The role of the Adora2B adenosine receptor in mediation of intestinal mucin production and epithelial defence

Introduction: Loss of mucosal barrier integrity and inappropriate perpetuation of the inflammatory response are key features of ulcerative colitis (UC). Increased permeability and disruption of the mucus gel layer (MGL) lining the colonic intestinal epithelium is observed prior to the onset of colitis in UC and murine colitis models. Therefore, understanding how the integrity of the MGL is maintained during health and disease may lead to novel therapeutic approaches for UC. The major constituents of the MGL are mucins which are secreted by the intestinal epithelium. Extracellular adenosine signalling can mediate mucin hypersecretion in asthma and other inflammatory respiratory conditions. The literature suggests that signalling via the Adora2a adenosine receptor (A2AAR) and Adora2b adenosine receptor (A2BAR) is protective in acute gastrointestinal inflammation such as colitis, although the mechanism by which this occurs, especially for the A2BAR, is unclear. We hypothesised that as in the respiratory epithelium, adenosine signalling in the gut may stimulate mucin synthesis thus enhancing the protective efficacy of the MGL.Methods Three representative human colonic epithelial cell lines (HT29-MTX-E12, Caco-2 and T84) were treated with A2 receptor agonists, selective antagonists and inhibitors of second messenger pathways. RT-PCR was performed to evaluate MUC2 and MUC5AC expression. Caco-2 cells transfected with MUC5AC promoter were treated as above and promoter activity evaluated by Dual-Luciferase™ assay. Two models to approximate GI mucosal injury were established using a HT29-MTX-E12 polarised monolayer and murine gastrointestinal organoids. After induction of injury with either scratch wound assay or exposure to interferon-gamma models were treated with A2B selective receptor agonist. For both models MUC2 and MUC5AC expression was evaluated by RT-PCR as previously. For the HT29-MTX-E12 model MUC5AC secretion was also evaluated by immunofluorescence staining with a MUC5AC specific antibody. Human colorectal healthy and neoplastic tissue was collected and RT-PCR performed to assess A2 receptor and mucin expression. Results: Treatment of Caco-2 and T84 colonic epithelial cell lines with A2B selective receptor agonist induced upregulation of MUC2 and MUC5AC, which was abolished by blockade of receptor signalling and inhibition of second messenger pathways. Similar results of these treatments were observed on MUC5AC promoter activity. In healthy HT29-MTX-E12 cells treatment with A2B agonist induced modest increases in MUC2 and MUC5AC expression, whereas significant increases were observed in a HT29-MTX-E12 model of epithelial injury. In this model of epithelial injury, selective A2BAR agonist also induced increased MUC5AC fluorescence intensity. In a murine enteroid model of gastrointestinal inflammation cellular injury appeared to increase expression of Muc5ac, although Muc2 expression was reduced. Finally, in neoplastic colorectal tissue a correlation between A2B receptor expression and MUC5AC expression was detected, supporting a role for the receptor in upregulation of MUC5AC production. Conclusion: Our results reveal that production of the mucins MUC2 and MUC5AC in the colonic epithelium is selectively modulated by the A2BAR via a classical G-protein-coupled receptor (GPCR) signalling pathway. In two advanced models of gastrointestinal injury, differential effects of A2BAR activation are observed. In a scratch wound assay model, A2BAR activation stimulates increased mucin expression. In a murine enteroid injury model, however, variable effects on mucin expression are observed. In neoplasia, there is a positive correlation between A2B receptor and MUC5AC expression. These novel findings further elucidate mechanisms of intestinal epithelial defence and may provide a basis for future pharmacologically targeted therapeutic interventions for IBD.