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