Mechanical induction of an epithelial cell chymase associated with wound edge migration.
Chymase is a chymotrypsin-like serine protease predominantly produced by mast cells. In this study, human cutaneous and gingival keratinocytes, ovary surface epithelia, and a porcine epithelial cell line were assayed by homology-based cloning, and the amplified DNA fragment was identified as a chymase. In vitro, chymase could not be induced by serum or cytokine treatment alone. Chymase was activated 3-fold within 60 min in basal media by scratch wounding cultured monolayers and further potentiated over 10-fold at 18 h by additional serum and cytokine treatment. Chymase activity was cell-associated and found to peak within 24 h of wounding and then steadily decreased as cultures healed, reaching baseline levels before confluence was reestablished. Affinity column purified enzyme effectively degraded fibronectin and was found by Western blot analysis using a human chymase antibody to be of about 30 kDa. Immunostaining revealed chymase activation at the wound edge colocalizing with reactive oxygen species generation. Specifically, chymase activation was attenuated by inhibition of nitric oxide, superoxide, and peroxynitrite. Exogenous peroxynitrite but not hydrogen peroxide also resulted in chymase activation in unwounded monolayers. Disruption of cytoskeletal stress fibers by cytochalasin D attenuated both wound-activated chymase and reactive oxygen species generation. Chymase inhibitor chymostatin reduced the loss of cell-cell contacts and the onset of porcine and human skin epithelial cell migration at the wound edge. This shows that an epithelial chymase is rapidly activated by a ligand-independent mechanism following mechanical stress via cytoskeletal and reactive oxygen species signaling and is associated with the onset of epithelial cell migration.
Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver V6T 1Z3, British Columbia, Canada.
James D
JD
Firth
Veli-Jukka
VJ
Uitto
Edward E
EE
Putnins
D000595:Amino Acid Sequence; D000818:Animals; D002465:Cell Movement; D053818:Chymases / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013481:Superoxides / Q000378:metabolism; D014945:Wound Healing
D017382:Reactive Oxygen Species; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D053818:Chymases