Inflammation can be defined as a complex set of interactions among soluble factors and cells that arise in any tissue in response to traumatic, infectious, postischemic, toxic, or autoimmune injury (30
). The inflammatory process normally leads to recovery; however, if not properly phased, it could lead to tissue damage. Inflammation is commonly seen in many pulmonary disorders, including high altitude pulmonary edema (31
) and hypoxia-induced pulmonary hypertension (32
Inflammatory pathways are regulated by a limited number of transcription factors, the most important being NF-κB (33
). Upon cellular stimulation by a variety of mediators, including cytokines, bacterial toxins, or oxidative stress, a signal transduction cascade is activated, leading to the phosphorylation of IκBα on Ser32 and -36 by the multimeric IKK complex (34
). Phosphorylation of IκBα is followed by ubiquitination via the E3 ligase SCFβTRCP
and is targeted for proteasomal degradation by the 26S proteasome (35
). Once IκBα is degraded, NF-κB translocates to the nucleus and binds to the promoter regions of several proinflammatory genes, inducing their expression and thus amplifying the inflammatory response. The extent of NF-κB activation depends on diverse factors, including the variable E3 activity of the SCFβTRCP
complex, which is regulated by a reversible covalent modification with the ubiquitin-like protein Nedd8 (36
In the current study, we have assessed the role of Ado as an antiinflammatory molecule released during HPC. Our studies revealed that cells subjected to HPC display a significant attenuation of NF-κB activation compared with those exposed to anoxia alone. A soluble mediator found to be released during HPC was identified as Ado. These findings are consistent with our findings in vivo that lungs of mice subjected to HPC prior to more severe hypoxia display no signs of perivascular hemorrhage, a precursor to NF-κB–mediated inflammation (37
A recent study by Majumdar and Aggarwal (39
) identified Ado as a suppressor of NF-κB activity induced by TNF-α and concluded that it may contribute to its role in suppression of inflammation of the immune system. We further showed that Ado analogs such as NECA are capable of inhibiting NF-κB activation as measured by IκBα degradation by the potent NF-κB activator TNF-α. The exact mechanism by which Ado inhibits NF-κB activation was previously unknown. Our studies focused on one pathway involved in the regulation of NF-κB activation, the ubiquitination of IκB via the E3 ligase SCFβTRCP
. Ado and NECA analogs displayed a dose-dependent deneddylation of Cul-1 (Figure A). It has been demonstrated that the SCF complex is active only when Cul-1 is covalently modified by the ubiquitin-like protein Nedd8; therefore, the neddylation status of Cul-1 directly reflects NF-κB activation (40
). We further examined not only the neddylation status of Cul-1 but also, by direct immunoblotting against Nedd8, whether these effects were inhibited by the general Ado receptor antagonist 8-PT.
The COP9 signalosome (CSN), an 8-subunit complex that regulates protein ubiquitination, posttranscriptionally modifies the cullin subunit of E3-ubiquitin ligases by cleaving off the covalently coupled protein Nedd8 (5
). Of the 8 different subunits of CSN, CSN5/JAB1, which has metalloprotease activity, has been established to be important in the role of deneddylating Cul-1. The cleavage of the Nedd8 moiety is catalyzed not by a conventional thiol protease, but rather by metalloisopeptidase activity centered within the CSN5 subunit (5
). In our studies, downregulation of CSN5/JAB1 by siRNA inhibited NECA-induced Cul-1 deneddylation and resulted in a loss of preconditioning as measured by NF-κB activation (Figure , E and F). These findings confirm the role of Ado released during HPC in conferring an antiinflammatory phenotype through influences on CSN5-mediated neddylation of Cul-1.
