Signal transduction in cellular senescence.
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Cell Cycle↗
Biomedical subjects
L M Obeid
Publications and source records attributed to L M Obeid.
Ceramide inhibits phospholipase D in a cell-free system.
Recent evidence in whole cells has implicated ceramide in the regulation of phospholipase D (PLD). In intact HL-60 cells, phorbol myristate acetate (PMA) activated PLD as measured by [3H]palmitate-labeled phosphatidylcholine conversion to phosphatidylethanol in the presence of 2% ethanol. C6-Ceramide completely inhibited PLD activation after 4 h of treatment and was maximally active at 10 microM. The activity was structurally specific in that the structural analogs 4,5-dihydro-C6-ceramide and dioctanoylglycerol were inactive. Although ceramide inhibited PMA-induced activation of PLD, it did not inhibit translocation of protein kinase C (PKC) to the membrane in response to PMA. In a cell-free system, we confirmed that PLD is activated by guanosine 5'-O-(3-thiotriphosphate (GTPgammaS); however, ceramide had no effect on this activity under a variety of conditions. Activation of PLD by GTPgammaS was synergistically enhanced by the addition of PKC activators. This upstream effect was inhibited rapidly and specifically by ceramide (30 microM). Recombinant ARF plus PKCalpha substituted for crude cytosol in the activation of PLD, and this activity was inhibited by C6-ceramide. Taken together, these data show that ceramide interferes with PKC-mediated activation of PLD.
Adenosine Triphosphate↗
Ceramide inactivates cellular protein kinase Calpha.
Ceramide mediates the effects of extracellular agents on cellular growth, differentiation and apoptosis. In this study, we explored the mechanisms by which ceramide induces its cellular effects. In Molt-4 cells, phorbol 12-myristate 13-acetate (PMA) induced retinoblastoma gene product (Rb) phosphorylation, and ceramide inhibited this effect, suggesting an inhibitory effect of ceramide on the protein kinase C (PKC) pathway, the primary target of PMA. Molt-4 cells contained primarily PKCalpha and betaII isoforms of PKC. To determine the effects of ceramide on PKC, we developed an immunoprecipitation assay for PKCalpha activity. Exposure of Molt-4 cells to C6-ceramide resulted in a concentration and time-dependent inhibition of immunoprecipitated protein kinase Calpha (PKCalpha). Initial inhibition was observed as early as 4.5 h after treatment of cells with C6-ceramide, and the activity was completely lost by 13 h. Inhibition of PKCalpha activity was seen at concentrations of ceramide as low as 5 microM with maximal effects occurring at a concentration of 15 microM. Both C2 and C6-ceramide were inhibitory, but C2 and C6 dihydroceramides were not. Ceramide did not directly inhibit PKCalpha in vitro or modulate the levels of PKCalpha protein, suggesting that ceramide acted indirectly. Moreover, ceramide did not inhibit PMA-induced translocation of PKCalpha. Taken together, these results suggested that ceramide caused inactivation of PKCalpha. Since PKC requires phosphorylation for activity, we determined the effects of ceramide on phosphorylation of PKCalpha. C6-ceramide inhibited basal and PMA-induced phosphorylation of PKCalpha. In addition, okadaic acid, a potent phosphatase inhibitor, slightly stimulated PKC activity and blocked the effects of ceramide on PKCalpha inhibition. These results demonstrate that ceramide causes inhibition/inactivation of PKCalpha and suggest these effects of ceramide may be mediated by a protein phosphatase.
Biological Transport↗
Bcl-2 interrupts the ceramide-mediated pathway of cell death.
Ceramide, a product of sphingomyelin turn-over, has been proposed as a novel lipid second messenger with specific roles in mediating antiproliferative responses including apoptosis and cell cycle arrest. In this study, we examine the relationship between the ceramide-mediated pathway of growth suppression and the bcl-2 protooncogene. In ALL-697 leukemia cells, the addition of the chemotherapeutic agent vincristine resulted in a time-dependent growth suppression characterized by marked apoptosis. The effects of vincristine on cell death were preceded by a prolonged and sustained accumulation of endogenous ceramide levels reaching -10.4 pmol ceramide/nmol phospholipids at 12 hr following the addition of vincristine--an increase of 220% over vehicle-treated cells. Overexpression of bcl-2 resulted in near total protection of cell death in response to vincristine. However, the ceramide response to vincristine was not modulated by overexpression of bcl-2, indicating that bcl-2 does not interfere with ceramide formation. Overexpression of bcl-2 prevented apoptosis in response to ceramide, suggesting that bcl-2 acts at a point downstream of ceramide. On the other hand, bcl-2 did not interfere with the ability of ceramide to activate the retinoblastoma gene product or to induce cell cycle arrest, suggesting that the effects of ceramide on cell cycle arrest can be dissociated from the effects on apoptosis. These studies suggest that ceramide and bcl-2 partake in a common pathway of cell regulation. The results also cast ceramide as a gauge of cell injury rather than an "executor" of cell death with clearly dissociable biological outcomes of its action depending on downstream factors.
