Abstract
Purpose :
Disruption of anatomical barriers and direct intercellular contacts between stromal and epithelial cells during corneal wound healing can lead to dysregulated induction of matrix metalloproteinases (MMPs) and melting. The goal of this study was to determine whether fibroblasts induce corneal keratinocytes to express MMP9 via galectin-3, a multimeric lectin with pleiotropic biological functions.
Methods :
Primary human corneal fibroblasts and immortalized human corneal keratinocytes cells were maintained in DMEM medium supplemented with 10% serum and keratinocyte serum-free medium, respectively. MMP9 expression and secretion were determined by qPCR and gel zymography, respectively. Galectin-3 expression was abrogated using siRNA and protein levels were monitored by western blot analysis. Plasma membrane protein extracts were purified from fibroblasts by centrifugation. Inhibition of N-linked glycosylation was achieved using tunicamycin.
Results :
As shown by qPCR and gel zymography, coculture of fibroblasts with keratinocytes induced the expression and secretion of MMP9 compared to keratinocytes alone. The secretion of MMP9 in coculture was down-regulated approximately 5-fold following galectin-3 silencing in corneal keratinocytes. Addition of plasma membrane extracts isolated from fibroblasts to cultures of corneal keratinocytes was sufficient to promote proteolytic activity. In these experiments, use of plasma membrane extracts from fibroblasts treated with tunicamycin failed to stimulate secretion of MMP9, suggesting that N-linked surface glycoproteins mediate the stimulatory effect in corneal fibroblasts.
Conclusions :
Our results indicate that endogenous galectin-3 in corneal keratinocytes and N-glycosylated surface proteins in fibroblasts regulate MMP9 secretion during stromal-epithelial interactions. Inhibition of galectin-3 may have therapeutic potential in reducing the complications associated with corneal melting.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.