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Platelet-Activating Factor Overturns the Transcriptional Repressor Disposition of Sp1 in the Expression of MMP-9 in Human Corneal Epithelial Cells

¹From the Departments of Biochemistry and Molecular Biology and ²Ophthalmology, Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana.

PURPOSE. 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 NFB, 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 NFB, 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 –600NFB 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.

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