Antineoplastic Agents, Phytogenic↗
prICE: a downstream target for ceramide-induced apoptosis and for the inhibitory action of Bcl-2.
The novel lipid second messenger, ceramide, specifically induced poly(ADP-ribose) polymerase cleavage through activation of the protease prICE. Over-expression of Bcl-2 inhibited ceramide-induced poly(ADP-ribose) polymerase proteolysis and protected cells from ceramide-induced death. These data provide the first insight into the mechanism by which ceramide mediates apoptosis and suggest a mechanism by which Bel-2 protects from cell death.
Apoptosis↗
Ceramide: an intracellular mediator of apoptosis and growth suppression.
Ceramide is an endogenous lipid molecule generated by hydrolysis of membrane sphingomyelin, in response to cellular stimulation by hormones and cytokines. Ceramide appears to have a role in mediating biological responses in a wide variety of cell types. These responses are generally considered anti-proliferative, but endpoints are varied and include differentiation, growth inhibition, senescence and apoptotic cell death. Mechanisms for ceramide action involve regulation of protein phosphorylation via stimulation of a serine/threonine protein phosphatase, a proline-directed kinase and possibly other direct and/or indirect targets.
Animals↗
Protein kinase C isoenzymes: regulation and function.
PKC is composed of an ever growing family of lipid dependent serine/threonine protein kinases. Collectively, these isoenzymes are known to mediate critical roles in signal transduction, tumour promotion and cell regulation. PKCs appear to function as switches in acute signal transduction paradigms as well as rheostats in long term cell regulation with multiple mechanisms operating in the regulation of these functions. Although these roles of PKC are well established, little is known about the regulation and roles of individual isoenzymes of PKC in vitro or in vivo.
Animals↗
Role of ceramide in cellular senescence.
Recently the sphingomyelin cycle, involving the hydrolysis of membrane sphingomyelin by an activated sphingomyelinase to generate ceramide, has emerged as a key pathway in cell differentiation and apoptosis in leukemic and other cell types. Here we investigate a role for this pathway in the senescence of WI-38 human diploid fibroblasts (HDF). We found that endogenous levels of ceramide increased considerably (4-fold) and specifically (compared with other lipids) as cells entered the senescent phase. Investigation of the mechanism of increased ceramide led to the discovery that neutral sphingomyelinase activity is elevated 8-10 fold in senescent cells. There were no changes in sphingomyelinase activity or ceramide levels as HDF entered quiescence following serum withdrawal or contact inhibition. Thus, the activation of the sphingomyelinase/ceramide pathway in HDF is due to senescence and supports the hypotheses that senescence represents a distinct program of cell development that can be differentiated from quiescence. Additional studies disclosed the ability of ceramide to induce a senescent phenotype. Thus, when exogenous ceramide (15 microM) was administered to young WI-38 HDF, it produced endogenous levels comparable to those observed in senescent cells (as determined by metabolic labeling studies). Ceramide concentrations of 10-15 microM inhibited the growth of young HDF and induced a senescent phenotype by its ability to inhibit DNA synthesis and mitogenesis. These concentrations of ceramide also induced retinoblastoma dephosphorylation and inhibited serum-induced AP-1 activation in young HDF, thus recapitulating basic biochemical and molecular changes of senescence. Sphingomyelinase and ceramide may thus be implicated as mediators of cellular senescence.
Base Sequence↗
Activation of a retinoblastoma-protein-dependent pathway by sphingosine.
