Co-inhibition of CD73 and a2bar improves long-term cigarette smoke exposure impaired airway wound repair

Adenosine (ADO) involvement in lung injury depends on the activation of its receptors (A1, A2A, A2B, A3). The A2A receptor (A2AAR) and A2B receptor (A2BAR) are best described to have both tissue-protective and tissue-destructive processes. However, no approach has been effective in delineating the mechanism(s) involved with ADO shifting from its tissue-protective to tissue-destructive properties in chronic airway injury. Using cigarette smoke (CS) as our model of injury, we hypothesized that chronic CS exposure increases CD73-generated ADO which, activates low affinity A2BAR, subsequently impairing airway wound repair. To test this hypothesis, we chronically exposed human bronchial epithelial cell line, Nuli-1 cells, to 5% CS extract (CSE) for three years establishing a long-term CSE exposure model (LTC). We found significant morphological changes, decreased proliferation, and migration resulting in impaired airway wound closure in LTC. Further investigations showed that CSE exposure upregulates CD73 and A2BAR expression, increases ADO production, inhibits PKC alpha activity and phosphorylation of MEK, ERK, p90RSK and CREB. Moreover, when both or either of A2BAR and CD73 are knocked down in LTC, PKC alpha is activated and there is an increase in phosphorylation of MEK, ERK, p90RSK and CREB. Collectively, long-term CSE exposure upregulates CD73 expression and increases ADO production, which promotes A2BAR activation and subsequent diminution of PKC alpha activity and ERK signaling pathway, and inhibition of airway wound repair. Moreover, the data suggested that targeting both A2BAR and CD73 as potential therapeutic targets may be more efficacious in improving chronic CS impaired wound repair than either alone. Nuli-1 cells were treated 1 day in normal media, and then two days in media containing 5% CSE, cells (designated LTC) were passaged every 3 days. Control Nuli-1 cell (designated LTM) culture in normal media and passaged at same time as LTC. Both LTM and LTC are cultured for more than 3 years.