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K. Han, J.-H. Chang, D. T. Azar; Keratocyte-associated MT1-MMP Modulates bFGF-induced VEGF Expression. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4967.
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To evaluate the pro-angiogenic role of stromal fibroblast-associated MT1-MMP.
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.
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.
Our data suggest that keratocyte-associated MT1-MMP modulates bFGF-induced VEGF expression via Ras- and ERK- signal transduction pathways.
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