Published online before print June 19, 2008, doi: 10.1161/ATVBAHA.108.168682
© 2008 American Heart Association, Inc.
Cell Biology/Signaling |
Sphingomyelin Synthase 2 Deficiency Attenuates NF
B Activation
From the Department of Anatomy and Cell Biology, Department of Microbiology and Immunology, Department of Medicine, State University of New York Downstate Medical Center (T.K.H., C.H., J.L., Z.L., C.R., R.W.), Brooklyn; and Lilly Research Laboratories (M.K., H.H.B., D.A.P., M.-S.K., G.C.), Eli Lilly & Company, Indianapolis, Ind.
Correspondence to Xian-Cheng Jiang, PhD, SUNY Downstate Medical Center, 450 Clarkson Ave, Box 5, Brooklyn, NY 11203. E-mail XJiang{at}downstate.edu or Raj Wadgaonkar, PhD, SUNY Downstate Medical Center. E-mail: rwadgaonker@downstate.edu
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Abstract |
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Background— NF
B has long been regarded as a proatherogenic factor, mainly because of its regulation of many of the proinflammatory genes linked to atherosclerosis. Metabolism of sphingomyelin (SM) has been suggested to affect NFB activation, but the mechanism is largely unknown. SMS2 regulates SM levels in cell plasma membrane and lipid rafts and has a potential to regulate NFB activation.
Methods and Results— To investigate the role of SMS2 in NFB activation we used macrophages from SMS2 knockout (KO) mice and SMS2 siRNA-treated HEK 293 cells. We found that NFB activation and its target gene expression are attenuated in macrophages from SMS2 KO mice in response to lipopolysaccharide (LPS) stimulation and in SMS2 siRNA- treated HEK 293 cells after tumor necrosis factor (TNF)–
simulation. In line with attenuated NFB activation, we found that SMS2 deficiency substantially diminished the abundance of toll like receptor 4 (TLR4)-MD2 complex levels on the surface of macrophages after LPS stimulation, and SMS2 siRNA treatment reduced TNF--stimulated lipid raft recruitment of TNF receptor-1 (TNFR1) in HEK293 cells. SMS2 deficiency decreased the relative amounts of SM and diacylglycerol (DAG) and increased ceramide, suggesting multiple mechanisms for the decrease in NFB activation.
Conclusions— SMS2 is a modulator of NFB activation, and thus it could play an important role in NFB-mediated proatherogenic process.
Key Words: sphingomyelin synthase 2 • sphingomyelin • lipid rafts • NFB • atherosclerosis
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Introduction |
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Atherosclerosis is an inflammatory disease. The accumulation of macrophage-derived foam cells in the vessel wall is always accompanied by the production of a wide range of chemokines, cytokines, and growth factors.1 These factors regulate the turnover and differentiation of immigrating and resident cells, eventually influencing plaque development. One of the key regulators of inflammation is NF
B,2 which has long been regarded as a proatherogenic factor, mainly because of its regulation of many of the proinflammatory genes linked to atherosclerosis.3,4
Sphingomyelin (SM) is one of the major lipids on the plasma membrane and is enriched in lipid rafts, which are considered microdomains of plasma membrane critical for signal transduction.5,6 Depletion of cholesterol from rafts causes a redistribution of TNF- receptor 1 to nonraft plasma membrane, preventing NFB activation7 or ligand-induced RhoA activation,8 and such treatment also inhibits proinflammatory signals mediated by TLRs.9 Studies also suggest that NFB activation is triggered by SM-derived ceramide.10,11 On the contrary, it has been shown that ceramide is not necessary or even inhibits NFB activation.12
SM biosynthesis might also affect NFB activation. SM is synthesized by sphingomyelin synthase (SMS), which transfers the phosphorylcholine moiety from phosphatidylcholine (PC) onto ceramide, producing SM and diacylglycerol (DAG).13 Luberto et al14 found that D609, a nonspecific SMS inhibitor, blocks TNF-- and phorbol ester–mediated NFB activation that was concomitant with decreased levels of SM and DAG. This did not affect the generation of ceramide, suggesting SM and DAG derived from SM synthesis are involved in NFB activation. However, D609 is widely used to inhibit PC-phospholipase C (PC-PLC), a well-known regulator of NFB activation via DAG signaling.15 Thus it remains unclear which pathway D609 inhibits to cause a diminished NFB activation.
