S156C Mutation in Tissue Inhibitor of Metalloproteinases-3 Induces Increased Angiogenesis

Jian Hua Qi,Ganying Dai,Philip Luthert,Shyam Chaurasia,Joe Hollyfield,Bernhard H.F Weber,Heidi Stöhr,Bela Anand-Apte

doi:10.1074/jbc.m109.013763

Abstract

Tissue Inhibitor of metalloproteinases-3 (TIMP-3) is a potent matrix-bound angiogenesis inhibitor. Mutations in TIMP-3 cause Sorsby Fundus Dystrophy, a dominant inherited, early onset macular degenerative disease, with choroidal neovascularization causing a loss of vision in the majority of patients. Here we report that expression of S156C TIMP-3 mutation in endothelial cells results in an abnormal localization of the protein, increased glycosylation, decreased matrix metalloproteinase inhibitory activity, and increased vascular endothelial growth factor (VEGF) binding with a consequent increase in VEGF-dependent migration and tube formation. These enhanced signaling events appear to be mediated as a consequence of a post-transcriptionally regulated increase in the expression of membrane-associated VEGFR-2 in endothelial cells of Timp-3(156/156) mutant mice as well as in human Sorsby fundus dystrophy eyes. Understanding the mechanism(s) by which mutant TIMP-3 can induce abnormal neovascularization provides important insight into the pathophysiology of a number of diseases with increased angiogenesis.

Highlights

Tissue inhibitor of metalloproteinases-3 (TIMP-3)2 is a member of the TIMP family of proteins and is an endogenous inhibitor of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM), and ADAM with thrombospondin domains (ADAMTS) family of enzymes
Western blot analysis using monoclonal antibodies against TIMP-3 determined that in endothelial cells WT TIMP-3 was predominantly localized in the extracellular matrix (ECM) in a 23-kDa form (Fig. 1B, upper panel) with a very small fraction secreted into the conditioned medium (CM) (Fig. 1B, lower panel), confirming previous reports that TIMP-3 is an ECM-bound protein in endothelial cells [8]
To determine whether the higher molecular weight form of mutant TIMP-3 was a result of glycosylation, samples were treated with peptide N-glycosidase F before analyses

Summary

Introduction

Tissue inhibitor of metalloproteinases-3 (TIMP-3)2 is a member of the TIMP family of proteins and is an endogenous inhibitor of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM), and ADAM with thrombospondin domains (ADAMTS) family of enzymes. We report that expression of S156C TIMP-3 mutation in endothelial cells results in an abnormal localization of the protein, increased glycosylation, decreased matrix metalloproteinase inhibitory activity, and increased vascular endothelial growth factor (VEGF) binding with a consequent increase in VEGF-dependent migration and tube formation.

Results

Conclusion