Abstract
Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.
Highlights
The sphingomyelin pathway is initiated by the hydrolysis of sphingomyelin to generate the second messenger ceramide.[1]
Environmental stress and inflammatory cytokines[1,18,19,20,21,22,23,24,25,26,27] stimulate stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) signaling, which involves the sequential activation of members of the mitogen-activated protein kinase (MAPK) family, including MAPK/ERK kinase kinase (MEKK)1/MAPK kinase (MKK)[4], and/or SAPK/ERK kinase (SEK)1/MKK7, JNK, and c-jun
The repression of JNK signaling by overexpression of the S270A mutant or the JNK1-DN-mutant negatively regulated Neutral sphingomyelinase (nSMase)[1] activation and ceramide signaling in zebrafish cells
Summary
NSMase[1] phosphorylation in response to stress stimuli. We used zebrafish ZE cells as a model to investigate the role of nSMase[1], in addition to Jurkat T cells, which represent a more physiologically relevant human model. Exposing MAPK8- and/or MAPK9-knocked-down cells to heat shock at 43.5 1C for 1 h, UV irradiation at 5 mJ/cm[2] and 254 nm, 1 mM H2O2, and 50 ng/ml anti-Fas antibody induced morphological and biochemical features typical of apoptosis, as determined using DAPI staining (Figure 6d) and a caspase-3 activity assay (Figure 6e) These effects were JNK1/2 depletion- and time-dependent. We confirmed that pretreating Jurkat T cells with 40 mM caspase inhibitor (carboxybenzyl-VAD-fluoromethyl ketone (z-VAD-fmk)) for 1 h prevented stress-induced apoptosis under conditions such as heat shock at 43 1C for 1 h, UV irradiation at 5 mJ/cm[2, 1] mM H2O2, and treatment with 50 ng/ml of anti-Fas antibody (Supplementary Figure S12). Our findings based on subcellular fractionation and immunocytochemical staining indicate that nSMase[1] and its phosphorylated form localized primarily to the Golgi apparatus, ER, and nuclear fractions in Jurkat T cells under stress conditions and upon Fas stimulation
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