Nicotinamide Adenine Dinucleotide Phosphate Reduced Oxidase 5 (Nox5) Regulation by Angiotensin II and Endothelin-1 is Mediated via Calcium/Calmodulin-dependent, Rac-1-independent Pathways in Human Endothelial Cells (original) (raw)

Circ Res. Author manuscript; available in PMC 2011 Jun 22.

Published in final edited form as:

PMCID: PMC3119893

NIHMSID: NIHMS301315

Augusto C Montezano,1 Dylan Burger,1 Tamara M Paravicini,1 Andreia Z Chignalia,1 Hiba Yusuf,1 Mahmoud Almasri,1 Ying He,1 Glaucia E Callera,1 Gang He,1 Karl-Heinz Krause,2 David Lambeth,3 Mark T Quinn,4 and Rhian M Touyz1

Augusto C Montezano

1Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada

Dylan Burger

1Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada

Tamara M Paravicini

1Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada

Andreia Z Chignalia

1Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada

Hiba Yusuf

1Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada

Mahmoud Almasri

1Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada

Ying He

1Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada

Glaucia E Callera

1Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada

Gang He

1Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada

Karl-Heinz Krause

2Department of Geriatrics, Geneva University Hospitals, Switzerland

David Lambeth

3Department of Pathology Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA

Mark T Quinn

4Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana, USA

Rhian M Touyz

1Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada

1Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada

2Department of Geriatrics, Geneva University Hospitals, Switzerland

3Department of Pathology Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA

4Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana, USA

Correspondence: Rhian M Touyz MD, PhD OHRI/University of Ottawa, 451 Smyth Road Ottawa, K1H 8M5, Ontario. Phone: (613) 562-5800 ext 8241, Fax: (613) 562-5487 ac.awattou@zyuotr

Abstract

Rationale

Although Nox5, (Nox2 homologue), has been identified in the vasculature, its regulation and functional significance remain unclear.

Objectives

To test if vasoactive agents regulate Nox5 through Ca2+/calmodulin-dependent processes and whether Ca2+ -sensitive Nox5, associated with Rac-1, generates superoxide (•O2−) and activates growth and inflammatory responses via MAP kinases in human endothelial cells (ECs).

Methods and results

Cultured ECs, exposed to AngII and ET-1 in the absence and presence of diltiazem (Ca2+ channel blocker), calmidazolium (calmodulin inhibitor) and EHT1864 (Rac-1 inhibitor), were studied. Nox5 was downregulated with siRNA. AngII and ET-1 increased Nox5 expression (mRNA and protein). Effects were inhibited by actinomycin D and cycloheximide and blunted by diltiazem, calmidazolium and low extracellular Ca2+ ([Ca2+]e). Ang II and ET-1 activated NADPH oxidase, an effect blocked by low [Ca2+]e, but not by EHT1864. Nox5 knockdown abrogated agonist-stimulated •O2− production and inhibited phosphorylation of ERK1/2, but not p38MAPK or SAPK/JNK. Nox5 siRNA blunted AngII-induced, but not ET-1-induced, upregulation of PCNA and VCAM-1, important in growth and inflammation.

Conclusions

Human ECs possess functionally active Nox5, regulated by AngII and ET-1 through Ca2+/calmodulin-dependent, Rac-1-independent mechanisms. Nox5 activation by AngII and ET-1 induces ROS generation and ERK 1/2 phosphorylation. Nox5 is involved in ERK1/2-regulated growth and inflammatory signaling by AngII but not by ET-1. We elucidate novel mechanisms whereby vasoactive peptides regulate Nox5 in human ECs and demonstrate differential Nox5-mediated functional responses by AngII and ET-1. Such phenomena link Ca2+/calmodulin to Nox5 signaling, potentially important in the regulation of endothelial function by AngII and ET-1.

Keywords: Reactive oxygen species, vascular cells, vasoactive peptides, ERK1/2

Introduction

Reactive oxygen species (ROS) play a pivotal role as signaling molecules in the regulation of vascular function. Under physiological conditions, ROS modulate cell growth and vasodilation whilst in pathological conditions they have been implicated in vascular cell proliferation, contraction, migration and inflammation and as such may be important in vascular diseases (1–3). Among the many enzymatic sources of vascular ROS, the non-phaogocytic Nox family of NADPH oxidases are particularly important (4–6). The Nox family, based on homologues of the prototype Nox2 (gp91phox), comprises seven members, Nox1–Nox7 (7,8).