Two SMS genes, SMS1 and SMS2, have been cloned and characterized for their cellular localizations.16,17 SMS1 is found in the trans-golgi apparatus, whereas SMS2 is predominantly found at the plasma membrane.16 We and other investigators have shown that SMS1 and SMS2 expression positively correlate with levels of SM in lipid rafts.18–20 Furthermore, SMS1 has been implicated in the regulation of lipid raft SM level and raft functions such as FAS receptor clustering,18 endocytosis, and apoptosis.19 However, the role of SMS2, the major SMS on the plasma membrane, in cell signaling, including NFB activation, is unknown.
In this report, we studied the role of SMS2 in NFB activation by using SMS2 KO mouse macrophages and SMS2 siRNA-treated HEK293 cells. In both cells, we found that SMS2 deficiency significantly attenuates NFB activation. We conclude that SMS2 is a modulator of NFB activation and may play important roles in inflammation during atherogenesis.
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Methods |
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See the online-only Data Supplement (available online at http://atvb.ahajournals.org) for expanded Methods for: (1) SMS2 KO mouse preparation; (2) Cell culture and transfection; (3) Cell surface receptor analysis by fluorescence-activated-cell sorter (FACS); (4) TNF-
binding and TNFR1 internalization assay; (5) Lipid analyses by LC/MS/MS; (6) mRNA analyses; SMS activity assay; (7) Lysenin treatment and cell mortality measurement; (8) Luciferase assay; (9) Immunocytochemistry; (10) Lipid raft isolation; and (11) statistical analysis.
Nuclear and Cytoplasmic Protein Preparation
The method is previously described by Dignam.21 Briefly, cells were washed in cold PBS and lysed in buffer (10 mmol/L Hepes pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, 0.01% NP-40) containing protease inhibitors. Nuclei were pelleted by centrifugation at 650g for 5 minutes at 4°C and the supernatant was collected as the cytoplasmic fraction. Nuclei were then resuspended in a buffer containing (10 mmol/L Hepes pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl, and 0.5 mmol/L DTT) and incubated on ice for 30 minutes with continuous agitation. The extract was recovered after centrifugation for 10 minutes at 12 000 rpm at 4°C. Proteins were separated on SDS-PAGE gels (Bio-Rad) and Western blots were conducted with specific antibodies to p65 (NFB) or IkB–. Antihistone 3 (H3) and anti-GAPDH were used as nuclear and cytoplasmic control, respectively.
Electromobility-Shift Assay
Nuclear extracts (6 µg) from macrophages were incubated on ice for 30 minutes with a [32P]-labeled oligonucleotide comprising the proximal NFB binding regions of the murine iNOS promoter (5'-CCAACTGGGGACTCTCCCTTTGGGAACA-3'),25 in 25 mmol/L HEPES (pH 7.9), 100 mmol/L KCl, 4% ficoll, 5 µmol/L ZnCl2, 0.1 mmol/L DTT, 0.05% NP-40, 5 mmol/L MgCl2, 1 µg/mL BSA, and 50 ng/uL poly dI-dC in a final volume of 15 µL. Competition analysis was performed with 50-fold excess of unlabeled oligonucleotides. For supershift, samples were incubated with 2 µg of antibodies for an additional 30 minutes on ice. Antibodies (all from Santa Cruz) to p65, p50, p300, C-rel, and Mitf were used in supershift assay. The reaction products were separated by 5% PAGE at 4°C and visualized by autoradiography.
