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F. Taheri, H.E. Bazan; Transcriptional Regulation of Gelatinase B Expression in Corneal Epithelial Cells by Platelet-Activating Factor (PAF): I. Induction of NFB, Sp1, and AP-2 Activity . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3833.
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Purpose: PAF is a potent lipid mediator that accumulates in cornea after injury. We found that PAF produces an imbalanced induction of gelatinase B (MMP-9) compared to that of its inhibitors TIMP-1 and TIMP-2 in rabbit corneal epithelial cells (RCEC) [Exp Eye Res 2002 Mar;74(3):393-402]. MMP-9 is an inducible matrix metalloproteinase essential for remodeling the extracellular matrix. Overexpression of MMP-9 interferes with effective re-epithelialization of wounded cornea. As control of MMP-9 overall expression and activity depends largely on transcriptional regulation, we investigated the effect of PAF on the activity of transcription factors reported to have a binding site in the promoter region of the MMP-9 gene in the first stage of this study. Methods: Induction of transcription factors AP-1, AP-2, Sp1, and NFΚB activity by PAF was determined by electrophoretic mobility shift assay (EMSA) and by transient transfection studies using a short-lived green fluorescence protein (d2EGFP) as a reporter gene driven by the consensus sequence of any of the four transcription factors. Primary cultures of RCEC as well as a human corneal epithelial cell line HCEC were treated with 100 nM cPAF (a nonhydrolyzable analog of PAF) and nuclear extracts were collected at different times to be examined by EMSA. Transiently transfected RCEC and HCEC were studied by fluorescence microscopy for induction of d2EGFP driven by any of the consensus sequences of the four transcription factors upon treatment with PAF. The phorbol ester PMA was used as a positive control. Results: The binding activity of NFΚB in EMSA was increased 50% after 2-hr stimulation of RCEC with 100 nM cPAF. This increase was 40% for Sp1, while the activity of AP-2 was twice the basal level as determined by EMSA. The specificity of these reactions was confirmed by adding excess unlabled oligos or by using a slightly mutated oligo. Fluorescence microscopy showed that at least 8-hr treatment with PMA or 12 hr with PAF was required for the induction of expression of d2EGFP driven by the consensus sequence of any of the three transcription factors NFΚB, Sp1, and AP-2. Conclusion: PAF can activate some of the transcription factors that have a consensus sequence in the promoter region of MMP-9; i.e. NFΚB, Sp1, and AP-2. To determine the regulatory sequences in the MMP-9 promoter region that are under PAF control, it will be important in the second stage of this study to analyze the promoter region of this gene by deletion of the binding sites for any of these transcription factors.
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