Elsevier

Experimental Cell Research

Volume 313, Issue 12, 15 July 2007, Pages 2680-2686
Experimental Cell Research

Research Article
Reactive nitrogen and oxygen species activate different sphingomyelinases to induce apoptosis in airway epithelial cells

https://doi.org/10.1016/j.yexcr.2007.04.002Get rights and content

Abstract

Airway epithelial cells are constantly exposed to environmental insults such as air pollution or tobacco smoke that may contain high levels of reactive nitrogen and reactive oxygen species. Previous work from our laboratory demonstrated that the reactive oxygen species (ROS), hydrogen peroxide (H2O2), specifically activates neutral sphingomyelinase 2 (nSMase2) to generate ceramide and induce apoptosis in airway epithelial cells. In the current study we examine the biological consequence of exposure of human airway epithelial (HAE) cells to reactive nitrogen species (RNS). Similar to ROS, we hypothesized that RNS may modulate ceramide levels in HAE cells and induce apoptosis. We found that nitric oxide (NO) exposure via the NO donor papa-NONOate, failed to induce apoptosis in HAE cells. However, when papa-NONOate was combined with a superoxide anion donor (DMNQ) to generate peroxynitrite (ONOO), apoptosis was observed. Similarly pure ONOO-induced apoptosis, and ONOO-induced apoptosis was associated with an increase in cellular ceramide levels. Pretreatment with the antioxidant glutathione did not prevent ONOO-induced apoptosis, but did prevent H2O2-induced apoptosis. Analysis of the ceramide generating enzymes revealed a differential response by the oxidants. We confirmed our findings that H2O2 specifically activated a neutral sphingomyelinase (nSMase2). However, ONOO exposure did not affect neutral sphingomyelinase activity; rather, ONOO specifically activated an acidic sphingomyelinase (aSMase). The specificity of each enzyme was confirmed using siRNA to knockdown both nSMase2 and aSMase. Silencing nSMase2 prevented H2O2-induced apoptosis, but had no effect on ONOO-induced apoptosis. On the other hand, silencing of aSMase markedly impaired ONOO-induced apoptosis, but did not affect H2O2-induced apoptosis. These findings support our hypothesis that ROS and RNS modulate ceramide levels to induce apoptosis in HAE cells. However, we found that different oxidants modulate different enzymes of the ceramide generating machinery to induce apoptosis in airway epithelial cells. These findings add to the complexity of how oxidative stress promotes lung cell injury.

Introduction

ROS and RNS are components of tobacco smoke, environmental pollutants, and are products of inflammation that affect lung epithelial cell survival and function. Recent work from our laboratory demonstrated a novel mechanism by which airway epithelial cells escape apoptosis via an enhanced caspase-3 and acidic sphingomyelinase interaction induced by the RNS nitric oxide [1]. In addition, our laboratory also showed that the RNS, peroxynitrite (ONOO), induced cross-linking of the epidermal growth factor receptor and impaired downstream receptor signaling in human airway epithelial (HAE) cells [2]. We have also shown that exposure of HAE cells to the ROS, hydrogen peroxide (H2O2), activates a neutral sphingomyelinase (nSMase) to generate ceramide and induce apoptosis [3], [4], [5], [6], [7]. RNS, like ROS, may also damage lung tissue via the generation of ceramide, an inducer of apoptosis [8]. Indeed, RNS exposure in different cell types has generated ceramide and promoted apoptosis [8], [9].

Ceramide may be generated through the hydrolysis of sphingomyelin by sphingomyelinases (SMases). Neutral sphingomyelinase 2 (nSMase2) and acidic sphingomyelinase (aSMase) belong to a family of sphingomyelinase enzymes, which were initially characterized on the basis of the pH optima of activation [10]. nSMase2 has been demonstrated to be a redox-sensitive enzyme while aSMase may be modulated by UV- and gamma-radiation [4], [11], [12], [13]. Activation of both enzymes is associated with the generation ceramide and induction of apoptosis. Although there is a link between RNS exposure and lung tissue injury, it is unclear how RNS modulate the ceramide machinery in HAE cells to induce apoptosis [14], [15], [16], [17]. In the current study we found that the RNS ONOO, like the ROS H2O2, generates ceramide and induces apoptosis in HAE cells. However, unlike H2O2, ONOO generates ceramide through a different ceramide-generating enzyme, aSMase. Characterizing how different oxidants affect apoptosis may provide better therapies for pulmonary ailments.

Section snippets

Reagents

Cell culture media, buffers, and fetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA). C6-ceramide and cardiolipin were from Matreya Inc. (Pleasant Gap, PA). Recombinant sn-1,2-diacylglycerol kinase (Escherichia coli), peroxynitrite, papa-NONOate, and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) were from Calbiochem (La Jolla, CA). N-methyl-[14C] sphingomyelin and [3H]-palmitic acid were from Amersham Biosciences (Piscataway, NJ). [γ32P] adenosine triphosphate (ATP) (5 mCi) was from ICN

Do RNS induce apoptosis in HAE cells as hydrogen peroxide?

Recent work from our laboratory demonstrated that exposure of HAE cells to the ROS, H2O2, induces apoptosis via the activation of nSMase2 and the generation of ceramide [3], [4], [5], [7]. To determine if RNS exposure mediates a similar response in HAE cells, cells were exposed to NO (via papa-NONOate) for 24 h and apoptosis was assessed. Papa-NONOate exposure did not induce apoptosis in HAE cells, while H2O2 exposure did (Fig. 1A). C6-ceramide was used as a positive control for the induction

Discussion

Our findings are the first to show how different oxidants modulate different components of the ceramide generating machinery to generate ceramide and induce apoptosis in airway epithelial cells (Fig. 7). Previously we demonstrated the role of nSMase2 in H2O2-mediated apoptosis in HAE cells. In the current study, we expand our findings on oxidative stress and apoptosis in HAE cells by examining the biological consequence of RNS exposure. Nitric oxide exposure did not promote an apoptotic

Acknowledgments

The authors would like to thank the members of the Goldkorn laboratory for their support and critical reading of the manuscript. S.S. Castillo was supported by the training grant T32-HL07013 and grants HL-71871 (T. G.) and HL-66189 (T.G.) from NIH and from a PM External Research Program grant. Additionally, this investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 RR-12088-01 from the National Center for Research

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