Role Of Ceramide Research Articles

Role of ceramide 1 in the molecular organization of the stratum corneum lipids

The main barrier of the skin is formed by the lipids in the apical skin layer, the stratum corneum (SC). In SC mainly ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL) are present. The CER are composed of at least six different fractions. CER 1 has an exceptional molecular structure as it contains a linoleic acid linked to a long-chain ω-hydroxy acid (C > 30). The SC lipids are organized in two lamellar phases with periodicities of approximately 6 and 13 nm, respectively. Recent studies revealed that ceramides isolated from pig SC mixed with cholesterol in confined ratios mimic stratum corneum lipid phase behavior closely (Bouwstra, J.A., et al. 1996. J. Lipid Res. 37: 999–1011). In this paper the role of CER 1 for the SC lipid lamellar organization was studied. For this purpose lipid phase behavior of mixtures of CHOL and total ceramide fraction was compared with that of mixtures of CHOL and a ceramide mixture lacking CER 1. These studies showed that in the absence of CER 1 almost no long periodicity phase was formed over a wide CHOL/CER molar ratio. A model is proposed for the molecular arrangement of the two lamellar phases. This model is based on the dominant role CER 1 plays in the formation of the long periodicity phase, electron density distribution calculations, and observations, such as i) the bimodal distribution of the fatty acid chain lengths of the ceramides, ii) the phase separation between long-chain ceramides and short-chain ceramides in a monolayer approach, and iii) the absence of swelling of the lamellae upon increasing the water content organization in SC. In this molecular model the short periodicity phase is composed of only two high electron density regions indicating the presence of only one bilayer, similar to that often found in phospholipid membranes. The molecular arrangement in the long periodicity phase is very exceptional. This phase most probably consists of two broad and one narrow low electron density regions. The two broad regions are formed by partly interdigitating ceramides with long-chain fatty acids of approximately 24–26 C atoms, while the narrow low-electron density region is formed by fully interdigitating ceramides with a short free fatty acid chain of approximately 16 to 18 C atoms.—Bouwstra, J. A., G. S. Gooris, F. E. R. Dubbelaar, A. M. Weerheim, A. P. IJzerman, and M. Ponec. Role of ceramide 1 in the molecular organization of the stratum corneum lipids. J. Lipid Res. 1998. 39: 186–196.

Signal transduction and the regulation of apoptosis: roles of ceramide.

Knowledge about the molecular regulators of apoptosis is rapidly expanding. Cell death signals emanating from death receptors or internal cell injury detectors launch a number of signaling pathways which converge on several key families of proteins including specialized proteases and endonucleases which play a critical role in the execution of the death order. In this review, we summarize recent discoveries relating to the signaling pathways involved, the death receptors, the caspase family of apoptotic proteases, Bcl-2 family members, the sphingolipid ceramide, and the tumor suppressor p53. In particular, we focus on the role played by ceramide as a coordinator of the stress response and as a candidate biostat in the detection of cell injury.

An accessory role for ceramide in interleukin-1beta induced prostaglandin synthesis.

Interleukin-1beta (IL-1) is a potent inducer of prostaglandin E2 (PGE2) synthesis. We previously showed that ceramide accumulates in fibroblasts treated with IL-1 and that it enhances IL-1-induced PGE2 production. The present study was undertaken to determine the mechanism(s) by which ceramide and IL-1 interact to enhance PGE2 production by examining their respective effects on the rate-limiting enzymes in PGE2 synthesis, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A2 (cPLA2). IL-1-induced PGE2 synthesis required approximately 8 h even though COX-1 was constitutively expressed (both mRNA and protein) and enzymatically active in untreated cells. Conversely, COX-2 mRNA was barely detectable in untreated cells but within 2 h, ceramide or IL-1 alone induced a 5 and 20 fold increase in COX-2 mRNA, respectively. However, IL-1 induced COX-2 protein synthesis was only detectable 6-7 h after maximal COX-2 mRNA induction; COX-2 protein accumulation was not induced by ceramide alone. Ceramide however, reduced the length of time required for IL-1 to induce COX-2 protein accumulation and increased COX-2 protein accumulation. IL-1 induced a 15 fold increase in COX-1 mRNA including an alternatively spliced form of COX-1. IL-1, but not ceramide induced cPLA2 mRNA and protein expression which corresponded with the initiation of PGE2 synthesis. These observations indicate that, (1) while either ceramide or IL-1 rapidly induced COX-2 mRNA, COX-2 protein only accumulated in IL-1 treated cells after a delay of 6-7 h, (2) IL-1-induced PGE2 synthesis required both COX-2 and cPLA2 protein synthesis and, (3) ceramide enhanced (temporally and quantitatively) IL-1-induced COX-2 protein