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Results |
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The Effect of SMS2 Deficiency on SMS Activity, Cellular, and Plasma Membrane SM Levels
To investigate the relationship between SMS2 and SM synthesis, we used gene knockout (KO) and knockdown approaches, respectively. SMS2 KO mice were established by conventional approaches (supplemental Figure IA). The resulting heterozygous mice were crossed, and SMS2 knockout (KO) homozygous mice were obtained (supplemental Figure IB). The targeted allele segregated in a Mendelian fashion. SMS2 KO mice display no obvious abnormalities, grow into adult hood, and breed normally under conventional diet and environment. As expected, SMS2 KO macrophages have no SMS2 mRNA (Figure 1A) and have significantly reduced SMS activity (18%, P<0.05), compared with controls (Figure 1C). Similarly, in HEK293 cells, SMS2 siRNA treatment significantly reduced SMS2 mRNA (80%, P<0.001; Figure 1B) and SMS activity (60%, P<0.001; Figure 1D), compared with control siRNA treated cells. To determine whether SMS2 is involved in de novo SM biosynthesis in cells, we incubated cells with [14C]serine, a component used for SM biosynthesis, for 2 hours, and measured [14C]SM levels in total cell lipid extracts. We found that, compared with controls, SMS2 KO macrophages and SMS2 knockdown HEK293 cells had significantly reduced intracellular [14C]SM (30% and 50%, P<0.01, respectively; Figure 1E and 1F), demonstrating that SMS2 is involved in de novo SM synthesis.
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We next sought to measure cellular SM, DAG, PC, and ceramide levels in SMS2-deficient cells and their controls by LC/MS/MS (supplemental Methods). As indicated in supplemental Table I, macrophages and SMS2 siRNA treated HEK293 cells contained significantly less SM than controls (18% and 29%, P<0.01, respectively). Interestingly, the amount of DAG, a concomitant product of SM synthesis, was significantly decreased in SMS2 KO macrophages (20%, P<0.01) but not in SMS2 siRNA HEK293 cells. The amount of ceramide was significantly increased in both SMS2 KO macrophages and SMS2 knockdown HEK293 cells (18% and 43%, P<0.01, respectively). There were no changes in the levels of PC. These results suggested that SMS2 activity is important in regulating cellular SM, DAG, and ceramide.
To investigate the consequences of SMS2 deficiency on plasma membrane SM levels in intact cells, we measured sensitivity of cells to lysenin, a SM-specific cytotoxic protein.22 Lysenin recognizes and binds SM only when it forms aggregates or domains.23 As indicated in Figure 1G and 1H, both SMS2 KO macrophages and SMS2 siRNA-treated HEK293 cells showed significantly less sensitivity to lysenin-mediated cytolysis than their corresponding controls (P<0.01), highlighting the critical and physiological role of SMS2 in regulating SM levels in cell membrane microdomains.
SMS2 Deficiency Attenuates NFB Activation and NFB-Regulated Gene Expression
To determine the role of SMS2 in NFB activation, ligand-induced NFB activation was compared in SMS2 KO macrophages and SMS2 knockdown HEK293 cells with their corresponding controls. As shown in Figure 2, SMS2 KO macrophages had significantly decreased levels of NFB in their nuclei compared with controls after LPS stimulation (71±16%, KO versus WT after 10 minutes LPS stimulation, P<0.01; 62±11%, KO versus WT after 30 minutes LPS stimulation, P<0.01. n=3; Figure 2A). We then used Western blot to measure cytoplasmic IB, which must be degraded for NFB to become activated, and found that its degradation is attenuated (Figure 2B). These results indicated that SMS2 deficiency diminishes IB degradation leading to reduced nuclear translocation of NFB.
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We did similar experiments with HEK293 cells. SMS2 or control siRNA-transfected cells were treated with TNF- for various time points. As shown in Figure 2C and 2D, SMS2 knockdown cell nuclei contain significantly less NFB (67±9%, KO versus WT after 10 minutes TNF- stimulation, P<0.01; 70±19%, KO versus WT after 30 minutes TNF- stimulation, P<0.01. n=3), whereas the cytoplasmic fraction contains more IB, than corresponding controls. These results again indicate a linkage between SMS2 activity and NFB activation. We also treated HEK293 cells with SMS1 siRNA and found that SMS1 knockdown also attenuates NFB activation (supplemental Figure IIA and IIB).
To confirm the above findings and to directly visualize the nuclear translocation of NFB, immunocytochemistry was used. In support of our earlier findings, after LPS treatment, NFB was localized in the nucleus in almost all of the wild-type macrophages, whereas nuclear localization was greatly diminished in SMS2 KO macrophages (Figure 2E). Similarly, in SMS2 knockdown HEK293 cells, after TNF- stimulation, the translocation of NFB to the nucleus was also substantially reduced compared with controls (Figure 2F). Moreover, we found that SMS1 knockdown also attenuates NFB activation in HEK293 cells after TNF- stimulation (supplemental Figure III).
