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 MI mAChR and the a3|34 and a7 nAChRs act synergistically in mediating chemotaxis via Ca2+-dependent alterations in the phosphorylation status of cadherins and sedentary integrins. M4 mAChR and a3 nAChR facilitate migration by upregulating migratory integrins through protein kinase (PK) CS-dependent pathway, whereas a9 nAChR prompts assembly/disassemblyof cell-cell and cell- substrate bonds. The aS-containing ?3 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062136-08
Application #
7748970
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Hagan, Ann A
Project Start
2000-12-01
Project End
2011-12-31
Budget Start
2010-01-01
Budget End
2011-12-31
Support Year
8
Fiscal Year
2010
Total Cost
$267,978
Indirect Cost
Name
University of California Irvine
Department
Type
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Grando, Sergei A (2012) Muscarinic receptor agonists and antagonists: effects on keratinocyte functions. Handb Exp Pharmacol :429-50
Chernyavsky, Alexander I; Kalantari-Dehaghi, Mina; Phillips, Courtney et al. (2012) Novel cholinergic peptides SLURP-1 and -2 regulate epithelialization of cutaneous and oral wounds. Wound Repair Regen 20:103-13
Chernyavsky, Alexander I; Arredondo, Juan; Skok, Maryna et al. (2010) Auto/paracrine control of inflammatory cytokines by acetylcholine in macrophage-like U937 cells through nicotinic receptors. Int Immunopharmacol 10:308-15
Marchenko, Steve; Chernyavsky, Alexander I; Arredondo, Juan et al. (2010) Antimitochondrial autoantibodies in pemphigus vulgaris: a missing link in disease pathophysiology. J Biol Chem 285:3695-704
Chernyavsky, Alexander I; Arredondo, Juan; Galitovskiy, Valentin et al. (2010) Upregulation of nuclear factor-kappaB expression by SLURP-1 is mediated by alpha7-nicotinic acetylcholine receptor and involves both ionic events and activation of protein kinases. Am J Physiol Cell Physiol 299:C903-11
Chernyavsky, Alexander I; Arredondo, Juan; Qian, Jing et al. (2009) Coupling of ionic events to protein kinase signaling cascades upon activation of alpha7 nicotinic receptor: cooperative regulation of alpha2-integrin expression and Rho kinase activity. J Biol Chem 284:22140-8
Hsu, Daniel K; Chernyavsky, Alexander I; Chen, Huan-Yuan et al. (2009) Endogenous galectin-3 is localized in membrane lipid rafts and regulates migration of dendritic cells. J Invest Dermatol 129:573-83
Chernyavsky, Alex I; Arredondo, Juan; Piser, Timothy et al. (2008) Differential coupling of M1 muscarinic and alpha7 nicotinic receptors to inhibition of pemphigus acantholysis. J Biol Chem 283:3401-8
Chernyavsky, Alex I; Arredondo, Juan; Vetter, Douglas E et al. (2007) Central role of alpha9 acetylcholine receptor in coordinating keratinocyte adhesion and motility at the initiation of epithelialization. Exp Cell Res 313:3542-55
Hasse, Sybille; Chernyavsky, Alex I; Grando, Sergei A et al. (2007) The M4 muscarinic acetylcholine receptor plays a key role in the control of murine hair follicle cycling and pigmentation. Life Sci 80:2248-52

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