Dataset Information

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 (Cul)-RING ligases, which play a central role in the stabilization of NF-?B and hypoxia-inducible factor (HIF). In these studies, we identify the human deneddylase-1 (SENP8) as a key regulator of Cul neddylation response in vitro and in vivo. Using human microvascular endothelial cells (HMECs), we examined inflammatory responses to LPS or TNF-? by assessing Cul neddylation status, NF-?B and HIF-1? stabilization, and inflammatory cytokine secretion. HMECs with an intact neddylation pathway showed a time-dependent induction of Cul-1 neddylation, nuclear translocation of NF-?B, stabilization of HIF-1?, and increased NF-?B/HIF-? promoter activity in response to LPS. HMECs lacking SENP8 were unable to neddylate Cul-1 and subsequently were unable to activate NF-?B or HIF-1?. Pharmacological targeting of neddylation (MLN4924) significantly abrogated NF-?B responses, induced HIF-1? promoter activity, and reduced secretion of TNF-?-elicited proinflammatory cytokines. MLN4924 stabilized HIF and abrogated proinflammatory responses while maintaining anti-inflammatory IL-10 responses in vivo following LPS administration. These studies identify SENP8 as a proximal regulator of Cul neddylation and provide an important role for SENP8 in fine-tuning the inflammatory response. Moreover, our findings provide feasibility for therapeutic targeting of the Culs during inflammation.

SUBMITTER: Ehrentraut SF

PROVIDER: S-EPMC3529810 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Central role for endothelial human deneddylase-1/SENP8 in fine-tuning the vascular inflammatory response.

Ehrentraut Stefan F SF Kominsky Douglas J DJ Glover Louise E LE Campbell Eric L EL Kelly Caleb J CJ Bowers Brittelle E BE Bayless Amanda J AJ Colgan Sean P SP

Journal of immunology (Baltimore, Md. : 1950) 20121203 1

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 (Cul)-RING ligases, which play a central role in the stabilization of NF-κB and hypoxia-inducible factor (HIF). In these studies, we identify the human deneddylase-1 (SENP8) as a key regulator of Cul neddylation response in vitro and in vivo. Using human microvascular endothelial cells (HMECs), we exa ...[more]

PMID: 23209320

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Project description:AimsHypoxia induces vascular inflammation by a mechanism not fully understood. Emerging evidence implicates O-GlcNAc transferase (OGT) in inflammation. This study explored the role of OGT in hypoxia-induced vascular endothelial inflammatory response.Methods and resultsHypoxia was either induced (1% O2 chamber) or mimicked by exposure to hypoxia-mimetic agents in cultured endothelial cells. Hypoxia increased hypoxia-inducible factor (HIF-1?) and inflammatory response (gene and protein expression of interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and E-selectin) but, surprisingly, reduced OGT protein (not mRNA) levels. Hypoxia-mimetic CoCl2 failed to reduce OGT when proteasome inhibitors were present, suggesting proteasome involvement. Indeed, CoCl2 enhanced 26S proteasome functionality evidenced by diminished reporter (Ub(G76V)-GFP) proteins in proteasome reporter cells, likely due to increased chymotrypsin-like activities. Mechanistically, ?-TrCP1 mediated OGT degradation, since siRNA ablation of this E3 ubiquitin ligase stabilized OGT. Administration of the oxidative stress inhibitors reversed both proteasome activation and OGT degradation. Furthermore, up-regulation of OGT by stabilization, overexpression, or activation mitigated CoCl2-elicited inflammatory response. These observations were recapitulated in a mouse (C57BL/6J) model mimicking hypoxia, in which lung tissues presented higher levels of HIF-1?, proteasome activity, and inflammatory response, but lower levels of OGT (n = 5/group, hypoxia vs. normoxia, P < 0.05). However, administration of an activator of OGT (glucosamine: 1 mg/g/day, vehicle: saline, ip, 5 days) abolished the up-regulation of proteasome activity and inflammatory response (n = 5/group, the treated vs. untreated hypoxia groups, P < 0.05).Conclusions26S proteasome-mediated OGT reduction contributed to hypoxia-induced vascular endothelial inflammatory response. Modulation of OGT may represent a new approach to treat diseases characterized by hypoxic inflammation.

Project description:As a key homeostasis regulator in mammals, the MERTK receptor tyrosine kinase is crucial for efferocytosis, a process that requires remodeling of the cell membrane and adjacent actin cytoskeleton. Membrane and cytoskeletal reorganization also occur in endothelial cells during inflammation, particularly during neutrophil transendothelial migration (TEM) and during changes in permeability. However, MERTK's function in endothelial cells remains unclear. This study evaluated the contribution of endothelial MERTK to neutrophil TEM and endothelial barrier function. In vitro experiments using primary human pulmonary microvascular endothelial cells found that neutrophil TEM across the endothelial monolayers was enhanced when MERTK expression in endothelial cells was reduced by siRNA knockdown. Examination of endothelial barrier function revealed increased passage of dextran across the MERTK-depleted monolayers, suggesting that MERTK helps maintain endothelial barrier function. MERTK knockdown also altered adherens junction structure, decreased junction protein levels, and reduced basal Rac1 activity in endothelial cells, providing potential mechanisms of how MERTK regulates endothelial barrier function. To study MERTK's function in vivo, inflammation in the lungs of global Mertk-/- mice was examined during acute pneumonia. In response to P. aeruginosa, more neutrophils were recruited to the lungs of Mertk-/- than wildtype mice. Vascular leakage of Evans blue dye into the lung tissue was also greater in Mertk-/- mice. To analyze endothelial MERTK's involvement in these processes, we generated inducible endothelial cell-specific (iEC) Mertk-/- mice. When similarly challenged with P. aeruginosa, iEC Mertk-/- mice demonstrated no difference in neutrophil TEM into the inflamed lungs or in vascular permeability compared to control mice. These results suggest that deletion of MERTK in human pulmonary microvascular endothelial cells in vitro and in all cells in vivo aggravates the inflammatory response. However, selective MERTK deletion in endothelial cells in vivo failed to replicate this response.

Dataset Information

Central role for endothelial human deneddylase-1/SENP8 in fine-tuning the vascular inflammatory response.

Publications

Central role for endothelial human deneddylase-1/SENP8 in fine-tuning the vascular inflammatory response.

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