Connecting Hsp70, sphingolipid metabolism and lysosomal stability

Heat shock protein 70 (Hsp70) is an evolutionary highly conserved molecular chaperone. Upon cancer-associated translocation to the lysosomal compartment, it promotes cell survival by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced death. We have recently shown that Hsp70 stabilizes lysosomes by binding to the endo-lysosomal lipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingolipid catabolism. The Hsp70–BMP interaction enhances the activity of acid sphingomyelinase, an important enzyme that hydrolyzes sphingomyelin. Importantly, treatment with recombinant Hsp70 effectively reverts the dramatic increase in lysosomal volume and decrease in lysosomal stability in cells from patients with Niemann-Pick disease, a genetic disorder associated with reduced acid sphingomyelinase activity. These findings give new insight into the mechanisms controlling lysosomal stability and integrity, and open new exciting possibilities for the treatment of cancer as well as Niemann-Pick disease.     

Production of multiple brain-like ganglioside species is dispensable for fas-induced apoptosis of lymphoid cells

Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells. The present study was undertaken to clarify the role of this enzyme in the generation of gangliosides during apoptosis triggered by Fas ligation. The issue was addressed by using aSMase-deficient and aSMase-corrected cell lines derived from Niemann-Pick disease (NPD) patients. Fas cross-linking elicited a rapid production of large amounts of complex a- and b-series species of gangliosides with a pattern and a chromatographic behavior as single bands reminiscent of brain gangliosides. The gangliosides were synthesized within the first ten minutes and completely disappeared within thirty minutes after stimulation. Noteworthy is the observation that GD3 was not the only ganglioside produced. The production of gangliosides and the onset of apoptotic hallmarks occurred similarly in both aSMase-deficient and aSMase-corrected NPD lymphoid cells, indicating that aSMase activation is not accountable for ganglioside generation. Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process. In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.     

Accurate Differentiation of Neuronopathic and Nonneuronopathic Forms of Niemann-Pick Disease by Evaluation of the Effective Residual Lysosomal Sphingomyelinase Activity in Intact Cells

Abstract: Niemann-Pick disease types A and B are two clinical forms of an inherited lysosomal storage disorder characterized by accumulation of sphingomyelin due to deficient activity of the lysosomal enzyme, acid sphingomyelinase. Patients with both types have hepatosplenomegaly, but only those with type A have nervous system involvement leading to death in early infancy. The residual activities of lysosomal sphingomyelinase in types A and B have never been well characterized because of limitations in both in vitro enzymatic assays and loading tests on intact cells. To evaluate the effective level of sphingomyelinase activity, intact, living cultured Epstein-Barr virus-transformed lymphoid cells were incubated with a radiolabeled sphingomyelin that was first associated to human low-density lipoproteins. This lipoprotein-associated sphingomyelin was targeted to lysosomes, thereby permitting selective hydrolysis by the lysosomal sphingomyelinase. Short-term pulse-chase experiments allowed the determination of the initial rates of degradation; in normal cells, the half-time of sphingomyelin degradation averaged 4.5 h. Whereas cells from the severe neuronopathic type A form of Niemann-Pick disease exhibited about 0.15% residual sphingomyelinase activity, cells from patients with the visceral type B form exhibited about 4%, i.e., 27 times more. Cells from heterozygous Niemann-Pick subjects showed about 70% residual activity. These results provide the first approach to measuring the effective activity of a lysosomal enzyme and represent an accurate method for the differential diagnosis of Niemann-Pick disease types A and B. They also support the hypothesis of relationships among the effective in situ residual enzyme activity, the amount of stored substrate, and the severity of the ensuing lysosomal storage disorder.     

Uptake and metabolism of radioactively labeled sphingomyelin in cultured skin fibroblasts from controls and patients with Niemann-Pick disease and other lysosomal storage diseases

