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

Kylie A Hotchkiss,Anthony W Ashton,Edward L Schwartz

doi:10.1074/jbc.m212670200

Kylie A Hotchkiss, Anthony W Ashton + Show 1 more

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https://doi.org/10.1074/jbc.m212670200

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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 alpha 5 beta 1 or alpha v beta 3, whereas VEGF-induced endothelial cell migration was only blocked by the alpha v beta 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 alpha 5 beta 1 and, in intact cells, increased the co-localization of focal adhesion kinase with alpha 5 beta 1. Thymidine phosphorylase and 2-deoxyribose-induced focal adhesion kinase phosphorylation was blocked by the antibodies to alpha 5 beta 1 and alpha v beta 3, directly linking the migration and signaling components of thymidine phosphorylase and 2-deoxyribose action. Cell surface expression of alpha 5 beta 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.

Highlights

Angiogenesis, the process of generating new capillaries from preexisting blood vessels, is a fundamental, tightly regulated process in reproduction and wound healing [1]
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
Both VEGF and thymidine phosphorylase (TP) have been previously shown to be chemoattractants for Human venous endothelial cells (HUVEC), mechanistic studies have only been extensively reported for VEGF

Summary

Introduction

Angiogenesis, the process of generating new capillaries from preexisting blood vessels, is a fundamental, tightly regulated process in reproduction and wound healing [1]. The integrin family protein heterodimers are the major cellular receptors for ECM and mediate cell-matrix interactions that activate multiple signaling pathways important for regulating endothelial cell responses, including cell attachment, adhesion, migration, survival, proliferation, and angiogenesis [4]. The integrin-matrix interaction is critical to the angiogenic process, and its disruption forms the basis for several experimental therapeutic approaches to the treatment of cancer [7] Integrins function as both outside-in and inside-out mediators of cell signaling due to their ability to regulate the activities of cytoplasmic kinases, growth factor receptors, and ion channels This activation is an early event associated with the assembly of focal adhesions and is accompanied by the recruitment of other cytoplasmic and cytoskeletal proteins to the focal adhesions, including talin, paxillin, vinculin, and actin [6] It is these interactions that place FAK in a central role in mediating cell attachment and migration

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