Cezanne Regulates Inflammatory Responses to Hypoxia in Endothelial Cells by Targeting TRAF6 for Deubiquitination

Abstract

T issues are exposed to hypoxia followed by reoxygenation during ischemia-reperfusion, which occurs in several clinical settings, including organ transplantation, percutaneous coronary intervention, and bypass grafting.1,2 Hypoxiareoxygenation promotes inflammation by activating nuclear factor κB (NF-κB) transcription factors, which induce adhesion proteins (eg, vascular cell adhesion moleucle-1 [VCAM-1], E-selectin) and other inflammatory molecules.3-9 NF-κB transcription factors are regulated by an intricate signaling network that governs their intracellular localization and transcriptional activity. 10 In the basal state, NF-κB dimers are inactivated by binding to inhibitor of κB (IκB) proteins, which sequester NF-κB in the cytoplasm by masking its nuclear localization sequence. IκB kinase promotes NF-κB activation by phosphorylating IκBα, a modification that targets it for ubiquitin-mediated degradation, and by phosphorylating RelA NF-κB subunits to enhance DNA binding and transcriptional activation. 11 In This Issue, see p 1521Editorial, see p 1526Recent studies have shed light on the signaling events that control NF-κB activation by hypoxia. In normoxic conditions, IκB kinase proteins are targeted for hydroxylation by prolyl hydroxylase domain 1, a modification that leads to their Rationale: Hypoxia followed by reoxygenation promotes inflammation by activating nuclear factor κB transcription factors in endothelial cells (ECs). This process involves modification of the signaling intermediary tumor necrosis factor receptor-associated factor 6 with polyubiquitin chains. Thus, cellular mechanisms that suppress tumor necrosis factor receptor-associated factor 6 ubiquitination are potential therapeutic targets to reduce inflammation in hypoxic tissues. Objective:In this study, we tested the hypothesis that endothelial activation in response to hypoxia-reoxygenation can be influenced by Cezanne, a deubiquitinating enzyme that cleaves ubiquitin from specific modified proteins. Methods and Results: Studies of cultured ECs demonstrated that hypoxia (1% oxygen) induced Cezanne viap38 mitogen-activated protein kinase-dependent transcriptional and post-transcriptional mechanisms. Hypoxiareoxygenation had minimal effects on proinflammatory signaling in unmanipulated ECs but significantly enhanced Lys63 polyubiquitination of tumor necrosis factor receptor-associated factor 6, activation of nuclear factor κB, and expression of inflammatory genes after silencing of Cezanne. Thus, although hypoxia primed cells for inflammatory activation, it simultaneously induced Cezanne, which impeded signaling to nuclear factor κB by suppressing tumor necrosis factor receptor-associated factor 6 ubiquitination. Similarly, ischemia induced Cezanne in the murine kidney in vascular ECs, glomerular ECs, podocytes, and epithelial cells, and genetic deletion of Cezanne enhanced renal inflammation and injury in murine kidneys exposed to ischemia followed by reperfusion. Conclusions:

Beyond Impressions

“…In the study by Feng et al, Cezanne was upregulated by low flow and its expression was necessary to stabilize NF-κB-induced HIF1α protein expression. The inability of Cezanne to impact disturbed-flow induced-upregulation of NF-κB is an apparent contradiction of previous findings 27, 29 . The authors address this conundrum by suggesting shear stress may activate NF-kB in a manner that is immune to Cezanne mediated de-ubiquitination.…”

The role of the ubiquitin-editing enzyme A20 in diseases of the central nervous system and other pathological processes

“…Cezanne also bares a single conserved zinc finger motif that is similar to the A20 zinc finger motifs conferring E3 ubiquitin ligase activity to A20 (Evans et al, 2001; Ma and Malynn, 2012). Expression of all Cezanne molecules, like that of A20, is induced by NF-κB creating thereby a negative feedback loop for NF-κB signaling (Luong et al, 2013). …”

“…This, together with the fact that their cell and tissue specific expression profiles differ, imply that A20 and Cezanne molecules might have to some extent overlapping, yet simultaneously unique functions as regulators of NF-κB signaling. Cezanne 1 is ubiquitously expressed in various cell types and tissues, though the brain seems to exhibit the highest Cezanne 1 mRNA levels (Hu et al, 2013; Luong et al, 2013). Cezanne 2 expression is instead restricted to the central nervous system (CNS), where it is particularly abundant in the cerebral cortex and in the cerebellum (Hu et al, 2013).…”

“…These observations are of interest as A20 is specific for the canonical NF-κB pathway and no other negative regulator of the non-canonical NF-κB signaling has been identified this far. Nonetheless, some other works have identified Cezanne 1, whose expression is up-regulated by hypoxia, as a negative regulator of the canonical NF-κB signaling, among others in an ischemia-reperfusion model (Enesa et al, 2008; Luong et al, 2013). These initial findings, together with the fact that Cezanne 1 regulates nuclear localization of DJ-1/Parkin 7, mutated forms of which are associated with the early-onset form of PD (McNally et al, 2011; Saito et al, 2014), may suggest potential roles for Cezanne proteins in the regulation of NF-κB activity in inflammatory responses to ischemia, and in neurodegenerative diseases.…”

Response by Feng et al to Letter Regarding Article, “Mechanical Activation of Hypoxia-Inducible Factor 1α Drives Endothelial Dysfunction at Atheroprone Sites”

“…However, although Cezanne can be inhibited by high levels of exogenous H 2 O 2 8 , it can function in conditions where endogenous ROS are abundant, for example, during ischemia/reperfusion. 9 It is, therefore, not surprising that Cezanne can stabilize HIF1α in conditions of disturbed flow, associated with augmented ROS. More broadly, the influence of ROS on enzymatic activity is highly context specific and varies according to the particular species of reactive molecule, their levels, and intracellular localization.…”