To investigate whether the inhibition of NFB activation affects its transcriptional activity, we carried out a reporter gene assay in SMS2 knockdown HEK293 cells using a B-luciferase plasmid (supplemental Methods). Stimulation of control siRNA-treated cells with TNF- for 8 hours resulted in a nearly 10-fold induction in luciferase activity (Figure 3A) compared with untreated cells. However, in the SMS2 knockdown cells, there was a significant reduction (P<0.01) in the induction of luciferase activity (Figure 3A). This result implies that SMS2 depletion might affect the expression of many NFB-regulated genes. We also found SMS1 knockdown in HEK293 cells reduces B-luciferase expression levels (supplemental Figure IV).
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(20 ng/mL) for the indicated time. D and E, EMSA assay for mouse iNOS promoter fragment (probe) binding ability of NF
To evaluate the physiological role of NFB attenuation caused by the SMS2 deficiency in macrophages, we evaluated LPS-induced expression of iNOS, a proinflammatory gene whose expression is regulated by NFB.24 The mRNA and protein levels of iNOS in LPS-stimulated macrophages were determined by real-time polymerase chain reaction (PCR) and Western blot, respectively. As shown in Figure 3, for both durations of LPS treatment, the induction in iNOS mRNA (Figure 3B) and protein levels (83±7%, KO versus WT after 6 hours LPS stimulation, P<0.01; 65±8%, KO versus WT after 24 hours LPS stimulation, P<0.01. n=3; Figure 3C) were significantly lower in SMS2 KO than in controls, suggesting the regulation of an inflammatory process by SMS2.
To investigate whether the suppression in iNOS gene expression was attributable to lack of binding of NFB to the iNOS promoter, we conducted EMSA using a native iNOS promoter fragment carrying NFB binding sites.25 NFB binding was indicated by a supershift with antibodies to the p50 or p65 subunits and there was no supershift when 3 control antibodies (C-Rel, p300, and Mitf) were used (Figure 3D). As shown in Figure 3E, after LPS stimulation, the NFB (p50/p65) promoter binding activity was markedly diminished in SMS2 KO macrophages compared with control (79±12%, KO versus WT after 10 minutes LPS stimulation, P<0.01; 71±8%, KO versus WT after 30 minutes LPS stimulation, P<0.01. n=3). This result suggests that the reduction in iNOS transcription (Figure 3B and 3C) was attributable to the decrease in NFB available to bind the iNOS promoter. We also noted an unknown shifted complex with diminished NFB binding in SMS2 KO macrophages (Figure 3D and 3E). This unknown complex could not be supershifted by p50, p65, C-Rel, p300, or Mitf antibodies (Figure 3D).
SMS2 Deficiency Impairs TNFR1 Recruitment to Lipid Rafts and TLR4-MD2 Complex Recruitment to Plasma Membrane
Lipid rafts play essential role in TNFR1 clustering and NFB activation.7 Hence, we investigated whether SMS2 knockdown affects TNF- mediated receptor clustering to lipid rafts. We isolated lipid rafts based on their insolubility in 1% Triton X-100 buffer at 4°C and centrifugation on discontinuous sucrose density gradient. Lipid rafts were found in light fractions enriched in the raft marker Src kinase lyn (Figure 4A). The transferrin receptor, CD71, is a nonraft marker. As shown on Figure 4, before stimulation, raft regions contain a small amount of TNFR1. The recruitment of TNFR1 into raft regions was greatly increased on TNF- stimulation in control siRNA-treated cells at both time points (5 minutes and 15 minutes, Figure 4B). However, in SMS2 knockdown cells, the recruitment of TNFR1 to the lipid rafts was significantly reduced (81±9%, SMS2 siRNA versus control siRNA after 5 minutes TNF- stimulation, P<0.01; 60±10%, SMS2 siRNA versus control siRNA after 15 minutes TNF- stimulation, P<0.01; n=3; Figure 4B). SMS2 siRNA did not affect total cellular TNFR1 levels (Figure 4C). These results suggest that SMS2 deficiency-mediated SM depletion in plasma membrane lipid rafts interferes with TNFR1 clustering.
