Purpose: To integrate structural and functional information about epidermal keratinocytes (KCs) during epithelialization with the knowledge on cholinergic mechanisms guiding KCs through epithelialization steps. Rationale: Epithelialization is a self-regulated process in which cell activities mediating adhesion and migration are controlled, in part, by a single """"""""pace-maker"""""""" system featuring autocrine, juxtacrine and paracrine acetylcholine (ACh) as a chemokine for cell motility. Background: KCs are simultaneously stimulated through two distinct types of cholinergic signaling pathways: the metabolic events, elicited by ACh binding to G protein-coupled muscarinic receptors (mAChRs), and the ionic events, mediated by ACh-gated ion channels (or nAChRs). This diversity allows the single cytotransmitter ACh to exert diverse effects on KCs at various stages of skin epithelialization. Working Hypotheses: The M1 mAChR and the alpha3beta4 and alpha7 nAChRs act synergistically in mediating chemotaxis via Ca2+dependent alterations in the phosphorylation status of cadherins and sedentary integrins. M4 mAChR and alpha3 nAChR facilitate migration by upregulating migratory integrins through protein kinase (PK) CS-dependent pathway, whereas alpha9 nAChR prompts assembly/disassembly of cell-cell and cell- substrate bonds. The alpha5-containing alpha3 nAChR increases the strength of cell-cell adhesion. M3 elicits stable cell-substrate attachment and terminates migration due to inactivation of the Rho-dependent events.
Specific Aims : 1) Identify ACh receptor subtypes regulating distinct cellular activities of KCs required for normal epithelialization. 2) Identify the ACh receptor-coupled signal transduction pathways mediating selective regulation of cellular activities comprising the keratinocyte migratory function. Methodology: A combination of molecular biological (real-time PCR; small interfering RNA; ACh receptor knockout mice), immunological (immunohistochemistry, Western blot, ELISA, flow cytometry), and pharmacological approaches will be employed to elucidate cholinergic regulation of epithelialization, using the assays of keratinocyte chemotaxis, chemokinesis, and cell-cell and cell-substrate attachments. Significance: Results of this study will lay a groundwork for designing therapeutic regimens able to treat the skin disease caused by abnormal epithelialization. DESCRIPTION: Recent research shows that the locally produced and released hormone-like molecule, ACh, may initiate, guide, and sustain skin cell motility in the wound bed. This makes ACh and drugs acting at the ACh receptors potentially powerful tools for wound healing. This study will identify potential therapeutic agents among cholinergic drugs acting upon specific types of ACh receptors to activate signaling pathways mediating keratinocyte functions during wound epithelialization.
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