Vascular cells possess multiple Noxes including Nox1, Nox2, Nox4 (9–12) and the recently identified Nox5 (7,13–15). Common to all vascular Noxes is their ability to generate ROS. However the specific species generated, their intracellular distribution, their requirement for interaction with other NADPH oxidase subunits (p22phox, p47phox, p67phox and p40phox) and the small G protein Rac-1, and their mode of regulation differ. Whereas Nox1 and Nox2 (which localize mainly in the plasma membrane) generate O2−, Nox4 (which localizes in focal adhesions and endoplasmic reticulum) seems to produce primarily H2O2 (16–18). Vascular Noxes are expressed in a cell-specific manner, with endothelial cells expressing mainly Nox2 and Nox4; vascular smooth muscle cells, Nox1, Nox2 and Nox4; and adventitial fibroblasts, Nox2 and Nox4 (19–21). Regulation of vascular Noxes, through de novo protein synthesis of Nox homologues (22,23) and phosphorylation of NADPH oxidase regulatory subunits (24,25), is multifactorial, involving physical factors (stretch, pressure), chemical factors (pH, O2) and vasoactive agents (angiotensin II (Ang II), endothelin-1 (ET-1), aldosterone and growth factors) (26,27).

Recent evidence indicates that vascular cells also possess Nox5 of which four splice variants Nox5α, Nox5β, Nox5γ and Nox5δ have been identified (15,28). The Nox5 gene is present in humans, but not in rodents. Unlike other vascular Noxes, Nox5 possesses an amino-terminal calmodulin-like domain with four binding sites for Ca2+ (EF hands) and does not require p22phox or other subunits for its activation (13,14,29). Whether Nox5 regulation involves the small G protein Rac-1, important for other Noxes, is unclear. Binding of Ca2+ to Nox5 induces a conformational change leading to enhanced ROS generation (29). The functional significance of vascular Nox5 is unknown, although it has been implicated in endothelial cell proliferation and angiogenesis (15), in PDGF-induced proliferation of vascular smooth muscle cells (30) and in oxidative damage in atherosclerosis (31). Vascular Nox5 is activated by thrombin, PDGF and ionomycin through PKC and cAMP response element-binding protein (CREB) (15,28,30,32). However, it is unknown whether Ang II and ET-1, important vasoactive agonists that regulate vascular contraction, dilation, growth and fibrosis, influence Nox5. Considering that these agonists signal through increased [Ca2+]i, as we previously demonstrated (33), we questioned whether Ang II and ET-1 regulate Nox5 through Ca2+- and calmodulin-dependent mechanisms and whether Rac-1 plays a role in its activation. In addition we sought to evaluate whether Nox5-based NADPH oxidase is functionally important in redox signaling involved in growth and inflammatory responses by Ang II and ET-1 in human endothelial cells.

Methods

Cell Culture

Human microvascular endothelial cells (ECs) (Human Microvascular Endothelial Cells adult dermis – HMVECad, #C-011-5C, Cascade Biologics, low passage) were studied. Cells were stimulated with Ang II or ET-1 (0.1 μmol/L) for 2 to 24 hours. In some studies, cells were exposed to the following inhibitors 30 minutes prior to stimulation: diltiazem (Ca2+ channel blocker, 0.1 μmol/L), calmidazolium (selective calmodulin inhibitor, 0.1 μmol/L), EHT1864 (Rac-1 inhibitor, 1 μmol/L), PD98059 (MEK1/2 inhibitor, 1 μmol/L), actinomycin D (transcription inhibitor, 10 μmol/L) and cycloheximide (protein synthesis inhibitor, 1 μmol/L). Cells were also exposed to reduced Ca2+ media containing 50% less Ca2+ than the normal media.

