Purpose: To understand how epidermal keratinocytes (KC) control their own migratory function and exploit this physiologic mechanism to affect wound closure with cholinergic drugs in future clinical studies. Background: KC, similarly to neurons, form local communication networks wherein acetylcholine (ACh) regulates vital functions of KC, including migration, by activating different types of ACh receptors (AChRs). Rationale: Keratinocyte migration is a self-regulated process in which cell activities mediating migration are controlled, in part, by a single """"""""pace-maker"""""""" system featuring autocrine, juxtacrine and paracrine ACh as a chemokine for cell movement. The differential control of cellular activities mediating migration are mediated by different types of keratinocyte nicotinic and muscarinic AChRs (nAChRs and mAChRs), respectively. Major goal: To understand contribution of each keratinocyte AChR type to wound re-epithelialization. Working Hypotheses: 1) The chemotactic effect of ACh on KC is mediated by activation of alpha3 nAChR. 2) The m4 mAChR stimulates migration, alpha7 nAChR inhibits it, and alpha9 nAChR controls assembly/disassembly of focal adhesions in crawling KC.
Specific Aims : 1) To identify keratinocyte AChRs that mediate chemotaxis of KC toward ACh gradient in experiments with receptor-selective cholinergic agonists and antagonists that will either elicit or block, respectively, directional migration of human KC and murine KC grown from the alpha3, alpha7, alpha9 nAChR or m4 mAChR knockout mice. 2) To identify contribution of each AChR type to cholinergic control of the basic intracellular events mediating migration of KC over specific extracellular matrix proteins. The wild type, and AChR-deficient KC will be used in pharmacologic and molecular biological assays of ionic and metabolic events coupled by nAChR and mAChR types. All experiments will be performed in an in vitro model of skin re- epithelialization that allows studies of pharmacologic compounds on random, and concentration gradient-and DC electric field- elicited directional migration of KC. We will demonstrate AChR- specific cholinergic effects on membrane potential, intracellular Ca2+, hydrogen, and cAMP levels, as well as actin filament polymerization, assembly/disassembly of focal adhesions, and integrin receptor expression in crawling KC. Significance: We will show how nAChRs and mAChRs differentially modulate the function and migration of KC in wound healing which will offer novel solutions for management of skin wounds that fail to heal.
Showing the most recent 10 out of 23 publications