Role of ceramide in tumour necrosis factor-mediated apoptosis and nuclear factor-kappa B activation.

Conference Article| November 01 1997 Role of ceramide in tumour necrosis factor-mediated apoptosis and nuclear factor-KB activation B. B. Aggarwal; B. B. Aggarwal 1 1Cytokine Research Section, Department of Molecular Oncology, University of Texas M. D. Anderson Cancer Centre, 1515 Holcombe Blvd, Houston, TX 77030, U.S.A. 1To whom correspondence should be addressed Search for other works by this author on: This Site PubMed Google Scholar M. Higuchi M. Higuchi 2 1Cytokine Research Section, Department of Molecular Oncology, University of Texas M. D. Anderson Cancer Centre, 1515 Holcombe Blvd, Houston, TX 77030, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1997) 25 (4): 1166–1171. https://doi.org/10.1042/bst0251166 Article history Received: July 02 1997 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation B. B. Aggarwal, M. Higuchi; Role of ceramide in tumour necrosis factor-mediated apoptosis and nuclear factor-KB activation. Biochem Soc Trans 1 November 1997; 25 (4): 1166–1171. doi: https://doi.org/10.1042/bst0251166 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: A-SMase, acidic SMase, NF, nuclear factor, N-SMase, neutral SMase, PTK, protein tyrosine kinase, PTPase, protein tyrosine phosphatase, SMase, sphingomyelinase, SR33557, (2-isopropyl-1-{4-[3-N-methyl-N-(3,4-dimethoxyphenethyl)-amino]-propyloxy}benzensulphonyl) indolizine, TNF, tumour necrosis factor This content is only available as a PDF. © 1997 Biochemical Society1997 Article PDF first page preview Close Modal You do not currently have access to this content.

Role of ceramide in suramin-induced cancer cell death

Suramin is an experimental antineoplastic agent which is currently being tested in clinical trials for its utility in treating breast and prostate cancer. Recent in vitro studies from our laboratory report a disruption in glycolipid metabolism and cell death in suramin-treated neurons. Evidence presented in this study proposes to consolidate the neurotoxic and cytotoxic effects of suramin. Electron microscopic studies, bis-benzimide staining and DNA gel electrophoresis of suramin and C 2ceramide treatment revealed apoptotic cells in human breast, prostate and rat neuron like cell lines. Apoptotic cell death was preceded by an elevation in intracellular ceramide.

2003 The androgen-sensitive LNCaP prostate cancer cells undergo apoptosis in response to C2-ceramide treatment: A role of ceramide in induction of apoptosis

2003 The androgen-sensitive LNCaP prostate cancer cells undergo apoptosis in response to C2-ceramide treatment: A role of ceramide in induction of apoptosis

