Nox4 and redox signaling mediate TGF-β-induced endothelial cell apoptosis and phenotypic switch

F Yan,Y Wang,X Wu,H M Peshavariya,G J Dusting,F Jiang,M Zhang

doi:10.1038/cddis.2013.551

Abstract

Transforming growth factor-β (TGF-β) triggers apoptosis in endothelial cells, while the mechanisms underlying this action are not entirely understood. Using genetic and pharmacological tools, we demonstrated that TGF-β induced a moderate apoptotic response in human cultured endothelial cells, which was dependent upon upregulation of the Nox4 NADPH oxidase and production of reactive oxygen species (ROS). In contrast, we showed that ectopic expression of Nox4 via viral vectors (vNox4) produced an antiapoptotic effect. TGF-β caused ROS-dependent p38 activation, whereas inhibition of p38 blunted TGF-β-induced apoptosis. However, vNox4, but not TGF-β, activated Akt, and inhibition of Akt attenuated the antiapoptotic effect of vNox4. Akt activation induced by vNox4 was accompanied by inactivation of the protein tyrosine phosphatase-1B (PTP1B) function and enhanced vascular endothelial growth factor receptor (VEGFR)-2 phosphorylation. Moreover, we showed that TGF-β enhanced Notch signaling and increased expression of the arterial marker EphrinB2 in a redox-dependent manner. In summary, our results suggest that Nox4 and ROS have pivotal roles in mediating TGF-β-induced endothelial apoptosis and phenotype specification. Redox mechanisms may influence endothelial cell functions by modulating p38, PTP1B/VEGFR/Akt and Notch signaling pathways.

Highlights

Transforming growth factor-b (TGF-b) expression induced endothelial oxidative stress and dysfunction.[11]
In light of some recent results showing that increased reactive oxygen species (ROS) production may have a prominent role in promoting cell apoptosis in response to TGF-b,17,23 we hypothesize that Nox4-dependent redox regulation may mediate TGF-b-induced endothelial cell apoptosis
Using caspase 3/7 activity assay, western blot and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, we further demonstrated that the effects of TGF-b on apoptosis were time dependent, with the strongest effects being observed at 24 and 48 h (Figures 1c–f)

Summary

Introduction

TGF-b expression induced endothelial oxidative stress and dysfunction.[11]. Remarkably, the biological effects of TGF-b in endothelial cells are often dichotomous (either protective or detrimental), depending on the cellular context and the type of endothelial cells involved.[4,7,12,13]. Stimulation of the ALK1 receptor phosphorylates Smad1/5/8, whereas stimulation of ALK5 triggers phosphorylation of Smad2/3 These activated Smad proteins work together with Smad[4] as a transcriptional activating complex to regulate the expression of various target genes,[1,3] whereas activation of ALK1 and ALK5 may induce distinct cellular effects in endothelium.[14] In addition to these canonical signaling pathways, emerging evidence suggests that NADPH oxidase-dependent redox mechanisms may be involved in mediating the biological actions of TGF-b. Several studies have pointed to a critical role of activation of the p38 mitogen-activated protein kinase (MAPK) in mediating TGF-b-induced endothelial apoptosis.[21,22] so far there is little information about whether TGF-b-induced apoptosis in endothelial cells involves redox-dependent signaling mechanisms. The second aim of this study is to explore the potential involvement of TGF-b-induced redox regulation in changes of the arterial and venous phenotype specification of endothelial cells

Objectives

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Results

Conclusion