The metabolism of [stearoyl-1-14C]- and [choline-methyl-14C]sphingomyelin, [stearoyl-1-14C]ceramide-1-phospho-N,N-dimethylethanolamine (demethylsphingomyelin) and [choline-methyl-14C]phosphatidylcholine was measured 1, 3 and 5 days after uptake from the media of cultured skin fibroblasts. This was done to measure the relative contributions of lysosomal sphingomyelinase and plasma membrane phosphocholine transferase on the metabolism of sphingomyelin, a component of all cell membranes. By using cell lines from controls and from patients with Niemann-Pick disease and other lysosomal storage diseases, it was concluded that a significant portion (10-15%) of the observed degradation of sphingomyelin is due to exchange of the phosphocholine moiety producing phosphatidylcholine. Although cell lines from type A and B Niemann-Pick disease have only 0-2% of lysosomal sphingomyelinase activity measured in vitro, three cell lines from type B Niemann-Pick disease could metabolize 54.4% of the labeled sphingomyelin by day 3 while cell lines from type A Niemann-Pick disease could only metabolize 18.5% by day 3. This compares to 86.7% metabolized in control cells by day 3. Cells from one patient with juvenile Niemann-Pick disease and one with type D Niemann-Pick disease metabolized sphingomyelin normally while cells from two other patients with juvenile or type C Niemann-Pick disease could only metabolize 58.2% by day 3. Cells from patients with I-cell disease and 'lactosylceramidosis' also demonstrated decreased metabolism of sphingomyelin (55.1 and 54.9% by day 3, respectively). Cells from the patient with Farber disease accumulated [14C]stearic acid-labeled ceramide produced from [14C]sphingomyelin. Studies with choline-labeled sphingomyelin and phosphatidylcholine demonstrated that phosphocholine exchange takes place in either direction in the cells, and this is normal in Niemann-Pick disease. Studies in cells from patients with all clinical types of sphingomyelinase deficiency have led to new methods for diagnosis and prognosis and to a better understanding of sphingomyelin metabolism.     

Niemann-Pick human lymphoblasts are resistant to phthalocyanine 4-photodynamic therapy-induced apoptosis.

Stress-induced activation of sphingomyelinase (SMase) leading to generation of ceramide, a lipid mediator, has been associated with apoptosis in several malignant and nonmalignant cell lines. Photodynamic therapy (PDT), with the phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N(CH3)2], is an oxidative stress associated with increased ceramide generation and subsequent induction of apoptosis in various cell types. We assessed the role of SMase in photocytotoxicity. Normal human lymphoblasts accumulated ceramide and underwent apoptosis after Pc 4-PDT. In contrast, Niemann-Pick disease (NPD) lymphoblasts, which are deficient in acid sphingomyelinase (ASMase) activity, failed to respond to Pc 4-PDT with ceramide accumulation and apoptosis, suggesting that ASMase may be a Pc 4-PDT target. NPD lymphoblasts were exposed to exogenous bacterial sphingomyelinase (bSMase) to test whether these defects are reversible. Treatment of NPD cells with bSMase itself led to elevated ceramide formation, which did not translate into induction of apoptosis. However, a combination of Pc 4-PDT + bSMase induced a significant apoptotic response. Thus, the combined treatment of Pc 4-PDT + bSMase, rather than bSMase alone, was required to restore apoptosis in NPD cells. These data support the hypothesis that SMase is a proapoptotic factor determining responsiveness of cells to Pc 4-PDT.     

Posttranslational regulation of acid sphingomyelinase in niemann-pick type C1 fibroblasts and free cholesterol-enriched chinese hamster ovary cells

Niemann-Pick type C disease is characterized by the accumulation of cholesterol and other lipids within the lysosomal compartment, a process that is often accompanied by a reduction in acid sphingomyelinase activity. These studies demonstrate that a CHO cell mutant (CT-60), which accumulates lysosomal cholesterol because of a defective NP-C1 protein, has approximately 5-10% of the acid sphingomyelinase activity of its parental cell line (25-RA) or wild type (CHO-K1) cells. The cholesterol-induced reduction in acid sphingomyelinase activity can be reproduced in CHO-K1 cells by incubation in the presence of low density lipoprotein (LDL) and progesterone, which impairs the normal egress of LDL-derived cholesterol from the lysosomal compartment. Kinetic analysis of sphingomyelin hydrolysis in cell extracts suggests that the CT60 cells have a reduced amount of functional acid sphingomyelinase as indicated by a 10-fold reduction in the apparent V(max). Western blot analysis using antibodies generated to synthetic peptides corresponding to segments within the carboxyl-terminal region of acid sphingomyelinase demonstrate that both the CT60 and the LDL/progesterone-treated CHO-K1 cells possess near normal levels of acid sphingomyelinase protein. Likewise, Niemann-Pick type C fibroblasts also displayed normal acid sphingomyelinase protein but negligible levels of acid sphingomyelinase activity. These data suggest that cholesterol-induced inhibition is a posttranslational event, perhaps involving cofactor mediated modulation of enzymatic activity or alterations in acid sphingomyelinase protein trafficking and maturation.     

