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Roles of reactive oxygen species in angiopoietin-1/tie-2 receptor signaling

Rania Harfouche^*, Nelly Abdel Malak^*, Ralf P. Brandes, Aly Karsan, Kaikobad Irani and Sabah N. A. Hussain^*,1

^* Critical Care and Respiratory Divisions, Royal Victoria Hospital and Meakins-Christie Laboratories, McGill University, Montreal, Québec, Canada;
Institut für Kardiovaskuläre Physiologie Klinikum der J. W. Goethe-Universität, Frankfurt am Main, Germany;
Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and
Department of Pathology and Laboratory Medicine, University of British Columbia, and Departments of Medical Biophysics and Pathology and Laboratory Medicine, British Columbia Cancer Agency, Vancouver, British Columbia, Canada

¹ Correspondence: Room L3.05, Critical Care Division, Royal Victoria Hospital, 687 Pine Ave. West, Montreal, Quebec H3A 1A1, Canada. E-mail: sabah.hussain{at}muhc.mcgill.ca

SPECIFIC AIMS

Recent studies have indicated that reactive oxygen species (ROS) play important roles as second messengers in regulating signaling of receptor tyrosine kinases (RTKs) such as those of vascular endothelial growth factor (VEGF) in endothelial cells (ECs). These roles are mediated through selective regulation of protein phosphorylation of the mitogen-activated protein kinases (MAPKs), the phosphatidylinositol 3'-kinase (PI-3 kinase), protein kinase B (AKT), and other signaling pathways. It has also been reported that the NADPH oxidase enzyme complex is an important molecular source of ROS generated in response to activated receptors. Tie-2 receptors are RTKs selectively expressed in ECs and play an important role in embryonic vascular development and promote differentiation, tube formation, sprouting, migration, adherence, and survival of ECs. Angiopoietin-1 (Ang-1) has recently been identified as the main endogenous agonist for tie-2 receptors and exposure to Ang-1 triggers autophosphorylation of these receptors and the association of phosphatases (Shp2), adaptor proteins (Grb14, Grb7, Grb2), the p85 subunit of the PI3-K, and Dok-R (a novel docking protein) with tyrosine-phosphorylated tie-2 receptors. Our group recently reported that the extracellular signal-regulated kinases (Erk1/2) and the p38 MAPKs are activated by Ang-1 exposure in ECs and that the Erk1/2 pathway promotes EC survival, whereas the p38 MAPKs enhance apoptosis. Despite recent progress in understanding tie-2 receptor signaling, little is known about the mechanisms linking these receptors to downstream effectors such as MAPK family proteins.

In this study, we evaluated the importance of ROS in signaling and the prochemotactic effects of the Ang-1/tie-2 receptor system in human umbilical vein endothelial cells (HUVECs). We also assessed the contribution of the NADPH oxidase to ROS generation in response to tie-2 receptor activation by Ang-1 in endothelial cells.

PRINCIPAL FINDINGS

1. Tie-2 receptor activation by Ang-1 triggers the production of ROS; the importance of NADPH oxidase
Ang-1 (50 ng/mL) elicited a significant rise in ROS production, as assessed by both lucigenin-derived chemiluminescence (LDCL) and 5-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence (Fig. 1 ). This effect of Ang-1 was abrogated in cells infected with adenoviruses expressing superoxide disumutase (SOD) and in cells pretreated with pure SOD protein, indicating that superoxide (O₂^–) is the main ROS induced in response to Ang-1 treatment in HUVECs. ROS production in response to Ang-1 was also attenuated in cells expressing a dominant negative form of Rac-1 (Rac1N17) and in cells pretreated with a peptide (gp91ds-tat) that selectively inhibits NADPH oxidase activity. These results suggest that Rac-1 and NADPH oxidase are involved in enhanced production of ROS in response to Ang-1 exposure.

View larger version (22K): [in this window] [in a new window]   Figure 1. A) ROS production, as measured by lucigenin-derived chemiluminescence (LDCL), in response to Ang-1 (50 ng/mL) and Ang-1 solvent. B) Rate of ROS production, measured by LDCL, in response to solvent and Ang-1. *P < 0.05 compared with that measured with solvent. C) Representative examples of DCFDA fluorescence measured in response to Ang-1 (50 ng/mL) and solvent in HUVECs. D) Changes in DCFDA fluorescence measured after 15 min of Ang-1 solvent or Ang-1 (50 ng/mL). *P < 0.05 compared with that measured with solvent.