Immunofluorescence Microscopy

Cells were incubated with primary antibody overnight at 4°C (anti-Nox 5 from D. Lambeth and K. Heinz Kraus, 1:500), anti-Nox 2 (from M. Quinn, 1:500), anti-Nox 4 (Santa Cruz, Ca., 1:500) and DAPI (Molecular Probes, 1:2000). Proteins were detected with anti-rabbit secondary antibody (Alexa fluor 488, Molecular Probes, 1:1000). Immunofluorescence images were acquired and analyzed (Stallion High Speed Digital Microscopy Workstation, Slidebook, Zeiss).

Nox5 mRNA detection

Quantitative real-time PCR (Applied Biosystems) was used to analyze mRNA expression of Nox5. Expression of Nox5 was interpolated from a standard curve (constructed from an independent sample of pooled EC cDNA) and expressed relative to 18S.

Western Blotting

Western blotting was used to examine expression of Nox1, Nox2, Nox4, Nox5, L-type Ca2+ channel, VCAM-1 and PCNA (proliferating cell nuclear antigen), GAPDH, β-actin and activation (phosphorylation) of ERK 1/2, p38MAP kinase, SAPK/JNK and CREB. In some experiments, positive Nox5 controls (Nox 5-overexpressing HEK 293 cells (15,29), ovarian tumour cell lines (gift from B. Vanderhyden, OHRI, Univ of Ottawa) and negative controls (rat vascular smooth muscle cells) were included.

Measurement of intracellular free Ca2+ concentration ([Ca2+]i)

Endothelial cell [Ca2+]i was measured using the fluorescent probe fura-2AM (Molecular Probes, OR) as we previously described (35). [Ca2+]i responses were measured in cells exposed to Ang II, ET-1 and the L-type Ca2+ channel agonist 1-4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl) phenyl] pyridine-3-carboxylic acid (Bay K8644, 0.1 μmol/L) in the presence of extracellular Ca2+. In some studies, cells were exposed 30 minutes prior to stimulation to diltiazem (Ca2+ channel blocker, 0.1 μmol/L).

Measurement of NAD(P)H Oxidase Activity

The lucigenin-derived chemiluminescence assay was used to determine NAD(P)H oxidase activity in total EC homogenates as previously described (35). Activity was expressed as arbitrary units/mg protein.

Nox 5 siRNA Transfection Studies

To examine the role of Nox5 in ROS production and signaling, ECs were transiently transfected with small interfering RNA (siRNA) against human Nox5 (Santa Cruz Biotechnology). Gene silencing was monitored by Nox5 protein expression. After 48h of siRNA transfection, cells were stimulated with Ang II or ET-1, and production of ROS, phosphorylation of ERK1/2, p38 MAP kinase and SAPK/JNK and expression of PCNA and VCAM-1 were measured.

Immunoprecipitation

To evaluate whether Nox5 associates with calmodulin, we immunoprecipitated Nox5 (anti-Nox5 antibody from Santa Cruz Biotechnology) and probed for calmodulin in basal and stimulated conditions.

See online supplement for detailed Methods section

Data Analysis

Effects of Ang II and ET-1 were determined relative to vehicle, with the control normalized to 100%. Results are presented as mean±SEM and compared by ANOVA or by the Student t test when appropriate. Values of P<0.05 were considered to be significant.

Results

Subcellular distribution of Nox5

Cellular localization of Nox2, Nox4 and Nox5 was assessed by immunofluorescence. In ECs Nox 5 was localized primarily in the perinuclear area (Online figures IA, IB). In contrast, Nox2 and Nox4 were distributed in both the cytosol and the plasma membrane (Online figures IC, ID). Using the same antibody (Lambeth antibody) as that for the immunofluorescence studies, we show by western blotting that Nox5 is indeed expressed (75 kDa) in human endothelial cells as well as in Nox5-overexpressing HEK cells, ovarian tumour cell lines and human vascular smooth muscle cells, but not in native HEK cells

Effects of Ang II and ET-1 on Nox 5 expression in human endothelial cells - Role of Ca2+/calmodulin

Exposure of ECs to Ang II and ET- increased Nox5 expression at both the gene and protein levels as assessed by real-time PCR and western blotting (Figures 1A, ​1B, online figures II and III). Figure 1A demonstrates expression of Nox5 in human endothelial cells, Nox5-overexpessing HEK cells and ovarian cell lines (positive controls), but not in HEK293 cells and rat vascular smooth muscle cells (negative controls). Expression of Nox4, (Figures 1A, ​1B), Nox1 (Online figure IVA, IVB) and Nox2 (Online figure VA, VB), was unaltered by Ang II and ET-1. Exposure of cells to another vascoactive agent, aldosterone (10−7 mol/L) did not significantly influence Nox5 expression (Online figure VI).

