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

Abstract

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. 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. Studies of cultured ECs demonstrated that hypoxia (1% oxygen) induced Cezanne via p38 mitogen-activated protein kinase-dependent transcriptional and post-transcriptional mechanisms. Hypoxia-reoxygenation 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. We conclude that inflammatory responses to ischemia are controlled by a balance between ubiquitination and deubiquitination, and that Cezanne is a key regulator of this process. Our observations have important implications for therapeutic targeting of inflammation and injury during ischemia-reperfusion.