Central role of α9 acetylcholine receptor in coordinating keratinocyte adhesion and motility at the initiation of epithelialization

Epithelialization, a major component of wound healing, depends on keratinocyte adhesion and migration. Initiation of migration relies upon the ability of keratinocytes to free themselves from neighboring cells and basement membrane. The local cytotransmitter acetylcholine (ACh) controls keratinocyte adhesion and locomotion through different classes of ACh receptors (AChR). In this study, we explored signaling pathways downstream of the alpha 9 AChR subtype that had been shown to control cell shape and cytoplasm mobility. inactivation of alpha 9 signaling by pharmacologic antagonism and RNA interference in keratinocyte cultures and null mutation in knockout mice delayed wound re-epithelialization in vitro and in vivo, respectively, and diminished the extent of colony scattering and cell outgrowth from the megacolony. Although keratinocytes at the leading edge elongated, produced filopodia and moved out, most of them remained anchored to the substrate by long cytoplasmic processes that stretched during their migration instead of retracting the uropod. Since the velocity of keratinocyte migration was not altered, we investigated the role of alpha 9 in assembly/disassembly of the cell-cell and cell-matrix adhesion complexes. Stimulation of alpha 9 upregulated in a time-dependent fashion phosphorylation of the adhesion molecules comprising focal adhesions (FAK, paxillin) and intercellular junctions (beta-catenin, desmoglein 3) as well as cytokeratins. Stimulation of alpha 9 was associated with activation of phospholipase C, Src, EGF receptor kinase, protein kinase C, Rac and Rho, whereas inhibition of this receptor interfered with phosphorylation of adhesion and cytoskeletal proteins, and also altered cell-cell cohesion. We conclude that signaling through alpha 9 AChR is critical for completion of the very early stages of epithelialization. By activating alpha 9 AChR, ACh can control the dynamics and strength of cell-cell cohesion, disabling of a trailing uropod and disassembly and reassembly of focal adhesions, thus facilitating crawling locomotion. (c) 2007 Elsevier Inc. All rights reserved.