Induction of Gelatinase B (MMP–9) Promoter Activity by Platelet–Activating Factor in Human Corneal Epithelial Cells Requires Multiple Transcription Factor Binding Sites Including NFB (–600) and Sp1 (–558)

Abstract

Abstract: : Purpose: MMP–9 is an important matrix metalloproteinase involved in remodeling of corneal epithelium during wound healing. Expression of the gene encoding MMP–9 is elevated after corneal injuries in epithelial cells. One important inducer is platelet activating factor (PAF), an inflammatory mediator that accumulates in corneal epithelium following injuries. Here, we studied the promoter region of human MMP–9 gene to determine the induction efficiency of PAF–stimulated expression of MMP–9 and the role of different transcription factors in the responsiveness of the promoter. Methods: Human corneal epithelial cells (HCEC) were transfected using FuGene 6 reagent with luciferase reporter constructs fused to the wild type (–2172/+54) or 5' deleted fragments of the human 92–kDa gelatinase B promoter (including –1511, –1112, –670, –460, –324, and –8). Transiently transfected cells were either left untreated or stimulated with 100nM PAF for 1–3 days and the expression of luciferase was tested. HCEC were stimulated by PAF and nuclear extracts were examined by electrophoretic mobility shift assay (EMSA) for the DNA binding activity of NFΚB, Sp1, AP–1, and AP–2. Transiently transfected HCEC with an NFΚB–d2EGFP plasmid were also stimulated with PAF and monitored up to 12 hours for the expression of GFP. Results: Expression of luciferase in transiently transfected HCEC was highly elevated by the presence of the segment spanning –324 to –8, which carries the TATA and GT boxes, and some transcription factor binding sites including AP–1. Another increase in the expression of the gene was observed when the segment spanning –1112 to –670 was present. PAF treatment of transiently transfected cells showed that the region spanning –670 to –460 is very crucial in the induction of promoter activity by PAF. We also found an increase in the binding activity of NFΚB, Sp1 and AP–2 by EMSA. Expression of NFΚB driven destabilized green fluorescent protein (NFΚB–d2EGFP) was also observed in HCEC within 8–12 hours after stimulation with 100nM PAF, indicating the activation of NFΚB in these cells. Conclusions: Induction of the promoter activity by PAF was found to be dependent on a segment spanning –670 to –460, which contains several transcription factor binding sites including NFΚB (–600), Sp1 (–558), PEA3 (–540) and AP–1 (–533). Upregulation of NFΚB and Sp1 DNA binding activity and d2EGFP over–expression by NFΚB motif confirm the contribution of these two transcription factors in PAF–mediated induction of MMP–9 in HCEC.

Keywords: gene/expression • extracellular matrix • transcription factors