, then causes degradation of RIP, and further inhibits IKK activation21. Therefore, A20 deficient mice exhibit severe inflammatory responses in multiple organs because of enhanced and prolonged activation of NF-κB after TNF or LPS stimulation. CezanneGT/GT mice did not show any phenotypic changes under the basal condition1, suggesting its role in NF-κB activation may be modest compared with CYLD and A20. These issues including the specificity of each DUB need to be clarified in the future studies.
Lastly, it is important to discuss other types of post-translational modification, which work together with ubiquitination and regulate NF-κB activation. SUMO-1 modification of NEMO is required for NF-κB activation in response to genotoxic stress inducers23. First, DNA damage activates PARP-1, and poly(ADP-ribose) generated by PARP-1 forms a complex with NEMO, PIAS4 and ATM kinase in the nucleus. PIAS4 interacts with NEMO and preferentially stimulates site-selective modification of NEMO by SUMO-1. DNA damage also activates ATM, which phosphorylates NEMO. Both SUMOylation and phosphorylation of NEMO promote its translocation to the cytoplasm and is incorporated into the IKK complex. At the same time, activated ATM translocates to the cytosol and the plasma membrane and induces a TRAF6-Ubc13-mediated K63-linked polyubiquitin-dependent signaling, involving TAB2, TAK1, and BIRC2 (Baculoviral IAP repeat-containing protein 2). K63-linked ubiquitination of BIRC2 promotes NEMO monoubiquitination at K285, which is crucial for IKKβ phosphorylation and IKK complex activation24. These data suggest that both NEMO SUMOylation and monoubuitination are crucial for subsequent DNA damage-mediated NF-κB activation25. These data also suggest the importance of a cross-talk between ubiquitination and SUMOylation in NF-κB signaling. Of note, TRAF6 can also be SUMOylated26, but its functional role in NF-κB activation remains unclear.
In particular, since the present study use hypoxia/re-oxygeneration to activate NF-κB, the role of SUMOylation under the hypoxic condition should be discussed27. Hydroxylation by prolyl hydroxylase domain (PHD) proteins of hypoxia-inducible transcription factor 1 (HIF1) serves as a recognition signal for the ubiquitin ligase VHL, which causes polyubiquitination and degradation by the 26S proteasome. Recently, it has been reported that hypoxia induces HIF1 SUMOylation, and VHL recognizes and ubiquitinates SUMOylated HIF1α and degradates it. However, in the presence of the SUMO-specific isopeptidase SENP1, HIF1α is deSUMOylated and escapes degradation27, 28. This is a good example that shows co-orporation of SUMOylation and ubiquitination regulating hypoxia-mediated signaling. As the authors have indicated, the possible involvement of PHD on IKKβ degradation has been reported, and hypoxia decreases PHD-dependent hydroxylation of IKKβ and at the same time increases IKKβ expression, resulting in NF-κB activation28. However, although the degradation and ubiquitination processes of IKKβ have been well described, the contribution of SUMOylation on these events has not been reported.
In conclusion, Luong et al. have nicely shown that Cezanne via its deubiquinating activity inhibits ischemia/reperfusion-mediated renal inflammation and injury. The TRAF6 ubiquitination has been suggested as the target of Cezanne1. However, the role of other DUBs for the interplay between NF-κB activation and ubiquitination signaling should be studied. In addition, the contribution of other post-translational modifications such as SUMOylation and hydroxylation on NF-κB activation will be important to understand. It is clear that ischemia/reperfusion-mediated inflammation is highly regulated process that is critically modulated by post-translational modifications, that are likely organs specific.
Sources of funding
This study was supported by a grant from National Institutes of Health to Drs Bradford C. Berk (HL-064839), Jun-ichi Abe (HL-064839, HL-108551, HL-102746).
This is a commentary on article Luong le A, Fragiadaki M, Smith J, Boyle J, Lutz J, Dean JL, Harten S, Ashcroft M, Walmsley SR, Haskard DO, Maxwell PH, Walczak H, Pusey C, Evans PC. Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination. Circ Res.. 2013;112(12):1583-91.
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