Abstract
Purpose: :
To evaluate the pro-angiogenic role of stromal fibroblast-associated MT1-MMP.
Methods: :
Western blotting was performed to determine the protein phosphorylation patterns in cell lysates of bFGF-stimulated wild type, MT-MMP knock-out and MT1-MMP knock-in corneal fibroblasts. The activities and levels of Ras and ERK were examined in wild type, MT1-MMP knock-out and MT1-MMP knock-in cell lines using ras-RBD pull down and phospho-ERK antibodies western blot analysis. The expression of VEGF mRNA were determined by real-time-PCR, western blotting and immunohistochemistry. VEGF mRNA was assayed in the presence or absence of ERK and Ras inhibitors.
Results: :
Differences in the patterns of protein phosphorylation by bFGF on wild type, MT1-MMP knock-out and MT1-MMP knock-in corneal keratocyte cell lines were noted. ERK was up-regulated in wild type fibroblast as compared to that in MT1-MMP knock-out cells. The amount of GTP binding Ras, relating ERK signal transduction, was higher in wild type keratocyte than MT1-MMP knock-out cells. The HIF-1 protein level was decreased in MT1-MMP knockout keratocyte and was restored in MT1-MMP knock-in keratocytes. The level of bFGF-induced VEGF mRNA was higher in wild type than knock-out and ERK or Ras inhibitor treated keratocytes.
Conclusions: :
Our data suggest that keratocyte-associated MT1-MMP modulates bFGF-induced VEGF expression via Ras- and ERK- signal transduction pathways.
Keywords: neovascularization • cornea: basic science • cornea: stroma and keratocytes