Biomedical subjects

Lina M Obeid

Publications and source records attributed to Lina M Obeid.

At least 19 recordsLinked to original sources

PubMed2006

A house divided: ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death.

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., sphingosine. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acylsphingosine, has been implicated in cell death following a myriad of cellular stresses. Sphingosine itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. Sphingosine-1-phosphate, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.

Animals↗

PubMed2006

Sphingosine kinase-1 is cleaved by cathepsin B in vitro: identification of the initial cleavage sites for the protease.

Previous work has identified sphingosine kinase-1 (SK1) as a substrate for the cysteine protease cathepsin B in vitro. In this study, the mechanism of SK1 cleavage by cathepsin B was investigated. We identified two initial cleavage sites for the protease, the first at histidine 122 and the second at arginine 199. Mutation analysis showed that replacement of histidine 122 with a tyrosine maintained the activity of SK1 while significantly reducing cleavage by cathepsin B at the initial cleavage site. The efficacy of cleavage of SK1 at arginine 199, however, was not affected. These studies demonstrate that SK1 is cleaved by cathepsin B in a sequential manner after basic amino acids, and that the initial cleavages at the two identified sites occur independently of each other.

Amino Acids, Basic↗

PubMed2006

Protein kinase C-induced activation of a ceramide/protein phosphatase 1 pathway leading to dephosphorylation of p38 MAPK.

Recently we showed that, in human breast cancer cells, activation of protein kinase C by 4beta-phorbol 12-myristate 13-acetate (PMA) produced ceramide formed from the salvage pathway (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). In this study, we investigated intracellular signaling events mediated by this novel activated pathway of ceramide generation. PMA treatment resulted in transient activation of mitogen-activated protein kinases (ERK1/2, JNK1/2, and p38) followed by dephosphorylation/inactivation. Interestingly, fumonisin B1 (FB1), an inhibitor of the salvage pathway, attenuated loss of phosphorylation of p38, suggesting a role for ceramide in p38 dephosphorylation. This was confirmed by knock-down of longevity-assurance homologue 5, which partially suppressed the formation of C(16)-ceramide induced by PMA and increased the phosphorylation of p38. These results demonstrate a role for the salvage pathway in feedback inhibition of p38. To determine which protein phosphatases act in this pathway, specific knock-down of serine/threonine protein phosphatases was performed, and it was observed that knock-down of protein phosphatase 1 (PP1) catalytic subunits significantly increased p38 phosphorylation, suggesting activation of PP1 results in an inhibitory effect on p38. Moreover, PMA recruited PP1 catalytic subunits to mitochondria, and this was significantly suppressed by FB1. In addition, phospho-p38 resided in PMA-stimulated mitochondria. Upon PMA treatment, a mitochondria-enriched/purified fraction exhibited significant increases in C(16)-ceramide, a major ceramide specie, which was suppressed by FB1. Taken together, these data suggest that accumulation of C(16)-ceramide in mitochondria formed from the protein kinase C-dependent salvage pathway results at least in part from the action of longevity-assurance homologue 5, and the generated ceramide modulates the p38 cascade via PP1.

Catalytic Domain↗

PubMed2006

Necessary role for the Lag1p motif in (dihydro)ceramide synthase activity.

Lag1 (longevity assurance gene 1) homologues, a family of transmembrane proteins found in all eukaryotes, have been shown to be necessary for (dihydro)ceramide synthesis. All Lag1 homologues contain a highly conserved stretch of 52 amino acids known as the Lag1p motif. However, the functional significance of the conserved Lag1p motif for (dihydro)ceramide synthesis is currently unknown. In this work, we have investigated the function of the motif by introducing eight point mutations in the Lag1p motif of the mouse LASS1 (longevity assurance homologue 1 of yeast Lag1). The (dihydro)ceramide synthase activity of the mutants was tested using microsomes in HeLa cells and in vitro. Six of the mutations resulted in loss of activity in cells and in vitro. In addition, our results showed that C18:0 fatty acid CoA (but not cis-C18:1 fatty acid CoAs) are substrates for LASS1 and that LASS1 in HeLa cells is sensitive to fumonisin B1, an in vitro inhibitor of (dihydro)ceramide synthase. Moreover, we mutated the Lag1p motif of another Lag homologue, human LASS5. The amino acid substitutions in the human LASS5 were the same as in mouse LASS1, and had the same effect on the in vitro activity of LASS5, suggesting the Lag1p motif appears to be essential for the enzyme activity of all Lag1 homologues.