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Deficiency of SMS1 has been shown to block raft-mediated internalization of ALP in mouse lymphoma cells (S49AR).19 We next sought to investigate the effects of SMS2 gene knockdown on TNF--induced TNFR1 endocytosis, a process that might be related to NFB activation.26 As shown in Figure 5, although there are no changes in total TNFR1 on cell surface (Figure 5A) nor in the specific binding of TNF- to surface receptor (Figure 5B), the internalization of TNF--TNFR1 complex, after binding, is impaired in SMS2 knockdown cells (Figure 5C). This result provides additional evidence for the dysfunction of lipid rafts and TNFR1 as a result of SMS2 deficiency.
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In macrophages, we investigated LPS-induced cell surface recruitment of TLR4 and its coreceptor MD2, a consequence of signaling upstream of NFB activation.27 FACS analysis showed that, after LPS stimulation, SMS2 KO macrophages contained fewer TLR4-MD2 complexes on the cell surface than control macrophages (Figure 5D). This result indicates SMS2 is needed for LPS induced cell surface TLR4-MD2 complex recruitment.
It is conceivable that SMS2 deficiency should also influence signal pathways other than NFB. To investigate this possibility, we did Western blot for MAP kinases, p38, and p42/44, in SMS2 KO and WT macrophages after LPS stimulation. We found that both phospho-p38 and phosphor-p42/44, the active form of the kinases, are decreased in KO macrophages, whereas total protein levels are increased (supplemental Figure V).
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Discussion |
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In this study, we show a novel and essential role of SMS2 in modulating NF
B activation. This is based on the following observations: in both SMS2 KO mouse macrophages and SMS2 knockdown HEK293 cells, (1) SMS activity, de novo SM synthesis, cellular and plasma membrane SM levels were significantly decreased, (2) ligand-induced NFB activation, including IB degradation and NFB nuclear translocation, as well as transcriptional activation, were significantly attenuated, and (3) LPS-induced membrane recruitment of TLR4-MD2 complex and TNF--induced raft association of TNFR1 were impaired in SMS2 KO macrophages and SMS2 siRNA-treated HEK293 cells, respectively. SMS2 makes an important contribution to the de novo SM biosynthesis and total cellular SM levels. Based on their relative proximity to the site of ceramide biosynthesis, it has been suggested that SMS1 might be involved in the de novo SM biosynthesis whereas SMS2 is involved in the remodeling of plasma membrane structure.28 However, our previous report20 and this study clearly indicate that SMS2 also participates in de novo SM biosynthesis (Figure 1E and 1F). In support of our data, a recent report indicated that both SMS1 and SMS2 are required for SM homeostasis and growth in human HeLa cells.29 SMS1 and SMS2 are coexpressed in a variety of cells with different ratios, suggesting that the genes contribute variably to cellular SM depending on the cell type. Intriguingly, in some cells, such as Huh 7 cells and macrophages, SMS2 contributes only 20% of the total SMS activity measured in vitro, whereas, SMS2 depletion disproportionately reduces cellular SM levels (supplemental Table I). This suggests that, in vivo, SMS1 and SMS2 activities depend on their local environments, such as availability of substrates.
SM synthesis by SMS2 is important for maintaining plasma membrane structure. In our previous study, we found that knockdown of SMS2 caused a depletion of SM in membrane lipid rafts.20 Our present work supports these observations, and shows that intact SMS2 KO macrophages (Figure 1G) and SMS2 siRNA treated HEK293 cells (Figure 1H) have a stronger resistance to lysenin-mediated lysis than that of controls. The results suggest the physiological role of SMS2 in the formation or maintenance of SM-enriched lipid microdomains or lipid rafts on the plasma membrane. Consistent with our observation, studies of SMS2 function in sperm cell also suggest that SMS2 is important for reconstruction of plasma membrane structure.30
SMS2 deficiency could alter signal transduction mediated by lipid raft-associated receptors. As reported, the interaction of SM and cholesterol drives the formation of plasma membrane rafts,5 and the relative proportions of both SM and cholesterol appear critical for the stability and function of lipid rafts.5,18,19 In the present study, we found that on stimulation by TNF-, the recruitment of TNFR1 receptor to lipid rafts after ligand stimulation was blocked in SMS2 knockdown cells (Figure 4B) suggesting a mechanism for the modulation of NFB activity by SMS2. This finding is in agreement with previous reports where raft association of TNFR1 found to be crucial for TNF--mediated NFB activation in human fibrosarcoma cells.7 Similar to earlier report that the activity of SMS1 is required for effective raft mediated endocytosis,19 we found that SMS2 knockdown also reduced ligand-induced internalization of the TNFR1 receptor (Figure 5C). We also found that LPS-induced plasma membrane recruitment of TLR4-MD-2 complex was diminished in SMS2 KO macrophages (Figure 5D). Taken together, these findings strongly suggest the critical role of SMS2 synthesized SM for the normal function of TNFR1 and TLR4 receptors on the plasma membrane following stimulation by their respective ligands.