Ceramide: A Novel Lipid Mediator of Apoptosis

Sphingolipids are now recognized as key signal transducing lipids with a role in various antiproliferative pathways, including differentiation, cell cycle arrest, senescence, and, of primary relevance of apoptosis discussed in this chapter, apoptosis. Recent progress in defining the signal transduction mechanisms regulating apoptosis stems from the recognition of the role of the sphingolipid, ceramide, as a novel lipid second messenger mediating apoptosis. Hydrolyzation of sphingomyelin and generation of ceramide and choline phosphate occurs when the sphingomyelin cycle is activated by the binding of an extracellular ligand to its receptor activates a sphingomyelinase. The chapter focuses on the role of ceramide with an introduction to the sphingomyelin cycle is followed by a discussion of inducers, biochemical and cellular effects, and biological outcomes. The topic of signal transduction in apoptosis is addressed, with particular emphasis on the role of sphingomyelin hydrolysis and ceramide generation in apoptosis. The chapter also discusses inducers, targets, and the mechanism of action of ceramide in apoptosis. Finally, the chapter concludes with a discussion of the implications of the results, an analysis of current challenges in the field, and a discussion of the potential therapeutic possibilities arising from manipulation of the apoptotic program.

Inhibitors of Sphingolipid Synthesis Modulate Interferon (IFN)-γ-Induced Intercellular Adhesion Molecule (ICAM)-1 and Human Leukocyte Antigen (HLA)-DR Expression on Cultured Normal Human Keratinocytes: Possible Involvement of Ceramide in Biologic Action of IFN-γ

To elucidate the participation of sphingosine and ceramide in the biologic action of cytokines on epidermal keratinocytes, we studied whether inhibitors of sphingolipid synthesis modulate interferon (IFN)-gamma-induced intercellular adhesion molecule (ICAM)-1 and human leukocyte antigen (HLA)-DR expression on cultured normal human keratinocytes. Pretreatment of keratinocytes with L-cycloserine or fumonisin B1, but not 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), significantly suppressed both ICAM-1 and HLA-DR expression induced by IFN-gamma. Because the synthesis of all kinds of sphingolipids is blocked by L-cycloserine and all except that of sphinganine by fumonisin B1, whereas PDMP inhibits the synthesis of glucosylceramide and glycosphingolipids, the result suggests the participation of ceramide and/or sphingosine in IFN-gamma-induced ICAM-1 and HLA-DR expression. Exogenous C2-ceramide reversed the effects of L-cycloserine and fumonisin B1. On the other hand, sphingosine reversed the effect of L-cycloserine, but not of fumonisin B1. These results indicate that ceramide participates in this pathway, as fumonisin B1, but not L-cycloserine, inhibits the synthesis of ceramide from sphingosine. In addition, reverse transcriptase polymerase chain reaction showed that L-cycloserine reduced the mRNA for ICAM-1, HLA-DR alpha, and HLA-DR beta induced by IFN-gamma, and C2-ceramide and sphingosine antagonized the effect of L-cycloserine. Furthermore, the degradation rate of fluorescent sphingomyelin into ceramide in keratinocytes was increased by IFN-gamma, suggesting that IFN-gamma activates sphingomyelin hydrolysis in keratinocytes. These observations suggest the possible role of ceramide in IFN-gamma-induced ICAM-1 and HLA-DR expression on keratinocytes. Ceramide may function as an endogenous modulator mediating the cytokine signals in keratinocytes.