Amino Acid Sequence↗

PubMed2006

Insulin-like growth factors mediate heterotrimeric G protein-dependent ERK1/2 activation by transactivating sphingosine 1-phosphate receptors.

Although several studies have shown that a subset of insulin-like growth factor (IGF) signals require the activation of heterotrimeric G proteins, the molecular mechanisms underlying IGF-stimulated G protein signaling remain poorly understood. Here, we have studied the mechanism by which endogenous IGF receptors activate the ERK1/2 mitogen-activated protein kinase cascade in HEK293 cells. In these cells, treatment with pertussis toxin and expression of a Galpha(q/11)-(305-359) peptide that inhibits G(q/11) signaling additively inhibited IGF-stimulated ERK1/2 activation, indicating that the signal was almost completely G protein-dependent. Treatment with IGF-1 or IGF-2 promoted translocation of green fluorescent protein (GFP)-tagged sphingosine kinase (SK) 1 from the cytosol to the plasma membrane, increased endogenous SK activity within 30 s of stimulation, and caused a statistically significant increase in intracellular and extracellular sphingosine 1-phosphate (S1P) concentration. Using a GFP-tagged S1P1 receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that IGF-1 and IGF-2 induced GFP-S1P receptor internalization and that the effect was blocked by pretreatment with the SK inhibitor, dimethylsphingosine. Treating cells with dimethylsphingosine, silencing SK1 expression by RNA interference, and blocking endogenous S1P receptors with the competitive antagonist VPC23019 all significantly inhibited IGF-stimulated ERK1/2 activation, suggesting that IGFs elicit G protein-dependent ERK1/2 activation by stimulating SK1-dependent transactivation of S1P receptors. Given the ubiquity of SK and S1P receptor expression, S1P receptor transactivation may represent a general mechanism for G protein-dependent signaling by non-G protein-coupled receptors.

Animals↗

PubMed2006

Tailoring structure-function and targeting properties of ceramides by site-specific cationization.

In the course of our studies on compartment-specific lipid-mediated cell regulation, we identified an intimate connection between ceramides (Cers) and the mitochondria-dependent death-signaling pathways. Here, we report on a new class of cationic Cer mimics, dubbed ceramidoids, designed to act as organelle-targeted sphingolipids (SPLs), based on conjugates of Cer and dihydroceramide (dhCer) with pyridinium salts (CCPS and dhCCPS, respectively). Ceramidoids having the pyridinium salt unit (PSU) placed internally (alpha and gamma- CCPS) or as a tether (omega-CCPS) in the N-acyl moiety were prepared by N-acylation of sphingoid bases with different omega-bromo acids or pyridine carboxylic acid chlorides following capping with respective pyridines or alkyl bromides. Consistent with their design, these analogs, showed a significantly improved solubility in water, well-resolved NMR spectra in D(2)O, broadly modified hydrophobicity, fast cellular uptake, and higher anticancer activities in cells in comparison to uncharged counterparts. Structure-activity relationship (SAR) studies in MCF-7 breast carcinoma cells revealed that the location of the PSU and its overall chain length affected markedly the cytotoxic effects of these ceramidoids. All omega-CCPSs were more potent (IC(50/48 h): 0.6-8.0 microM) than their alpha/gamma-CCPS (IC(50/48 h): 8-20 microM) or D-erythro-C6-Cer (IC(50/48 h): 15 microM) analogs. omega-DhCCPSs were also moderately potent (IC(50/48 h): 2.5-12.5 microM). Long-chain omega-dhCCPSs were rapidly and efficiently oxidized in cells to the corresponding omega-CCPSs, as established by LC-MS analysis. CCPS analogs also induced acute changes in the levels and composition of endogenous Cers (upregulation of C16-, C14-, and C18-Cers, and downregulation of C24:0- and C24:1-Cers). These novel ceramidoids illustrate the feasibility of compartment-targeted lipids, and they should be useful in cell-based studies as well as potential novel therapeutics.

Antineoplastic Agents↗

PubMed2006

Yeast sphingolipids: recent developments in understanding biosynthesis, regulation, and function.

