Cezanne is a critical regulator of pathological arterial remodelling by targeting ß-catenin signalling.

AIMS: Pathological arterial remodelling including neointimal hyperplasia and atherosclerosis is the main underlying cause for occluding arterial diseases. Cezanne is a novel deubiquitinating enzyme, functioning as a NF-кB negative regulator, and plays a key role in renal inflammatory response and kidney injury induced by ischaemia. Here we attempted to examine its pathological role in vascular smooth muscle cell (VSMC) pathology and arterial remodelling. METHODS AND RESULTS: Cezanne expression levels were consistently induced by various atherogenic stimuli in VSMCs, and in remodelled arteries upon injury. Functionally, VSMCs over-expressing wild-type Cezanne, but not the mutated catalytically-inactive Cezanne (C209S), had an increased proliferative ability and mobility, while the opposite was observed in VSMCs with Cezanne knockdown. Surprisingly, we observed no significant effects of Cezanne on VSMC apoptosis, NF-κB signalling, or inflammation. RNA-sequencing and biochemical studies showed that Cezanne drives VSMC proliferation by regulating CCN family member 1 (CCN1) by targeting ß-catenin for deubiquitination. Importantly, local correction of Cezanne expression in the injured arteries greatly decreased VSMC proliferation, and prevented arterial inward remodelling. Interestingly, global Cezanne gene deletion in mice led to smaller atherosclerotic plaques, but with a lower level of plaque stability. Translating, we observed a similar role for Cezanne in human VSMCs, and higher expression levels of Cezanne in human atherosclerotic lesions. CONCLUSION: Cezanne is a key regulator of VSMC proliferation and migration in pathological arterial remodelling. Our findings have important implications for therapeutic targeting Cezanne signalling and VSMC pathology in vascular diseases.

Zebrafish Model for Functional Screening of Flow-Responsive Genes.

OBJECTIVE: Atherosclerosis is initiated at branches and bends of arteries exposed to disturbed blood flow that generates low shear stress. This mechanical environment promotes lesions by inducing endothelial cell (EC) apoptosis and dysfunction via mechanisms that are incompletely understood. Although transcriptome-based studies have identified multiple shear-responsive genes, most of them have an unknown function. To address this, we investigated whether zebrafish embryos can be used for functional screening of mechanosensitive genes that regulate EC apoptosis in mammalian arteries. APPROACH AND RESULTS: First, we demonstrated that flow regulates EC apoptosis in developing zebrafish vasculature. Specifically, suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) enhanced that rate of EC apoptosis (≈10%) compared with controls exposed to flow (≈1%). A panel of candidate regulators of apoptosis were identified by transcriptome profiling of ECs from high and low shear stress regions of the porcine aorta. Genes that displayed the greatest differential expression and possessed 1 to 2 zebrafish orthologues were screened for the regulation of apoptosis in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. A phenotypic change was observed in 4 genes; p53-related protein (PERP) and programmed cell death 2-like protein functioned as positive regulators of apoptosis, whereas angiopoietin-like 4 and cadherin 13 were negative regulators. The regulation of perp, cdh13, angptl4, and pdcd2l by shear stress and the effects of perp and cdh13 on EC apoptosis were confirmed by studies of cultured EC exposed to flow. CONCLUSIONS: We conclude that a zebrafish model of flow manipulation coupled to gene knockdown can be used for functional screening of mechanosensitive genes in vascular ECs, thus providing potential therapeutic targets to prevent or treat endothelial injury at atheroprone sites.

Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination.

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 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. CONCLUSIONS: 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.

Inhibition of NF-κB signaling in human dendritic cells by the enteropathogenic Escherichia coli effector protein NleE.

