Serum Tenascin-X Strongly Binds to Vascular Endothelial Growth Factor

Abstract

Interstitial extracellular matrix tenascin-X (iTNX) with about 450 kDa is prominently present in various tissues. Previously, we identified the serum form of TNX (sTNX) with 200 kDa in the mouse. In the present study, in order to investigate distinctive features and functions of sTNX, a plasmid encoding the recombinant mouse sTNX was constructed. As a control, we also constructed a plasmid encoding mouse 450-kDa iTNX and a plasmid encoding 250-kDa iTNX, which lacks the region of 200-kDa sTNX from 450-kDa iTNX. In cells stably expressing each recombinant TNX, a more than 7-fold larger amount of 200-kDa sTNX was released into conditioned medium than the amounts of 250-kDa iTNX and 450-kDa iTNX released into the medium. We previously reported that a splice isoform of iTNX (340-kDa iTNX) binds to vascular endothelial growth factor B (VEGF-B) as well as to VEGF-A. Therefore, the ability of VEGF-A and VEGF-B to bind to 200-kDa sTNX was examined by a co-immunoprecipitation assay in comparison with the binding abilities to 250-kDa iTNX and 450-kDa iTNX. It was found that sTNX strongly bound to VEGF-A and VEGF-B, compared with the binding abilities of other iTNX proteins. Based on the results of assays of incorporation of 5-ethynyl-2′-deoxyuridine (EdU), we found that purified recombinant 200-kDa sTNX both alone and in combination with VEGF-A or basic fibroblast growth factor (bFGF) can weakly promote DNA synthesis in proliferating vascular endothelial cells (UV♀2 cells). These results suggest that sTNX possesses weak activity for proliferation of endothelial cells.