Sphingolipids function as required membrane components of virtually all eukaryotic cells. Data indicate that members of the sphingolipid family of lipids, including sphingoid bases, sphingoid base phosphates, ceramides, and complex sphingolipids, serve vital functions in cell biology by both direct mechanisms (e.g., binding to G-protein coupled receptors to transduce an extracellular signal) and indirect mechanisms (e.g., facilitating correct intracellular protein transport). Because of the diverse roles these lipids play in cell biology, it is important to understand not only their biosynthetic pathways and regulation of sphingolipid synthesis, but also the mechanisms by which some sphingolipid species with specific functions are modified or converted to other sphingolipid species with alternate functions. Due to many factors including ease of culture and genetic modification, and conservation of major sphingolipid metabolic pathways, Saccharomyces cerevisiae has served as an ideal model system with which to identify enzymes of sphingolipid biosynthesis and to dissect sphingolipid function. Recent exciting developments in sphingolipid synthesis, transport, signaling, and overall biology continue to fuel vigorous investigation and inspire investigations in mammalian sphingolipid biology.

Animals↗

PubMed2006

Acid ceramidase but not acid sphingomyelinase is required for tumor necrosis factor-{alpha}-induced PGE2 production.

Sphingolipids are well established effectors of signal transduction downstream of the tumor necrosis factor (TNF) receptor. In a previous study, we showed that the sphingosine kinase/sphingosine 1-phosphate (S1P) pathway couples TNF receptor to induction of the cyclooxygenase 2 gene and prostaglandin synthesis (Pettus, B. J., Bielawski, J., Porcelli, A. M., Reames, D. L., Johnson, K. R., Morrow, J., Chalfant, C. E., Obeid, L. M., and Hannun, Y. A. (2003) FASEB J. 17, 1411-1421). In this study, the requirement for acid sphingomyelinase and sphingomyelin metabolites in the TNFalpha/prostaglandin E(2) (PGE(2)) pathway was investigated. The amphiphilic compound desipramine, a frequently employed inhibitor of acid sphingomyelinase (ASMase), blocked PGE(2) production. However, the action of desipramine was independent of its action on ASMase, since neither genetic loss of ASMase (Niemann-Pick fibroblasts) nor knockdown of ASMase using RNA interference affected TNFalpha-induced PGE(2) synthesis. Further investigations revealed that desipramine down-regulated acid ceramidase (AC), but not sphingosine kinase, at the protein level. This resulted in a time-dependent drop in sphingosine and S1P levels. Moreover, exogenous administration of either sphingosine or S1P rescued PGE(2) biosynthesis after desipramine treatment. Interestingly, knockdown of endogenous AC by RNA interference attenuated cyclooxygenase 2 induction by TNFalpha and subsequent PGE(2) biosynthesis. Taken together, these results define a novel role for AC in the TNFalpha/PGE(2) pathway. In addition, the results of this study warrant careful reconsideration of desipramine as a specific inhibitor for ASMase.

Animals↗

PubMed2006

Bioactive sphingolipids in the modulation of the inflammatory response.

Inflammation is viewed as a protective response against insults to the organism. It involves the recruitment of many cell types and the production of various inflammatory mediators in attempts to contain and reverse the insult. However, inflammation can lead to irreversible tissue destruction by itself and, therefore, can represent a disease state that causes significant morbidity and mortality. Understanding the molecular mechanisms controlling the inflammatory response is essential to formulate therapeutic strategies for the treatment of inflammatory conditions. In fact, substantial research has unveiled important aspects of the inflammatory machinery, both at the cellular and molecular levels. Recently, sphingolipids (SLs) have emerged as signaling molecules that regulate many cell functions, and ample evidence emphasizes their role in the regulation of inflammatory responses. Here, we review the role of bioactive SL as regulators and mediators of inflammatory responses.

Animals↗

PubMed2006

Modulation of total Akt kinase by increased expression of a single isoform: requirement of the sphingosine-1-phosphate receptor, Edg3/S1P3, for the VEGF-dependent expression of Akt3 in primary endothelial cells.

