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L M Obeid

Publications and source records attributed to L M Obeid.

At least 19 recordsLinked to original sources
PubMed2001

TNFalpha-induced glutathione depletion lies downstream of cPLA(2) in L929 cells.

Both glutathione (GSH) depletion and arachidonic acid (AA) generation have been shown to regulate sphingomyelin (SM) hydrolysis and are known components in tumor necrosis factor alpha (TNFalpha)-induced cell death. In addition, both have hypothesized direct roles in activation of N-sphingomyelinase (SMase); however, it is not known whether these are independent pathways of N-SMase regulation or linked components of a single ordered pathway. This study was aimed at differentiating these possibilities using L929 cells. Depletion of GSH with L-buthionin-(S,R)-sulfoximine (BSO) induced 50% hydrolysis of SM at 12 h. In addition, TNF induced a depletion of GSH, and exogenous addition of GSH blocked TNF-induced SM hydrolysis as well as TNF-induced cell death. Together, these results establish GSH upstream of SM hydrolysis and ceramide generation in L929 cells. We next analyzed the L929 variant, C12, which lacks both cytosolic phospholipase A(2) (cPLA(2)) mRNA and protein, in order to determine the relationship of cPLA(2) and GSH. TNF did not induce a significant drop in GSH levels in the C12 line. On the other hand, AA alone was capable of inducing a 60% depletion of GSH in C12 cells, suggesting that these cells remain responsive to AA distal to the site of cPLA(2). Furthermore, depleting GSH with BSO failed to effect AA release, but caused a drop in SM levels, showing that the defect in these cells was upstream of the GSH drop and SMase activation. When cPLA(2) was restored to the C12 line by expression of the cDNA, the resulting CPL4 cells regained sensitivity to TNF. Treatment of the CPL4 cells with TNF resulted in GSH levels dropping to levels near those of the wild-type L929 cells. These results demonstrate that GSH depletion following TNF treatment in L929 cells is dependent on intact cPLA(2) activity, and suggest a pathway in which activation of cPLA(2) is required for the oxidation and reduction of GSH levels followed by activation of SMases.

Animals↗
PubMed2001

Phytosphingosine as a specific inhibitor of growth and nutrient import in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, we have demonstrated a necessary role for sphingolipids in the heat stress response through inhibition of nutrient import (Chung, N., Jenkins, G. M., Hannun, Y. A., Heitman, J., and Obeid, L. M. (2000) J. Biol. Chem. 275, 17229-17232). In this study, we used a combination of pharmacological and genetic approaches to determine which endogenous sphingolipid is the likely mediator of growth inhibition. When cells were treated with exogenous phytosphingosine (PHS, 20 microm) or structurally similar or metabolically related molecules, including 3-ketodihydrosphingosine, dihydrosphingosine, C(2)-phytoceramide (PHC), and stearylamine, only PHS inhibited growth. Also, PHS was shown to inhibit uptake of uracil, tryptophan, leucine, and histidine. Again this effect was specific to PHS. Because of the dynamic nature of sphingolipid metabolism, however, it was difficult to conclude that growth inhibition was caused by PHS itself. By using mutant yeast strains defective in various steps in sphingolipid metabolism, we further determined the specificity of PHS. The elo2Delta strain, which is defective in the conversion of PHS to PHC, was shown to have slower biosynthesis of ceramides and to be hypersensitive to PHS (5 microm), suggesting that PHS does not need to be converted to PHC. The lcb4Delta lcb5Delta strain is defective in the conversion of PHS to PHS 1-phosphate, and it was as sensitive to PHS as the wild-type strain. The syr2Delta mutant strain was defective in the conversion of DHS to PHS. Interestingly, this strain was resistant to high concentrations of DHS (40 microm) that inhibited the growth of an isogenic wild-type strain, demonstrating that DHS needs to be converted to PHS to inhibit growth. Together, these data demonstrate that the active sphingolipid species that inhibits yeast growth is PHS or a closely related and yet unidentified metabolite.

Biological Transport↗
PubMed2001

Cystic fibrosis transmembrane regulator regulates uptake of sphingoid base phosphates and lysophosphatidic acid: modulation of cellular activity of sphingosine 1-phosphate.

