nSMase2 activation and trafficking are modulated by oxidative stress to induce apoptosis

Abstract

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. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript 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 ...

Identification of Novel Anionic Phospholipid Binding Domains in Neutral Sphingomyelinase 2 with Selective Binding Preference

“…For example, the PM localization of nSMase2 could be enhanced upon stimulation (e.g. TNF-␣, H 2 O 2 ) or cell confluence (11,15,16,44). A recent study also demonstrated that nSMase2 localization and activity were regulated by endocytosis (45).…”

“…Indeed, PA has emerged as a pivotal lipid messenger and can be produced by both phospholipase D and diacylglycerol kinase pathways. The identification of PA effectors can provide valuable insights into the cellular roles of PA. Interestingly, some of the known nSMase2 activators have also been reported to generate PA. For example, H 2 O 2 has been reported to induce nSMase2 activity in bronchial epithelial cells (44) and has also been shown to stimulate phospholipase D activity and generation of PA (47). Additionally, hypoxia can increase the intracellular level of PA through the action of DGK (48) and can also stimulate nSMase2 activity (49).…”

Aging Up-Regulates Expression of Inflammatory Mediators in Mouse Adipose Tissue

“…Both SMase and SPT seem to be regulated by the redox state. nSMase activity can be activated by decreasing total glutathione (GSH) and the GSH/glutathione disulfide (GSSG) ratio and increasing hydrogen peroxide, and it can be inhibited by increasing GSH (73)(74)(75)(76)(77). Aging is associated with increased oxidative stress and, in particular, the age-related loss of GSH and a reduced GSH/ GSSG ratio have been shown in different tissues (78 -81).…”

Isc1p Plays a Key Role in Hydrogen Peroxide Resistance and Chronological Lifespan through Modulation of Iron Levels and Apoptosis

“…In both conditions, the increased cell death was associated with apoptotic and oxidative stress markers ( Figure 11). Interestingly, the neutral sphingomyelinase 2, the mammalian orthologue of Isc1p, has been implicated in H 2 O 2 -and TNF-induced apoptosis (Luberto et al, 2002;Levy et al, 2006). However, it has also been suggested that ceramides containing different fatty acids may have distinct impacts in cell physiology, by differentially affecting the biophysical properties of the membrane lipid bilayer or by interacting with specific downstream components in signaling pathways (Cutler and Mattson, 2001).…”