Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation
Joseph Khoury, Juan C. Ibla, Andrew S. Neish, Sean P. Colgan
Joseph Khoury, Juan C. Ibla, Andrew S. Neish, Sean P. Colgan
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Research Article Vascular biology

Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation

Abstract

A major adaptive pathway for hypoxia is hypoxic preconditioning (HPC), a form of endogenous protection that renders cells tolerant to severe challenges of hypoxia. We sought to define the antiinflammatory properties of HPC. cDNA microarray analysis of lung tissue from mice subjected to hypoxia or HPC identified a cluster of NF-κB–regulated genes whose expression is attenuated by HPC. Studies using an NF-κB luciferase reporter assay confirmed a significant suppression of NF-κB activation during HPC. HPC-elicited activity was conferrable, as a soluble supernatant from HPC-treated cells, and the active fraction was purified and identified as adenosine (Ado). Guided by recent studies demonstrating bacterial inhibition of NF-κB through cullin-1 (Cul-1) deneddylation, we found a dose-dependent deneddylation of Cul-1 by Ado receptor stimulation predominantly mediated by the Ado A2B receptor subtype. Further, siRNA-mediated repression of CSN5, a subunit of the COP9 signalosome responsible for deneddylation of Cul-1, partially reversed HPC-mediated inhibition of NF-κB. Cul-1 deneddylation was evident in a murine model of HPC and lost in animals lacking extracellular Ado (Cd73–/– mice). Taken together, these results demonstrate that HPC induces extracellular accumulation of Ado and suppresses NF-κB activity through deneddylation of Cul-1. These results define a molecular regulatory pathway by which Ado provides potent antiinflammatory properties.

Authors

Joseph Khoury, Juan C. Ibla, Andrew S. Neish, Sean P. Colgan

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Figure 4

Exogenous Ado acts like HPC and results in the deneddylation of Cul-1, and siRNA-mediated repression of the CSN5 component of the COP9 signalosome inhibits Cul-1 deneddylation.

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Exogenous Ado acts like HPC and results in the deneddylation of Cul-1, a...
Antibodies against Cul-1 reveal 2 bands: a lower unmodified Cul-1 protein and an upper, neddylated form, Cul-1NEDD8. HeLa cell lysates were prepared as described in Methods, separated on 8% SDS-PAGE, and blotted against Cul-1, Nedd8, or actin (A–C). Log dose response of NECA (100 μM to 100 pM; 30 minutes) in HeLa (A) or CaLu-3 cells (B) shows potent deneddylation of Cul-1. Deneddylation could be seen with as little as 1 nM NECA; EC50 is estimated to be 30 nM. (C) Preincubation with 8-PT (10 nM; 15 minutes) inhibits the deneddylation effect on Cul-1 by NECA at both 100-μM and 10-μM doses (30 minutes). (D) Inhibition by HPC in cells overexpressing individual Ado receptors as compared to cells expressing endogenous levels of Ado receptors and examined for inhibition of NF-κB. Data are expressed as percent inhibition of NF-κB activation ± SEM for individual receptors; *P < 0.05, #P < 0.01. (E) siRNA targeted against the CSN5 component of the COP9 signalosome results in approximately 70% reduction in protein levels when blotted against a CSN5 antibody. (F) NECA-mediated (10 mM; 30 minutes) deneddylation of Cul-1 was inhibited in cells with knockdown of CSN5 (siCSN5) compared with cells that had not been knocked down (Empty). (G) CSN5 knockdown of cells (white bars, siCSN5) does not affect NF-κB activity in control or hypoxic cells, however, it causes HPC/hypoxia cells to lose their ability to attenuate NF-κB. *P < 0.05, HPC/hypoxia versus hypoxia alone.