Sphingolipids have been implicated in the regulation of cell growth, differentiation, and programmed cell death. Sphingosine 1-phosphate (SPP) has recently emerged as an important lipid messenger and a ligand for the endothelial differentiation gene receptor family of proteins through which it mediates its biologic effects. Recent studies in Saccharomyces cerevisiae in our laboratory implicated the yeast oligomycin resistance gene (YOR1), a member of the ATP binding cassette family of proteins, in the transport of SPP. The cystic fibrosis transmembrane regulator is a unique member of the ATP binding cassette transporter family and has high homology with YOR1. We therefore set out to investigate if this member of the family can regulate SPP transport. We demonstrate that C127/cystic fibrosis transmembrane regulator (CFTR) cells, expressing wild type CFTR, exhibited significantly higher uptake of sphingosine 1-phosphate than either cells expressing a mutant CFTR C127/DeltaF508 or C127/mock-transfected cells. This effect was specific, dose-dependent, and competed off by dihydrosphingosine 1-phosphate and lysophosphatidic acid. There was no difference in uptake of sphingosine, C(16)-ceramide, sphingomyelin, lysophingomyelin, phosphatidylcholine, lysophosphatidylcholine, or phosphatidic acid among the different cell lines. Pretreatment with forskolin or isobutylmethylxanthine to stimulate cAMP did not affect the uptake in any of the cell lines. Moreover, we found that mitogen-activated protein kinase activation by SPP was less responsive in C127/CFTR as compared with C127/mock-transfected cells, suggesting that uptake of SPP by CFTR may divert it from interacting with its cell surface receptors and attenuate signaling functions. Taken together, these data implicate CFTR in uptake of SPP and the related phosphorylated lipids dihydrosphingosine 1-phosphate and lysophosphatidic acid. This uptake influences the availability of SPP to modulate biologic activity via endothelial differentiation gene receptors. These studies may have important implications to cystic fibrosis.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Molecular mechanisms of ceramide-mediated telomerase inhibition in the A549 human lung adenocarcinoma cell line.

This study was aimed at identifying the molecular mechanisms by which ceramide inhibits telomerase activity in the A549 human lung adenocarcinoma cell line. C(6)-ceramide (20 microm) caused a significant reduction of telomerase activity at 24 h as detected using the telomeric repeat amplification protocol, and this inhibition correlated with decreased telomerase reverse transcriptase (hTERT) protein. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analyses showed that C(6)-ceramide significantly decreased hTERT mRNA in a time-dependent manner. Electrophoretic mobility shift and supershift assays demonstrated that the binding activity of c-Myc transcription factor to the E-box sequence on the hTERT promoter was inhibited in response to C(6)-ceramide at 24 h. These results were also confirmed by transient transfections of A549 cells with pGL3-Basic plasmid constructs containing the functional hTERT promoter and its E-box deleted sequences cloned upstream of a luciferase reporter gene. Further analysis using RT-PCR and Western blotting showed that c-Myc protein but not its mRNA levels were decreased in response to C(6)-ceramide at 24 h. The effects of ceramide on the c-Myc protein were shown to be due to a reduction in half-life via increased ubiquitination. Similar results were obtained by increased endogenous ceramide levels in response to nontoxic concentrations of daunorubicin, resulting in the inhibition of telomerase and c-Myc activities. Furthermore, the elevation of endogenous ceramide by overexpression of bacterial sphingomyelinase after transient transfections also induced the inhibition of telomerase activity with concomitant decreased hTERT and c-Myc protein levels. Taken together, these results show for the first time that both exogenous and endogenous ceramides mediate the modulation of telomerase activity via decreased hTERT promoter activity caused by rapid proteolysis of the ubiquitin-conjugated c-Myc transcription factor.

Adenocarcinoma↗
PubMed2001

Cloning and characterization of a novel human alkaline ceramidase. A mammalian enzyme that hydrolyzes phytoceramide.

