Molecular mechanisms of synergy of corneal muscarinic and nicotinic acetylcholine receptors in upregulation of E-cadherin expression.

Corneal epithelial erosion is one of the most common problems in clinical ophthalmology. Despite significant progress in understanding how the cornea heals, clinically available pharmacological therapies that can promote repair and prevent visual complications remain limited. We have recently demonstrated that the acetylcholine (ACh) axis of corneal epithelium plays an important role in regulation and coordination of distinct activities of corneal epithelial cells (CEC) mediating re-epithelialization, but mechanisms remained unclear. We hypothesized that the grounds for synergistic effects of corneal ACh receptors lie within the signaling pathways linking different receptors to specific elements of the CEC pro-epithelialization activities. In this study, we sought to elucidate the molecular mechanisms of cooperation of corneal muscarinic and nicotinic ACh receptors (mAChRs and nAChRs) in upregulation of E-cadherin expression. The roles of individual corneal mAChRs and nAChRs subtypes were investigated by in-cell western assay of the ACh-treated CEC, in which different ACh receptor genes were silenced by receptor-specific shRNAs. Functional inactivation of M3, but not M4, mAChR subtype, or α3 or α7, but not α9, nAChR subunit significantly inhibited E-cadherin expression. To gain a mechanistic insight, we blocked the key steps of the downstream signaling pathways. Results demonstrated that cholinergic agonists can upregulate E-cadherin expression by activating M3 mAChR, and α3β2 and α7 nAChRs via the common signaling cascade Ca(2+)-CaMKII-PKC-Ras-Raf-MEK-ERK. Activation of α7 nAChR can launch the Ras-Raf-MEK-ERK cascade both indirectly, through the Ca(2+)-CaMKII-PKC step, and directly, perhaps, due to its direct interaction with Ras. Although the biological significance of such redundancy remained to be elucidated, results of the present study point to a new direction to pharmacologically accelerate corneal re-epithelialization, and should have salient clinical implication.