We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H 2 O 2 ), induces apoptosis of human airway epithelial (HAE) cells. Under oxidant exposure, a lung sphingomyelinase (SMase) is activated and displays continued ceramide generation and proapoptotic signaling, thus leading to the pathological apoptosis that causes lung injury. In a search for a specific SMase that is modulated by oxidative stress, we recently cloned nSMase2 from monkey lung tissue and HAE cells. Here, we show that this nSMase2 is up-regulated by an oxidant (H 2 O 2 ) and is inhibited by an antioxidant (glutathione (GSH)). Moreover, nSMase2 subcellular localization is governed by oxidant exposure, which leads to its preferential trafficking to the plasma membrane, where it generates ceramide and induces apoptosis. On the other hand, exposure to GSH results in nSMase2 trafficking to the nucleus, where it neither generates ceramide nor induces apoptosis.
KeywordsNeutral sphingomyelinase 2; Apoptosis; Hydrogen peroxide; Glutathione; Ceramide; Lung Reactive oxidants such as hydrogen peroxide (H 2 O 2 ) cause lung injury and contribute to pulmonary diseases mainly through targeting human airway epithelial (HAE) cells. Yet, the cellular and molecular mechanisms that link these reactive oxidants to the development of lung injury and disease are not fully understood. We have shown that HAE cells exposed to reactive oxygen species (ROS) are stimulated to generate excessive ceramide, which functions as a potent inducer of apoptosis in these cells [1][2][3][4][5][6]. Although antioxidant defenses are constitutively expressed inmammalian cells [7], additional responses are mounted when the amount of environmental oxidants exceeds a threshold level, thereby becoming a threat to overall tissue integrity. Apoptosis is one such cellular adaptive response [8][9][10][11].The pro-apoptotic effects of ceramide are mediated by a variety of mechanisms [12][13][14][15]. Ceramide is synthesized primarily through a de novo pathway involving serine palmitoyl-☆ Abbreviations: BSA, bovine serum albumin; BSO, L-buthionine-SRsulfoximine; DAPI, 4′,6-diamidino-2-phenylindole; GSH, glutathione; H 2 O 2 , hydrogen peroxide; HAE, human airway epithelial; nSMase, neutral sphingomyelinase; ROS, reactive oxygen species; siRNA, small interfering RNA.
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Author ManuscriptCoA transferase and ceramide synthase or from membrane sphingomyelin breakdown by sphingomyelinases (SMases). Sphingomyelin (N-acylsphingosin-1-phosphocholine) is a phospholipid preferentially concentrated in the plasma membrane of mammalian cells [16]. Sphingomyelin catabolism occurs via the action of sphingomyelin-specific forms of phospholipase C termed sphingomyelinases (SMases), which hydrolyze the phosphodiester bond of sphingomyelin, yielding ceramide and phosphorylcholine. Ceramide then serves as a second messenger, leading to apoptotic DNA degradation. The main forms of SMases are distinguished by ...