Abstract
Purpose: :
Decorin, a small leucine-rich proteoglycan, regulates matrix assembly and wound healing in the cornea. EGF and EGFR are expressed in all 3 major corneal cells and play an important role in stromal healing following injury in vivo. EGF is known to increase keratocyte proliferation, migration and collagen synthesis in the corneal fibroblasts. The aims of this study were to test the effects of decorin on EGFR phosphorylation, EGFR internalization and EGF-mediated human corneal fibroblast (HCF) migration.
Methods: :
HCF cultures were generated from donor corneas in serum-containing medium. At 50% confluence, cultures were switched to serum-free conditions for 48 hours, and then treated with decorin (250nM) in the presence or absence of EGF (50ng/ml) for various time points (10-60 min). Samples were subjected to proteome array analysis screening for 42 different phosphorylated human receptor tyrosine kinases (RTKs), immunocytochemistry or immunoblotting to analyze EGFR phosphorylation and/or expression. Scratch assay determined the effects of decorin on EGF-mediated HCF migration.
Results: :
Decorin caused a rapid EGFR phophorylation as suggested by the detection of a prominent EGFR band in RTK blot in samples exposed to decorin for 10 minutes. This data suggested that decorin acts as a biological ligand for EGFR in HCF. Extended (15min, 30min or 60min) decorin exposure to HCF showed a gradual decline in the EGFR levels up to 30min and absence at 60min in immunoblot suggesting that deocrin binding to EGFR causes EGFR down-regulation. In scratch assay, EGF addition showed profound increase in HCF migration compared to vehicle (91±7%, p<0.001) whereas decorin (250 nM) addition significantly inhibited EGF-mediated HCF migration (89±9%, p<0.001), which was similar to the inhibitory effect of EGF antibody. This suggested that decorin modulates keratocyte migration via EGFR down-regulation.
Conclusions: :
Decorin acts as a biological ligand for EGFR in HCF and plays an important role in corneal healing modulating keratocyte function.
Keywords: cornea: stroma and keratocytes • cornea: basic science • proteoglycans/glycosaminoglycans