The retinoblastoma protein (Rb) is a tumour suppressor that is activated by dephosphorylation the function of which appears to be mediated, at least partly, through the inhibition of several transcription factors, such as E2F. We have recently described sphingosine, a sphingolipid-breakdown product, as a potent and specific inducer of Rb dephosphorylation resulting in inhibition of cell growth and a specific arrest in the G0/G1 phase of the cell cycle. Here we examine the role of Rb and its interaction with E2F in mediating the effects of sphingosine on cell growth. Sphingosine potently inhibited growth of lymphoblastic leukaemic cells, Molt-4, at submicromolar concentrations but showed a 10-fold reduced potency in inhibiting growth of retinoblastoma cells, WERI-Rb-1, which lack functional Rb. In addition, sphingosine's ability to inhibit growth of mink lung epithelial cells was significantly attenuated in cells overexpressing simian virus 40 large T antigen which binds Rb and related proteins. Sphingosine treatment of Molt-4 cells, but not WERI-Rb-1 cells, resulted in the loss of the specific E2F bands produced by the interaction of E2F and its specific DNA sequence element on gel-shift assays. The concentration (submicromolar) and kinetics (4 h) of sphingosine treatment were identical with those required to induce Rb dephosphorylation. In addition, at similar concentrations, sphingosine caused c-myc down-regulation in Molt-4 cells starting at 6 h after treatment. These results demonstrate that activation of Rb by sphingosine leads to sequestration of E2F by the active (hypophosphorylated) form of Rb with the resultant loss of its DNA-binding and genetranscribing abilities. A functional Rb is required to mediate the specific effects of sphingosine on growth arrest.
Amines↗
Retinoblastoma gene product as a downstream target for a ceramide-dependent pathway of growth arrest.
Ceramide, a lipid mediator, has been most closely associated with antiproliferative activities. In this study, we examine the mechanism by which ceramide induces growth suppression and the role of the retinoblastoma gene product (Rb) in this process. Withdrawal of serum from the serum-dependent MOLT-4 cells resulted in significant dephosphorylation of Rb, correlating with the induction of G0/G1 cell cycle arrest. Serum withdrawal resulted in marked elevation in the levels of endogenous ceramide (3-fold at 24 h and 8-fold at 96 h) with little changes in the endogenous levels of sphingosine. The addition of exogenous C6-ceramide resulted in a concentration- and time-dependent dephosphorylation of Rb. Exogenous ceramide was active at levels comparable to endogenous levels achieved with serum withdrawal. Peak activity of exogenous ceramide (at 6 h) correlated with the uptake of C6-ceramide by MOLT-4 cells. Next, a number of studies were conducted to determine whether Rb plays a role in ceramide-induced growth suppression. (i) C6-Ceramide was poorly active in growth suppression of retinoblastoma cells that lack Rb. (ii) Mink lung epithelial cells in which Rb had been sequestered by overexpression of large tumor antigen were resistant to the action of ceramide compared to cells transfected with large tumor antigen mutated in the Rb-binding pocket. (iii) Overexpression of the EIA adenoviral protein, which binds and sequesters Rb, resulted in protection from growth suppression and cell cycle arrest induced by ceramide. Thus, these studies demonstrate that Rb is a downstream target for ceramide and may function in a growth suppressor pathway resulting in cell cycle arrest.
Adenovirus E1A Proteins↗
Role for ceramide in cell cycle arrest.
The dependence of some cell types on serum factors for growth may represent a powerful, but poorly studied, model for antimitogenic pathways. In this study, we examine ceramide as a candidate intracellular mediator of serum factor dependence. In Molt-4 leukemia cells, serum withdrawal caused a significant arrest in cell cycle progression (80% of cells in G0/G1), accompanied by a modest apoptotic cell death (12%). Serum deprivation of these cells resulted in significant sphingomyelin hydrolysis (72%; corresponding to hydrolysis of 47 pmol/nmol phosphate), which was accompanied by a profound and progressive elevation (up to 10-15-fold) in endogenous levels of ceramide. Withdrawal of serum caused the activation of a distinct, particulate, and magnesium-dependent sphingomyelinase. The addition of exogenous C6-ceramide induced a dramatic arrest in the G0/G1 phase of the cell cycle comparable to the effects observed with serum withdrawal, albeit occurring much sooner. Unlike serum withdrawal, however, the addition of C6-ceramide resulted in more pronounced apoptosis. Because of the previously noted ability of exogenously added phorbol esters to inhibit ceramide-mediated apoptosis, we investigated the hypothesis that endogenous activation of the diacylglycerol/protein kinase C pathway may modulate the response to serum withdrawal. Indeed, serum withdrawal resulted in 3-4-fold elevation in endogenous diacylglycerol levels. The addition of exogenous diacylglycerols resulted in selective attenuation of ceramide's effects on apoptosis but not on cell cycle arrest. Thus, the combination of ceramide and diacylglycerol recapitulated the complex effects of serum withdrawal on cell cycle arrest and apoptosis. These studies identify a novel role for ceramide in cell cycle regulation, and they may provide the first evidence for an intracellular signal transduction pathway in mammalian cells mediating cell cycle arrest. These studies also underscore the importance of lipid second messengers and the significance of the interplay between glycerolipid-derived and sphingolipid-derived lipid mediators.
