PAF-induced furin and MT1-MMP expression is independent of MMP-2 activation in corneal myofibroblasts.
OBJECTIVE
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.
Ottino
Paulo
P
Department of Ophthalmology and Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA.
He
Jiucheng
J
Axelrad
Thomas W
TW
Bazan
Haydee E P
HE
D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D003319:Corneal Stroma / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D019084:Fluorescent Antibody Technique, Indirect; D045683:Furin / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D020778:Matrix Metalloproteinase 2 / Q000235:genetics* / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism
D000199:Actins; D010972:Platelet Activating Factor; D012333:RNA, Messenger; D019716:Tissue Inhibitor of Metalloproteinase-2; D045683:Furin; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9