Ceramidases are enzymes involved in regulating cellular levels of ceramides, sphingoid bases, and their phosphates. Based on sequence homology to the yeast alkaline ceramidases YPC1p (Mao, C., Xu, R., Bielawska, A., and Obeid, L. M. (2000) J. Biol. Chem. 275, 6876--6884) and YDC1p (Mao, C., Xu, R., Bielawska, A., Szulc, Z. M., and Obeid, L. M. (2000) J. Biol Chem. 275, 31369--31378), we report the identification and cloning of a cDNA encoding for a novel human alkaline ceramidase (aPHC) that hydrolyzes phytoceramide selectively. Northern blot analysis showed that aPHC was ubiquitously expressed, with the highest expression in placenta. Green fluorescent protein tagging showed that it was localized in both the Golgi apparatus and endoplasmic reticulum. Overexpression of aPHC in mammalian cells elevated in vitro ceramidase activity toward N-4-nitrobenz-2-oxa-1,3-diazole-C(12)-phytoceramide. Its expression in a yeast mutant strain devoid of any ceramidase activity restored the ceramidase activity and caused an increase in the hydrolysis of phytoceramide in yeast cells, thus leading to the decreased biosynthesis of sphingolipids. These data collectively suggest that, similar to the yeast phytoceramidase YPC1p, aPHC has phytoceramidase activity both in vitro and in cells; hence, it is a functional homolog of the yeast phytoceramidase YPC1p. However, in contrast to YPC1p, aPHC exhibited no reverse activity of ceramidase either in vitro or in cells. Biochemical characterization showed that aPHC had a pH optimum of 9.5, was activated by Ca(2+), but was inhibited by Zn(2+) and sphingosine. Substrate specificity showed that aPHC hydrolyzed phytoceramide preferentially. Together, these data demonstrate that aPHC is a novel human alkaline phytoceramidase, the first mammalian alkaline ceramidase to be identified as being specific for the hydrolysis of phytoceramide.

Alkaline Ceramidase↗
PubMed2001

Role of ceramide in mediating the inhibition of telomerase activity in A549 human lung adenocarcinoma cells.

This study was designed to analyze whether ceramide, a bioeffector of growth suppression, plays a role in the regulation of telomerase activity in A549 cells. Telomerase activity was inhibited significantly by exogenous C(6)-ceramide, but not by the biologically inactive analog dihydro-C(6)-ceramide, in a time- and dose-dependent manner, with 85% inhibition produced by 20 microm C(6)-ceramide at 24 h. Moreover, analysis of phosphatidylserine translocation from the inner to the outer plasma membrane by flow cytometry and of poly(ADP-ribose) polymerase degradation by Western blotting showed that ceramide treatment (20 microm for 24 h) had no apoptotic effects. Trypan blue exclusion, [(3)H]thymidine incorporation, and cell cycle analyses, coupled with clonogenic cell survival assay on soft agar, showed that ceramide treatment with a 20 microm concentration at 24 h resulted in the cell cycle arrest of the majority of the cell population at G(0)/G(1) with no detectable cell death. These results suggest that the inhibition of telomerase by ceramide is not a consequence of cell death but is correlated with growth arrest. Next, to determine the role of endogenous ceramide in telomerase modulation, A549 cells were transiently transfected with an expression vector containing the full-length bacterial sphingomyelinase cDNA (b-SMase). The overexpression of b-SMase, but not exogenously applied purified b-SMase enzyme, resulted in significantly decreased telomerase activity compared with controls, showing that the increased endogenous ceramide is sufficient for telomerase inhibition. Moreover, treatment of A549 cells with daunorubicin at 1 microm for 6 h resulted in the inhibition of telomerase, which correlated with the elevation of endogenous ceramide levels and growth arrest. Finally, stable overexpression of human glucosylceramide synthase, which attenuates ceramide levels by converting ceramide to glucosylceramide, prevented the inhibitory effects of C(6)-ceramide and daunorubicin on telomerase. Therefore, these results provide novel data showing for the first time that ceramide is a candidate upstream regulator of telomerase.

Adenocarcinoma↗
PubMed2001

Selective hydrolysis of a mitochondrial pool of sphingomyelin induces apoptosis.

