Platelet-activating factor overturns the transcriptional repressor disposition of

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 5Ј 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. (Invest Ophthalmol Vis Sci

A Novel Platelet Activating Factor Receptor Antagonist Reduces Cell Infiltration and Expression of Inflammatory Mediators in Mice Exposed to Desiccating Conditions after PRK

Purpose. To study the contribution of a novel PAF receptor antagonist LAU-0901 in the modulation of the increased inflammatory response in mice exposed to dessicating conditions (DE) after PRK. Methods. Eighty 13-14 week old female Balb/C mice were used. They were divided into two groups: One group was treated with LAU-0901 topical drops. The other group was treated with vehicle. In each group ten mice served as controls and ten were placed in DE. The other twenty mice underwent bilateral PRK and were divided in two additional groups: ten mice remained under normal conditions (NC) and the other ten were exposed to DE. After 1 week all animals underwent in vivo confocal microscopy, immunostaining and western blotting analysis. Results. Confocal microscopy showed an increased number of reflective structures in the corneal epithelium after PRK and exposure to DE in eyes treated with vehicle as compared to eyes treated with LAU-090). Significant decrease of COX-2 and Arginase I expression and reduced alpha SMA cells was observed after PRK and exposure to DE in eyes treated with LAU-0901. Discussion: Exposure of mice to a DE after PRK increases the epithelial turnover rate. PAF is involved in the inflammatory cell infiltration and expression of inflammatory cytokines that follow PRK under DE

EGF Stimulates Lipoxin A4 Synthesis and Modulates Repair in Corneal Epithelial Cells through ERK and p38 Activation

These findings provide a better understanding of the effect of epidermal growth factor on the wound-healing process in corneal epithelial cells and raise the possibility of the therapeutic use for lipoxygenase derivatives in corneal wound healing

Association of protein tyrosine phosphatases (PTPs)-1B with c-Met receptor and modulation of corneal epithelial wound healing. Invest Ophthalmol Vis Sci.

PURPOSE. The purpose of this study was to investigate the expression and activity of protein tyrosine phosphatases (PTPs) in epithelium during corneal wound healing and to investigate how PTPs regulate activation of the c-Met receptor and the receptor's proximal signaling. METHODS. Rabbit corneas were injured by gentle scraping of the surface, leaving the limbal epithelium intact, and epithelium was collected at 1, 2, 3, and 7 days after injury. In organ culture models, epithelium was removed and corneas were incubated with hepatocyte growth factor (HGF), with or without the PTP inhibitor bpV(phen), and the PI-3K inhibitors wortmannin and LY294002. Human corneal epithelial (HCE) cells were stimulated with HGF with or without bpV(phen). Total cell lysates and cytosolic and membrane fractions were analyzed by Western blot. PTP activities were measured with specific substrates. PTP1B and SHP-2 genes were knocked down by interference RNA (siRNA). RESULTS. PTP activity and expression increased during wound healing. The most abundant were SHP-2, PTP1B, and PTEN. HGF activated the c-Met receptor in HCE cells up to 30 minutes and was downregulated by 2 hours. Inhibition of PTPs increased HGF-promoted wound healing, HGF-activated phosphorylation of c-Met, and its downstream signal PI-3K/Akt, but not ERK1/2 or p70S6K. PTP1B and SHP-2 were bound to the c-Met. Part of the c-Met was colocalized in the endoplasmic reticulum with PTP1B. PTP1B phosphorylation increased when the c-Met receptor was deactivated, and gene knockdown of PTP1B increased c-Met activation. SHP-2 phosphorylation and binding to c-Met was higher during receptor activation, and SHP-2 gene silencing decreased receptor phosphorylation. CONCLUSIONS. Inhibition of PTP activity mimics the effect of HGF by activating the PI-3K/Akt signal involved in wound healing. PTP1B and SHP-2 are bound to the c-Met receptor to control its activity. Although the binding of PTP1B increases when there is a decrease in c-Met activation and acts as a negative regulator of the receptor, the increased binding and phosphorylation of SHP-2 coincide with maximal stimulation of c-Met, acting as a positive regulator. (Invest Ophthalmol Vis Sci

Neuroprotectin D1 Synthesis and Corneal Nerve Regeneration after Experimental Surgery and Treatment with PEDF plus DHA

This study revealed that pigment epithelial–derived growth factor in combination with docosahexaenoic acid (DHA) enhances the regeneration of corneal nerves damaged after surgery