Neutral SMase Activity Research Articles

NADPH Oxidase-Mediated Activation of Neutral Sphingomyelinase Is Responsible for Diesel Particulate Extract-Induced Keratinocyte Apoptosis.

Human epidermis is positioned at the interface with the external environment, protecting our bodies against external challenges, including air pollutants. Emerging evidence suggests that diesel particulate extract (DPE), a major component of air pollution, leads to impairment of diverse cellular functions in keratinocytes (KC). In this study, we investigated the cellular mechanism underlying DPE-induced KC apoptosis. We first addressed cell death occurring in KC exposed to DPE, paralleled by increased activation of NADPH oxidases (NOXs) and subsequent ROS generation. Blockade of NOX activation with a specific inhibitor attenuated the expected DPE-induced KC apoptosis. In contrast, pre-treatment with a specific inhibitor of reactive oxygen species (ROS) generation did not reverse DPE/NOX-mediated increase in KC apoptosis. We next noted that NOX-mediated KC apoptosis is mainly attributable to neutral sphingomyelinase (SMase)-mediated stimulation of ceramides, which is a well-known pro-apoptotic lipid. Moreover, we found that inhibition of NOX activation significantly attenuated DPE-mediated increase in the ratio of ceramide to its key metabolite sphingosine-1-phosphate (S1P), an important determinant of cell fate. Together, these results suggest that activation of neutral SMase serves as a key downstream signal for the DPE/NOX activation-mediated alteration in ceramide and S1P productions, and subsequent KC apoptosis.

Mevalonate inhibits acid sphingomyelinase activity, increases sphingomyelin levels and inhibits cell proliferation of HepG2 and Caco-2 cells

BackgroundSphingomyelin (SM) and cholesterol are two types of lipid closely related biophysically. Treating the cells with exogenous sphingomyelinase (SMase) induces trafficking of cholesterol from membrane to intracellular pools and inhibition of cholesterol synthesis. In the present work, we address a question whether increased cholesterol synthesis affects hydrolysis of SM by endogenous SMases.MethodsBoth HepG2 and Caco-2 cells were incubated with mevalonate. The SMase activity was determined and its mRNA examined by qPCR. The cellular levels of cholesterol, SM, and phosphatidylcholine (PC) were determined and cell proliferation rate assayed.ResultsWe found that mevalonate dose-dependently decreased acid but not neutral SMase activity in both HepG2 and Caco-2 cells with HepG2 cells being more sensitive to mevalonate. Kinetic examination in HepG2 cells revealed that acid SMase activity was increasing with cell proliferation, and such an increase was reversed by mevalonate treatment. Acid SMase mRNA was not significantly decreased and Western blot showed signs of proteolysis of acid SMase by mevalonate. After mevalonate treatment, the levels of cholesterol were significantly increased associated with increases in SM and PC. The cell growth was retarded by mevalonate and the effect was more obvious in HepG2 cells than in Caco-2 cells.ConclusionMevalonate can trigger a mechanism to enhance SM levels by inhibition of acid SMase. The effect may ensure the coordinate changes of SM and cholesterol in the cells. Mevalonate also affects cell growth with mechanism required further characterization.

Effects of inhibitors of key enzymes of sphingolipid metabolism on insulin-induced glucose uptake and glycogen synthesis in liver cells of old rats.

Sphingolipids play an important role in the development of insulin resistance. Ceramides are the most potent inhibitors of insulin signal transduction. Ceramides are generated in response to stress stimuli and in old age. In this work, we studied the possible contribution of different pathways of sphingolipid metabolism in age-dependent insulin resistance development in liver cells. Inhibition of key enzymes of sphingolipid synthesis (serine palmitoyl transferase, ceramide synthase) and degradation (neutral and acidic SMases) by means of specific inhibitors (myriocin, fumonisin B1, imipramine, and GW4869) was followed with the reduction of ceramide level and partly improved insulin regulation of glucose metabolism in "old" hepatocytes. Imipramine and GW4869 decreased significantly the acidic and neutral SMase activities, respectively. Treatment of "old" cells with myriocin or fumonisin B1 reduced the elevated in old age ceramide and SM synthesis. Ceramide and SM levels and glucose metabolism regulation by insulin could be improved with concerted action of all tested inhibitors of sphingolipid turnover on hepatocytes. The data demonstrate that not only newly synthesized ceramide and SM but also neutral and acidic SMase-dependent ceramide accumulation plays an important role in development of age-dependent insulin resistance.

