Role of matriptase in corneal epithelial homeostasis and repair - Project summary/Abstract: The role of the corneal epithelium is to: 1) act as a barrier against physical trauma, pathogens, and chemicals, 2) maintain a constant level of stromal hydration, and 3) serve as an optical interface focusing light onto the retina with optimal quality. A healthy corneal epithelium requires coordinated actions of multiple dynamic cellular processes and signaling pathways. To date, studies of extra- and pericellular proteases in eye and vision research have mostly centered on their harmful effects in corneal wound healing and infection. Based on our preliminary data, we hypothesize that the cell-surface anchored serine protease matriptase, in contrast, is essential for normal ocular surface function and promotes recovery from injury to the corneal epithelium. We generated matriptase hypomorphic mice and observed an abnormal irregular corneal surface cell pattern and increased inflammation. Our preliminary data using human corneal epithelial cells suggest that matriptase silencing causes decreased barrier function accompanied by impaired tight junction (TJ) integrity and aberrant proteolytic processing of the epithelial cell adhesion molecule (EpCAM). Under injury conditions, we identified the pro-form of hepatocyte growth factor (pro-HGF) as a candidate substrate for matriptase with key functions in corneal wound healing. Matriptase efficiently cleaves pro-HGF and converts it into the active form needed for activation of its cell-surface tyrosine kinase receptor c-Met and for subsequent stimulation of cell migration. Our hypothesis is that matriptase promotes corneal epithelial barrier function via regulation of tight junction formation and promotes tissue repair by activation of the HGF/c-Met signaling pathway. Aim 1: Determine the role of matriptase in corneal epithelial maintenance and function. Novel matriptase knockout mouse models as well as 3D cellular models of the human cornea will be utilized. We will use a balanced combination of hypothesis-driven targeted experimentation and unbiased approaches to comprehensively characterize the role of matriptase in corneal homeostasis and to identify critical pathways required for proper regeneration and differentiation, TJ formation, EpCAM processing, and barrier function. Aim 2: Identify functions for matriptase in corneal epithelial tissue repair. Matriptase-deletion mouse models and 3D epithelium/stroma cell models will be used to determine the impact of matriptase on corneal repair in experimental wound repair. Comparative RNA-Seq and Mass Spectrometry analysis will be performed to identify matriptase substrates and matriptase-mediated pathways in homeostasis and injury response. The impact of stimulation with Internalin B321 ( InlB321 ), an agonist of the HGF receptor c-Met, on cell migration/tissue repair in vitro and in vivo will be tested as a potential therapeutic strategy to treat corneal epithelial injury. Significance: Implementation of the approaches described in this proposal will provide a mechanistic understanding of the role of matriptase in corneal epithelial homeostasis and injury and identify actionable targets (factors/pathways) for therapeutic options to promote corneal epithelial repair leading to restoration of function.