Lysosomal involvement in cellular turnover of plasma membrane sphingomyelin

At least two isoenzymes of sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12), including lysosomal acid sphingomyelinase and nonlysosomal magnesium-dependent neutral sphingomyelinase, catalyse the degradation of sphingomyelin in cultured human skin fibroblasts. A genetically determined disorder of sphingomyelin metabolism, type A Niemann-Pick disease, is characterized by a deficiency of lysosomal acid sphingomyelinase. To investigate the involvement of lysosomes in the degradation of cellular membrane sphingomyelin, we have undertaken studies to compare the turnover of plasma membrane sphingomyelin in fibroblasts from a patient with type A Niemann-Pick disease, which completely lack acid sphingomyelinase activity but retain nonlysosomal neutral sphingomyelinase activity, with turnover in fibroblasts from normal individuals. Plasma membrane sphingomyelin was labeled by incubating cells at low temperature with phosphatidylcholine vesicles containing radioactive sphingomyelin. A fluorescent analog of sphingomyelin, N-4-nitrobenzo-2-oxa-1,3-diazoleaminocaproyl sphingosylphosphorylcholine (NBD-sphingomyelin) is seen to be readily transferred at low temperature from phosphatidylcholine liposomes to the plasma membranes of cultured human fibroblasts. Moreover, when kinetic studies were done in parallel, a constant ratio of [14C]oleoylsphingosylphosphorylcholine ( [14C]sphingomyelin) to NBD-sphingomyelin was taken up at low temperature by the fibroblast cells, suggesting that [14C]sphingomyelin undergoes a similar transfer. The comparison of sphingomyelin turnover at 37 degrees C in normal fibroblasts compared to Niemann-Pick diseased fibroblasts shows that a rapid turnover of plasma membrane-associated sphingomyelin within the first 30 min appears to be similar in both normal and Niemann-Pick diseased cells. This rapid turnover appears to be primarily due to rapid removal of the [14C]sphingomyelin from the cell surface into the incubation medium. During long-term incubation, an increase in the formation of [14C]ceramide correlating with the degradation of [14C]sphingomyelin is observed in normal fibroblasts. In contrast, the level of [14C]ceramide remains constant in Niemann-Pick diseased cells, which correlates with a higher level of intact [14C]sphingomyelin remaining in these cells compared to normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dimethylsulfoxide on sphingomyelinase activities in normal and Niemann-Pick type A, B and C fibroblasts

The effects of dimethylsulfoxide (DMSO) on sphingomyelinase activity measured at pH range 3.5-8.0 were examined in normal and Niemann-Pick disease type A, B and C fibroblasts culture. In normal cells, a minor activity was observed at pH 7.5, which was 3- to 4-fold lower than a major one at pH 5.0. Both activities at pH 5.0 and 7.5 were Mg2+-independent and localized to lysosomes. Niemann-Pick type C cells had 30-50% residual sphingomyelinase activity at both pH 5.0 and 7.5, as compared to normal control cells, whereas type A and B cells exhibited virtually no activity over the entire pH range examined. Treatment with 2% DMSO caused a marked increase in sphingomyelinase activities at pH 5.0 and 7.5 in normal and Niemann-Pick disease type C cells, while in type A and B cells, both activities remained virtually unchanged after DMSO treatment. The increase in sphingomyelinase activity at pH 5.0 induced in normal cells by DMSO resulted in an increase in the Vmax without a substantial change in the Km and was inhibited by the simultaneous addition of 10 micrograms/ml of cycloheximide. By comparison, a less than 2-fold increase in other lysosomal hydrolase activities was observed after DMSO treatment in all cell lines examined.     

The role of sphingomyelin in phosphatidylcholine metabolism in cultured human fibroblasts from control and sphingomyelin lipidosis patients and in Chinese hamster ovary cells.