Effect of Procysteine on aging-associated changes in hepatic GSH and SMase: evidence for transcriptional regulation of smpd3

In hepatocytes, aging-associated decline in GSH has been linked to activation of neutral SMase (nSMase), accumulation of bioactive ceramide, and inflammation. In this study, we seek to test whether dietary supplementation with the cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTC), would correct the aging-associated differences in hepatic GSH, nSMase, and ceramide. Young and aged mice were placed on a diet that either lacked sulfur-containing amino acids (SAAs) or had 0.5% OTC for 4 weeks. Mice fed standard chow were used as an additional control. SAA-deficient mice exhibited significant aging-associated differences in hepatic GSH, GSH/GSSG, ceramide, and nSMase. C24:1 ceramide, the major ceramide species in liver, was affected the most by aging, followed by the less abundant C16:0 ceramide. OTC supplementation eliminated the aging-associated differences in hepatic GSH and GSH/GSSG ratio. Surprisingly, however, instead of decreasing, the nSMase activity and ceramide increased in the OTC-fed mice irrespective of their age. These effects were due to elevated nSMase-2 mRNA and protein and appeared to be direct. Similar increases were seen in HepG2 cells following treatment with OTC. The OTC-fed aged mice also exhibited hepatic steatosis and triacylglyceride accumulation. These results suggest that OTC is a potent stimulant of nSMase-2 expression and that there may be unanticipated complications of OTC supplementation.

Neutral sphingomyelinase inhibition decreases ER stress-mediated apoptosis and inducible nitric oxide synthase in retinal pigment epithelial cells

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via the induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of ocular diseases characterized by retinal degeneration. Previous studies have revealed the sphingomyelinase/ceramide pathway in the regulation of NOS2 induction. Thus, the objective of this study was to determine the activity of the sphingomyelinase/ceramide pathway, assess nitric oxide production, and examine apoptosis in human retinal pigment epithelial (RPE) cells undergoing ER stress. Sphingomyelinase (SMase) activity; nuclear factor κB (NF-κB) activation; NOS2, nitrite/nitrate, and nitrotyrosine levels; and apoptosis were determined in cultured human RPE cell lines subjected to ER stress via exposure to tunicamycin. Induction of ER stress was confirmed by increased intracellular levels of ER stress markers including phosphorylated PKR-like ER kinase, C/EBP-homologous protein, and 78-kDa glucose-regulated protein. ER stress increased nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, and nitrotyrosine formation and caused apoptosis in RPE cell lines. Inhibition of neutral SMase (N-SMase) activity via GW 4869 treatment caused a significant reduction in nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, nitrotyrosine formation, and apoptosis in ER-stressed RPE cells. In conclusion, N-SMase inhibition reduced nitrative stress and apoptosis in RPE cells undergoing ER stress. Obtained data suggest that NOS2 can be regulated by N-SMase in RPE cells experiencing ER stress.

Diaphragm dysfunction in heart failure is accompanied by increases in neutral sphingomyelinase activity and ceramide content

Chronic heart failure (CHF) causes inspiratory (diaphragm) muscle weakness and fatigue that contributes to dyspnoea and limited physical capacity in patients. However, the mechanisms that lead to diaphragm dysfunction in CHF remain poorly understood. Cytokines and angiotensin II are elevated in CHF and stimulate the activity of the enzyme sphingomyelinase (SMase) and accumulation of its reaction product ceramide. In the diaphragm, SMase or ceramide exposure in vitro causes weakness and fatigue. Thus, elevated SMase activity and ceramide content have been proposed as mediators of diaphragm dysfunction in CHF. In the present study, we tested the hypotheses that diaphragm dysfunction was accompanied by increases in diaphragm SMase activity and ceramide content. Myocardial infarction was used to induce CHF in rats. We measured diaphragm isometric force, SMase activity by high-performance liquid chromatography, and ceramide subspecies and total ceramide using mass spectrometry. Diaphragm force was depressed and fatigue accelerated by CHF. Diaphragm neutral SMase activity was increased by 20% in CHF, while acid SMase activity was unchanged. We also found that CHF increased the content of C18 -, C20 -, and C24 -ceramide subspecies and total ceramide. Downstream of ceramide degradation, diaphragm sphingosine was unchanged, and sphingosine-1-phosphate level was increased in CHF. Our major novel finding was that diaphragm dysfunction in CHF rats was accompanied by higher diaphragm neutral SMase activity, which is expected to cause the observed increase in diaphragm ceramide content.