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Nox4 and Nox5 protein expression in human endothelial cells stimulated with Ang II (B) and ET-1 (C) for 2–24 hours. Representative immunoblots demonstrate Nox4, Nox5 and β-actin expression in human endothelial cells in control (C) and stimulated conditions (2–24 hrs). Top immunoblot (A) shows Nox5 expression in HEK cells that stably overexpress Nox5 (positive control), human endothelial cells (HMEC), human ovarian tumor cell line (positive control) and rat vascular smooth muscle cells (negative control). Bar graphs are means ± SEM from 7 experiments. Results were normalized to β-actin. Control (C) was taken as 100% and data presented as the percentage change relative to control conditions. *p<0.05 vs C.

To evaluate mechanisms whereby Ang II and ET-1 regulate Nox5, cells were pretreated with actinomycin D (transcription inhibitor) and cycloheximide (protein synthesis inhibitor). As shown in online figures VIIA and VIIB, upregulation of Nox5 by Ang II and ET-1 was inhibited by actinomycin D and cycloheximide.

Phosphorylation of CREB is important for Nox5 regulation. Here we show that CREB activation is significantly increased by Ang II and ET-1, with maximal responses obtained within 4 hours (Online figures VIIIA, VIIIB).

To assess the role of Ca2+/calmodulin in Ang II and ET-1-induced regulation of Nox5, cells were exposed to diltiazem (a L-type Ca2+ channel blocker) or calmidazolium (a calmodulin inhibitor) for 30 minutes prior to agonist stimulation (24 hrs). The presence of L-type Ca2+ channels in ECs was confirmed by western blotting. As shown in online figure IXA and IXB, human ECs possess L-type Ca2+ channels, which are regulated by Ang II and ET-1. Ang II-induced Nox5 expression was decreased in the presence of diltiazem, calmidazolium and reduced Ca2+ medium (Figures 2A, ​2B). Manipulating cellular Ca2+ levels by diltiazem or reduced Ca2+ medium also decreased ET-1-induced Nox5 expression (Figure 2C), whilst calmidazolium treatment only partially inhibited ET-1-induced effects (Figure 2D). Ca2+ channel activation by Bay K8644 significantly increased Nox5 expression (Online figure XC). To confirm that Ang II and ET-1 increase [Ca2+]i, at the concentrations used in our study, we used fura-2AM to assess Ca2+ transients in agonist-stimulated cells. As shown in online figure XA, Ang II and ET-1 significantly increased [Ca2+]i. Bay K8644 also induced a significant increase in EC [Ca2+]i (Online figure XB).

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Diltiazem, calmidazolium and low [Ca2+]e attenuate agonist-induced effects on Nox5 expression. Cells were exposed to diltiazem (L-type Ca2+ channel blocker) or calmidazolium (calmodulin inhibitor) for 30 minutes prior to agonist stimulation (24 hrs). In some experiments cells were grown in reduced Ca2+ culture media containing 50% of normal Ca2+ levels (Ca2+ 50%). Upper panels are representative immunoblots. Results were normalized to GAPDH. Control (C) was taken as 100% and data presented as the percentage change relative to control conditions. Data are means ± SEM from 5 experiments. *p<0.05 vs C.

Ang II and ET-1-induced ROS production is Ca2+-dependent

To determine if Ang II and ET-1 influence NADPH oxidase activation through Ca2+-dependent processes, oxidase activity was measured by enhanced lucigenin chemiluminescence in cells that were exposed to reduced [Ca2+]e. As shown in Figures 3A and ​3B, Ang II and ET-1 induced a significant increase in NADPH oxidase activation. This effect was significantly blunted when cells were grown in low Ca2+ -containing medium (Figure 3C, 3D) and in the presence of calmidazolium (Figures 4A, ​4B). To confirm an association between Nox5 and calmodulin, we immunoprecipitated Nox5 and probed for calmodulin. As shown in figure 4C, even in basal conditions, calmodulin associates with Nox5, an effect that is enhanced by Ang II and ET-1.

