Alexander A. Karakashian

Expression of neutral sphingomyelinase-2 (NSMase-2) in primary rat hepatocytes modulates IL-β-induced JNK activation

Neutral sphingomyelinase (NSMase) has been proposed to mediate interleukin (IL)‐1β signaling in liver. In this paper, we used adenovirus‐mediated gene transfer to inducibly express FLAG‐tagged mouse NSMase‐2 in primary rat hepatocytes in order to further elucidate the molecular nature of the NSMase involved. Initial studies confirmed that the EST clone used in these experiments encoded a Mg2+‐dependent NSMase. The in vitro activity of the heterologously expressed enzyme was inhibited in the presence of 0.5% Triton or 50 mM EDTA. In addition, the expression of this NSMase‐2 clone in primary hepatocytes led to increased cellular levels of ceramide, indicating that the enzyme is active in situ. Immunofluorescence studies in Hep G2 cells infected with NSMase‐2 expressing adenoviruses showed that the FLAG‐tagged NSMase‐2 was localized to the plasma membrane. Cell viability remained unchanged 72 h following infection and induction. The effect of NSMase‐2 expression on IL‐1β‐induced activation of c‐Jun N‐terminal kinase (JNK) was tested. Expression of NSMase‐2 increased JNK phosphorylation between 1.5‐ and 2‐fold over the basal level. Furthermore, NSMase‐2 was found to strongly increase the ability of IL‐1β to phosphorylate JNK. This potentiation was mediated by a phosphatase from the PP2A family, possibly by modulating the phosphorylation pattern of IL‐1β receptor‐associated kinase (IRAK). In conclusion, the data presented suggest that NSMase‐2 could be involved in IL‐1β‐induced JNK activation in hepatocytes.

Elevated sphingomyelinase activity and ceramide concentration in serum of patients undergoing high dose spatially fractionated radiation treatment: implications for endothelial apoptosis.

Spatially Fractionated high dose (Grid) Radiation Treatment (SFGRT) involves irradiation of bulky tumors with one high, grid-delivered, dose of 15 Gy followed by multiple consecutive doses of 2 Gy each. The goal of this study is to determine the effect of this treatment on serum ceramide content and to investigate possible involvement of ceramide in tumor regression after SFGRT. Serum ceramide and Secretory SMase (SSMase) were quantified in 11 patients before and at 24, 48 and 72 h after the dose of 15 Gy. Furthermore, LDL particles were isolated from the serum and their apoptotic ability was tested in human endothelial cells by TUNEL assay. Sixty seven per cent (6/8) of the patients with partial (PR) or complete (CR) response showed statistically significant increase in serum ceramide levels. Of the non-responders in the study, none showed an elevation in ceramide. S-SMase activity underwent similar changes. LDL particles from serum of patients collected 72 hours after SFGRT sensitized the endothelial cells to undergo apoptosis in response to 5 Gy radiation that by itself had only modest effect on cell death. Independent elevation of ceramide content of endothelial cells that were otherwise resistant to radiation-induced cell death also was sufficient to sensitize these cells to apoptosis. Serum S-SMase activity and ceramide content increase following SFGRT and correlate with the clinical response. Apparently, these changes are in the LDL-associated ceramide and may contribute to better tumor reduction after SFGRT, due to the ability of LDL–derived ceramide to sensitize endothelial cells for apoptosis.

Studies on the Role of Acid Sphingomyelinase and Ceramide in the Regulation of Tumor Necrosis Factor α (TNFα)-converting Enzyme Activity and TNFα Secretion in Macrophages

Acid sphingomyelinase (ASMase) has been proposed to mediate lipopolysaccharide (LPS) signaling in various cell types. This study shows that ASMase is a negative regulator of LPS-induced tumor necrosis factor α (TNFα) secretion in macrophages. ASMase-deficient (asm−/−) mice and isolated peritoneal macrophages produce severalfold more TNFα than their wild-type (asm+/+) counterparts when stimulated with LPS, whereas the addition of exogenous ceramides or sphingomyelinase reduces the differences. The underlying mechanism for these effects is not transcriptional but post-translational. The TNFα-converting enzyme (TACE) catalyzes the maturation of the 26-kDa precursor (pro-TNFα) to an active 17-kDa form (soluble (s)TNFα). In mouse peritoneal macrophages, the activity of TACE was the rate-limiting factor regulating TNFα production. A substantial portion of the translated pro-TNFα was not processed to sTNFα; instead, it was rapidly internalized and degraded in the lysosomes. TACE activity was 2–3-fold higher in asm−/− macrophages as compared with asm+/+ macrophages and was suppressed when cells were treated with exogenous ceramide and sphingomyelinase. Indirect immunofluorescence analyses revealed distinct TNFα-positive structures in the close vicinity of the plasma membrane in asm−/− but not in asm+/+ macrophages. asm−/− cells also had a higher number of early endosomal antigen 1-positive early endosomes. Experiments that involved inhibitors of TACE, endocytosis, and lysosomal proteolysis suggest that in the asm−/− cells a significant portion of pro-TNFα was sequestered within the early endosomes, and instead of undergoing lysosomal proteolysis, it was recycled to the plasma membrane and processed to sTNFα.