2. Regulation of tie-2 receptor signaling by ROS
We have previously shown that tie-2 receptor activation by Ang-1 in HUVECs triggers the phosphorylation of Erk1/2 and p38 members of the mitogen-activated protein kinases (MAPKs). In this study, we report that Ang-1 triggers a significant increase in the phosphorylation of p21-activated protein kinase 1 (PAK-1), particularly at Ser^199/204 and Thre⁴²³ and that this rise in phosphorylation was significantly attenuated in HUVECs overexpressing SOD, catalase, and Rac1N17. By comparison, Ang-1-induced phosphorylation of Erk1/2 was potentiated in HUVECs overexpressing SOD, catalase, and Rac1N17 and in cells incubated with SOD protein. We found that pretreatment with diphenyleneiodonium (DPI) and gp91ds-tat peptide also potentiated Ang-1-induced Erk1/2 phosphorylation. Regulation of Erk1/2 phosphorylation by ROS appears to be mediated through direct effects on either MEK or at Erk1 and 2 themselves since Ang-1-induced Raf-1 phosphorylation was not influenced by antioxidants. Unlike the potentiation of Ang-1-induced Erk1/2, antioxidants significantly attenuated VEGF-induced Erk1/2 phosphorylation in HUVECs, suggesting that ROS play opposite roles in regulating Erk1/2 phosphorylation in Ang-1 and VEGF signaling. In addition to regulation of Erk1/2 activation, NADPH oxidase-derived ROS regulate the status of p38 phosphorylation. Overexpression of antioxidant enzymes, Rac1N17 and pretreatment with selective inhibitors of NADPH oxidase activity augmented basal p38 phosphorylation and resulted in no further phosphorylation of p38 proteins in response to Ang-1 or VEGF exposure. By comparison, these interventions had no influence on Ang-1-induced AKT phosphorylation indicating that activation of AKT as a result of tie-2 receptor stimulation is redox-insensitive.

3. ROS promote the chemotactic effects of Ang-1
Previous studies have indicated that Ang-1 promotes EC chemotaxis through tie-2 receptor activation. In this study, we report that the chemotactic effects of Ang-1 on cultured ECs was significantly attenuated in cells overexpressing SOD, catalase, Rac1N17, and in cells pretreated with DPI and gp91ds-tat peptide. These results indicate that NADPH oxidase-derived ROS play an important role in promoting the chemotactic effects of Ang-1 in HUVECs.

CONCLUSIONS AND SIGNIFICANCE

In nonphagocytes, increased ROS production has been observed when various classes of receptors are activated, including G-protein coupled, cytokine, and RTKs. In ECs, activation of PDGF, angiotensin II, and VEGF receptors trigger the acute release of O₂^– and H₂O₂. Using two independent methods of ROS detection, we found that Ang-1 triggers the formation of ROS in HUVECs. The observations that ROS levels were strongly attenuated by overexpression of SOD and by pre-incubation with exogenous SOD, suggest that O₂^– anions are the main ROS species produced in response to Ang-1 exposure in HUVECs.

Recent studies have confirmed the importance of NADPH oxidase-derived ROS in mediating various signaling events in non-phagocytes. Following receptor stimulation by ligands such as PDGF or VEGF, the vascular NADPH oxidase complex is activated through several mechanisms including p47^phox phosphorylation and activation of Rac1, resulting in the association of these two proteins with membrane-bound p22^phox and gp91^phox subunits. The assembled NADPH oxidase complex will then produce O₂^– anions that target diverse signaling molecules. We propose that Ang-1 exposure in HUVECs triggers the activation of NAPDH oxidase, resulting in enhanced O₂^– production. This proposal is supported by the fact the kinetics of ROS generation triggered by Ang-1 (Fig. 1) are similar to those described for vascular NADPH oxidase activation by growth factors such VEGF. Moreover, our finding that DPI and a selective blocker of the association of gp91^phox with p47^phox (gp91ds-tat peptide) strongly attenuated Ang-1-induced ROS generation implicate the NADPH oxidase in producing ROS in response to Ang-1 exposure. Our data suggest that the Rac-1 protein is necessary for Ang-1-induced NADPH oxidase in HUVECs since selective inhibition of the activity of this protein resulted in strong attenuation of Ang-1-induced ROS production. We should emphasize also that the PI-3 kinase, AKT, and SAPK/JNK pathways are not involved in NADPH oxidase activation by Ang-1 since selective inhibitors of these pathways did not alter ROS levels. In this regard, the Ang-1/tie-2 receptor system is clearly different from the VEGF/VEGF receptor pathway, which depends on Rac-1 activation by the PI-3 kinase pathway for ROS generation in ECs.