Apoptosis↗
Ceramide: a stress signal and mediator of growth suppression and apoptosis.
A novel pathway termed the sphingomyelin cycle has been identified whereby membrane sphingomyelin is hydrolyzed in response to multiple extracellular stimuli (such as tumor necrosis factor alpha) which cause activation of regulated sphingomyelinases. The product, ceramide, has emerged as a second messenger that mediates many of the cellular effects of these extracellular stimuli. An intriguing relation exists between activation of the sphingomyelin cycle and the action of multiple stress stimuli that induce growth arrest and programmed cell death. Exogenously administered ceramide mimics these growth-suppressing effects, including the induction of apoptosis. This review will highlight the role of the sphingomyelin cycle in signal transduction and will focus on the role and function of ceramide in the regulation of cell growth in general and apoptosis specifically.
Animals↗
Ceramide: an endogenous regulator of apoptosis and growth suppression.
The action of several cytokines is mediated via activation of the sphingomyelin cycle of signal transduction. Ceramide, the product of this pathway, is emerging as an intracellular messenger that mediates effects on terminal differentiation, apoptosis and cell-cycle arrest. Here, Marina Pushkareva, Lina Obeid and Yusuf Hannun discuss the regulation and mechanism of action of this stress-activated pathway.
Animals↗
Ceramide: an intracellular signal for apoptosis.
Recent investigation of the roles of sphingolipids in signal transduction and cell regulation is shedding a new light on the mechanisms of growth suppression and apoptosis. A sphingomyelin cycle has been identified whereby the action of certain extracellular agents (such as tumor necrosis factor alpha) results in activation of a sphingomyelinase, which cleaves membrane sphingomyelin, to generate cellular ceramide. Ceramide, in turn, has emerged as a candidate intracellular mediator for the action of these extracellular agents, and has multiple cellular and biochemical targets. In particular, ceramide is a potent and specific suppressor of cell growth and an inducer of apoptosis. Further studies on this signal transduction pathway should provide new understanding of the physiological functions of ceramide and promise significant insight into a novel biochemical pathway regulating apoptosis.
Apoptosis↗
Identification of a defect in the phospholipase D/diacylglycerol pathway in cellular senescence.
Normal cells become senescent in culture after a limited number of population doublings becoming unable to respond to mitogens. This raises the possibility of defects in mitogenic signaling pathways in cellular senescence. In contrast to young human diploid fibroblasts (HDF), their senescent counterparts failed to undergo protein kinase C translocation in response to serum stimulation. On the other hand, phorbol 12-myristate 13-acetate was equally active in inducing protein kinase C translocation in young and senescent HDF. This suggested a defect in generation of the endogenous activator of protein kinase C, diacylglycerol. Stimulation of young HDF with serum resulted in 3-4-fold generation of diacylglycerol (DAG). In contrast, senescent cells displayed insignificant DAG formation in response to serum. The mechanism of DAG generation was investigated next. In young HDF, serum induced a 5-fold activation of the phospholipase D (PLD) pathway as measured by the incorporation of exogenous ethanol into phosphatidylethanol, which is a measure of the transphosphatidylation reaction of PLD. In contrast, PLD in senescent cells was not activated by serum. Since senescent cells demonstrate significant elevations in the level of endogenous ceramide, the impact of ceramide on the PLD/DAG pathway was also investigated. A soluble analog of ceramide, C6-ceramide, was found to inhibit serum-stimulated DAG accumulation and PLD activation in young cells. These data demonstrate for the first time a defect in PLD activation in cellular senescence and suggest that ceramide may be responsible for the inhibition of this pathway.
Cells, Cultured↗
Role of ceramide-activated protein phosphatase in ceramide-mediated signal transduction.