Intestinal dendritic cells (DCs) send processes between epithelial cells into the gut lumen to sample pathogens. Noninvasive enteropathogenic Escherichia coli (EPEC) colonize the gut using a type three secretion system (T3SS) to inject effector proteins into epithelial cells. We hypothesized that EPEC might also inject proteins into DC processes to dampen immune recognition. Using a T3SS-linked fluorescence resonance energy transfer-based system we show that EPEC injects effectors into in vitro grown human myeloid DCs. Injected cells emit a blue signal due to cleavage of the green fluorescence resonance energy transfer-based substrate CCF2/AM by ß-lactamase. When cultured with a mutant EPEC unable to translocate effector proteins, myeloid DCs show rapid activation of NF-κB, secrete large amounts of proinflammatory cytokines and increase expression of CD80, CD83, and CD86, whereas wild-type EPEC barely elicits cytokine production and shuts off nuclear translocation of NF-κB p65. By deleting effector protein genes, we identified NleE as being critical for this effect. Expression of NleE in HeLa cells completely prevented nuclear p65 accumulation in response to IL1-ß, and luciferase production in an NF-κB reporter cell line. DCs cocultured with wild-type EPEC or NleE-complemented strains were less potent at inducing MLR. EPEC was also able to inject effectors into DCs sending processes through model gut epithelium in a transwell system and into Peyer's patch myeloid DCs. Thus, EPEC translocate effectors into human DCs to dampen the inflammatory response elicited by its own pathogen-associated molecular patterns.

Role of nuclear factor kappaB in cardiovascular health and disease.

Cardiovascular pathologies are still the primary cause of death worldwide. The molecular mechanisms behind these pathologies have not been fully elucidated. Unravelling them will bring us closer to therapeutic strategies to prevent or treat cardiovascular disease. One of the major transcription factors that has been linked to both cardiovascular health and disease is NF-kappaB (nuclear factor kappaB). The NF-kappaB family controls multiple processes, including immunity, inflammation, cell survival, differentiation and proliferation, and regulates cellular responses to stress, hypoxia, stretch and ischaemia. It is therefore not surprising that NF-kappaB has been shown to influence numerous cardiovascular diseases including atherosclerosis, myocardial ischaemia/reperfusion injury, ischaemic preconditioning, vein graft disease, cardiac hypertrophy and heart failure. The function of NF-kappaB is largely dictated by the genes that it targets for transcription and varies according to stimulus and cell type. Thus NF-kappaB has divergent functions and can protect cardiovascular tissues from injury or contribute to pathogenesis depending on the cellular and physiological context. The present review will focus on recent studies on the function of NF-kappaB in the cardiovascular system.

The A20 gene protects kidneys from ischaemia/reperfusion injury by suppressing pro-inflammatory activation.

Ischaemia followed by reperfusion (I/R) can induce inflammation and injury and is a risk factor for delayed graft function and rejection of transplanted kidneys. Inflammation is regulated by NF-kappaB transcription factors which induce pro-inflammatory molecules in endothelial cells (EC). We examined whether A20, a negative regulator of NF-kappaB, can protect kidneys from I/R injury. To mimic the fluctuations in endothelial oxygenation that occur during I/R we exposed cultured human umbilical vein EC (HUVEC) to hypoxia (1% O(2) for 4 h) followed by re-oxygenation (21% O(2) for 1 h-24 h). We observed transient expression of pro-inflammatory molecules (E-selectin, VCAM-1 and IL-8) and sustained expression of A20 in HUVEC exposed to hypoxia/re-oxygenation. The effect of A20 on endothelial responses to hypoxia/re-oxygenation was assessed. We observed that pre-treatment of HUVEC with an adenovirus containing A20 (Ad-A20) suppressed activation of NF-kappaB and induction of pro-inflammatory molecules by hypoxia/re-oxygenation, whereas a control adenovirus had little or no effect. Thus the induction of A20 may form a negative feedback loop in pro-inflammatory signalling in cells exposed to hypoxia/re-oxygenation. To validate our cell culture experiments we examined the role of A20 in renal responses to I/R. We observed that A20 was induced in rat kidneys exposed to I/R. Moreover, pre-treatment of animals with Ad-A20 significantly reduced acute tubular necrosis, renal expression of VCAM-1 and NF-kappaB activation in response to I/R, whereas pre-treatment with control adenovirus did not. Our observations suggest that A20 maintains physiological homeostasis in kidneys exposed to I/R by protecting them from inflammation and injury.