The role of ceramides 1 and 2 in the stratum corneum lipid organisation

A mixture of ceramide 1 and ceramide 2 (CER(1 + 2)) was isolated from pig stratum corneum and mixed in various molar ratios with cholesterol (CHOL) or with CHOL and palmitic acid (PA). The mixtures were hydrated in a buffer solution of pH 5.0 and their phase behaviour was studied by wide- and small-angle X-ray diffraction. The small-angle diffraction curve of the CHOL/CER(1 + 2) mixture at a molar ratio of 0.4 revealed the presence of only one peak at a spacing of 6.7 nm. Increasing the amount of CHOL to a molar ratio of 0.6 was accompanied by a shift of this peak to a smaller spacing (5.7 nm) and the appearance of two weak peaks at 11.8 and 4.1 nm spacings. Increasing the CHOL content to an equimolar ratio resulted in the appearance of two lamellar phases with periodicities of 5.5 and 12 nm, respectively. In a CHOL/CER(1 + 2) mixture at a molar ratio of 2 the periodicities of the two phases were 5.6 and 12 nm, respectively. From these observations it was concluded that the CHOL/CER(1 + 2) mixtures exerted similar phase behaviour, as reported earlier for intact SC (Bouwstra et al. (1995) J. Lipid Res. 36, 496–504) and for mixtures (Bouwstra et al. (1996) J. Lipid Res., in press) prepared from CHOL and total ceramide fraction (CER) isolated from pig stratum corneum. However, in the CHOL/CER mixtures a lower relative amount of CHOL was required to acquire these lamellar phases, indicating that at low CHOL contents, CER 3, 4, 5 and 6 play a crucial role in the formation of the lamellar phases. Furthermore, the solubility of CHOL in the mixtures increased in the presence of CER 1, suggesting its important role for the barrier function of the skin. When palmitic acid (PA) was included, the phase behaviour of the CHOL/CER(1 + 2)/PA mixture was more complex. Next to two lamellar phases, an additional phase with a spacing of 3.77 nm was observed, never seen in intact stratum corneum. In the absence of CHOL, the wide-angle diffraction pattern of the CER(1 + 2) revealed one sharp reflection at 0.456 nm and two diffuse reflections at 0.430, 0.417 nm and 0.395 nm, indicating the presence of a crystalline sublattice. In an equimolar mixture of CHOL/CER(1 + 2) no sharp 0.456 nm reflection was observed indicating a more disordered packing. Furthermore, phase separation of CHOL occurred, this conclusion is based on the presence of reflections corresponding to polycrystalline cholesterol monohydrate. These findings indicate that the lateral packing of mixtures of CHOL/CER(1 + 2) is more complex than that of the CHOL/CER mixtures that reveals a hexagonal lateral packing.

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.

Regulation of Cytochrome P450 2C11 (CYP2C11) Gene Expression by Interleukin-1, Sphingomyelin Hydrolysis, and Ceramides in Rat Hepatocytes

Interleukin-1 triggers the down-regulation of several hepatic cytochrome P450 gene products, but the cellular signaling pathways involved are not known. We have examined the role of sphingomyelin hydrolysis to ceramide in the suppression of CYP2C11, a major constitutive form of cytochrome P450, by interleukin-1. Treatment of rat hepatocytes cultured on matrigel with interleukin-1 beta caused a rapid turnover of sphingomyelin and an increase in cellular ceramide, with no change in cellular phosphatidylcholine. The ceramide was composed mainly of a D-erythro-sphingosine backbone, suggesting that it was derived from sphingolipid hydrolysis rather than from increased de novo synthesis. Treatment of the cells with either N-acetyl-D-erythro-sphingosine (C2-ceramide) or bacterial sphingomyelinase suppressed the expression of CYP2C11 and induced the expression of the interleukin-1-responsive alpha 1-acid glycoprotein mRNA. In contrast, the acute-phase gene beta-fibrinogen, which is induced by interleukin-6 but not by interleukin-1, did not respond to C2-ceramide. N-Acetyl-D-erythro-sphinganine mimicked the effect of C2-ceramide on CYP2C11, but not on alpha 1-acid glycoprotein expression. These results are consistent with a role for ceramide or a related sphingolipid in mediating the down-regulation of CYP2C11, the induction of alpha 1-acid glycoprotein, and perhaps other cellular effects of interleukin-1 in hepatocytes.

Stereoselectivity of induction of the retinoblastoma gene product (pRb) dephosphorylation by D-erythro-sphingosine supports a role for pRb in growth suppression by sphingosine.