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Regulation of NADPH oxidase by Ca2+. NADPH oxidase is activated by Ang II (A) and ET-1 (B) as assessed by enhanced lucigenin (5 μmol/L) chemiluminescence. Reduced Ca2+ culture media (Ca2+ 50%) attenuated Ang II (C) and ET-1 (D)-induced activation of NADPH oxidase. *p<0.05 vs other groups

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Regulation of NADPH oxidase activity and Nox5 by calmodulin. Ang II- and ET-1-induced activation of NADPH oxidase is inhibited by calmidazolium (fig 4A and 4B). Data are means ± SEM from 6 experiments. *p<0.05 vs other groups. Figure 4C. Ang II and ET-1 promote association between Nox5 and calmodulin. Nox5 was immunoprecipitated (IP) and then probed for calmodulin by western blotting (WB). Data are means ± SEM from 6 experiments. *p<0.05 vs control (Ctl). IGG, immunoglobulin G, and Nox5, negative and positive controls respectively.

siRNA knockdown of Nox5 decreases ROS production in human ECs

Since Nox5 is a Ca2+-dependent Nox homologue and because we found that agonist stimulated NADPH oxidase is Ca2+-sensitive, we questioned whether Nox5-based NADPH oxidase contributes to ROS production in ECs. Using siRNA Nox5 was downregulated in ECs. Significant reduction in expression of Nox5, but not Nox4, was evident after 48 and 72 hours of incubation with siRNA. Since the knockdown of Nox5 was optimal at 48 hours (p<0.05), this time point was used for further experiments (Figure 5A).

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Nox5 is important in ROS generation stimulated by Ang II and ET-1. siRNA knockdown of Nox5 decreases ROS production. Nox5, but not Nox4, was downregulated with siRNA. Significant reduction in expression of Nox5, but not Nox4, was evident after 48 hours as shown by western blot (A). RNA interference knockdown of Nox5 abrogated ROS generation induced by Ang II and ET-1 in ECs (B,C). Data are means ± SEM from 5 experiments. *p<0.05 vs control (C) and non-silenced (NS) conditions. Figure 5D, effects of EHT1864 on Ang II- and ET-1-induced activation of NADPH oxidase. Cells were exposed to the Rac1 inhibitor for 30 mins prior to Ang II and ET-1 stimulation. Data are means ± SEM from 5 experiments. *p<0.05 vs control (C).

Knockdown of Nox5 by RNA interference abrogated ROS generation induced by Ang II and ET-1 (Figures 5B, ​5C). EHT1864, a Rac1 inhibitor, did not interfere with Ang II and ET-1-induced NADPH oxidase activation (Figure 5D), but decreased Ang II and ET-1-stimulated Rac-1 translocation, as evidenced by decreased membrane expression of Rac-1 (Online figure XI).

siRNA knockdown of Nox5 attenuates Ang II and ET-1-induced effects on ERK1/2, but not p38MAP kinase or SAPK/JNK, phophorylation and differentially influences VCAM-1 and PCNA expression

To assess the functional significance of Nox5-based NADPH oxidase in ECs, we evaluated the effects of Ang II and ET-1 on ERK1/2 phosphorylation and growth and inflammatory responses. As shown in figure 6A, downregulation of Nox5 is associated with significantly reduced activation of ERK1/2, but had no effect on phosphorylation of p38MAP kinase and SAPK/JNK (Online figures XIIA, XIIB). Short-term (5 minutes) exposure to Ang II and ET-1 increased ERK 1/2 phosphorylation. In Nox5 siRNA-transfected cells, neither Ang II nor ET-1 increased ERK 1/2 activation (Figure 6A). Treatment with calmidazolium also blocked Ang II and ET-1-induced activation of ERK 1/2 (Figure 6B). Ang II and ET-1 increased expression of VCAM-1 and PCNA, molecular markers of inflammation and cell growth. Downregulation of Nox5 by siRNA (Figures 7A–D) and ERK 1/2 inhibition (Online figures XIII) blunted Ang II-induced, but not ET-1-induced upregulation of VCAM-1 and PCNA.

