Extracellular matrix tenascin‐X in combination with vascular endothelial growth factor B enhances endothelial cell proliferation

Abstract

An extracellular matrix tenascin‐X (TNX) is highly expressed in muscular tissues, especially heart and skeletal muscle, and is also prominent around blood vessels. The precise in vivo role of TNX remains to be elucidated. To identify proteins that interact with TNX in the extracellular environment, we searched for TNX‐binding proteins using a yeast two‐hybrid system. We used mouse TNX‐specific fibronectin type III repeats (mTNX/FNIII 13‐25 ) as a bait for the screening. We found that vascular endothelial growth factor B (VEGF‐B) binds to mTNX/FNIII 13‐25 . This interaction was confirmed by pull‐down assays and co‐immunoprecipitation assays. The full‐length mTNX, as well as mTNX/FNIII 13‐25 , interacted with both alternative splice isoforms VEGF‐B 186 and VEGF‐B 167 . Furthermore, the full‐length mTNX also bound to VEGF‐A. The minimal region of TNX that interacts with VEGF‐B was mapped to the FNIII repeats (FNIII 13‐25 ) but not to the other characteristic domains of TNX. The TNX‐binding site of VEGF‐B was located in the N‐terminal 115‐amino acid region. mTNX/FNIII 13‐25 did not prevent the interaction of VEGF‐B with VEGFR‐1 (VEGF receptor 1), and VEGF‐B could simultaneously bind to both mTNX/FNIII 13‐25 and VEGFR‐1. A conditioned medium from transfected 293T cells coexpressing full‐length TNX and VEGF‐B could promote DNA synthesis in bovine endothelial cells in which VEGFR‐1 were expressed. VEGFR‐1 phosphorylation triggered by VEGF‐B 186 were increased in cells plated with mTNX/FNIII 13‐25 or full‐length mTNX, compared with cells plated with VEGF‐B 186 alone. TNX interacts with VEGF‐B and enhances the ability of VEGF‐B to stimulate cell proliferation. This enhanced mitogenecity is caused by increased signals mediated by the VEGFR‐1 receptor. This finding suggests a role for TNX in the regulation of the development of blood vessels such as vasculogenesis and angiogenesis.