Sphingosine has been shown to inhibit cell growth in many cell lines although the mechanism of this effect remains obscure. More recently, D-erythro-sphingosine has been shown to act as an early inducer of dephosphorylation of the retinoblastoma gene product (pRb) in the lymphoblastic leukemia cell line MOLT-4 [Chao, R., Khan, W., & Hannun, Y.A. (1992) J. Biol. Chem., 267, 23459-23462]. In the current study, the role of the natural D-erythro-sphingosine in regulation of cell growth and pRb dephosphorylation was evaluated using chemically synthesized pure isomers of sphingosine. Of the four possible stereoisomers of sphingosine, D-erythro-sphingosine was most active in inducing dephosphorylation of pRb protein with an EC50% of 0.6 microM whereas its enantiomer L-erythro-sphingosine was 8-fold less potent with an EC50% of 5 microM. The dose responses for inhibition of cell growth were nearly identical to the EC50% for pRb dephosphorylation with D-erythro-sphingosine causing 50% inhibition at 0.6 microM whereas L-erythro-sphingosine was 5-6-fold less potent. All of the stereoisomers were taken up by the cells, and the greater potency of D-erythro-sphingosine was not due to differences in cellular uptake. The metabolism of D-erythro-sphingosine was also studied to evaluate the possible role of sphingosine metabolites on regulation of retinoblastoma protein. Evidence is provided against a role for ceramide or sphingosine 1-phosphate as mediators of the effects of sphingosine on pRb dephosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Growth-dependent regulation of cellular ceramides in human T-cells

The role of ceramide, a putative lipid second messenger in the regulation of cell growth, was investigated in T-lymphocytes. An inverse relationship between the cellular concentrations of ceramide and the proliferative capacity of human T-lymphocytes was observed for cells treated with either interleukin-2 or phorbol ester plus ionomycin. The same relationship between cellular ceramide concentrations and DNA synthesis also was observed for cells derived from a cultured T-cell line, the Jurkat T-cells. Alternative approaches for modulating the cellular ceramide concentrations were employed to determine the relationship between sphingolipids and cell growth. Treatment of normal T-lymphocyte cultures with exogenous cell-permeable ceramide analogues or sphingosine stereoisomers decreased DNA synthesis. A similar effect was seen with stearylamine. Cells treated with d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of UDP-glucosyl:ceramide transferase, accumulated cellular ceramide concentrations and had decreased DNA synthesis. These results define a correlation between the concentration of cellular ceramides and the capacity of T-lymphocytes to proliferate. However, the addition of bacterial sphingomyelinase to the T-cell medium caused an increase in ceramide concentrations (presumably at the plasma membrane), which did not affect cell growth. These results support the hypothesis that functionally distinct pools of ceramide may reside within the T-cell.

Role of ceramide in mitogenesis induced by exogenous sphingoid bases.

Dihydrosphingosine, an intermediate in the de novo synthesis of ceramide, induced proliferation of Swiss 3T3 cells. The proliferative effects of this lipid were much more potent than those of sphingosine, a break-down product of ceramide. The maximal proliferative response to dihydrosphingosine occurred at relatively low concentrations (1 microM), while sphingosine produced its maximal effect at much higher concentrations (15 microM). The cell-permeable ceramide, N-hexanoylsphingosine (C6-ceramide), which was also a mitogen in these cells (at 1 microM), caused a striking morphological change when added to the cells at concentrations of 5-10 microM. This shape change was reversible with the removal of ceramide. Exogenous dihydrosphingosines and sphingosines have at least two metabolic fates in Swiss 3T3 cells, conversion to ceramide or to sphingosine 1-phosphate. Surprisingly, both the synthetic threo- isomer and the naturally occurring erythro- isomer of dihydrosphingosine and sphingosine (D-erythro-sphingosine, L-threo-sphingosine, DL-threo-dihydrosphingosine, and DL-erythro-dihydrosphingosine) were readily phosphorylated in intact Swiss 3T3 cells. This substrate specificity may be an indication of a sphingosine kinase activity which is distinct from that of platelets or rat brain. Although sphingosine 1-phosphate and ceramide were both produced upon the addition of sphingosine and dihydrosphingosine, no sphingosine 1-phosphate was produced when Swiss 3T3 cells were treated with mitogenic concentrations of C6-ceramide. These data are consistent with the formation of ceramide and not sphingosine 1-phosphate being required for the mitogenesis produced by exogenous sphingoid bases.