Human fibroblasts in culture take up exogenous [choline-Me-3H,32P]sphingomyelin (SM) from the medium and incorporate it into cellular SM and phosphatidylcholine [Spence, Clarke & Cook (1983) J. Biol. Chem. 258, 8595-8600]. The ratio of [3H]choline/[32P]Pi is similar in SM and phosphatidylcholine, indicating that the phosphocholine (P-Cho) moiety is transferred intact. Similar results are obtained with Niemann-Pick (NP) cells which are deficient in lysosomal sphingomyelinase activity, suggesting that the P-Cho transfer may not be mediated by the lysosomal sphingomyelinase and that alternative pathways of sphingomyelin catabolism are present in cultured cells. In this study we have shown that: (1) the P-Cho pool in control and NP cells incubated with exogenous labelled SM has a specific radioactivity intermediate between that of SM and PtdCho; (2) expansion of the intracellular P-Cho pool by incubation with exogenous choline reduces the incorporation of [3H]choline from SM into PtdCho; and (3) incorporation of P-Cho from SM into PtdCho is decreased at the non-permissive temperature in Chinese hamster ovary cells with a temperature-sensitive mutation in the cytidylyltransferase reaction. These results suggest that incorporation of P-Cho from SM into PtdCho involves a reaction sequence in which P-Cho is hydrolysed from SM by a sphingomyelinase, followed by incorporation of P-Cho into PtdCho via the cytidine pathway of biosynthesis (SM----P-Cho----CDP-Cho----PtdCho). The appreciable incorporation of P-Cho from SM into PtdCho in sphingomyelinase-deficient NP cells suggests a more substantial or effective lysosomal sphingomyelinase activity in intact cells than is measured in vitro, and/or a significant contribution by other sphingomyelinase activities in these cells.     

Activation of sphingosine kinase by tumor necrosis factor-alpha inhibits apoptosis in human endothelial cells

Human umbilical vein endothelial cells (HUVEC), like most normal cells, are resistant to tumor necrosis factor-alpha (TNF)-induced apoptosis in spite of TNF activating sphingomyelinase and generating ceramide, a known inducer of apoptosis. Here we report that TNF activates another key enzyme, sphingosine kinase (SphK), in the sphingomyelin metabolic pathway resulting in production of sphingosine-1-phosphate (S1P) and that S1P is a potent antagonist of TNF-mediated apoptosis. The TNF-induced SphK activation is independent of sphingomyelinase and ceramidase activities, suggesting that TNF affects this enzyme directly other than through a mass effect on sphingomyelin degradation. In contrast to normal HUVEC, in a spontaneously transformed endothelial cell line (C11) TNF stimulation failed to activate SphK and induced apoptosis as characterized by morphological and biochemical criteria. Addition of exogenous S1P or increasing endogenous S1P by phorbol ester markedly protected C11 cell line from TNF-induced apoptosis. Conversely, N, N-dimethylsphingosine, an inhibitor of SphK, profoundly sensitized normal HUVEC to killing by TNF. Thus, we demonstrate that the activation of SphK by TNF is an important signaling for protection from the apoptotic effect of TNF in endothelial cells.     

CD40 signals apoptosis through FAN-regulated activation of the sphingomyelin-ceramide pathway

The possibility that the sphingomyelin (SM)-ceramide pathway is activated by CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily and that plays a critical role in the regulation of immune responses has been investigated. We demonstrate that incubation of Epstein-Barr virus-transformed lymphoid cells with an anti-CD40 antibody acting as an agonist results in the stimulation of a neutral sphingomyelinase, hydrolysis of cellular SM, and concomitant ceramide generation. In addition, SM degradation was observed in acid sphingomyelinase-deficient cells, as well as after ligation by soluble CD40 ligand. The anti-CD40 antibody, as well as the soluble CD40 ligand induced a decrease in thymidine incorporation and morphological features of apoptosis, which were mimicked by cell-permeant or bacterial sphingomyelinase-produced ceramides. Stable expression of a dominant-negative form of the FAN protein (factor associated with neutral sphingomyelinase activation), which has been reported to mediate tumor necrosis factor-induced activation of neutral sphingomyelinase, significantly inhibited CD40 ligand-induced sphingomyelinase stimulation and apoptosis of transformed human fibroblasts. Transformed fibroblasts from FAN knockout mice were also protected from CD40-mediated cell death. Finally, anti-CD40 antibodies were able to co-immunoprecipitate FAN in control fibroblasts but not in cells expressing the dominant-negative form of FAN, indicating interaction between CD40 and FAN. Altogether, these results strongly suggest that CD40 ligation can activate via FAN a neutral sphingomyelinase-mediated ceramide pathway that is involved in the cell growth inhibitory effects of CD40.     

Niemann-Pick disease type B: prenatal diagnosis and enzymatic and chemical studies on fetal brain and liver.