Aging in rat causes hepatic hyperresposiveness to interleukin‐1β which is mediated by neutral sphingomyelinase‐2

The process of aging has recently been shown to substantially affect the ability of cells to respond to inflammatory challenges. We demonstrate that aging leads to hepatic hyperresponsiveness to interleukin 1β (IL‐1β), and we examine the factors that could be responsible for this phenomenon. IL‐1β‐induced phosphorylation of c‐jun N‐terminal kinase (JNK) in hepatocytes isolated from aged rats was 3 times more potent than that in hepatocytes from young rats. Moreover, JNK was activated by substantially lower doses of IL‐1β. These age‐related changes in JNK phosphorylation correlated with diminished IL‐1β–induced degradation of interleukin‐1 receptor–associated kinase‐1 (IRAK‐1). Expression levels of IL1β receptor I, total JNK, IRAK‐1, and transforming growth factor‐β–activated kinase‐1 (TAK‐1) were not affected by aging. However, increased neutral sphingomyelinase activity was observed in hepatocytes from old animals, which we show is caused by induction of the plasma membrane localized neutral sphingomyelinase‐2 (NSMase‐2). We provide evidence that NSMase‐2 is both required and sufficient for the onset of IL‐1β hyperresponsiveness during aging. Overexpression of NSMase‐2 in hepatocytes from young rats leads both to a reduction in IRAK‐1 degradation and potentiation of JNK phosphorylation, mimicking that seen in hepatocytes from old animals. More importantly, inhibition of NSMase activity in hepatocytes from aged rats using either scyphostatin or short interfering ribonucleic acid (siRNA) leads to reversion to the “young” phenotype of IL‐1β response. Conclusion: These results show that the process of aging causes increased basal NSMase‐2 activity in hepatocytes, which in turn leads to IRAK‐1 stabilization, JNK potentiation, and ultimately IL‐1β hyperresponsiveness. (HEPATOLOGY 2007.)

Ceramide- and ERK-dependent pathway for the activation of CCAAT/enhancer binding protein by interleukin-1β in hepatocytes

Interleukin-1β (IL-1β) is a major inducer of liver acute-phase protein expression in response to infection. Several transcription factors, including CCAAT/enhancer binding protein (C/EBP), are known mediators in this process, although the mechanisms by which they modulate IL-1βs action are not completely understood. Activation of sphingomyelinase (SMase) and the subsequent generation of ceramide are early steps in the IL-1β signaling cascade. In this study, we investigate the role of ceramide in the IL-1β regulation of C/EBP in primary hepatocytes. The C/EBP DNA binding activity was found to increase in a dose-dependent manner after stimulation with IL-1β and exogenous addition of C2-ceramide or treatment with SMase. These changes were accompanied by an increase in the nuclear content of C/EBPβ. Both IL-1β and ceramide led to extracellular signal-regulated kinase 1/2 (ERK1/2) activation as early as 15 min after treatment. Furthermore, the increase of cellular ceramide content resulted in increased phosphorylation of C/EBPβ at serine 105 at later time points. Concurrently, the cytosolic levels of C/EBPβ decreased, suggesting that IL-1β and ceramide induced nuclear translocation of C/EBPβ. Ceramide-induced C/EBPβ phosphorylation, translocation, and DNA binding were suppressed by the addition of PD98059, an inhibitor of ERK1/2 phosphorylation. These results suggest that ceramide and ERK mediate a pathway in the IL-1β signaling cascade, which results in rapid posttranslational activation of C/EBPβ.

