A new mechanism in the inflammation pathway has been identified by HMS researchers working in mice. Specific adenosine receptors inactivated a subunit of the E3-SCF ubiquitin ligase complex, which prevented activation of the pro-inflammatory transcription factor nuclear factor kappa B (NF-kappa B). The findings could advance tissue-specific anti-inflammatory therapies, according to the study, reported in the March issue of the Journal of Clinical Investigation.
Joseph Khoury, a cell and molecular biologist and a former HMS instructor; Juan Ibla, HMS instructor in anesthesia at Children’s Hospital Boston; and colleagues used a model involving oxygen deprivation to induce inflammation in the lungs. In mice, 10 minutes of breathing five percent oxygen induces hypoxia and increases markers of inflammation, including NF-kappa B.
Activation of NF-kappa B can be suppressed by habituating the mice to low oxygen levels with a regimen of hypoxia preconditioning. The mice receive three 10-minute sessions in a chamber containing eight percent oxygen prior to their exposure to the hypoxic five percent oxygen treatment. Preconditioning prolongs survival in the otherwise deadly hypoxic environment. Though the adaptive properties of the regimen make it a good model for studying endogenous protection from inflammation, its molecular mechanisms have been unclear.
Analyzing lung tissue using microarrays and high-performance liquid chromatography, the researchers found that hypoxia preconditioning decreased NF-kappa B expression and promoted the release of the anti-inflammatory molecule adenosine. Based on previous work showing that adenosine directly suppresses NF-kappa B, the team sought to identify other players in the pathway. In particular, they investigated adenosine’s relationship to the uncoupling, or deneddylation, of the Cullin-1 (Cul-1) subunit in the E3-SCF complex, which activates NF-kappa B by turning off its inhibitor I kappa B. The researchers found that adenosine dose-dependently deneddylated Cul-1 from Nedd8, an effect they mimicked with an adenosine receptor analog. Overexpression of individual adenosine receptors revealed that A2B and, to a lesser extent, A1 receptors were particularly involved in the preconditioning-induced suppression of NF-kappa B. Other studies indicate that A2B receptors can protect against bone marrow and vascular inflammation, but not in chronic inflammatory conditions.
To verify their in vitro findings that adenosine causes Cul-1 deneddylation, Khoury, Ibla, and their colleagues compared lung water content, a measure of inflammation, between wild-type mice and mutants lacking the critical enzyme for adenosine formation. Following hypoxia preconditioning, the visibly swollen lungs of the mutant mice had about a 136 percent increase in water content compared to the wild types’ lungs. Immunoblotting revealed that preconditioning offered no protection against hypoxia-induced NF-kappa B expression and higher levels of neddylated Cul-1 in the adenosine-deficient mice.
Deneddylation equals anti-inflammation, summarized Khoury and Ibla of their novel finding that adenosine’s anti-inflammatory actions are due at least in part to deneddylating Cul-1.
