Original Contribution
Catalase potentiates interleukin-1β-induced expression of nitric oxide synthase in rat vascular smooth muscle cells

https://doi.org/10.1016/j.freeradbiomed.2004.11.022Get rights and content

Abstract

The role of reactive oxygen species (ROS) in regulating the expression of the inducible nitric oxide synthase (iNOS) was studied in rat aortic vascular smooth muscle cells (VSMC). We hypothesized that ROS regulate iNOS expression through the mitogen-activated protein kinases ERK and p38MAPK. We found that interleukin-1β (IL-1β) stimulated the production of hydrogen peroxide (H2O2) which could be inhibited by loading the cells with the H2O2-scavenging enzyme catalase. Inhibition of the upstream ERK1,2 activator MEK1,2 with U0126 prevented IL-1β-stimulated iNOS expression, while the p38MAPK inhibitor SB03580 potentiated iNOS expression. Loading the cells with catalase enhanced ERK activation and iNOS expression but had no effect on p38MAPK activation or PDGF-induced ERK activation. These data indicated that H2O2 negatively regulates iNOS expression through ERK inhibition independently of p38MAPK. The present results outline a novel role for H2O2 in suppressing signaling pathways leading to gene expression such as iNOS in VSMC in response to cytokines.

Introduction

Vascular diseases including atherosclerosis, restenosis following balloon angioplasty and vascular hyporesponsiveness during sepsis are associated with the upregulation of the inducible isoform of nitric oxide synthase (iNOS) and the concomitant increase in the production of nitric oxide (NO) [1], [2]. Although iNOS expression is documented in different cell types of the vascular wall including macrophages, fibroblasts, and smooth muscle cells (VSMCs), the significance of its expression in VSMCs during vascular injury is unclear. The expression of iNOS has been proposed to be beneficial in the absence of a functional endothelium because NO suppresses adhesion of leukocytes and platelets, dilates blood vessels, and inhibits VSMC proliferation into the intima [1]. However, high concentrations of NO as produced through iNOS are deleterious to tissues, resulting in oxidative, nitrosative, and nitrative modifications [3]. This is compounded by recent reports indicating that iNOS expression may exacerbate atherosclerosis since mice lacking the iNOS gene exhibit decreased atherosclerotic lesion formation when crossed with ApoE-/- mice [4]. It is clear that in many inflammatory settings the timing, amplitude, and location of iNOS expression are critical for understanding the impact of NO production.

Interleukin-1β (IL-1β) is a proinflammatory cytokine implicated in the early stages of restenosis, the development of atherosclerosis, and ischemic heart disease in humans as well as experimental animal models [5], [6], [7], [8]. It is a potent inducer of iNOS expression in VSMC [9], [10], [11] through complex regulation by various signal transduction pathways including the mitogen-activated protein kinases (MAPKs) [2], [12], [13]. In this regard, recent studies indicate that the extracellular signal-regulated kinases (ERKs) are essential because pharmacological and antisense inhibition of ERK1,2-kinase (MEK 1,2) as well as forced overexpression of a dominant-negative MEK abolished IL-1β-mediated iNOS expression [12], [14]. The proximal signal transduction pathways eliciting ERK activity upon IL-1β stimulation are unclear at the present time but detailed studies of more distal events clearly indicated that ERK is necessary for the sustained activation of the transcription factor nuclear factor–κB (NF-κB), a critical step for iNOS expression [14].

A feature common to a number of cytokines and growth factors is their ability to induce the intracellular production of reactive oxygen species (ROS) including superoxide anion (O2radical dot) and hydrogen peroxide (H2O2), which in turn regulate various signaling pathways such as ERK [15]. Seminal work by Sundaresan and co-workers revealed that the transitory increase in H2O2 production induced by platelet-derived growth factor (PDGF) in VSMCs was necessary to obtain full activation of ERK [16]. In contrast, studies by Jiang and Brecher suggested that IL-1β-induced ROS production may limit ERK activation upon engagement of the IL-1β receptor, because pretreatment of the cells with antioxidants potentiated ERK activity [12]. Although pharmacological treatment with antioxidants provides a foundation for a role of ROS in negatively regulating ERK activity and iNOS expression upon cytokine stimulation, they do not provide critical information as to the specific nature of the ROS involved. In the present study, we investigated the effect of the H2O2-scavenging enzyme catalase on IL-1β induction of iNOS expression and its relationship with ERK activation. Our results are consistent with the endogenous production of H2O2 providing negative regulation of iNOS expression through modulation of ERK activity (Fig. 6).

Section snippets

Materials

Rat recombinant interleukin-1β was purchased from Sigma (St. Louis, MO). U0126 and SB203580 were purchased from Calbiochem (San Diego, CA). Antibodies to iNOS were purchased from Upstate Biotechnologies (Waltham, MA), antibodies to ERK and p38MAPK were from Cell Signaling Technologies (Beverly, MA), antibody to α-actin was from Sigma. NF-κB consensus oligonucleotide and T4 polynucleotide kinase were purchased from Promega (Madison, WI). All other reagents were purchased from Sigma.

Cell culture

Cells were

IL-1β stimulated the production of hydrogen peroxide in vascular smooth muscle cells

To examine whether IL-1β stimulates the production of hydrogen peroxide (H2O2) in VSMCs, the oxidation of intracellular 2′7′-dichlorofluorescin-diacetate to dichlorofluorescein was examined by confocal microscopy. H2DCF is sensitive to H2O2-mediated oxidation, but insensitive to superoxide (O2radical dot). As shown in Figs. 1A and 1B, there was a significant increase in fluorescence intensity upon IL-1β stimulation (50 ng/ml) compared to unstimulated cells indicating DCF formation. Sundaresan and

Discussion

IL-1β inhibits vascular contraction mostly by the production of iNOS-derived NO [10], [25]. However, the expression of iNOS in VSMC may also be compensatory in an endothelium-denuded vessel [1], [26], [27]. Previous studies demonstrated that ERK activation is necessary for IL1β-mediated iNOS expression in VSMC [12], [14]. There is also a large body of literature showing that mitogen-activated protein kinases including ERK are regulated by endogenous ROS production [15], [16], [28], [29], [30].

Acknowledgments

This work was supported in part by the National Institutes of Health Grants CA89366 to D.J. and HL49426 to H.S., B.G. is supported by a Predoctoral Training Grant, T32-HL-07194, from the National Heart, Lung, & Blood Institute.

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