Our previous results have indicated that the major cellular pool of sphingomyelin present on the outer leaflet of the plasma membrane is not involved in the ceramide pathway of apoptosis. Thus, in this study we aimed at defining which intracellular pools of sphingomyelin and ceramide are involved in cell death. The bacterial sphingomyelinase (SMase) gene fused with green fluorescent protein was subcloned into mammalian vectors containing sequences that target the fusion proteins to cytoplasm, plasma membrane, mitochondria, Golgi apparatus, endoplasmic reticulum, or nucleus. Transfection of MCF7 breast cancer cells showed for all constructs an increase in SMase activity ranging from 2- to 60-fold, concomitant with an increase in total cellular ceramide levels (10-100%) as compared with vector-transfected cells. Next, the effect of overexpression of the SMase on cell death was examined. Results demonstrate that only when bacterial SMase was targeted to mitochondria did cells undergo apoptosis; its targeting to the other intracellular compartments was ineffective. Further, the results show that apoptosis induced by mitochondrial targeting of bacterial SMase requires SMase catalytic activity, is prevented by the overexpression of Bcl-2, and is mediated by inducing cytochrome c release. These results demonstrate that ceramide induces cell death specifically when generated in mitochondria. The results highlight the significance of compartment-specific lipid-mediated cell regulation, and they offer a novel general approach for these studies.

Amino Acid Sequence↗
PubMed2000

Regulation of cyclin-dependent kinase 2 activity by ceramide.

Cyclin-dependent kinases have been implicated in the inactivation of retinoblastoma (Rb) protein and cell cycle progression. Recent studies have demonstrated that the lipid molecule ceramide is able to induce Rb hypophosphorylation leading to growth arrest and cellular senescence. In this study, we examined the underlying mechanisms of Rb hypophosphorylation and cell cycle progression utilizing the antiproliferative molecule ceramide. C6-Ceramide induced a G0/G1 arrest of the cell cycle in WI38 human diploid fibroblasts. Employing immunoprecipitation kinase assays, we found that ceramide specifically inhibited cyclin-dependent kinase CDK2, with a mild effect on CDC2 and significantly less effect on CDK4. The effect of ceramide was specific such that C6-dihydroceramide was not effective. Ceramide did not directly inhibit CDK2 in vitro but caused activation of p21, a major class of CDK-inhibitory proteins, and led to a greater association of p21 to CDK2. Using purified protein phosphatases, we showed that ceramide activated both protein phosphatase 1 and protein phosphatase 2A activities specific for CDK2 in vitro. Further, calyculin A and okadaic acid, both potent protein phosphatase inhibitors, together almost completely reversed the effects of ceramide on CDK2 inhibition. Taken together, these results demonstrate a dual mechanism by which ceramide inhibits the cell cycle. Ceramide causes an increase in p21 association with CDK2 and through activation of protein phosphatases selectively regulates CDK2. These events may lead to activation of Rb protein and subsequent cell cycle arrest.

CDC2-CDC28 Kinases↗
PubMed2000

Cloning and characterization of a Saccharomyces cerevisiae alkaline ceramidase with specificity for dihydroceramide.

In a previous study, we reported that the Saccharomyces cerevisiae gene YPC1 encodes an alkaline ceramidase with a dual activity, catalyzing both hydrolysis and synthesis of yeast ceramide (Mao, C., Xu, R., Bielawska, A., and Obeid, L. M. (2000) J. Biol. Chem. 275, 6876-6884). In this study, we have identified a YPC1 homologue in S. cerevisiae that also encodes an alkaline ceramidase. We show that these two ceramidases have different substrate specificity, such that YPC1p preferentially hydrolyzes phytoceramide, whereas the new ceramidase YDC1p hydrolyzes dihydroceramide preferentially and phytoceramide only slightly. Neither enzyme hydrolyzes unsaturated mammalian-type ceramide. In contrast to YPC1p, YDC1p had only minor in vitro reverse activity of catalyzing dihydroceramide formation from a free fatty acid and dihydrosphingosine and no activity with phytosphingosine. Overexpression of YDC1p had no reverse activity in non-stressed yeast cells, but like YPC1p suppressed the inhibition of growth by fumonisin B1 albeit more modestly. Deletion of YDC1 and YPC1 or both did not apparently affect growth, suggesting neither gene is essential. However, the Deltaydc1 deletion mutant but not the Deltaypc1 deletion mutant was sensitive to heat stress, indicating a role for dihydroceramide but not phytoceramide in heat stress responses, and suggesting that the two enzymes have distinct physiological functions.