Long-term food restriction prevents aging-associated sphingolipid turnover dysregulation in the brain

Abnormalities of sphingolipid turnover in the brain during normal aging and age-related neurological disorders were associated with the neurons loss and cognitive malfunction. Calorie restriction (CR) prevented age-related deficits in hippocampal long-term potentiation and improved cognitive function at old age. In the paper we investigated the ceramide and sphingomyelin (SM) levels in the brain regions, which are critical for learning and memory of 3- and 24-month-old rats, as well as the correction of sphingolipid turnover in the brain of old rats, by means of the CR diet and modulators of SM turnover. Using the [methyl-14C-choline]SM, the neutral, but not the acid SMase activity has been observed to increase in both the hippocampus and brain cortex of 24-month-old rats with respect to 3-month-old animals. Age-dependent changes of neutral SMase activities were associated with ceramide accumulation and SM level drop in the brain structures studied. Treatment of the rats with the CR diet or N-acetylcysteine (NAC) or α-tocopherol acetate, but not an inhibitor of acid SMase imipramine, reduced the ceramide content and neutral SMase activity in the hippocampus of 24-month-old animals with respect to control rats of the same age. These results suggest that redox-sensitive neutral SMase plays important role in SM turnover dysregulation in both the hippocampus and neocortex at old age and that the CR diet can prevent the age-dependent accumulation of ceramide mainly via neutral SMase targeting.

Stichoposide D의 백혈병 세포주에서 세라마이드 생성을 통한 세포 사멸 유도 및 항암 작용

Marine triterpene glycosides are physiologically active natural compounds isolated from sea cucumbers(holothurians). It was demonstrated that they have a wide range of biological activities, including antifungal, cytotoxic, and antitumor effects. A previous study showed that stichoposide C (STC) isolated from Thelenota anax induces apoptosis through generation of ceramide by activation of acid sphingomyelinase (SMase) and neutral SMase in human leukemia cells. In this study, we investigated whether STD, a structural analog of STC, can induce apoptosis and examined the molecular mechanisms for its activity. It was found that STC and STD induce apoptosis in a dose- and time-dependent manner and lead to the activation of caspase-8, mitochondrial damage, activation of caspase-9, and activation of caspase-3 in K562 and HL-60 cells. STC activates acid SMase and neutral SMase, which results in the generation of ceramide. Specific inhibition of acid SMase or neutral SMase partially blocked STC-induced apoptosis, but not STD-induced apoptosis. In contrast, STD generates ceramide through the activation of ceramide synthase. Specific inhibition of ceramide synthase partially blocked STD-induced apoptosis, but not STC-induced apoptosis. Moreover, STC and STD markedly reduced tumor growth of HL-60 xenograft tumors and increased ceramide generation in vivo. These results indicate that STC and STD can induce apoptosis and have antitumor activity through the different molecular mechanisms, because they have a different sugar residue attached to aglycones. Thus, these results suggest that their actions are affected by a sugar residue attached to aglycones and they can be used as anticancer agents in the treatment of leukemia.

Heart failure increases neutral sphingomyelinase activity and ceramide content in rat diaphragm

