Original ContributionCatalase potentiates interleukin-1β-induced expression of nitric oxide synthase in rat vascular smooth muscle cells
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 (O2−) 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 (O2−). 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.
References (43)
- et al.
Regulation of inducible nitric oxide synthase gene expression in vascular smooth muscle cells
Gen. Pharmacol.
(1999) - et al.
The cytokine responsive vascular smooth muscle cell enhancer of inducible nitric oxide synthase. Activation by nuclear factor-kappa B
J. Biol. Chem.
(1995) - et al.
Modulation of catalase peroxidatic and catalatic activity by nitric oxide
Free Radic. Biol. Med.
(2001) - et al.
Induction of nitric oxide synthase mRNA expression. Suppression by exogenous nitric oxide
J. Biol. Chem.
(1995) Nitric oxide and septic shock
Gen. Pharmacol.
(1997)- et al.
Nanotransducers in cellular redox signaling: modification of thiols by reactive oxygen and nitrogen species
Trends Biochem. Sci.
(2002) - et al.
p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy
J. Biol. Chem.
(1998) - et al.
Cytosolic peroxiredoxin attenuates the activation of JNK and p38 but potentiates that of ERK in HeLa cells stimulated with tumor necrosis factor-alpha
J. Biol. Chem.
(2004) - et al.
Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo+
Mol. Cell.
(2002) - et al.
Cytokine-induced activation of nuclear factor-kappa b is inhibited by hydrogen peroxide through oxidative inactivation of ikappa B kinase
J. Biol. Chem.
(2001)