FoxO Proteins Mediate Hypoxic Induction of Connective Tissue Growth Factor in Endothelial Cells

Julia Wessel,Margarete Goppelt-Struebe,Sven Kroening,Christina Warnecke,Jana Samarin,Iwona Cicha

doi:10.1074/jbc.m109.049650

Abstract

Hypoxia, a driving force in neovascularization, promotes alterations in gene expression mediated by hypoxia-inducible factor (HIF)-1alpha. Connective tissue growth factor (CTGF, CCN2) is a modulator of endothelial cell growth and migration, but its regulation by hypoxia is poorly understood. Therefore, we analyzed signaling pathways involved in the regulation of CTGF by hypoxia in endothelial cells. Exposure to low oxygen tension or treatment with the hypoxia-mimetic dimethyloxalyl glycine (DMOG) stabilized HIF-1alpha and up-regulated CTGF in human umbilical vein endothelial cells and in a murine microvascular endothelial cell line. Induction of CTGF correlated with a HIF-dependent increase in protein and mRNA levels, and nuclear accumulation of the transcription factor FoxO3a. By contrast, gene expression and cellular localization of FoxO1 were not significantly altered by hypoxia. Expression of CTGF was strongly reduced by siRNA silencing of FoxO1 or FoxO3a. Furthermore, nuclear exclusion of FoxO1/3a transcription factors by inhibition of serine/threonine protein phosphatases by okadaic acid inhibited CTGF expression, providing evidence for both FoxO proteins as regulators of CTGF expression. The DMOG-stimulated induction of CTGF was further increased when endothelial cells were co-incubated with transforming growth factor-beta, an activator of Smad signaling. Activation of RhoA-Rho kinase signaling by the microtubule-disrupting drug combretastatin A4 also enhanced the DMOG-induced CTGF expression, thus placing CTGF induction by hypoxia in a network of interacting signaling pathways. Our findings provide evidence that FoxO1, hypoxia-stimulated expression of FoxO3a and its nuclear accumulation are required for the induction of CTGF by hypoxia in endothelial cells.

Highlights

These proteins are secreted and sequestered in the extracellular matrix, where they interact with cell surface receptors such as integrins, and with growth factors, proteases, cytokines, or extracellular matrix proteins
In mouse renal epithelial cells, hypoxia-inducible factor (HIF)-1␣ was described as transcription factor directly activating CTGF transcription [22], whereas an increase of CTGF mRNA stability by factors binding to the 3Ј-untranslated region was observed in a chondrosarcoma cell line [18]
The cells were exposed to low oxygen tension or the hypoxia mimetic dimethyloxalyl glycine (DMOG), which leads to the accumulation of active HIF-1␣ by inhibition of 2-oxoglutarate-dependent dioxygenases including HIF prolyl hydroxylases and the asparagyl hydroxylase FIH-1 [23]

Summary

Introduction

These proteins are secreted and sequestered in the extracellular matrix, where they interact with cell surface receptors such as integrins, and with growth factors, proteases, cytokines, or extracellular matrix proteins. Regulation of FoxO Expression and Intracellular Localization by Hypoxia—Incubation of glEND.2 cells with DMOG led to a time-dependent increase of the expression of FoxO3a protein, whereas FoxO1 levels remained essentially unaltered (Fig. 3A). We provided evidence that CTGF is reg- phosphatases by vanadate did not affect FoxO3a expression or ulated by FoxO proteins in endothelial cells [8, 7, 27].

Results

Conclusion