Hydrogen peroxide prolongs nuclear localization of NF-kappaB in activated cells by suppressing negative regulatory mechanisms.

NF-kappaB transcription factors induce pro-inflammatory molecules (e.g. IL-8) in response to cytokines (e.g. TNFalpha, IL-1beta) or other stimuli. In the basal state, they are sequestered in the cytoplasm by inhibitory IkappaB proteins. Pro-inflammatory signaling triggers polyubiquitination of intermediaries (e.g. RIP1), which activate IkappaB kinases that trigger Ser phosphorylation and degradation of IkappaBalpha, thereby promoting nuclear translocation of NF-kappaB. A negative feedback loop exists whereby NF-kappaB drives resynthesis of IkappaBalpha, which promotes export of NF-kappaB from the nucleus to the cytoplasm. This process relies on Cezanne, a deubiquitinating cysteine protease that stabilizes resynthesized IkappaBalpha by removing polyubiquitin from modified intermediaries. H(2)O(2) is generated during inflammation. Here we examined the effects of H(2)O(2) on NF-kappaB dynamics and pro-inflammatory activation in cultured cells co-stimulated with TNFalpha or IL-1beta. Quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay revealed that H(2)O(2) enhanced the induction of IL-8 by TNFalpha or IL-1beta. We demonstrated by using assays of NF-kappaB nuclear localization and by imaging of live cells expressing a fluorescent form of NF-kappaB that H(2)O(2) prolonged NF-kappaB nuclear localization in cells co-stimulated with TNFalpha or IL-1beta by suppressing its export from the nucleus. We provide evidence that H(2)O(2) suppresses NF-kappaB export by prolonging polyubiquitination of signaling intermediaries, which promotes Ser phosphorylation and destabilization of newly synthesized IkappaBalpha proteins. Finally, we observed that the catalytic activity of Cezanne and its ability to suppress RIP1 polyubiquitination and NF-kappaB transcriptional activity were inhibited by H(2)O(2). We conclude that H(2)O(2) prolongs NF-kappaB activation in co-stimulated cells by suppressing the negative regulatory functions of Cezanne and IkappaBalpha.

NF-kappaB suppression by the deubiquitinating enzyme Cezanne: a novel negative feedback loop in pro-inflammatory signaling.

Transcription factors belonging to the NF-kappaB family regulate inflammation by inducing pro-inflammatory molecules (e.g. interleukin (IL)-8) in response to cytokines (e.g. tumor necrosis factor (TNF) alpha, IL-1) or other stimuli. Several negative regulators of NF-kappaB, including the ubiquitin-editing enzyme A20, participate in the resolution of inflammatory responses. We report that Cezanne, a member of the A20 family of the deubiquitinating cysteine proteases, can be induced by TNFalpha in cultured cells. Silencing of endogenous Cezanne using small interfering RNA led to elevated NF-kappaB luciferase reporter gene activity and enhanced expression of IL-8 transcripts in TNFalpha-treated cells. Thus we conclude that endogenous Cezanne can attenuate NF-kappaB activation and the induction of pro-inflammatory transcripts in response to TNF receptor (TNFR) signaling. Overexpression studies revealed that Cezanne suppressed NF-kappaB nuclear translocation and transcriptional activity by targeting the TNFR signaling pathway at the level of the IkappaB kinase complex or upstream from it. These effects were not observed in a form of Cezanne that was mutated at the catalytic cysteine residue (Cys209), indicating that the deubiquitinating activity of Cezanne is essential for NF-kappaB regulation. Finally, we demonstrate that Cezanne can be recruited to activated TNFRs where it suppresses the build-up of polyubiquitinated RIP1 signal adapter proteins. Thus we conclude that Cezanne forms a novel negative feedback loop in pro-inflammatory signaling and that it suppresses NF-kappaB activation by targeting RIP1 signaling intermediaries for deubiquitination.