Central Role for Endothelial Human Deneddylase-1/SENP8 in Fine-Tuning the Vascular Inflammatory Response

Abstract

A deeper understanding of the mechanisms that control responses to inflammation is critical to the development of effective therapies. We sought to define the most proximal regulators of the Cullin-Ring-Ligases (CRL), which play a central role in the stabilization of NF-κB and HIF. In these studies, we identify the human deneddylase-1 (SENP8) as a key regulator of Cullin neddylation response in vitro and in vivo. Using human microvascular endothelial cells (HMEC), we examined inflammatory responses to LPS or TNF-α by assessing Cullin-neddylation status, NF-κB and HIF-1α stabilization and inflammatory cytokine secretion. HMEC with an intact neddylation pathway showed a time-dependent induction of Cullin-1 (Cul-1) neddylation, nuclear translocation of NF-κB, stabilization of HIF-1α and increased NF-κB/HIF-α promoter activity in response to LPS. HMEC cells lacking SENP8 were unable to neddylate Cul-1 and subsequently were unable to activate NF-κB or HIF-1α. Pharmacological targeting of neddylation (MLN4924) significantly abrogated NF-κB responses, induced HIF-1α promoter activity and reduced secretion of TNF-α-elicited pro-inflammatory cytokines. MLN4924 stabilized HIF and abrogated pro-inflammatory responses while maintaining anti-inflammatory IL-10 response in vivo, following LPS administration. These studies identify SENP8 as a proximal regulator of Cullin neddylation and provide an important role for SENP8 in fine-tuning of the inflammatory response. Moreover, our findings provide feasibility for therapeutic targeting of the Cullins during inflammation.

Inhibition of atherogenesis by the COP9 signalosome subunit 5 in vivo

“…Also, MLN4924 reduced MAPK signaling in inflammatory-elicited macrophages. These data are in accord with reports of MLN4924 effects on HIF-1α stabilization in microvascular endothelial cells (26) and MAPK activation in T cells, the latter linked to MLN-induced stabilization of SHC, a negative regulator of ERK signaling that was identified as a target for NEDDylation (67). Interestingly, the pathways identified in our study to be affected by MLN are linked with each other, e.g., reduced NF-κB activity, as observed upon MLN4924, was inversely linked with M2 polarization (68), and Arginase-1 has been reported to be regulated by HIF-2α (69), which is stabilized by NEDDylation (58).…”

“…A preference for MLN4924 to reduce ICAM-1 and VCAM-1 but not P-selectin levels is consistent with our observation that it markedly reduced monocyte arrest on endothelium. As mentioned, MLN4924 increases LPSinduced HIF-1α levels in endothelial cells, which corresponds with a previous observation of increased HIF activity in the kidney of LPS-treated mice (26). Interestingly, endothelial HIF-1α had been found to promote atherosclerosis and enhance leukocyte adhesion, and myeloid HIF-1α was seen to promote or not affect atherosclerotic lesion formation (60,72,73).…”

“…With respect to our study, this could imply that increased HIF-1α may begin to counteract initial NF-κB-associated atheroprotective effects of MLN4924 implicated in early atherosclerosis and explain the absence of atheroprotection in more advanced lesions. A protective role for MLN4924 in early atherogenesis and endothelial dysfunction is supported by the notion that this compound improves endothelial barrier integrity and disturbed flow/ oxidized lipoprotein-elicited NEDDylation-dependent endothelial dysfunction (26,74).…”

“…Inhibition of NEDDylation by the pharmacological agent MLN4924, which blocks E1 activity (24), results in an increase of phosphorylated IκB-α (p-IκB-α) and consequently reduced NF-κB activation in B-cells, myeloid leukemia cells, macrophages, and endothelial cells (25)(26)(27)(28). Similarly, signal-induced turnover of IκB-α was shown to be reduced by the deNEDDylating protein CSN5 in endothelial cells (29) and cervical cancer cells (30), reducing NF-κB activation (29).…”

NEDDylation promotes endothelial dysfunction: A role for HDAC2

“…Evidence suggests that NEDDylation is tightly associated with ubiquitination and the regulation of cellular protein turnover [3], although NEDDylation has other functions as well. A paucity of literature exists with regard to NEDDylation and the regulation of vascular function, but two recent studies have shown the inhibition of NEDDylation to be effective in attenuating NFkB-mediated proinflammatory cytokine release by LPS-activated macrophages and by human microvascular endothelial cells [6,7]. Furthermore, knockdown of the SENP8 NEDDylase in human microvascular endothelial cells blocks LPS-mediated activation of NFKB and HIF1α [6].…”