Platelet-activating factor overturns the transcriptional repressor disposition of Sp1 in the expression of MMP-9 in human corneal epithelial cells.
OBJECTIVE
Matrix metalloproteinase (MMP)-9 is induced in corneal epithelial cells stimulated with platelet-activating factor (PAF), and interferes with the normal reepithelialization of wounded cornea. Here the transcriptional regulation of MMP-9 gene expression by PAF was investigated in human corneal epithelial cells (HCECs).


METHODS
DNA-binding activity of NFkappaB, Sp1, and AP-1 was determined in quiescent and PAF-stimulated HCECs by electrophoretic mobility shift assay (EMSA). A series of 5' deleted human MMP-9 promoter-luciferase reporter constructs was transiently transfected into HCECs, and luciferase activity was examined after stimulation with PAF. Mutagenesis and specific deletions of some elements in the MMP-9 promoter were also introduced and analyzed. Phosphorylation of Sp1 and MEK/ERK pathway proteins was examined by Western blot analysis. Activation of Sp1 and MMP-9 was also determined by ELISA and zymography, respectively, in the absence or presence of the MEK inhibitor PD98059.


RESULTS
DNA-binding activity of NFkappaB, Sp1, and AP-1 was upregulated by PAF with a peak at 1 hour after stimulation. A region spanning -670 to -460 relative to the transcription start point was required for the induction of the MMP-9 promoter by PAF. Mutation of the -79AP-1 or -600NFkappaB motif reduced the activity of MMP-9 promoter and the induction of gene expression by PAF. In untreated HCECs, mutation of the -558Sp1 motif upregulated gene expression, but it caused a significant decrease in the promoter activity induced by PAF. Inhibition of MEK activity eliminated the PAF-induced phosphorylation and activation of Sp1 and abolished the upregulation of MMP-9 expression and activity.


CONCLUSIONS
These findings demonstrate that collaboration between several regulatory elements is required for the induction of MMP-9 promoter activity by PAF and that PAF overturns the repressor effect of Sp1 through activation of the MEK/ERK signaling cascade.
Taheri
Faramarz
F
Department of Biochemistry, Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112-2223, USA.
Bazan
Haydee E P
HE
D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000201:enzymology; D005419:Flavonoids / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D010972:Platelet Activating Factor / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection; D000097782:NF-kappaB-Inducing Kinase
D017871:Calcium-Calmodulin-Dependent Protein Kinases; D005419:Flavonoids; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D020780:Matrix Metalloproteinase 9; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D010972:Platelet Activating Factor; D017346:Protein Serine-Threonine Kinases; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D000097782:NF-kappaB-Inducing Kinase; C482128:MAP2K2 protein, human; C482125:MAP2K1 protein, human; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one