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siRNA knockdown of Nox5 decreases ERK 1/2 activation. Downregulation of Nox5 and inhibition of calmodulin were associated with significantly reduced activation of ERK1/2. Short-term (5 minutes) exposure to Ang II and ET-1 increased ERK 1/2 phosphorylation. In Nox5 siRNA-transfected cells (A) and cells exposed to calmidazolium (B), neither Ang II nor ET-1 increased ERK 1/2 activation. Data are means ± SEM from 5 experiments. *p<0.05 vs control (C) and non-silenced (NS) conditions.

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siRNA knockdown of Nox5 decreases VCAM-1 and PCNA expression induced by Ang II, but not by ET-1. Downregulation of Nox5 was associated with significantly reduced expression of VCAM-1 (A) and PCNA (B) induced by Ang II. Nox5 siRNA did not affect ET-1-induced effects on VCAM-1 (C) and PCNA (B) expression. Data are means ± SEM from 5 experiments. *p<0.05 vs control (C) and non-silenced (NS) conditions.

Discussion

Major findings from our study demonstrate that 1) human ECs possess a functionally active Nox5-based NADPH oxidase that localizes in the perinuclear region, 2) Ang II and ET-1 regulate Nox5 through Ca2+- and calmodulin-dependent and Rac-1-independent processes, 3) Nox5, which associates with calmodulin, is involved in ROS generation and ERK1/2, but not p38MAPK or SAPK/JNK, signaling by Ang II and ET-1 and 4) Nox5 is involved in PCNA and VCAM regulation by Ang II but not by ET-1. Our findings elucidate novel mechanisms whereby vasoactive peptides regulate Nox5 in human endothelial cells (Figure 8). Such phenomena link Ca2+/calmodulin to Nox5-mediated ROS production and ERK1/2 signaling, important factors in the regulation of endothelial function by agonists that signal through G protein-coupled receptors, such as Ang II and ET-1. We also highlight the fact that Nox5 is differentially involved in functional responses to Ang II and ET-1. Whereas Nox5 is involved in growth and inflammatory responses induced by Ang II, this is not so for ET-1. Moreover regulation of MAP kinases is highly specific, since activation of ERK1/2, but not p38MAP kinase or SAPK/JNK, by Ang II and ET-1 involves Nox5-sensitive processes.

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Schematic demonstrating mechanisms of Nox5 regulation by Ang II and ET-1 in human endothelial cells. Binding of Ang II and ET-1 to their respective receptors (ATR, ETR), stimulates Ca2+ influx through L-type Ca2+ channels and activation of calmodulin, which interact with Ca2+/calmodulin-binding domains to activate Nox5. Activated Nox5 results in generation of superoxide (•O2−) and phosphorylation (p) of ERK1/2. These processes are independent of Rac-1. Stimulation of ECs by Ang II and ET-1 induces upregulation of PCNA (proliferating cell nuclear antigen) and VACM-1 (vascular cell adhesion molecule-1) important in growth and inflammation respectively. Whereas Ang II stimulates these responses through Nox5/•O2−/ERK1/2, ET-1 influences PCNA and VCAM-1 through Nox5-independent processes.

Endothelial cells are involved in maintaining normal vascular function and integrity. Under pathological conditions, the phenotype of endothelial cells changes from being vasodilatory, anti-inflammatory and antithrombotic to pro-inflammatory, angiogenic, adhesive and contractile. Critical to these processes is an increased bioavailability of ROS, generated in response to activation of Nox-based NADPH oxidases (36). Human ECs possess multiple Nox homologues including Nox1, Nox2, Nox4 and Nox5 (9–15), the functional significance of which still remains elusive. Petry et al demonstrated that in human ECs Nox2 and Nox4 are abundantly expressed and that they contribute equally to ROS production (37). Nox1 is less prominent than other Noxes in ECs and does not appear to be important in ROS generation, since Nox1 depletion did not alter ROS levels under basal conditions (9,38). In human lung endothelial cells, Nox2 and Nox4 play a physiological role in hyperoxia-induced ROS production and migration (39). Nox5, although extensively expressed in lymphatic cells, prostate and testes, has only recently been identified in ECs. We demonstrate here that Nox5 has a predominantly intracellular distribution in human ECs, mainly in the perinuclear region, whereas Nox2 and Nox4 localize in the plasmalemmal area. Nox5 appears to concentrate in the endoplasmic reticulum, while Nox4 has been identified in focal adhesions (13–15). The differential distribution of Nox homologues in ECs may relate to functional diversity and possibly to Nox-specific generation of distinctive reactive oxygen species. For example, Nox4 seems to generate mainly H2O2 whereas Nox5 produces •O2−, H2O2 as well as NO (15,37,40). Moreover although Nox2 and Nox4 require other NADPH oxidase subunits, particularly p22phox, for their regulation, Nox5 does not seem to have an obligatory need for these regulatory subunits (13–15). Finally, different Nox homologues appear to activate different kinases and signaling pathways. For example in Nox2 overexpressing cells, p38MAPK is phosphorylated in response to Ang II, whereas in Nox4 overexpressing cells, ERK1/2 and JNK are activated (41,42)

