Investigative ophthalmology & visual science · 2005
PAF-induced furin and MT1-MMP expression is independent of MMP-2 activation in corneal myofibroblasts.
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Ottino Paulo, He Jiucheng, Axelrad Thomas W, Bazan Haydee E P
Аннотация
PURPOSE: Corneal stromal myofibroblasts express the platelet-activating factor (PAF) receptor, but its role is unclear. In the present study, the effect of PAF on induction of metalloproteinases (MMPs) was investigated.
METHODS: Rabbit corneal myofibroblasts were identified by immunodetection of alpha-smooth muscle (alpha-SM)-actin. MT1-MMP, MMP-2, MMP-9, and tissue inhibitor of matrix metalloproteinase (TIMP)-2 were detected by immunofluorescence. Cells were treated with 100 nM cPAF, with or without the PAF antagonist BN 50730 or the furin inhibitor nona-D-arg-NH(2). Gene-expression levels for furin, urokinase plasminogen activator, MMP-2, MMP-9, MT1-MMP, and TIMP-2 were determined by real-time PCR. Protein expression was assessed by Western blot. MMP-2 and -9 activity was determined by gelatin zymography. Active MT1-MMP levels were measured by ELISA.
RESULTS: cPAF triggered significantly increased MT1-MMP, MMP-2, MMP-9, and TIMP-2 mRNA expression, followed by increased active MT1-MMP protein expression at 12 hours, whereas TIMP-2 protein increased at 24 hours. PAF also induced furin gene expression, followed by increased protein expression. Nona-D-arg-NH(2) blocked cPAF induction of MT1-MMP activity. PAF-treated myofibroblasts showed increased active MMP-9 protein, but unchanged MMP-2 activity. Pretreatment with BN 50730 blocked PAF-induced transcription and translation of these proteins.
CONCLUSIONS: PAF, through a receptor-mediated mechanism, induces a specific pattern of furin, MMP, and TIMP-2 expression in corneal myofibroblasts. MMP-2 activity was unchanged by PAF treatment. These results suggest that in response to the inflammatory mediator PAF, induction of MT1-MMP is independent of MMP-2 activity in corneal myofibroblasts. Thus, PAF-mediated changes in extracellular matrix composition surrounding the myofibroblasts could be important in regulating the corneal scarring process. Moreover, PAF antagonists could be useful in maintaining corneal transparency.
METHODS: Rabbit corneal myofibroblasts were identified by immunodetection of alpha-smooth muscle (alpha-SM)-actin. MT1-MMP, MMP-2, MMP-9, and tissue inhibitor of matrix metalloproteinase (TIMP)-2 were detected by immunofluorescence. Cells were treated with 100 nM cPAF, with or without the PAF antagonist BN 50730 or the furin inhibitor nona-D-arg-NH(2). Gene-expression levels for furin, urokinase plasminogen activator, MMP-2, MMP-9, MT1-MMP, and TIMP-2 were determined by real-time PCR. Protein expression was assessed by Western blot. MMP-2 and -9 activity was determined by gelatin zymography. Active MT1-MMP levels were measured by ELISA.
RESULTS: cPAF triggered significantly increased MT1-MMP, MMP-2, MMP-9, and TIMP-2 mRNA expression, followed by increased active MT1-MMP protein expression at 12 hours, whereas TIMP-2 protein increased at 24 hours. PAF also induced furin gene expression, followed by increased protein expression. Nona-D-arg-NH(2) blocked cPAF induction of MT1-MMP activity. PAF-treated myofibroblasts showed increased active MMP-9 protein, but unchanged MMP-2 activity. Pretreatment with BN 50730 blocked PAF-induced transcription and translation of these proteins.
CONCLUSIONS: PAF, through a receptor-mediated mechanism, induces a specific pattern of furin, MMP, and TIMP-2 expression in corneal myofibroblasts. MMP-2 activity was unchanged by PAF treatment. These results suggest that in response to the inflammatory mediator PAF, induction of MT1-MMP is independent of MMP-2 activity in corneal myofibroblasts. Thus, PAF-mediated changes in extracellular matrix composition surrounding the myofibroblasts could be important in regulating the corneal scarring process. Moreover, PAF antagonists could be useful in maintaining corneal transparency.
Предметные рубрики MeSH
ActinsAnimalsBlotting, WesternCorneal StromaEnzyme ActivationEnzyme-Linked Immunosorbent AssayFibroblastsFluorescent Antibody Technique, IndirectFurinGene Expression Regulation, EnzymologicMatrix Metalloproteinase 2Matrix Metalloproteinase 9Matrix Metalloproteinases, Membrane-AssociatedMetalloendopeptidasesPlatelet Activating FactorRNA, MessengerRabbitsReverse Transcriptase Polymerase Chain ReactionTissue Inhibitor of Metalloproteinase-2