Luberto et al14 indicated that, in the absence of SMS activity cellular ceramide inhibits NFB activation, but under high SMS, the resulting DAG signal stimulates NFB. Here we demonstrate that SMS2 deficiency shifts the cellular ceramide and DAG balance in favor of ceramide (supplemental Table I). Cellular DAG functions as activator of both conventional and novel protein kinase C,31,32 a family of serine/threonine kinases that regulate a diverse set of cellular processes, including NFB activation.33,34 Several pathways can lead to the generation of DAG.31 Because of the absence of specific SMS inhibitor, whether the DAG generated by SMS regulates cellular functions is unknown. In this study, in line with a decreased activity of NFB, we provide a direct evidence for a significant reduction in macrophage DAG levels as a consequence of SMS2 deficiency. The absence of the reduction of DAG level in SMS2 knockdown HEK293 cells may reflect the intrinsic difference between these cell type and mouse macrophages.
SMS2 deficiency may also influence signal transduction pathways other than NFB activation. The activation of MAP kinases was attenuated in SMS2 KO macrophages (supplemental Figure V). Moreover, in the EMSA analysis, in addition to NFB, an unknown shifted complex was noted (Figure 3D and 3E). This unknown complex could not be supershifted by any of the anti-NFB (p50/p65), or with antibodies against the other NFB family proteins C-Rel and p300 (Figure 3D). The identification of this complex and its relationship to SMS2 and NFB warrant further investigation.
SMS1 and SMS2 expression positively correlate with levels of SM in lipid rafts.18–20 SMS1 is involved in the regulation of lipid raft SM level and raft functions.18,19 In this study, we show that SMS1 knockdown in HEK293 cells also attenuates NFB activation (supplemental Figures II through IV). In HEK293 cells the expression of SMS1 and SMS2 is almost 1:1 (Hailemariam and Jiang, unpublished observation, 2008). Hence their contribution to total SMS activity and cellular SM content is proportional. In mouse macrophages, the mRNA of SMS1 to SMS2 is 4:1 (Hailemariam and Jiang, unpublished observation, 2008). As a result, SMS2 contributes to lesser proportion of the total cellular SMS activity in these cells. In either cell types because of the difference in their subcellular localization, we believe that each of SMS1 or SMS2 is responsible for a local pool of cellular SM and such pool cannot be compensated by each other. As SMS2 is plasma membrane associated, its contribution to this pool of SM is substantial independent of its role in the total SMS activity. This is strongly suggested by the lysenin sensitivity assays in both cell types (Figure 1G and Figure 1H).
In conclusion, SMS2 physiologically contributes to de novo SM biosynthesis and plasma membrane SM levels and also affects the metabolism of DAG and ceramide. Perturbations to the balance of these molecules by SMS2 inhibition caused blunted NFB responses to inflammatory/immunologic stimuli. Thus, regulation of SMS2 activity may have an important impact on inflammation and thus influence atherogenic processes.
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Acknowledgments |
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The authors are grateful to Thomas Beyer for his support for lipid analysis.
Sources of Funding
This work was supported by an American Heart Association Heritage Affiliate Grant-in-Aid (to Dr Jiang) and National Institute of Health grants (HL-64735 and HL-69817 to Dr Jiang).
Disclosures
None.
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Footnotes |
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T.K.H. and C.H. contributed equally to this study.
Original received February 7, 2008; final version accepted May 22, 2008.
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