Akt kinase is an important downstream effector of VEGF in primary endothelial cells (EC), promoting angiogenesis by increased cellular survival, motility and tubulogenesis. Akt1 is the founding member of a family of serine threonine kinases thought to have overlapping function. We sought to determine if other Akt family members were also regulated by VEGF in EC. We show that treatment of EC with the angiogenic inducers VEGF or sphingosine-1-phosphate (S1P) results in an increased stabilization of Akt3 mRNA, concurrent with a PI3 kinase-dependent, Akt1-independent increase in both the protein and its phosphorylation. Given the similarity of Akt3 regulation by VEGF and S1P, the sensitivity of VEGF stimulation to the Gi-protein uncoupling reagent, pertussis toxin was tested and shows that VEGF stimulation requires Gi-protein signaling. We show that the VEGF stimulates the expression of Edg3/S1P3 (S1P3) and that expression of this Gi-protein-coupled receptor is both sufficient and necessary for the expression of Akt3. Blockade of a single isoform does not overtly affect cellular function, whereas inhibition of both kinases results in an increase in apoptosis and a down-regulation of cyclin D3. These results suggest a model whereby extracellular cues maintain total Akt kinase levels through the regulation of specific isoform expression providing a fail-safe mechanism to maintain necessary levels of Akt kinase activity.

Cells, Cultured↗

PubMed2006

Potent antitumor activity of a novel cationic pyridinium-ceramide alone or in combination with gemcitabine against human head and neck squamous cell carcinomas in vitro and in vivo.

In this study, a cationic water-soluble ceramide analog L-threo-C6-pyridinium-ceramide-bromide (L-t-C6-Pyr-Cer), which exhibits high solubility and bioavailability, inhibited the growth of various human head and neck squamous cell carcinoma (HNSCC) cell lines at low IC50 concentrations, independent of their p53 status. Consistent with its design to target negatively charged intracellular compartments, L-t-C6-Pyr-Cer accumulated mainly in mitochondria-, and nuclei-enriched fractions upon treatment of human UM-SCC-22A cells [human squamous cell carcinoma (SCC) of the hypopharynx] at 1 to 6 h. In addition to its growth-inhibitory function as a single agent, the supra-additive interaction of L-t-C6-Pyr-Cer with gemcitabine (GMZ), a chemotherapeutic agent used in HNSCC, was determined using isobologram studies. Then, the effects of this ceramide, alone or in combination with GMZ, on the growth of UM-SCC-22A xenografts in SCID mice was assessed following the determination of preclinical parameters, such as maximum tolerated dose, clearance from the blood, and bioaccumulation. Results demonstrated that treatment with L-t-C6-Pyr-Cer in combination with GMZ significantly prevented the growth of HNSCC tumors in vivo. The therapeutic efficacy of L-t-C6-Pyr-Cer/GMZ combination against HNSCC tumors was approximately 2.5-fold better than that of the combination of 5-fluorouracil/cis-platin. In addition, liquid chromatography/mass spectroscopy analysis showed that the levels of L-t-C6-Pyr-Cer in HNSCC tumors were significantly higher than its levels in the liver and intestines; interestingly, the combination with GMZ increased the sustained accumulation of this ceramide by approximately 40%. Moreover, treatment with L-t-C6-Pyr-Cer/GMZ combination resulted in a significant inhibition of telomerase activity and decrease in telomere length in vivo, which are among downstream targets of ceramide.

Animals↗

PubMed2006

Differentiation-associated expression of ceramidase isoforms in cultured keratinocytes and epidermis.

Ceramides (Cers) accumulate within the interstices of the outermost epidermal layers, or stratum corneum (SC), where they represent critical components of the epidermal permeability barrier. Although the SC contains substantial sphingol, indicative of ceramidase (CDase) activity, which CDase isoforms are expressed in epidermis remains unresolved. We hypothesized here that CDase isoforms are expressed within specific epidermal compartments in relation to functions that localize to these layers. Keratinocytes/epidermis express all five known CDase isoforms, of which acidic and alkaline CDase activities increase significantly with differentiation, persisting into the SC. Conversely, neutral and phytoalkaline CDase activities predominate in proliferating keratinocytes. These differentiation-associated changes in isoform activity/protein are attributed to corresponding, differentiation-associated changes in mRNA levels (by quantitative RT-PCR). Although four of the five known CDase isoforms are widely expressed in cutaneous and extracutaneous tissues, alkaline CDase-1 occurs almost exclusively in epidermis. These results demonstrate large, differentiation-associated, and tissue-specific variations in the expression and activities of all five CDase isoforms. Because alkaline CDase-1 and acidic CDase are selectively upregulated in the differentiated epidermal compartment, they could regulate functions that localize to the distal epidermis, such as permeability barrier homeostasis and antimicrobial defense.