The findings were tested in vivo to further elucidate the pathway by which Ado released during HPC confers antiinflammatory protection via NF-κB inhibition. Our in vivo findings are consistent with those of in vitro studies, particularly those showing that Cul-1 is deneddylated following HPC and that stabilization of IκB is maintained. Morphologically and histologically, evidence of antiinflammation was indicated by the gross size and water content of the lung as well as the protection conferred against hemorrhage. Hemorrhage has been well established to be a precursor to NF-κB–mediated inflammation (37
). Hemorrhage consists of not only red blood cells, but also plasma, which contributes to pulmonary edema. H&E staining did not reveal plasma infiltration in the alveolar spaces, but edema was evidenced in the wet/dry ratio. With regard to the direct role of NF-κB inhibition in this model, it is difficult to assess the exact contribution of Ado. For example, a number of drugs that have been shown to inhibit NF-κB activation (e.g., pyrrolidine dithiocarbamate) can result in tissue phenotypes and outcomes that closely resemble HPC (41
). However, none of the known NF-κB inhibitors used in in vivo settings have sufficient specificity to convincingly distinguish the role of NF-κB in preconditioning. A recent study has shed new light on this topic. Frantz et al. utilized NF-κB p50 subunit knockout mice to investigate the role of NF-κB in myocardial preconditioning (42
). These studies revealed a crucial role for p50 in damage resulting from myocardial infarction. Multiple levels of analysis revealed that NF-κB is central to endpoint tissue damage associated with infarction and, likewise, that inhibition of NF-κB is central to the phenotype of ischemic preconditioning. Taken together, these results strongly support our ongoing hypothesis that Ado-mediated inhibition of NF-κB promotes a protective phenotype during preconditioning.
As proof of principle, Cd73–/– mice, which lack the ectonucleotidase to convert AMP to extracellular Ado, do not precondition to hypoxia as compared with wild-type littermates. In these animals, lung water is not decreased, Cul-1 is not deneddylated, and there are no changes in IκB levels. Taken together, our results clarify the importance of Ado as an antiinflammatory molecule released during HPC and its role downstream on the inactivation of the NF-κB pathway.
Cumulatively, these results provide new insight into the mechanisms of actions of Ado and, by association, antiinflammatory aspects of lung HPC. Whether such findings are universal for tissues other than lung is not clear at present. Since individual tissues and cells are known to express different Ado receptor profiles (10
), it is likely that specific tissues responses will depend on the relative density of individual Ado receptors. Studies using transient transfection assays identified primarily Ado A2B, and to a lesser extent A1 receptors, in Cul-1 deneddylation. Our previous studies have implicated A2B receptors in vascular and inflammatory protection (14
). These observations have been made primarily through the use of available pharmacological inhibitors, as well as molecular tools for in vitro settings. More specific inhibitors as well as A2B receptor–null mice have now become available, which will allow some of these questions to be answered, and the results, at least in part, support our ongoing hypothesis that Ado liberated at sites of inflammatory hypoxia protects via A2B receptor activation. In particular, Yang et al. demonstrated that A2B receptor–null mice are prone to inflammation and that acute inflammatory endpoints (e.g., leukocyte adhesion to the vasculature) are augmented in sepsis models (45
). These studies revealed the likelihood that both bone marrow and vascular A2B receptors contribute to this phenotype. Conversely, in more chronic models of inflammation, A2B receptors may be detrimental to disease progression. For instance, Sun et al. utilized the highly specific A2B receptor antagonist CVT-6883 and revealed that following systemic administration, both wild-type and Ado deaminase–deficient animals showed less pulmonary fibrosis and fewer inflammatory endpoints (46
). More work will be necessary to define potential differences between acute and chronic responses mediated by the A2B receptor.
Neddylation/deneddylation responses are highly conserved and appear to be universal in all cell types examined (27
). Deneddylation reactions on cullin targets via CSN-associated proteolysis is increasingly implicated as a central point for cullin-mediated E3 ubiquitination (47
). Notably, other pathways for deneddylation have been reported. For example, the recent identification of the Nedd8-specific proteases NEDP1 and DEN1 (48
) have provided new insight into this emerging field. NEDP1/DEN1 appear to have isopeptidase activity capable of directly deneddylating cullin targets (49
). Determining whether Ado receptor activation might influence NEDP1/DEN1 activity should prove interesting. Overall, our findings here that Ado deneddylates Cul-1 provide new insight into the use of selective Ado receptor analogs as antiinflammatory-based therapies.