Extracellular agonists such as tumor necrosis factor-alpha (TNF-alpha) activate the sphingomyelin cycle leading to the generation of ceramide. Ceramide has been suggested as an important mediator of the effect of TNF-alpha on growth inhibition, c-myc down-regulation, apoptosis, and the activation of the nuclear factor kappa B. Although there is no clearly defined intracellular target for ceramide activity, previous studies have demonstrated the existence of a ceramide-activated protein phosphatase (CAPP) in vitro. Since c-myc is an early downstream cellular target for TNF-alpha, we examined the role of ceramide and CAPP in c-myc down-regulation. In intact HL-60 cells ceramide induced down-regulation of c-myc RNA levels. C2-ceramide was active at 1-10 microM and caused 40-80% inhibition of c-myc RNA levels at 30-120 min of treatment. In nuclear run-on studies, C2-ceramide induced a block to transcription elongation of the c-myc transcript without affecting transcription through the first exon. Therefore, ceramide appeared to inhibit c-myc expression via a mechanism identical with that of TNF-alpha. HL-60 cells contained CAPP which was inhibited by okadaic acid (0.1-10 nm). CAPP in HL-60 cells was activated by D-erythro-ceramide but not D-erythro-dihydroceramide. The specificity of activation of CAPP by ceramide in vitro was matched by a similar specificity of c-myc down-regulation in cells. Moreover, okadaic acid inhibited the effects of ceramide and TNF-alpha on c-myc down-regulation. On the other hand, okadaic acid did not inhibit the ability of phorbol 12-myristate 13-acetate to down-regulate c-myc, demonstrating the existence of at least two distinct pathways in the regulation of c-myc expression. These results demonstrate that CAPP is important for ceramide-induced down-regulation of c-myc in myeloid leukemia cells. The implications of these findings in further delineating a sphingomyelin signaling pathway with important anti-proliferative effects are discussed.
Ceramides↗
Regulation of protein kinase C and role in cancer biology.
Protein kinase C (PKC) is a family of closely related lipid-dependent and diacyglycerol-activated isoenzymes known to play an important role in the signal transduction pathways involved in hormone release, mitogenesis and tumor promotion. Reversible activation of PKC by the second messengers diacylglycerol and calcium is an established model for the short term regulation of PKC in the immediate events of signal transduction. PKC can also be modulated long term by changes in the levels of activators or inhibitors for a prolonged period or by changes in the levels of functional PKC isoenzymes in the cell during development or in response to hormones and/or differentiation factors. Indeed, studies have indicated that the sustained activation or inhibition of PKC activity in vivo may play a critical role in regulation of long term cellular events such as proliferation, differentiation and tumorigenesis. In addition, these regulatory events are important in colon cancer, where a decrease in PKC activators and activity suggests PKC acts as an anti-oncogene, in breast cancer, where an increase in PKC activity suggests an oncogenic role for PKC, and in multidrug resistance (MDR) and metastasis where an increase in PKC activity correlates with increased resistance and metastatic potential. These studies highlight the importance and significance of regulation of PKC activity in vivo.
Animals↗
Specific role for protein kinase C beta in cell differentiation.
A critical role for protein kinase C (PKC) in signal transduction events has been well established. On the other hand, emerging evidence also suggests a role for regulation of PKC levels in mediating long term cellular functions. In human leukemia cell line HL-60, the action of 1,25-dihydroxyvitamin D3 results in transcriptional up-regulation of PKC beta (within 8-12 h) (L. M. Obeid et al., J. Biol. Chem., 265: 2370-2374, 1990). In this study, the role of PKC beta in the regulation of proliferation and differentiation was studied. 1,25-Dihydroxyvitamin D3 caused an increase in PKC beta I, beta II, and, to a lesser extent, PKC alpha, as determined by Western blot analysis. This increase was accompanied by inhibition of proliferation and induction of differentiation. The addition of a 25-base pair antisense oligonucleotide directed against the 5' coding sequence of PKC beta attenuated up-regulation of PKC beta I and beta II levels in response to 1,25-dihydroxyvitamin D3. This antisense oligonucleotide, but not sense oligonucleotide or antisense oligonucleotide to PKC alpha, caused inhibition of 1,25-dihydroxyvitamin D3-induced differentiation by 25-45%. On the other hand, inhibition of cell proliferation by 1,25-dihydroxyvitamin D3 was minimally affected by the addition of antisense oligonucleotides. These results identify a role for PKC beta in cell differentiation and underscore the significance of transcriptional activation of PKC as a mechanism for long-term regulation of PKC. The results also distinguish signaling pathways involved in cell differentiation from those involved in antiproliferation.
Base Sequence↗