Amidohydrolases↗
PubMed2000

Functional dichotomy of protein kinase C (PKC) in tumor necrosis factor-alpha (TNF-alpha ) signal transduction in L929 cells. Translocation and inactivation of PKC by TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is capable of inducing a variety of biologic responses through multiple signaling pathways. Because of the potential role of protein kinase C (PKC) in apoptosis, we examined the effects and mechanisms of TNF-alpha on PKC regulation, specifically on PKC alpha. In L929 murine fibroblasts, TNF-alpha (0.5- 5 nm) caused potent inhibition of PKC alpha activity and induced translocation of PKC alpha from the cytosol to the membrane. Treatment of cells with TNF-alpha also induced dephosphorylation of PKC alpha as detected by a mobility shift on SDS-polyacrylamide gel and inhibition of PKC phosphorylation as probed by anti-phospho-PKC antibodies. Since PKC is activated directly by diacylglycerol and inactivated indirectly by ceramide, we next examined the roles of these lipid mediators in the regulation of PKC alpha. Addition of TNF-alpha led to accumulation of both ceramide and diacylglycerol. Fumonisin B(1), an inhibitor of ceramide synthase, and glutathione, an inhibitor of neutral sphingomyelinase, both reversed the effect of TNF-alpha on PKC alpha activity, suggesting that ceramide production is necessary for the action of TNF-alpha. The diacylglycerol mimic phorbol 12-myristate 13-acetate was sufficient to cause translocation of PKC alpha, but not the mobility shift. Okadaic acid at 2 nm, a potent protein phosphatase inhibitor, blocked the effects of TNF-alpha on PKC alpha activity, but not on PKC alpha translocation, thus demonstrating that dephosphorylation and translocation are independent processes. These results demonstrate that PKC alpha acts as a downstream target for TNF-alpha and that different lipid-mediated pathways in TNF-alpha signaling lead to opposing signals in the regulation of PKC alpha activity.

Animals↗
PubMed2000

Sphingolipids signal heat stress-induced ubiquitin-dependent proteolysis.

Sphingolipids are essential eukaryotic membrane lipids that are structurally and metabolically conserved through evolution. Sphingolipids have also been proposed to regulate eukaryotic stress responses as novel second messengers. Here we show that, in Saccharomyces cerevisiae, phytosphingosine, a putative sphingolipid second messenger, mediates heat stress signaling and activates ubiquitin-dependent proteolysis via the endocytosis vacuolar degradation and 26 S proteasome pathways. Inactivation of serine palmitoyltransferase, a key enzyme in generating endogenous phytosphingosine, prevents proteolysis during heat stress. Addition of phytosphingosine bypasses the requirement for serine palmitoyltransferase and restores proteolysis. Phytosphingosine-induced proteolysis requires multiubiquitin chain formation through the stress-responsive lysine 63 residue of ubiquitin. We propose that heat stress increases phytosphingosine and activates ubiquitin-dependent proteolysis.

Acyltransferases↗
PubMed2000

Cloning of an alkaline ceramidase from Saccharomyces cerevisiae. An enzyme with reverse (CoA-independent) ceramide synthase activity.

Ceramide is not only a core intermediate of sphingolipids but also an important modulator of many cellular events including apoptosis, cell cycle arrest, senescence, differentiation, and stress responses. Its turnover may be tightly regulated. However, little is known about the regulation of its metabolism because most enzymes responsible for its synthesis and breakdown have yet to be cloned. Here we report the cloning and characterization of the yeast gene YPC1 (YBR183w) by screening Saccharomyces cerevisiae genes whose overexpression bestows resistance to fumonisin B1. We demonstrate that the yeast gene YPC1 encodes an alkaline ceramidase activity responsible for the breakdown of dihydroceramide and phytoceramide but not unsaturated ceramide. YPC1 ceramidase activity was confirmed by in vitro studies using an Escherichia coli expression system. Importantly, YPC1p also has reverse activity, catalyzing synthesis of phytoceramide from palmitic acid and phytosphingosine. This ceramide synthase activity is CoA-independent and is resistant to fumonisin B1, thus explaining why YPC1 was cloned as a fumonisin B1-resistant gene.