Patients with Niemann-Pick disease type A have a severe neurovisceral disease caused by a deficiency of lysosomal sphingomyelinase activity in all tissues examined. The patients with the type B form have signs and symptoms related to storage of sphingomyelin in the spleen, liver, and lungs, while neurologically they remain normal. They also have a severe deficiency of lysosomal sphingomyelinase activity in all tissues previously examined. Here the brain and liver of a fetus with Niemann-Pick disease type B are examined for enzymatic anc chemical changes. Despite careful analysis, no measurable lysosomal sphingomyelinase could be measured in either organ. Lipid changes were comparable to those observed in fetuses aborted with Niemann-Pick disease type A. The affected child in this family is now age 3 and remains neurologically normal but continues to show organ enlargement and lung infiltration of lipids. It appears that the lack of neurological involvement in type B patients cannot be due to an obvious presence of significant lysosomal sphingomyelinase activity in brain.     

Effects of dimethylsulfoxide on sphingomyelinase in cultured human fibroblasts and correction of sphingomyelinase deficiency in fibroblasts from Niemann-Pick patients

The effects of dimethylsulfoxide (DMSO) on sphingomyelinase activity were studied using human skin fibroblasts from normal individuals and Niemann-Pick patients. Sphingomyelinase activity in normal fibroblasts increased up to 230% of controls by 2% DMSO while the cell growth was inhibited. Other lysosomal hydrolases showed a rather smaller extent of increment in activity. There was no direct effect of DMSO in cell-free system and no evidence of any activating factor of sphingomyelinase in homogenates. Sphingomyelinase deficiency in fibroblasts from a Niemann-Pick patient (type C) was corrected by 2% DMSO with regaining its enzyme activity.     

Stress-induced apoptosis is impaired in cells with a lysosomal targeting defect but is not affected in cells synthesizing a catalytically inactive cathepsin D

The role of cathepsin D in stress-induced cell death has been investigated by using ovine fibroblasts exhibiting a missense mutation in the active site of cathepsin D. The cathepsin D (lysosomal aspartic protease) deficiency did not protect cells against toxicity induced by doxorubicin and other cytotoxic agents, neither did it protect cells from caspase activation. Moreover, the cathepsin D inhibitor, pepstatin A, did not prevent stress-induced cell death in human fibroblasts or lymphoblasts. The possible role of lysosomal ceramide or sphingosine-mediated activation of cathepsin D in apoptosis was also excluded by using human cells either overexpressing or deficient in acid ceramidase. However, a normal lysosomal function seems to be required for efficient cell death, as indicated by the finding that fibroblasts from patients with mucolipidosis II were partially resistant to staurosporine, sphingosine and TNF-induced apoptosis, suggesting a key role of lysosomes in cell death.     

Ceramide is the key mediator of oxidative stress-induced apoptosis in retinal photoreceptor cells

Nitric oxide and reactive oxygen species play a critical role in photoreceptor apoptosis. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. Here, we demonstrate that the sphingolipid ceramide is the key mediator of oxidative stress-induced apoptosis in 661W retinal photoreceptor cells. Treatment of 661W cells with the nitric oxide donor, sodium nitroprusside, activates acid sphingomyelinase. As a result, sphingomyelin is hydrolysed, which leads to an increase in the concentration of ceramide. We also show that ceramide is responsible for the activation of the mitochondrial apoptotic pathway in 661W photoreceptor cells and subsequent activation of the caspase cascade. Furthermore, we show for the first time that ceramide is responsible for the increased Ca2+ levels in the mitochondria and cytosol that precedes activation of the calpain-mediated apoptotic pathway. Additionally, we provide evidence that ceramide also activates the endolysosomal protease cathepsin D pathway. In summary, our findings show that ceramide controls the cell death decisions in photoreceptor cells and highlight the relevance of acid sphingomyelinase as a potential therapeutic target for the treatment of retinal pathologies.     

Apoptosis and signalling in acid sphingomyelinase deficient cells

Background

Recent evidence suggests that the activation of a non-specific lipid scramblase during apoptosis induces the flipping of sphingomyelin from the cell surface to the cytoplasmic leaftet of the plasma membrane. Inner leaflet sphingomyelin is then cleaved to ceramide by a neutral sphingomyelinase. The production of this non-membrane forming lipid induces blebbing of the plasma membrane to aid rapid engulfment by professional phagocytes. However contrary evidence suggests that cells which are deficient in acid sphingomyelinase are defective in apoptosis signalling. This data has been interpreted as support for the activation of acid sphingomyelinase as an early signal in apoptosis.