Amidohydrolases↗

PubMed2006

Oxidized LDL immune complexes induce release of sphingosine kinase in human U937 monocytic cells.

The transformation of macrophages into foam cells is a critical event in the development of atherosclerosis. The most studied aspect of this process is the uptake of modified LDL through the scavenger receptors. Another salient aspect is the effect of modified LDL immune complexes on macrophages activation and foam cell formation. Macrophages internalize oxidized LDL immune complexes (oxLDL-IC) via the Fc-gamma receptor and transform into activated foam cells. In this study we examined the effect of oxLDL-IC on sphingosine kinase 1 (SK1), an enzyme implicated in mediating pro-survival and inflammatory responses through the generation of the signaling molecule sphingosine-1-phosphate (S1P). Intriguingly, oxLDL-IC, but not oxLDL alone, induced an immediate translocation and release of SK1 into the conditioned medium as evidenced by fluorescence confocal microscopy. Immunoblot analysis of cell lysates and conditioned medium revealed a decrease in intracellular SK1 protein levels accompanied by a concomitant increase in extracellular SK1 levels. Furthermore, measurement of S1P formation showed that the activity of cell-associated SK decreased in response to oxLDL-IC compared to oxLDL alone, whereas the activity of SK increased extracellularly. Blocking oxLDL-IC binding to Fc-gamma receptors resulted in decreased levels of extracellular S1P. The data also show that cell survival of human U937 cells exposed to oxLDL-IC increased compared to oxLDL alone. Exogenously added S1P further increased cell survival induced by oxLDL-IC. Taken together, these findings indicate that S1P may be generated extracellularly in response to modified LDL immune complexes and may therefore promote cell survival and prolong cytokine release by activated macrophages.

Antigen-Antibody Complex↗

PubMed2006

Golgi alkaline ceramidase regulates cell proliferation and survival by controlling levels of sphingosine and S1P.

Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, promotes cell proliferation and survival whereas its precursor, sphingosine, has the opposite effects. However, much remains unknown about their regulation. Here we identify a novel human ceramidase (haCER2) that regulates the levels of both sphingosine and S1P by controlling the hydrolysis of ceramides. haCER2 is localized to the Golgi complex and is highly expressed in the placenta. High ectopic expression of haCER2 caused fragmentation of the Golgi complex and growth arrest in HeLa cells due to sphingosine accumulation. Low ectopic expression of haCER2 increased S1P without sphingosine accumulation, promoting cell proliferation in serum-free medium. This proliferative effect was suppressed by dimethylsphingosine, an inhibitor of the S1P formation, or by the RNA interference (RNAi) -mediated inhibition of S1P(1,) a G-protein-coupled receptor for S1P. The RNAi-mediated down-regulation of haCER2 enhanced the serum deprivation-induced growth arrest and apoptosis of HeLa cells, which was inhibited by addition of exogenous S1P. Serum deprivation up-regulated both haCER2 mRNA and activity in HeLa cells. haCER2 mRNA is also up-regulated in some tumors. Taken together, these results suggest that haCER2 is important for the generation of S1P and S1P-mediated cell proliferation and survival, but that its overexpression may cause cell growth arrest due to an accumulation of sphingosine.

Alkaline Ceramidase↗

PubMed2006

Cationic long-chain ceramide LCL-30 induces cell death by mitochondrial targeting in SW403 cells.

Ceramides are sphingolipid second messengers that are involved in the mediation of cell death. There is accumulating evidence that mitochondria play a central role in ceramide-derived toxicity. We designed a novel cationic long-chain ceramide [omega-pyridinium bromide D-erythro-C16-ceramide (LCL-30)] targeting negatively charged mitochondria. Our results show that LCL-30 is highly cytotoxic to SW403 cells (and other cancer cell lines) and preferentially accumulates in mitochondria, resulting in a decrease of the mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 and caspase-9. Ultrastructural analyses support the concept of mitochondrial selectivity. Interestingly, levels of endogenous mitochondrial C16-ceramide decreased by more than half, whereas levels of sphingosine-1-phosphate increased dramatically and selectively in mitochondria after administration of LCL-30, suggesting the presence of a mitochondrial sphingosine kinase. Of note, intracellular long-chain ceramide levels and sphingosine-1-phosphate remained unaffected in the cytosolic and extramitochondrial (nuclei/cellular membranes) cellular fractions. Furthermore, a synergistic effect of cotreatment of LCL-30 and doxorubicin was observed, which was not related to alterations in endogenous ceramide levels. Cationic long-chain pyridinium ceramides might be promising new drugs for cancer therapy through their mitochondrial preference.

