Priming of STAT1 and STAT3 for cytokine-triggered degradation by the proteasome upon A2Aadenosine receptor (A2AAR) expression

The A2A adenosine receptor (A2AAR) functions as a key non-redundant suppressor of inflammatory responses in vivo. However, whether it regulates activation of the JAK-STAT pathway utilised by many pro-inflammatory cytokines is unknown. Using a vascular endothelial cell model system, I have demonstrated that adenovirus-mediated expression of the human A2AAR conferred an ability of IFNα, leptin and a soluble IL- 6 receptor-α/IL-6 (sIL-6Rα/IL-6) trans-signalling complex to promote a time-dependent reduction in the levels of STAT proteins that was entirely due to proteasomal degradation. In terms of functional consequences, degradation was sufficient to attenuate sIL-6Rα/IL-6-stimulated STAT3-dependent up-regulation of vascular endothelial growth factor receptor-2 (VEGFR-2) and enhance eNOS expression. Degradation required JAK activity since A) it was blocked by preincubation with JAK inhibitor. B) STAT1 but not STAT3 was resistant to both tyrosine phosphorylation and down-regulation in response to leptin and C) a Tyr705→Phe mutated STAT3 was also resistant to cytokine-triggered degradation, suggesting that JAK-mediated phosphorylation of this residue is required to produce the effect. Consistent with this hypothesis, sIL-6Rα/IL-6 treatment of A2AAR-expressing cells resulted in the accumulation of polyubiquitylated endogenous and epitope-tagged recombinant wild-type but not Tyr705→ Phe-mutated STAT3. In addition the results show that inhibition of proteasome function was sufficient to block the inhibitory effect of the A2AAR on STAT3 phosphorylation, demonstrating that priming of STATs for degradation is the only mechanism responsible for the reduced cytokine-stimulated STAT phosphorylation observed in A2AAR-expressing cells. To date there is only one E3 ligase known for mediating STAT degradation which is SLIM protein. However, our results suggest the involvement of another E3 ubiquitin ligase in HUVECs, since we have been unable to detect SLIM message or protein in HUVECs under conditions in which STAT degradation occurs. In addition, while Tyr-phosphorylation is clearly the critical step in targeting STATs for degradation in A2AAR-expressing cells, it is unclear as to whether it functions simply as a classical phosphodegron, or whether the nuclear translocation that occurs as a result of phosphorylation is also important for localising the phosphorylated STAT dimer with the relevant E3 ubiquitin ligase. Together, these observations suggest a model whereby expression of the A2AAR in endothelial cells primes JAK-phosphorylated STATs for polyubiquitylation and subsequent degradation by the proteasome following cytokine treatment, and represents a previously unappreciated mechanism by which G-protein-coupled receptors can negatively regulate responsiveness to specific JAK-STAT-mobilising adipocytokines acting on the vascular endothelium