Molecular mechanisms controlling Nox5 have not been fully elucidated, but PKC, c-Abl, CREB and phosphatidylinositol (4,5)-bisphosphate have been implicated (32,43–45). Considering the unique feature of Nox5 in that it possesses a calmodulin-like domain with four binding sites for Ca2+, we questioned whether Ang II and ET-1, which increase [Ca2+]i, regulate Nox5 through Ca2+ and calmodulin-dependent processes. Both agonists, important in endothelial function, significantly increased mRNA and protein expression of Nox5, but not Nox1, Nox2 or Nox4, in a time-dependent manner. Nox5 regulation appears to be both at the transcriptional and translational levels, since actinomycin D and cycloheximide prevented agonist-induced increase in Nox5 expression. These Nox5 effects are not generalized phenomena, as other vasoactive agonists, such as aldosterone, did not influence expression of Nox5 in our study. We also showed that in human ECs Ang II and ET-1 significantly increased [Ca2+]i, in part through L-type Ca2+ channels, and that this phenomenon influences Nox5 regulation. This is based on the findings that 1) agonist-induced increases in Nox5 expression are reduced when cells are cultured in low Ca2+-containg medium, 2) the upregulation of Nox5 by Ang II and ET-1 is attenuated in cells pretreated with the L-type Ca2+ channel blocker diltiazem, 3) Ang II and ET-1-stimulated Nox5 expression is decreased in ECs exposed to the calmodulin inhibitor calmidazolium and 4) the L-type Ca2+ channel agonist Bay K8644 stimulates Ca2+ influx and increases Nox5 expression. These data indicate that Ang II and ET-1 promote Ca2+ influx through L-type Ca2+ channels, which we show here to be functionally present in human ECs, and that this results in increases in [Ca2+]i that modulate Nox5. This process probably also involves calmodulin as evidenced by the findings that calmidazolium attenuated agonist-induced Nox5 responses and that Ang II and ET-1 promote Nox5:calmodulin association.

Although Nox5 activation appears to be independent of the NADPH oxidase subunits, p22phox, p47phox and p67phox (13,14,29), it is unknown whether Rac-1, important in the regulation of other Noxes, is involved in Nox5 regulation (46). Here we show that the Rac-1 inhibitor, EHT1864, does not influence Nox5-based NADPH oxidase-derived ROS generation in response to Ang II and ET-1, although it inhibited agonist-stimulated Rac1 membrane translocation. These findings suggest that Nox5, at least in human ECs, does not have an obligatory need for Rac-1 for its activation. Our findings are in keeping with recent data demonstrating that whereas Nox1, Nox2 and Nox3 contain Rac-binding sites, Nox5 does not (47).

To elucidate the functional significance of Nox5 we examined ROS generation and MAP kinase activation by Ang II and ET-1 in human ECs in which Nox5 was knocked down with siRNA.Although Nox5 was not completely knocked down activation of NADPH oxidase was inhibited. Reasons for this are complex but may relate to the fact that perhaps Nox5 downregulation influences other NADPH oxidase subunits that may impact on activity of the oxidase. It is also possible that Nox5 might interact with other Nox isoforms, which in the context of Nox5 downregulation, would inhibit Nox5-associated Nox activity. Such considerations require further examination.

