Thymidine Phosphorylase and 2-Deoxyribose Stimulate Human Endothelial Cell Migration by Specific Activation of the Integrins α5β1 and αVβ3

Abstract

Thymidine phosphorylase is an angiogenic factor that is frequently overexpressed in solid tumors, in rheumatoid arthritis, and in response to inflammatory cytokines. Our previous studies showed that cells expressing thymidine phosphorylase stimulated endothelial cell migration in vitro. This was a consequence of the intracellular metabolism of thymidine by thymidine phosphorylase and subsequent extracellular release of 2-deoxyribose. The mechanisms by which 2-deoxyribose might mediate thymidine phosphorylase-induced cell migration in vitro, however, are obscure. Here we show that both thymidine phosphorylase and 2-deoxyribose stimulated the formation of focal adhesions and the tyrosine 397 phosphorylation of focal adhesion kinase in human umbilical vein endothelial cells. Although similar actions occurred upon treatment with the angiogenic factor vascular endothelial growth factor (VEGF), thymidine phosphorylase differed from VEGF in that its effect on endothelial cell migration was blocked by antibodies to either integrin ␣ 5 ␤ 1 or ␣ v ␤ 3 , whereas VEGFinduced endothelial cell migration was only blocked by the ␣ v ␤ 3 antibody. Further, thymidine phosphorylase and 2-deoxyribose, but not VEGF, increased the association of both focal adhesion kinase and the focal adhesion-associated protein vinculin with integrin ␣ 5 ␤ 1 and, in intact cells, increased the co-localization of focal adhesion kinase with ␣ 5 ␤ 1 . Thymidine phosphorylase and 2-deoxyribose-induced focal adhesion kinase phosphorylation was blocked by the antibodies to ␣ 5 ␤ 1 and ␣ v ␤ 3 , directly linking the migration and signaling components of thymidine phosphorylase and 2-deoxyribose action. Cell surface expression of ␣ 5 ␤ 1 was also increased by thymidine phosphorylase and 2-deoxyribose. These experiments are the first to demonstrate a direct effect of thymidine phosphorylase and 2-deoxyribose on signaling pathways associated with endothelial cell migration.

Accumulation of thymidine-derived sugars in thymidine phosphorylase overexpressing cells

“…Cells with a high TP can secrete angiogenic factors at high levels, including the vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), but the role of sugars in the activation of these factors remains unclear [6,13]. TdR-derived sugars accumulated in the cell fractions of the membrane and cytoskeleton, which may be related to glycation of proteins by the formation of AGE.…”

“…In various in vitro studies, TP had a chemotactic effect on endothelial cells [5,6,7,8]. In vivo, TP induced angiogenesis using various cancer cell lines that were transfected with TP [9,10].…”

“…The metabolite dR has previously shown angiogenic activity [6,10] and could elongate the sprouting in the aortic ring assay [19]. In addition, dR induced endothelial cell migration in various studies [5,8].…”

The Nucleoside Derivative 5′-O-Trityl-inosine (KIN59) Suppresses Thymidine Phosphorylase-triggered Angiogenesis via a Noncompetitive Mechanism of Action

“…It is assumed that the products of the enzyme reaction are angiogenic, in partic- ular 2-deoxy-D-ribose that is obtained by dephosphorylation of 2-deoxy-␣-D-ribose-1-phosphate. It has been proposed that the extracellular release of 2-deoxy-D-ribose forms a chemotactic gradient that mediates endothelial cell migration (25,28,29). Recent observations also suggest that 2-deoxy-D-ribose induces angiogenesis by generating oxygen radical species, which induce the secretion of oxidative stress-responsive angiogenic factors, like vascular endothelial cell growth factor, interleukin-8, and matrix metalloproteinase-1 (6,28).…”

Angiogenic Factor Thymidine Phosphorylase Increases Cancer Cell Invasion Activity in Patients with Gastric Adenocarcinoma

“…These pathways could be divided into those that are focal adhesion related or growth factor related. Hotchkiss et al (25) reported that TP and D-dRib stimulate the formation of focal adhesions and activate specific integrins for endothelial cell migration. We did not observe FAK activation in our experiments.…”