Central role of alpha9 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 alpha9 AChR subtype that had been shown to control cell shape and cytoplasm mobility. Inactivation of alpha9 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 alpha9 in assembly/disassembly of the cell-cell and cell-matrix adhesion complexes. Stimulation of alpha9 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 alpha9 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 alpha9 AChR is critical for completion of the very early stages of epithelialization. By activating alpha9 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.
Chernyavsky
Alex I
AI
Department of Dermatology, University of California Irvine, C340 Medical Sciences I, Irvine, CA 92697, USA.
Arredondo
Juan
J
Vetter
Douglas E
DE
Grando
Sergei A
SA
D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D006801:Humans; D007231:Infant, Newborn; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D010641:Phenotype; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013997:Time Factors; D014945:Wound Healing / Q000187:drug effects
D003598:Cytoskeletal Proteins; D011978:Receptors, Nicotinic; C443079:nAChR alpha9; D017228:Hepatocyte Growth Factor; D011505:Protein-Tyrosine Kinases
J Biol Chem. 2005 Nov 25;280(47):39220-8
16150734
Brain Res. 1984 Dec 24;324(2):305-12
6529621
J Invest Dermatol. 1996 Apr;106(4):590-7
8617990
J Cell Biol. 1999 Nov 29;147(5):1009-22
10579721
Am J Pathol. 2000 Oct;157(4):1377-91
11021840
J Neurosci. 2005 Nov 23;25(47):10905-12
16306403
J Invest Dermatol. 1993 Dec;101(6):804-10
8245509
J Cell Sci. 2004 Aug 1;117(Pt 17):3759-68
15252114
Cell Motil Cytoskeleton. 1995;30(2):108-21
7606804
Exp Cell Res. 2004 Apr 1;294(2):534-49
15023540
Curr Opin Cell Biol. 2003 Oct;15(5):590-7
14519394
J Cell Sci. 2004 Mar 15;117(Pt 8):1339-50
14996911
Skin Pharmacol. 1993;6(2):135-47
8352951
Nat Cell Biol. 2003 Mar;5(3):236-41
12598902
Cell Motil Cytoskeleton. 2003 May;55(1):14-25
12673595
Mol Cell Biol. 1997 Dec;17(12):6906-14
9372922
Cell Calcium. 2006 Apr;39(4):349-55
16451809
Biochem J. 2005 Oct 15;391(Pt 2):359-70
15975092
FASEB J. 2006 Oct;20(12):2093-101
17012261
Mol Biol Cell. 2002 Jun;13(6):1857-70
12058054
Eur J Immunol. 1999 Jul;29(7):2233-40
10427986
J Invest Dermatol. 2004 Nov;123(5):937-49
15482483
Mol Pharmacol. 2002 Jan;61(1):150-9
11752216
Cell Adhes Commun. 1999;7(2):125-38
10427965
Br J Dermatol. 1990 Jun;122(6):777-84
2369558
Curr Opin Cell Biol. 1999 Apr;11(2):274-86
10209151
Neuropharmacology. 2000 Oct;39(13):2515-24
11044723
J Invest Dermatol. 1999 Jan;112(1):67-71
9886266
Cell. 1994 Nov 18;79(4):705-15
7954834
Neuron. 1999 May;23(1):93-103
10402196
Cell Death Differ. 1996 Oct;3(4):357-71
17180106
Acta Otolaryngol. 1999 Mar;119(2):185-8
10320073
J Cell Sci. 2004 Nov 1;117(Pt 23):5665-79
15494367
Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3501-6
11248107
Front Biosci. 2002 Jan 1;7:d143-50
11779709
J Cell Biol. 1989 Oct;109(4 Pt 1):1495-509
2677020
J Cell Physiol. 1998 Aug;176(2):255-65
9648913
J Biol Chem. 2002 May 24;277(21):18694-702
11907028
J Invest Dermatol. 1976 Jul;67(1):39-57
778296
J Cell Biol. 2006 Dec 4;175(5):721-7
17130286
Mol Pharmacol. 2003 May;63(5):1067-74
12695535
Endocrinology. 2002 Mar;143(3):744-6
11861491
Mol Pharmacol. 1973 Sep;9(5):630-9
4788157
J Cell Biol. 2002 Oct 28;159(2):325-36
12391028
J Cell Sci. 2003 Sep 1;116(Pt 17):3543-56
12865436
J Invest Dermatol. 1997 Apr;108(4):482-7
9077478
Mol Pharmacol. 1999 Feb;55(2):248-54
9927615
J Investig Dermatol Symp Proc. 1999 Sep;4(2):137-44
10536988
Mol Cell Biol. 2004 Dec;24(24):10558-72
15572663
J Cell Biol. 2004 Jul 19;166(2):261-72
15263021
J Neurosci Res. 1996 May 15;44(4):350-4
8739154
J Cell Biol. 1999 Mar 22;144(6):1235-44
10087266
J Cell Sci. 1997 Jan;110 ( Pt 1):23-33
9010781
J Invest Dermatol. 2006 Sep;126(9):1948-65
16912692
Hear Res. 2000 Mar;141(1-2):117-28
10713500
J Biol Chem. 2004 Jul 2;279(27):27965-70
15107421
Cell Adhes Commun. 1998 Jan;5(1):1-12
9638337
Exp Cell Res. 2004 Feb 1;293(1):1-13
14729052
J Invest Dermatol. 2006 Nov;126(11):2458-72
16810300
J Physiol Paris. 1998 Oct-Dec;92(5-6):345-9
9789835
J Comp Neurol. 1996 May 20;369(1):31-55
8723701
Genomics. 2001 May 1;73(3):272-83
11350119
Nat Rev Cancer. 2002 Jun;2(6):442-54
12189386
Exp Dermatol. 2006 Apr;15(4):265-82
16512874