Amidohydrolases↗
PubMed2000

Yeast sphingosine-1-phosphate phosphatases: assay, expression, deletion, purification, and cellular localization by GFP tagging.

DHS-1-P phosphatases cloned from yeast represent novel lipid phosphatases, which were not thought to exist in yeast. Identification and characterization of YSR2 and YSR3 have demonstrated that the DHS-1-P phosphatase is an important mediator in the biosynthesis of sphingolipids and in the maintenance of the balance of signaling lipid molecules ceramide, sphingosine, and sphingosine-1-P. Methods introduced here for purification, activity assay, in vivo labeling, and cellular localization using GFP tagging are expected to facilitate our understanding of this enzyme.

Cell Compartmentation↗
PubMed1999

The dihydrosphingosine-1-phosphate phosphatases of Saccharomyces cerevisiae are important regulators of cell proliferation and heat stress responses.

We have identified YSR2 and YSR3 of Saccharomyces cerevisiae as genes encoding dihydrosphingosine-1-phosphate phophatases which are involved in regulation of sphingolipid metabolism [Mao, Wadleigh, Jenkins, Hannun and Obeid (1997) J. Biol. Chem. 272, 28690-28694]. In this study, we explored the physiological roles that these enzymes may have in S. cerevisiae. Deletion of either YSR2, YSR3 or both did not affect viability or growth rate of yeast cells. However, overexpression of YSR2 significantly prolonged the doubling time of cell growth, whereas overexpression of YSR3 affected cell growth only slightly. Cell cycle analysis suggested that overexpression of either YSR2 or, to a lesser extent, YSR3 caused cell cycle arrest at the G1 phase. Disruption of YSR2, but not YSR3, conferred increased thermotolerance. On the other hand, overexpression of either YSR2 or YSR3 diminished thermotolerance. Using labelled dihydrosphingosine and dihydrosphingosine-1-P (DHS-1-P), we found that overexpression of YSR2 significantly increased ceramide formation, whereas deletion of YSR2, YSR3, or both, accumulated DHS-1-P, and deletion of YSR2 decreased ceramide formation. Together, these results show that the phenotypes of YSR2 are associated with changes in endogenous levels of the different sphingolipids. Green fluorescent protein tagging showed that in the exponentially growing cells, YSR2 and YSR3 had the same cellular localization to endoplasmic reticulum. However, YSR2 and YSR3 differ in mRNA levels: YSR2 had significantly higher mRNA levels than YSR3. This discrepancy might result in the functional differences that these proteins exhibited. In addition, this study implicates sphingolipids and their metabolism in the regulation of growth and heat stress responses of the yeast S. cerevisiae.

Cell Division↗
PubMed1999

Phospholipase D in cellular senescence.

Cellular senescence appears to be an important part of organismal aging. Cellular senescence is characterized by flattened enlarged morphology, inhibition of DNA replication in response to growth factors, inability to phosphorylate the pRb tumor suppressor protein, inability to produce c-fos or AP-1 and overexpression of a variety of genes, notably p21 (CIP-1/WAF-1) and p16(INK). It is now clear that certain early mitotic signals become defective with the onset of senescence. Among these is the PLD/PKC pathway. Evidence suggests that activation of PLD and PKC is critical for mitogenesis. Recent data suggest that the defect in PLD/PKC in cellular senescence is a result of elevated cellular ceramide levels which inhibit PLD activation. It appears that the elevated ceramide is a result of neutral sphingomyelinase activation. Ceramide acts to inhibit the activation of PLD by possibly three mechanisms, inhibiting activation by Rho, translocation to the membrane and gene expression. Addition of ceramide to young cells not only inhibits PLD but also recapitulates all the standard measures of cellular senescence as described above.

Animals↗