Interleukin 1β Regulation of FoxO1 Protein Content and Localization EVIDENCE FOR A NOVEL CERAMIDE-DEPENDENT MECHANISM

Background: The FoxO1 transcription factor, which controls the hepatic metabolism, is regulated through Akt-mediated phosphorylation. Results: We show that stimulation of hepatocytes with the pro-inflammatory cytokine IL-1β leads to increased FoxO1 nuclear content through an Akt-independent mechanism involving neutral sphingomyelinase-2 and ceramide. Conclusion: IL-1β is a regulator of FoxO1. Significance: IL-1β may adversely affect hepatic metabolism by modulating FoxO1 regulation. FoxO1 transcription factor controls the glucose and lipid metabolism, as well as cell proliferation and stress response. Akt, activated by insulin and other growth factors, phosphorylates FoxO1 causing its nuclear export and activity suppression. In this manuscript, we show that IL-1β, a pro-inflammatory cytokine, has the opposite effects on FoxO1. IL-1β stimulation of primary rat hepatocytes and HEK293 cells overexpressing the IL-1β receptor (293-IL-1RI) results in increased nuclear and cytosolic FoxO1 protein but not mRNA levels. IL-1β stimulation also elevates the levels of a mutant FoxO1 that is resistant to Akt phosphorylation. This suggests that an Akt-independent mechanism is involved. Co-stimulation with insulin does not affect the IL-1β induction of FoxO1. The IL-1β effects on FoxO1 are counteracted, however, by the silencing or inhibition of neutral sphingomyelinase 2 (nSMase-2) using shRNAi, scyphostatin, or GW4869, as well as by the pharmacological inhibition of JNK and ERK. Reversely, the overexpression of nSMase-2 through adenovirus-mediated gene transfer potentiates, in a JNK- and ERK-dependent manner, the IL-1β effects. We also show that transcription of insulin-like growth factor-binding protein-1 mRNA, which requires active FoxO1, is stimulated by IL-1β and is suppressed by the inhibition of nSMase-2 and JNK. In conclusion, we propose that IL-1β regulates FoxO1 activity through a novel nSMase-2-dependent pathway.

Protein Phosphatase 2A and Neutral Sphingomyelinase 2 Regulate IRAK-1 Protein Ubiquitination and Degradation in Response to Interleukin-1β

The IL-1β signaling cascade is initiated by the phosphorylation of IL-1β receptor-associated kinase-1 (IRAK-1), followed by its ubiquitination and degradation. This paper investigates the regulation of IRAK-1 degradation in primary hepatocytes and in HEK cells overexpressing the IL-1β receptor. We provide evidence that protein phosphatase 2A (PP2A) is a negative regulator of the phosphorylation, Lys48-linked ubiquitination, and degradation of IRAK-1. PP2A catalytic activity increased within 30 min of stimulation with IL-1β. siRNA against PP2A catalytic subunit (PP2Ac) or treatment with pharmacological inhibitor, okadaic acid, enhanced IRAK-1 Lys48-linked ubiquitination and degradation. Direct interaction between PP2Ac and IRAK-1 was observed, suggesting that IRAK-1 might be a PP2A substrate. The mechanisms of PP2A activation by IL-1β involved neutral sphingomyelinase-2 (NSMase-2) and an accumulation of ceramide. Overexpression of NSMase-2 delayed IRAK-1 degradation in a PP2A-dependent manner, whereas NSMase-2 silencing had the opposite effect. The addition of sphingomyelinase, ceramide, or a proteasome inhibitor all led to retention of IRAK-1 at the cell membrane and to increased JNK phosphorylation. This study suggests that NSMase-2- and PP2A-dependent regulation of IRAK-1 degradation is a novel mechanism to fine tune the magnitude of IL-1β response.

Novel interconnections in lipid metabolism revealed by overexpression of sphingomyelin synthase-1