Antibiotics, Antineoplastic↗

PubMed2005

Loss of sphingosine kinase-1 activates the intrinsic pathway of programmed cell death: modulation of sphingolipid levels and the induction of apoptosis.

Activation of sphingosine kinase-1 (SK1) by overexpression or agonist stimulation promotes cell proliferation, survival, and anti-apoptosis. Studies on the function of endogenous SK1 are lacking. Endogenous SK1 has been shown to be down-regulated under stress, and knockdown of the enzyme reduces the percentage of viable MCF-7 breast cancer cells (Taha, T. A. et al. 2004. J. Biol. Chem. 279, 20546-20554). In this study, we examined the mechanisms by which SK1 loss affects the growth of cells. Knockdown of the enzyme by small interfering RNA caused cell cycle arrest and induced apoptosis. Cell death involved effector caspase activation, cytochrome c release and Bax oligomerization in the mitochondrial membrane, thus placing SK1 knockdown upstream of the mitochondrial pathway of apoptosis. SK1 knockdown also induced significant increases in ceramide levels in whole cells and in mitochondria enriched fractions of cells. Inhibition of de novo sphingolipid biosynthesis with myriocin significantly attenuated Bax oligomerization and downstream caspase activation after SK1 loss. These studies for the first time implicate endogenous SK1 as an important survival enzyme in MCF-7 cells and link the biological consequences of knocking down the enzyme to its biochemical role as a regulator of sphingolipid metabolism.

Adenocarcinoma↗

PubMed2005

Sphingosine kinase 1 is up-regulated in colon carcinogenesis.

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to form sphingosine 1-phosphate (S1P), which has the ability to promote cell proliferation and survival and stimulate angiogenesis. The SK1/S1P pathway also plays a critical role in regulation of cyclooxygenase-2 (COX-2), a well-established pathogenic factor in colon carcinogenesis. Therefore, we examined the expression of SK1 and COX-2 in rat colon tumors induced by azoxymethane (AOM) and the relationship of these two proteins in normal and malignant intestinal epithelial cells. Strongly positive SK1 staining was found in 21/28 (75%) of rat colon adenocarcinomas induced by AOM, whereas no positive SK1 staining was observed in normal mucosa. The increase in SK1 and COX-2 expression in AOM-induced rat colon adenocarcinoma was confirmed at the level of mRNA by real-time RT-PCR. In addition, it was found that 1) down-regulation of SK1 in HT-29 human colon cancer cells by small interfering RNA (siRNA) decreases COX-2 expression and PGE2 production; 2) overexpression of SK1 in RIE-1 rat intestinal epithelial cells induces COX-2 expression; and 3) S1P stimulates COX-2 expression and PGE2 production in HT-29 cells. These results suggest that the SK1/S1P pathway may play an important role in colon carcinogenesis, in part, by regulating COX-2 expression and PGE2 production.

Animals↗

PubMed2005

The mechanism of membrane targeting of human sphingosine kinase 1.

Sphingosine 1-phosphate is a bioactive sphingolipid that regulates cell growth and suppresses programmed cell death. The biosynthesis of sphingosine 1-phosphate is catalyzed by sphingosine kinase (SK) but the mechanism by which the subcellular localization and activity of SK is regulated in response to various stimuli is not fully understood. To elucidate the origin and structural determinant of the specific subcellular localization of SK, we performed biophysical and cell studies of human SK1 (hSK1) and selected mutants. In vitro measurements showed that hSK1 selectively bound phosphatidylserine over other anionic phospholipids and strongly preferred the plasma membrane-mimicking membrane to other cellular membrane mimetics. Mutational analysis indicates that conserved Thr54 and Asn89 in the putative membrane-binding surface are essential for lipid selectivity and membrane targeting both in vitro and in the cell. Also, phosphorylation of Ser225 enhances the membrane affinity and plasma membrane selectivity of hSK1, presumably by modulating the interaction of Thr54 and Asn89 with the membrane. Collectively, these studies suggest that the specific plasma membrane localization and activation of SK1 is mediated largely by specific lipid-protein interactions.

Amino Acid Sequence↗