Patients with chronic heart failure (CHF) have skeletal muscle weakness and elevated secretory sphingomyelinase activity. Sphingomyelinase (SMase) acts through ceramide to depress skeletal muscle force in vitro. We hypothesized that diaphragm weakness in CHF is accompanied by increases in SMase activity and tissue ceramide content. We studied rats 14–16 weeks after myocardial infarction (n = 5–10) and sham surgery (n = 10–13). CHF was confirmed by a decrease in left‐ventricular fractional shortening measured by echocardiography (Sham 38 ± 2%, CHF 28 ± 1%; P < 0.05). Skeletal muscle dysfunction in CHF was confirmed by decreased maximal tetanic force of diaphragm strips (in N/cm2: Sham 30 ± 0.6, CHF 26 ± 0.5; P < 0.05). CHF elicited a 27% increase (P < 0.05) in neutral SMase (N‐SMase) activity. Consistent with this finding, total ceramide content was elevated in CHF diaphragm (in pmol/mg protein: Sham, 280 ± 5 and CHF, 343 ± 16; P < 0.05)—subspecies heightened (P < 0.05) in CHF were C18, C18:1, C20, C20:1, C20:4, C22:1. Acid SMase activity was unchanged in CHF. Similarly, there were no changes in the content of either sphingosine or sphingosine‐1‐phosphate (P > 0.05). Our data suggests that increased diaphragm ceramide content is a consequence of N‐SMase activation in CHF. Activation of N‐SMase and heightening of ceramide content may contribute to skeletal muscle weakness.Funding support: NIH 1K99HL098453‐01, 4R00HL098453‐02

Identification and evaluation of neutral sphingomyelinase 2 inhibitors

Sphingomyelinase catalyzes the hydrolysis of sphingomyelin to generate ceramide, an important molecule involved in the regulation of various cellular responses. In this study, we partially purified the neutral sphingomyelinase2 (nSMase2) and identified the inhibitors, D-lyxophytosphingosine and D-arabino-phytosphingosine, which have an inhibitory effect on nSMase2 in a concentration-dependent manner. A Dixon plot of each phytosphingosines revealed their probable inhibitory pattern, i.e., apparent competitive inhibition. These compounds did not inhibit the Mg(2+)-independent neutral SMase activity, although the known nSMase2 inhibitor, GW4869, showed inhibitory effects on Mg(2+)-independent neutral SMase activity. Further, the two phytosphingosines specifically inhibited the ceramide generation regulated by nSMase2.

Effects of long-term fish oil-enriched diet on the sphingolipid metabolism in brain of old rats

An increase of the neutral sphingomyelinase (SMase) activity in the animal brain at old age is associated with a higher expression of the inflammatory marker, interleukin-1β and a cognitive decline. Dietary n − 3 polyunsaturated fatty acids ( n − 3 PUFA) are found to exhibit anti-inflammatory effects and normalize the age- or dementia-related cognitive function decline. In the paper we investigate the ceramide, sphingomyelin (SM) and phosphatidylserine (PS) levels in the hippocampus of 3-month-old-32-month-old rats as well as the correction of sphingolipid turnover in the brain of old rats by means of the dietetic fish oil and PS. To study the lipid turnover, the [ 14C]-labeled SM and palmitic acid were used. The [ 14C]ceramide level significantly increased while those of the [ 14C]SM, SM and PS levels decreased in the hippocampus of aged rats as compared with 3-month-old animals. Treatment of the rats with the fish oil or PS reduced the [ 14C]ceramide content and neutral SMase activity in the hippocampus of the 24-month-old animals with respect to control rats of the same age. These results suggest that dietary n − 3 PUFA can prevent the age-dependent abnormalities of sphingolipid turnover via increased PS content in the hippocampus of old rats.

Aggregated low density lipoprotein induces tissue factor by inhibiting sphingomyelinase activity in human vascular smooth muscle cells.

Our previous results demonstrated that aggregated low density lipoprotein (agLDL) induces tissue factor (TF) expression and activation through Rho A translocation in human vascular smooth muscle cells (VSMC). We also previously demonstrated that membrane sphingomyelin (SM) content is higher in agLDL-exposed VSMC than in control cells. The main enzymes regulating cellular SM content are the family of sphingomyelinases (Smases) that hydrolize SM to phosphorylcholine and ceramide (CER). We wished to investigate whether agLDL has the ability to modulate acidic- (A-) and neutral (N-) Smase activity and whether or not this effect is related to the upregulatory effect of agLDL on Rho A translocation and TF activation in human VSMC. By measuring generated [(14)C]-phosphorylcholine, we found that agLDL significantly decreased A-Smase and specially N-Smase activity. Pharmacological Smase inhibitors increased Rho A and TF. Specific loss-of-function of A-Smase or N-Smase 1 (N1-Smase) by siRNA treatment (500 nmol L(-1), 12 hours) dramatically increased membrane Rho A protein levels (5- and 3-fold, respectively). Concomitantly, TF protein expression and TF procoagulant activity were also increased. Inhibition of A-Smase or N-Smase activity by agLDL, siRNA-anti A- or N1-Smase or pharmacological treatment significantly increased the SM content of vascular cells. The inhibition of SM synthesis by fumonisin B(1) (FB(1)) prevented the upregulatory effect of agLDL on TF. These results demonstrate that inhibition of both A- and N1-Smase might explain the upregulatory effect of agLDL on TF activation, and suggest that this effect is related, at least in part, to membrane SM enrichment.