This study investigates the consequences of elevating sphingomyelin synthase 1 (SMS1) activity, which generates the main mammalian sphingolipid, sphingomyelin. HepG2 cells stably transfected with SMS1 (HepG2-SMS1) exhibit elevated enzyme activity in vitro and increased sphingomyelin content (mainly C22:0- and C24:0-sphingomyelin) but lower hexosylceramide (Hex-Cer) levels. HepG2-SMS1 cells have fewer triacylglycerols than controls but similar diacylglycerol acyltransferase activity, triacylglycerol secretion, and mitochondrial function. Treatment with 1 mm palmitate increases de novo ceramide synthesis in both cell lines to a similar degree, causing accumulation of C16:0-ceramide (and some C18:0-, C20:0-, and C22:0-ceramides) as well as C16:0- and C18:0-Hex-Cers. In these experiments, the palmitic acid is delivered as a complex with delipidated BSA (2:1, mol/mol) and does not induce significant lipotoxicity. Based on precursor labeling, the flux through SM synthase also increases, which is exacerbated in HepG2-SMS1 cells. In contrast, palmitate-induced lipid droplet formation is significantly reduced in HepG2-SMS1 cells. [14C]Choline and [3H]palmitate tracking shows that SMS1 overexpression apparently affects the partitioning of palmitate-enriched diacylglycerol between the phosphatidylcholine and triacylglycerol pathways, to the benefit of the former. Furthermore, triacylglycerols from HepG2-SMS1 cells are enriched in polyunsaturated fatty acids, which is indicative of active remodeling. Together, these results delineate novel metabolic interactions between glycerolipids and sphingolipids.

Neutral sphingomyelinase-2 is a redox sensitive enzyme: role of catalytic cysteine residues in regulation of enzymatic activity through changes in oligomeric state

Neutral sphingomyelinase-2 (nSMase-2) is the major sphingomyelinase activated in response to pro-inflammatory cytokines and during oxidative stress. It is a membrane-bound 655 amino acid protein containing 22 cysteine residues. In this study, we expressed recombinant mouse nSMase-2 protein in Escherichia coli, and investigated whether nSMase-2 is a redox sensitive enzyme. Our results demonstrate that nSMase-2 exists as both monomers and multimers that are associated with high and low enzymatic activity respectively. Mutational analysis of nSMase-2 identified within its C-terminal catalytic domain several oxidant-sensitive cysteine residues that were shown to be involved in enzyme oligomerization. Changing Cys(617) to Ser for example is a gain-of-function mutation associated with a decreased propensity for oligomerization. Alternatively, nSMase-2 expression in a bacterial strain that lacks endogenous thioredoxin, Rosetta-gami2, results in increased oligomer formation and lower enzyme activity. Phenotypic rescue was accomplished by treating nSMase-2 lysates with recombinant human thioredoxin. This indicates that nSMase-2 may be a novel substrate for thioredoxin. FRET analysis confirmed the presence of nSMase-2 multimers in mammalian HEK cells and their localization to the plasma membrane. In conclusion, our results identify nSMase-2 as a redox-sensitive enzyme, whose basal activity is influenced by thioredoxin-mediated changes in its oligomeric state.

Direct regulation of IGF-binding protein 1 promoter by interleukin-1β via an insulin- and FoxO-1-independent mechanism.

The level of insulin-like growth factor-binding protein 1 (IGFBP1), a liver-produced serum protein that regulates insulin-like growth factor-I bioactivity, glucose homeostasis, and tissue regeneration, increases during inflammation. This manuscript describes a novel pathway for the regulation of hepatic IGFBP1 mRNA and protein levels by interleukin (IL)-1β. Experiments with the luciferase reporter system show that IL-1β stimulates transcriptional activity from the 1-kb promoter region of IGFBP1. Although IL-1β stimulation suppresses the insulin activation of protein kinase B, the major upstream regulator of IGFBP1 mRNA transcription, the induction of IGFBP1 by IL-1β did not require an intact insulin response element. Furthermore, neither overexpression nor silencing of FoxO-1 had any effect on the IL-1β-induced increase in IGFBP1 mRNA levels and promoter activity. However, inhibition of the ERK MAP kinases effectively prevented the IL-1β effects. Inhibition of neutral sphingomyelinase, a key player in the IL-1β signaling cascade that acts upstream of ERK, also suppressed the IL-1β effects, while increasing the ceramide, through the addition of C2-ceramide or via treatment with exogenous sphingomyelinase, was sufficient to induce IGFBP1 promoter-driven luciferase activity. Studies in primary rat hepatocytes where the levels of neutral sphingomyelinase were either elevated or suppressed using adenoviral constructs affirmed the key role of neutral sphingomyelinase and ceramide (exerted likely through ERK activation) in the IL-1β-induced IGFBP1 production. Finally, the IL-1β effects on IGFBP1 mRNA production and protein secretion could be abolished by the addition of insulin, either at very late time points or at very high doses.