Mast cell tryptase activates extracellular-regulated kinases (p44/p42) in airway smooth-muscle cells: importance of proteolytic events, time course, and role in mediating mitogenesis.
We previously reported that mast cell tryptase is a potent mitogen for cultured airway smooth-muscle cells, but the early intracellular signals mediating this response are not known. In many cells, proliferative effects are mediated by a mitogen-activated protein kinase signaling pathway involving Raf-1, MAP kinase kinases (MEKs), and extracellular signal-regulated protein kinases (ERKs) 1 and 2. Therefore, we tested for tryptase-induced activation of ERK1 and 2 in cultured dog tracheal smooth-muscle cells. Tryptase, in nanomolar concentrations which potently stimulated DNA synthesis, increased dual phosphorylation of ERKs in cellular lysates as well as ERK2 kinase activity in immunoprecipitates. Pretreatment of cells with the MEK inhibitor PD098059 abolished tryptase-induced increases in DNA synthesis and attenuated increases in ERK2 activity. Irreversible inhibition of tryptase's proteolytic activity, using p-amidino phenylmethanesulfonyl fluoride, attenuated tryptase-induced increases in DNA synthesis and dual phosphorylation of ERKs by 76% and 40 to 60%, respectively. Tryptase also increased c-fos transcription as quantified in polymerase chain reactions. In concentrations that caused similar increases in DNA synthesis, tryptase and platelet-derived growth factor (PDGF-BB) increased ERK activity (and c-fos transcription) with markedly different kinetics, the tryptase-induced responses being slower in onset and more sustained. We conclude that tryptase-induced mitogenesis in airway smooth-muscle cells requires activation of ERK1 and 2; that these responses depend partially, but not completely, upon tryptase's properties as a protease; and that they are slower in onset and more sustained than those induced by PDGF-BB.
Brown
J K
JK
Pulmonary and Critical Care Medicine Section (111-D), Department of Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121, USA. jkbrown@itsa.ucsf.edu
Jones
C A
CA
Rooney
L A
LA
Caughey
G H
GH
D000818:Animals; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D048369:MAP Kinase Kinase 1; D008407:Mast Cells / Q000201:enzymology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000031:analogs & derivatives* / Q000494:pharmacology; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000009:adverse effects / Q000494:pharmacology*; D013997:Time Factors; D014132:Trachea / Q000166:cytology / Q000201:enzymology; D053802:Tryptases
D017931:DNA Primers; D004791:Enzyme Inhibitors; D005419:Flavonoids; D011480:Protease Inhibitors; D016760:Proto-Oncogene Proteins c-fos; D010664:Phenylmethylsulfonyl Fluoride; C027226:(4-amidinophenyl)methanesulfonyl fluoride; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D012697:Serine Endopeptidases; D053802:Tryptases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one