Neutral sphingomyelinase-induced ceramide accumulation by oxidative stress during carbon tetrachloride intoxication

Ceramide is a biologically active lipid causing apoptosis in a variety of cells. In this study, we examined the effect of CCl 4 on the ceramide metabolism and indicators of oxidative stress. After 12 h of oral administration of CCl 4 (4 ml/kg body weight as a 1:1 mixture of CCl 4 and mineral oil) to rats, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased. Antioxidants such as vitamins C and E were decreased in the liver and kidney. In addition, the ratio of GSH/GSSG in the liver, plasma, kidney, and brain decreased at 2 h. The total ceramide in the liver significantly increased as early as 2 h after CCl 4 administration. After 24 and 36 h, the total ceramide in plasma and the kidney was also augmented. In the brain, the total ceramide dramatically increased at 36 h. These results suggested that the increased ceramide in plasma was transferred to the kidney and the brain. The activity of neutral sphingomyelinase (SMase), which was reported to be enhanced by the decrease of GSH, was significantly increased after CCl 4 treatment in the liver, kidney, and brain. However, acid SMase activities were not increased in the liver and kidney. Thus, the activation of neutral SMase via oxidative stress induced the increase of ceramide during CCl 4 intoxication in not only the liver but also other tissues. These results suggested that the excess accumulation of ceramide causes damage in other organs including the kidney and brain during fulminant hepatic failure.

Acid sphingomyelinase inhibition suppresses lipopolysaccharide‐mediated release of inflammatory cytokines from macrophages and protects against disease pathology in dextran sulphate sodium‐induced colitis in mice

Lipopolysaccharide (LPS) and inflammatory cytokines cause activation of sphingomyelinases (SMases) and subsequent hydrolysis of sphingomyelin (SM) to produce a lipid messenger ceramide. The design of SMase inhibitors may offer new therapies for the treatment of LPS- and cytokine-related inflammatory bowel disease. We synthesized a series of difluoromethylene analogues of SM (SMAs). We report here the effects of the most potent SMase inhibitor, SMA-7, on the LPS-mediated release of tumour necrosis factor-alpha, interleukin-1beta and interleukin-6 from THP-1 macrophages and the pathology of dextran sulphate sodium (DSS)-induced colitis in mice. SMA-7 suppressed the LPS-induced cytokine release and nuclear factor-kappaB activation. LPS stimulation caused a four-fold increase in acid SMase activation, but little increase in neutral SMase activity. The presence of 10 microm SMA-7 caused acid SMase to remain at the control levels and reduced the formation of ceramide. HT-29 cells had significantly decreased cell viability when incubated with media from LPS-stimulated THP-1 macrophages. However, incubating the colon cells in media from both SMA-7 and LPS-treated macrophages caused little decrease in viability, suggesting that ceramide has a role in the LPS-stimulated signalling that releases cytotoxic factors against colon cells. Oral administration of SMA-7 to mice with 2% DSS in the drinking water, for 10 or 21 consecutive days, reduced significantly the cytokine levels in the colon and the severity of colonic injury. These findings suggest a central role for acid SMase/ceramide signalling in the pathology of DSS-induced colitis in mice, indicating a possible preventive or therapeutic role for SMase inhibitor in inflammatory bowel disease.