Hypothesis

An alternative explanation is put forward whereby the accumulation of intracellular sphingomyelin in sphingomyelinase deficient cells leads to the formation of intracellular rafts which lead to the sequestration of important signalling molecules that are normally present on the cell surface where they perform their function.

Testing the hypothesis

It is expected that the subcellular distribution of important signalling molecules is altered in acid sphingomyelinase deficient cells, leading to their sequestration in late endosomes / lysosomes. Other sphingolipid storage diseases such as Niemann-Pick type C which have normal acid sphingomyelinase activity would also be expected to show the same phenotype.

Implications of the hypothesis

If true the hypothesis would provide a mechanism for the pathology of the sphingolipid storage diseases at the cellular level and also have implications for the role of ceramide in apoptosis.     

Oxidative stress and Alzheimer disease: the autophagy connection?

Intraneuronal accumulation of amyloid beta-protein (Abeta) is believed to be responsible for degeneration and apoptosis of neurons and consequent senile plaque formation in Alzheimer disease (AD), the main cause of senile dementia. Oxidative stress, an early determinant of AD, has been recently found to induce intralysosomal Abeta accumulation in cultured differentiated neuroblastoma cells through activation of macroautophagy. Because Abeta is known to destabilize lysosomal membranes, potentially resulting in apoptotic cell death, this finding suggests the involvement of oxidative stress-induced macroautophagy in the pathogenesis of AD.     

Sphingomyelinase and nonspecific phosphodiesterase activities in Epstein-Barr virus-transformed lymphoid cell lines from Niemann-Pick disease A, B and C

Acid sphingomyelinase activity determined using the natural substrate, [choline-methyl-14C]sphingomyelin, or the chromogenic synthetic analogue, 2-N-(hexadecanoyl)amino-4-nitrophenylphosphorylcholine, was deficient in Epstein-Barr virus-transformed lymphoid cell lines from Niemann-Pick disease types A and B. In contrast, lines from Niemann-Pick disease type C and "sea-blue histiocyte syndrome" showed a sphingomyelinase activity within the normal range. Bis(4-methylumbelliferyl)phosphate and bis(4-methylumbelliferyl)pyrophosphate phosphodiesterase activities were not deficient in any Niemann-Pick disease cell line. These results demonstrate the validity of such cell lines as an experimental model system for enzymatic studies of Niemann-Pick disease.     

Butyrate-induced apoptosis in HCT116 colorectal cancer cells includes induction of a cell stress response

Short chain fatty acids (SCFA), principally butyrate, propionate, and acetate, are produced in the gut through the fermentation of dietary fiber by the colonic microbiotica. Butyrate in particular is the preferred energy source for the cells in the colonic mucosa and has been demonstrated to induce apoptosis in colorectal cancer cell lines. We have used proteomics, specifically 2D-DIGE and mass spectrometry, to identify proteins involved in butyrate-induced apoptosis in HCT116 cells and also to identify proteins involved in the development of butyrate insensitivity in its derivative, the HCT116-BR cells. The HCT116-BR cell line was characterized as being less responsive to the apoptotic effects of butyrate in comparison to its parent cell line. Our analysis has revealed that butyrate likely induces a cellular stress response in HCT116 cells characterized by p38 MAPK activation and an endoplasmic reticulum (ER) stress response, resulting in caspase 3/7 activation and cell death. Adaptive cellular responses to stress-induced apoptosis in HCT116-BR cells may be responsible for the development of resistance to apoptosis in this cell line. We also report for the first time additional cellular processes altered by butyrate, such as heme biosynthesis and dysregulated expression of nuclear lamina proteins, which may be involved in the apoptotic response observed in these cell lines.     

Involvement of the acid sphingomyelinase pathway in uva-induced apoptosis

The sphingomyelin-ceramide pathway is an evolutionarily conserved ubiquitous signal transduction system that regulates many cell functions including apoptosis. Sphingomyelin (SM) is hydrolyzed to ceramide by different sphingomyelinases. Ceramide serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase) activity was induced by UVA in normal JY lymphoblasts but was not detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. We also provide evidence that UVA can induce apoptosis by activating acid SMase in normal JY cells. In contrast, UVA-induced apoptosis was inhibited in MS1418 cells. Exogenous SMase and its product, ceramide (10-40 micrometer), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20-80 micrometer), induced apoptosis only in JY cells. These results suggest that UVA-induced apoptosis by SM is dependent on acid SMase activity. We also provide evidence that induction of apoptosis by UVA may occur through activation of JNKs via the acid SMase pathway.