Summary In mammals, ceramide, a key intermediate in sphingolipid metabolism and an important signaling molecule, is synthesized by a family of six ceramide synthases (CerS), each of which synthesizes ceramides with distinct acyl chain lengths. There are a number of common biochemical features between the CerS, such as their catalytic mechanism, and their stucture and intracellular localization. Different CerS also display remarkable differences in their biological properties, with each of them playing distinct roles in processes as diverse as cancer and tumor suppression, in the response to chemotherapeutic drugs, in apoptosis, and in neurodegenerative diseases.
The epidermal growth factor (EGF) receptor (EGFR) has been found to be overexpressed in several types of cancer cells, and the regulation of its oncogenic potential has been widely studied. The paradigm for EGFR down-regulation involves the trafficking of activated receptor molecules from the plasma membrane, through clathrin-coated pits, and into the cell for lysosomal degradation. We have previously shown that oxidative stress generated by H 2 O 2 results in aberrant phosphorylation of the EGFR. This leads to the loss of c-Cbl-mediated ubiquitination of the EGFR and, consequently, prevents its degradation. However, we have found that c-Cbl-mediated ubiquitination is required solely for degradation but not for internalization of the EGFR under oxidative stress. To further examine the fate of the EGFR under oxidative stress, we used confocal analysis to show that the receptor not only remains colocalized with caveolin-1 at the plasma membrane, but at longer time points, is also sorted to a perinuclear compartment via a clathrin-independent, caveolae-mediated pathway. Our findings indicate that although the EGFR associates with caveolin-1 constitutively, caveolin-1 is hyperphosphorylated only under oxidative stress, which is essential in transporting the EGFR to a perinuclear location, where it is not degraded and remains active. Thus, oxidative stress may have a role in tumorigenesis by not only activating the EGFR but also by promoting prolonged activation of the receptor both at the plasma membrane and within the cell. Activation of the epidermal growth factor (EGF)2 receptor (EGFR) by EGF results in the initiation of signal transduction cascades involved in cellular survival and proliferation. Therefore, to control cellular growth and tumorigenesis, the activation of the EGFR has to be tightly regulated in a process that includes degradation of the receptor. Binding of EGF to EGFR is rapidly followed by internalization of the membrane-bound receptor mainly through clathrin-coated pits and into early endosomes, which develop into late endosomes. There, the EGFR is either targeted to lysosomes for degradation or recycled to the plasma membrane (1-3). The inability of the EGFR to be down-regulated via clathrinmediated endocytosis and degradation has been linked to its oncogenicity (4, 5).H 2 O 2 -induced oxidative stress has been shown to activate and aberrantly phosphorylate the EGFR, which impedes the clathrin-mediated endocytosis and subsequent lysosomal degradation of the receptor (6 -8). This results in prolonged downstream activation of proliferative molecules such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) (9), and the lack of receptor turnover has been shown to mediate tumor promotion in non-neoplastic rat liver epithelial cells (10). To gain more insight into H 2 O 2 -induced EGFR signaling and hyperplastic responses, we examined the trafficking of the receptor under oxidative stress.Huang and Sorkin (11) have recently reported that knock-down of Grb2 by RNA interference inhibits clat...
We have generated a mouse that cannot synthesize very long acyl chain (C22-C24) ceramides (Pewzner-Jung, Y., Park, H., Laviad, E. L., Silva, L. C., Lahiri, S., Stiban, J., Erez-Roman, R., Brugger, B., Sachsenheimer, T., Wieland, F. T., Prieto, M., Merrill, A. H., and Futerman, A. H. (2010) J. Biol. Chem. 285, 10902-10910) due to ablation of ceramide synthase 2 (CerS2). As a result, significant changes were observed in the sphingolipid profile of livers from these mice, including elevated C16-ceramide and sphinganine levels. We now examine the functional consequences of these changes. CerS2 null mice develop severe nonzonal hepatopathy from about 30 days of age, the age at which CerS2 expression peaks in wild type mice, and display increased rates of hepatocyte apoptosis and proliferation. In older mice there is extensive and pronounced hepatocellular anisocytosis with widespread formation of nodules of regenerative hepatocellular hyperplasia. Progressive hepatomegaly and noninvasive hepatocellular carcinoma are also seen from approximately 10 months of age. Even though CerS2 is found at equally high mRNA levels in kidney and liver, there are no changes in renal function and no pathological changes in the kidney. High throughput analysis of RNA expression in liver revealed up-regulation of genes associated with cell cycle regulation, protein transport, cell-cell interactions and apoptosis, and down-regulation of genes associated with intermediary metabolism, such as lipid and steroid metabolism, adipocyte signaling, and amino acid metabolism. In addition, levels of the cell cycle regulator, the cyclin dependent-kinase inhibitor p21(WAF1/CIP1), were highly elevated, which occurs by at least two mechanisms, one of which may involve p53. We propose a functional rationale for the synthesis of sphingolipids with very long acyl chains in liver homeostasis and in cell physiology.
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 ...
Cigarette smoke (CS) induces a rapid, sustained upregulation of ceramide production in human bronchial epithelial cells, leading to increased apoptosis. Using loss-of-function and overexpression analyses, we show that neutral sphingomyelinase 2 (nSMase2) is required for CS-mediated ceramide generation and apoptosis. Glutathione (GSH), a crucial antioxidant in lung defense, blocks nSMase2 activity and thus inhibits apoptosis triggered by CS. We found that the exposure to CS, as with exposure to H(2)O(2), results in increased nSMase2 activation leading to ceramide generation and therefore increased apoptosis. Interestingly, exposure of cells to GSH abolishes nSMase2 activation caused by CS and leads to a decrease in CS-induced apoptosis. This suggests that the effects of CS oxidants on nSMase2 are counteracted by GSH. Our data support a model where CS induces nSMase2 activation thereby increasing membrane-sphingomyelin hydrolysis to ceramide. In turn, elevated ceramide enhances airway epithelial cell death, which causes bronchial and alveolar destruction and lung injury in pulmonary diseases.
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