The influence of ROS on phosphorylation of Erk1/2 pathway is highly dependent on the type of receptors being activated, the molecular sources, and the nature of ROS involved. In terms of VEGF exposure, we confirm here that ROS generation elicits strong activation of Erk1/2. By comparison, we demonstrate for the first time that Ang-1-induced ROS exert an inhibitory effect on Erk1/2 phosphorylation, since overexpression of SOD and pretreatment with NADPH oxidase inhibitors (DPI and gp91ds-tat) strongly potentiated Ang-1-induced Erk1/2 phosphorylation. The mechanisms behind the differential effects of ROS generated by tie-2 and VEGF receptor activation on Erk1/2 phosphorylation are not clear. ROS could target the Erk1/2 pathway at multiple levels, including the receptors responsible for inducing ROS release. However, we found that tie-2 receptor phosphorylation was not influenced by ROS. Another level at which ROS could modulate Erk1/2 phosphorylation is by targeting the activity of p21^Ras, an upstream component of the MAPK pathway. Although we did not assess the status of p21^Ras activation in our study, phosphorylation of a downstream target of p21^Ras action, namely Raf-1, in response to Ang-1 treatment was not influenced by antioxidants, suggesting that Ang-1-triggered ROS target the Erk1/2 pathway downstream from Raf-1, possibly at the level of MEK or Erk1/2 themselves and/or at the level of SHP-2, a dual specificity protein tyrosine phosphatase that promotes Erk1/2 phosphorylation in HUVECs.

Another important finding in our study is that Ang-1-induces significant phosphorylation of PAK-1 at Ser^199/204 and Thre⁴²³ residues in HUVECs. Previous studies have revealed that phosphorylation of these residues results in increased PAK-1 activity and that PDK-1 is the most likely mediator of Thre⁴²³ phosphorylation of PAK-1 by growth factor receptors. PAK-1 is an important modulator of many cellular functions including cell migration, proliferation and survival. We also found that increased PAK-1 phosphorylation in response to Ang-1 was attenuated in cells overexpressing SOD, catalase, and Rac1N17 and cells incubated with gp91ds-tat peptide indicating that PAK-1 activation by Ang-1 is triggered in part by ROS derived from NADPH oxidase. These results are similar to those reported in vascular cells exposed to growth factors such as PDGF and VEGF.

Another important finding in our study is that Ang-1-induced EC migration requires the activity of NADPH oxidase, since selective inhibitors of this enzyme significantly attenuate this response. These results are similar to those showing that VEGF-induced EC migration is mediated by a Rac-1-dependent NADPH oxidase. The mechanisms of action underlying ROS-mediated EC migration following Ang-1 are unclear. In the case of VEGF, parallel activation of both the PI-3 kinase/AKT and PAK pathways by ROS promote EC migration. Similarly, both the PI-3 kinase and PAK pathways are responsible for Ang-1-induced EC migration. We have excluded a role for the AKT pathway in ROS-induced EC migration since overexpression of antioxidants and pre-incubation with NADPH oxidase inhibitors had no influence on Ang-1-induced AKT phosphorylation. However, our results do not exclude the possibility that ROS generated by Ang-1 exposure may activate the PI-3 kinase, which in turn promotes EC migration. Another possible pathway activated by Ang-1 that may mediate the pro-migratory role of ROS includes the DOK-R and PAK proteins (p-21 activated kinase) (Fig. 2 ). This proposal is based on recent studies documenting the recruitment of DOK-R to phosphorylated tie-2 receptors and the subsequent activation of PAK. The inhibitory effect of antioxidants on Ang-1-induced PAK-1 phosphorylation in the current study strongly support the possibility that ROS generated in response to Ang-1 promote EC migration through increased PAK-1 phosphorylation and activation (Fig. 2) . The mechanisms through which ROS modulate PAK-1 phosphorylation in response to Ang-1 exposure remain to be determined.

View larger version (35K): [in this window] [in a new window]   Figure 2. Proposed mechanisms through which NADPH- oxidase-derived ROS modulate signaling pathways of the Ang-1/tie-2 receptor system. Interrupted arrows indicate pathways which have not been directly evaluated.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3621fje;

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