An Endothelial Cell Genetic Screen Identifies the GTPase Rem2 as a Suppressor of p19ARF Expression That Promotes Endothelial Cell Proliferation and Angiogenesis

Ruben Bierings,Miguel Beato,Michael J Edel

doi:10.1074/jbc.m707438200

Abstract

Angiogenesis requires an increase in endothelial cell proliferation to support an increase in mass of blood vessels. We designed an in vitro endothelial cell model to functionally screen for genes that regulate endothelial cell proliferation. A gain of function screen for genes that bypass p53 endothelial cell arrest identified Rem2, a Ras-like GTPase. We show that ectopic Rem2 suppresses p14(ARF) (human) or p19(ARF) (mouse) expression that leads to increased endothelial cell proliferation. Conversely, loss of ectopic Rem2 by RNA interference restores p19(ARF) expression in endothelial cells. We further show that Rem2-interacting 14-3-3 proteins are involved in the cell localization of Rem2, regulation of p19(ARF) expression, and endothelial cell proliferation. Finally, we demonstrate using the RIP1 tag2 mouse model of pancreatic disease that Rem2 is up-regulated in endothelial cells of stage IV disease. The data unravel a possible molecular mechanism for Rem2-induced angiogenesis and suggests Rem2 as a potential novel target for treating pathological angiogenesis.

Highlights

The Ink4a-Arf locus encodes two tumor suppressor proteins, p16INK4a and p19ARF (p14 ARF in humans) that up-regulate the activities of the retinoblastoma protein (Rb)3 and the p53 tran
Rad/Gem/ Kir (RGK) proteins interact with 14-3-3 and calmodulin and Gem regulates endothelial shape changes required for angiogenesis [12, 13]
It has been shown that 14-3-3, together with calmodulin, regulates the subcellular distribution of Rem2 between the cytoplasm and the nucleus and this distribution has been correlated to cell shape changes [12, 13, 15]

Summary

Introduction

The Ink4a-Arf locus encodes two tumor suppressor proteins, p16INK4a and p19ARF (p14 ARF in humans) that up-regulate the activities of the retinoblastoma protein (Rb)3 and the p53 tran-. Loss of endogenous Rem2 in p53 null-treated endothelial cells had no effect on colony formation assay suggesting that activation of Rem2 is needed before suppressing p19ARF expression (Fig. 3B, inset iii, fourth panel). To further establish that the Rem2-mediated effects on p19ARF are needed for proliferation we overexpressed p19ARF in Rem2 expressing mouse endothelial cell clones by retroviral infection (70% infection levels) and performed colony formation assay.

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Conclusion