Inhibition of lipopolysaccharide-induced release of interleukin-8 from intestinal epithelial cells by SMA, a novel inhibitor of sphingomyelinase and its therapeutic effect on dextran sulphate sodium-induced colitis in mice

Lipopolysaccharide (LPS) and inflammatory cytokines cause activation of sphingomyelinases (SMases) and subsequent hydrolysis of sphingomyelin (SM) to produce a lipid messenger ceramide. The use of SMase inhibitors may offer new therapies for the treatment of the LPS- and cytokines-related inflammatory bowel disease (IBD). We synthesized a series of difluoromethylene analogues of SM (SMAs). Here, we show that LPS efficiently increases the release of IL-8 from HT-29 intestinal epithelial cells by activating both neutral SMase and nuclear factor (NF)-κB in the cells. The addition of SMA-7 suppressed neutral SMase-catalyzed ceramide production, NF-κB activation, and IL-8 release from HT-29 cells caused by LPS. The results suggest that activation of neutral SMase is an underlying mechanism of LPS-induced release of IL-8 from the intestinal epithelial cells. Ceramide production following LPS-induced SM hydrolysis may trigger the activation of NF-κB in nuclei. Oral administration of SMA-7 (60 mg/kg) to mice with 2% dextran sulfate sodium (DSS) in their drinking water, for 21 consecutive days, reduced significantly the severity of colonic injury. This finding suggests a central role for SMase/ceramide signaling in the pathology of DSS-induced colitis in mice. The therapeutic effect of SMA-7 observed in mice may involve the suppression of IL-8 production from intestinal epithelial cells by LPS or other inflammatory cytokines.

Ursodeoxycholic acid differentially affects three types of sphingomyelinase in human colon cancer Caco 2 cells

We investigated the effects of UDCA on sphingomyelinase (SMase) in Caco 2 cells cultured in monolayer and polarized conditions. Alkaline SMase activity was high in polarized cells whereas, acid and neutral SMase activities were high in monolayer cells. In polarized cells, UDCA increased alkaline SMase expression and caspase 3 activity but had no effect on acid and neutral SMases. In monolayer cells, UDCA reduced both acid and neutral SMase activities, inhibited cell proliferation, but had little effect on alkaline SMase and caspase 3 activities. In conclusion, UDCA differentially affects SMase activity, cell proliferation, and apoptosis in colonic cells depending on the cell conditions.

Acid sphingomyelinase is induced by butyrate but does not initiate the anticancer effect of butyrate in HT29 and HepG2 cells

Butyric acid and sphingomyelin (SM) affect colonic tumorigenesis. We examined the potential link between butyrate stimulation and SM metabolism in colonic and hepatic cancer cell lines. After incubating HT29 and HepG2 cells with butyrate and other short-chain fatty acids, we found that butyrate increased acid but not neutral or alkaline sphingomyelinase (SMase) activity by 10- to 20-fold. The effects occurred after 16 h of incubation and were associated with reduced SM and phosphatidylcholine contents and increased ceramide levels. Northern blotting showed increased acid SMase mRNA levels in these cells after butyrate stimulation. Propionate was less potent, and acetate had no effect. No similar changes of acid phosphatase could be identified. At concentrations that increased acid SMase expression, butyrate inhibited cell proliferation, activated caspase 3, and induced apoptosis. However, the antiproliferative and apoptotic effects of butyrate preceded the changes of acid SMase and were not affected by knocking down acid SMase expression by small, interfering RNA. In addition, butyrate-induced acid SMase expression was not affected by blocking the caspase pathway. In conclusion, butyrate regulates SM metabolism by stimulating acid SMase expression in colon and liver cancer cells, but the increased acid SMase is not a critical mechanism for initiating the anticancer effects of butyrate.

Role for Mammalian Neutral Sphingomyelinase 2 in Confluence-induced Growth Arrest of MCF7 Cells