In Nox5 downregulated cells phosphorylation of ERK1/2, but not p38MAP kinase or SAPK/JNK, was significantly reduced, indicating the importance of Nox5 in redox- and ERK1/2 signaling by Ang II and ET-1. Moreover, these processes are Ca2+-sensitive, because exposure of cells to low Ca2+ attenuated ROS generation and ERK1/2 signaling. Hence not only does [Ca2+]i influence expression of Nox5, but it also regulates enzymatic activity of Nox5-based NADPH oxidase and ROS production. We showed that Ang II and ET-1 at concentrations above 0.01 μmol/L increase vascular cell [Ca2+]i to levels of ~ 1 μmol/L, the concentration at which Nox5 is highly sensitive to Ca2+. Upon increases in [Ca2+]i, Nox5 undergoes a conformational change leading to the activation of NADPH oxidation and electron transport (13,14,28,29). At lower [Ca2+]i Nox5 is regulated through protein kinase C (PKC)-dependent processes, which induce phosphorylation of residues 494 and 498 and enhanced enzyme activation (43,64).

The functional effects of Nox5 are not generalized phenomena and appear to have differential outcomes. Whereas Nox5 appears to be critical in growth and inflammatory responses by Ang II this may not be so for ET-1. This is supported by the findings that agonist-stimulated expression of PCNA and VCAM-1, molecular markers of growth and inflammation respectively, was suppressed by Nox5 downregulation and PD98059 only in Ang II-stimulated cells. The point of divergence in the signaling cascade for this differential response is downstream of ROS, since Nox5 is involved in ROS generation by both Ang II and ET-1.

Our findings have pathophysiological significance because Ang II and ET-1 play an important role in endothelial dysfunction and vascular damage and have been implicated in various cardiovascular pathologies associated with oxidative stress, including hypertension, atherosclerosis and ischemia-reperfusion injury. Guzik et al recently showed that in coronary arteries from patients with coronary artery disease, Nox5 protein and mRNA levels are increased and Ca2+-dependent NADPH-driven production of ROS in vascular membranes is augmented (31). They also demonstrated that Nox5 was expressed in the endothelium in the early stage lesions and in vascular smooth muscle cells in the advanced coronary lesions, suggesting that Nox5 is an important source of ROS in atherosclerosis (31).

In conclusion, our data demonstrate that human ECs possess functionally active Nox5 that is regulated at the transcriptional and translational levels by Ang II and ET-1 through Ca2+- and calmodulin-sensitive processes. Unlike other Noxes, Nox5 does not appear to have an obligatory need for Rac-1 for its activation, at least in human ECs. Regulation by Ang II and ET-1 occurs at multiple levels: acutely by activating Nox5-based NADPH oxidase enzymatic activity and chronically by regulating expression of Nox5 mRNA and protein. Moreover we show that Ang II and ET-1 signal through Nox5 via highly specific ERK1/2-mediated processes. These phenomena link Ca2+ to Nox5-based NADPH oxidase-derived ROS production and ERK1/2 signaling, crucial pathways in the regulation of vascular function by Ang II and ET-1. Such events may be involved in Ca2+- and redox-sensitive phenomena underlying endothelial function. Dysregulation of these processes, due to augmented Ang II and ET-1 signaling, may lead to Ca2+ overload and Nox5-derived oxidative stress which could contribute to endothelial dysfunction and vascular disease.

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Novelty and Significance

What is known?

What new information does this article contribute?

Supplementary Material

Supplemental Material

Acknowledgements

This study was funded by grant 44018 from the Canadian Institute of Health Research (CIHR). RMT is supported through a Canada Research Chair/Canadian Foundation for Innovation award. ACM is supported by a fellowship from the CIHR. DB is supported through a KRESCENT fellowship. TMP was supported by fellowships from the National Heart Foundation of Australia (O 04M 1727) and the Heart and Stroke Foundation of Canada.

Non-standard Abbreviations and Acronyms

EC Endothelial cells
[Ca2+]e Extracellular calcium levels
[Ca2+]i Intracellular calcium levels
ROS Reactive oxygen species
C Control
Ca2+ 50% Cell culture medium containing 50% reduced calcium levels
IP Immunoprecipitation assay
WB Western blotting
Ctl Control conditions
NS non-silenced conditions

Footnotes

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