Role of ceramide as a lipid mediator of 1 alpha,25-dihydroxyvitamin D3-induced HL-60 cell differentiation.

The treatment of HL-60 myelocytic leukemia cells with 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) resulted in the activation of a neutral sphingomyelinase and in sphingomyelin turnover (Okazaki, T., Bell, R., and Hannun, Y. (1989) J. Biol. Chem. 264, 19076-19080). In this paper, the effects of 1,25-(OH)2D3 on the product of sphingomyelin hydrolysis, ceramide, and the possible function of ceramide as a lipid mediator of the effects of 1,25-(OH)2D3 on HL-60 cell differentiation were investigated. Treatment of HL-60 cells with 1,25-(OH)2D3 resulted in a time- and dose-dependent increase in ceramide mass levels. Ceramide levels peaked at 2 h following treatment of HL-60 cells with 100 nM 1,25-(OH)2D3 with an increase of 41% over base line. The mass of generated ceramide (13 +/- 2 pmol/nmol of phospholipid) agreed with the mass of hydrolyzed sphingomyelin (17 +/- 4 pmol/nmol of phospholipid). Cell-permeable ceramides with shorter N-acyl chains induced HL-60 cell differentiation at subthreshold concentrations of 1,25-(OH)2D3. Higher concentrations of cell-permeable ceramides potently induced HL-60 cell differentiation independent of 1,25-(OH)2D3. A 2-h exposure of HL-60 cells to N-acetyl-sphingosine was sufficient to cause differentiation. Morphologically, N-acetylsphingosine caused a similar monocytic differentiation of HL-60 cells as did 1,25-(OH)2D3. Exogenous ceramide was further metabolized to sphingomyelin and other sphingolipids, but no conversion to sphingosine was detected. Moreover, sphingosine and its analogs failed to affect monocytic differentiation of HL-60 cells in response to subthreshold 1,25-(OH)2D3, indicating that the effect of ceramide was independent of sphingosine generation. These studies demonstrate that ceramide is a lipid mediator that may transduce the action of 1,25-(OH)2D3 on HL-60 cell differentiation.

Possible role of ceramide in defining structure and function of membrane glycolipids.

A possible role of ceramide in defining the carbohydrate structure of glycolipids and the expression of glycolipid function has been proposed, on the basis of our finding that the ceramide composition of "lacto-series" glycosphingolipid isolated from human erythrocytes shows a remarkable correlation with the terminal carbohydrate structure: (i) The ceramides of three glycosphingolipids with Lex (or x) determinant [Gal beta 1 leads to 4(Fuc alpha 1 leads to 3) GlcNAc] had almost exclusively 16:0 fatty acid; in contrast, the ceramide of its positional isomer H determinant had mainly 20--24:0 fatty acids. (ii) The ceramide of two glycosphingolipids with NeuAc alpha 2 leads to 6GAL structure was predominantly of 16:0 fatty acid, in contrast to that of its positional isomer NeuAc alpha 2 leads to 3Gal residue, in which the ceramide had 20--24:0 fatty acids. These results, together with our previous observation that ceramide composition of mouse lymphoma and myelocytic leukemia MI cells affects their antigenicity, suggest that ceramide structure may define the organization of glycosyltransferase for synthesis of the carbohydrate determinants and may affect the organization and orientation of the carbohydrate chain in membranes, eliciting or suppressing the reactivity to its ligand. Because these glycolipids with Lex and NeuAc alpha 2 leads to 6Gal structures are developmentally regulated and are highly expressed in certain tumors, ceramide composition may affect development, differentiation, and oncogenesis.