Recently, we reported that neutral sphingomyelinase 2 (nSMase2) functions as a bona fide neutral sphingomyelinase and that overexpression of nSMase2 in MCF7 breast cancer cells caused a decrease in cell growth (Marchesini, N., Luberto, C., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 13775-13783). In this study, the role of endogenous nSMase2 in regulating growth arrest was investigated. The results show that endogenous nSMase2 mRNA was up-regulated approximately 5-fold when MCF7 cells became growth-arrested at confluence, and total neutral SMase activity was increased by 119 +/- 41% with respect to control. Cell cycle analysis showed that up-regulation of endogenous nSMase2 correlated with G(0)/G(1) cell cycle arrest and an increase in total ceramide levels (2.4-fold). Analysis of ceramide species showed that confluence caused selective increases in very long chain ceramide C(24:1) (370 +/- 54%) and C(24:0) (266 +/- 81%) during arrest. The role of endogenous nSMase2 in growth regulation and ceramide metabolism was investigated using short interfering RNA (siRNA)-mediated loss-of-function analysis. Down-regulation of nSMase2 with specific siRNA increased the cell population of cells in S phase of the cell cycle by 59 +/- 14% and selectively reverted the effects of growth arrest on the increase in levels of very long chain ceramides. Mechanistically, confluence arrest also induced hypophosphorylation of the retinoblastoma protein (6-fold) and induction of p21(WAF1) (3-fold). Down-regulation of nSMase2 with siRNA largely prevented the dephosphorylation of the retinoblastoma protein and the induction of p21(WAF1), providing a link between the action of nSMase2 and key regulators of cell cycle progression. Moreover, studies on nSMase2 localization in MCF7 cells showed that nSMase2 distributed throughout the cells in subconfluent, proliferating cultures. In contrast, nSMase2 became nearly exclusively located at the plasma membrane in confluent, contact-inhibited cells. Hence, we demonstrate for the first time that nSMase2 functions as a growth suppressor in MCF7 cells, linking confluence to the G(0)/G(1) cell cycle check point.

Psyllium and fat in diets differentially affect the activities and expressions of colonic sphingomyelinases and caspase in mice.

Dietary fibre and fat affect colonic tumourigenesis and inflammation. Sphingomyelin metabolism may have implications for the pathogenesis of colonic tumours and ulcerative colitis. The present study examined the effects of psyllium and fat on the enzymes responsible for sphingomyelin metabolism and apoptosis in the colon. Mice were fed control, psyllium-containing (100 g/kg), high-fat (313 g/kg, 53 % energy as fat) or high-fat plus psyllium diets for 4 weeks. The activities of acid, neutral and alkaline sphingomyelinase (SMase), neutral ceramidase, and caspase 3, 8 and 9 in colonic mucosa were determined. The expressions of alkaline SMase and caspase 3 were examined. The psyllium-containing diet was found to increase significantly the activities of alkaline SMase and caspase 3 and decreased those of acid SMase and neutral ceramidase. The high-fat diet had opposite effects on these enzymes and attenuated the effects of psyllium. Western blotting showed that psyllium increased and high-fat decreased the levels of alkaline SMase and caspase 3 in colonic mucosa. The change in caspase 3 activity was positively correlated with that of alkaline SMase and negatively with acid SMase. No similar changes of acid and alkaline phosphatase activities in the colon or acid and neutral SMase activity in the liver were identified. In conclusion, colonic sphingomyelin metabolism and apoptosis were affected by psyllium and fat in an opposite manner. The results may have implications for colorectal tumourigenesis and inflammation.

Role of sphingomyelin-MAPKs pathway in heat-induced apoptosis.

The role of sphingomyelinase (SMase) activation and mitogen activated protein kinases (MAPKs) activation in cellular apoptosis was investigated during the hyperthermic treatment of HL-60 human leukemia cells. Treating the cells for 1 h at 43(o)C caused more than 50% of cellular apoptosis within several hours. The neutral-SMase activity in the cells treated for 1 h at 42(o)C was slightly increased but decreased in the cells treated at 43(o)C or 44(o)C for the same period whereas the acid SMase activity was slightly increased after heating the cells at 42(o)C and 43(o)C and markedly increased at 44(o)C for 1 h. Treatment of cells with inhibitors of SMase activation and ceramide formation significantly reduced the heat-induced apoptosis. Three major families of mitogen-activated protein kinases (MAPKs), i.e. ERK1/2, p38 and JNK, were activated by the hyperthermic treatment of cells. Inhibition of ERK1/2 with PD98059 exerted little effect on the heat-induced apoptosis and p38 inhibition with SB203580 slightly lessened apoptosis whereas, inhibition of JNK with SP600125 markedly suppressed the heat-induced apoptosis. These results indicate that heat-shock induced the activation of SMase, particularly acid-SMase, thereby causing apoptosis and that JNK played a pivotal role in heat-induced apoptosis in HL-60 leukemia cells.