©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulatory Role of Ceramide in Interleukin (IL)-1-induced E-selectin Expression in Human Umbilical Vein Endothelial Cells
CERAMIDE ENHANCES IL-1beta ACTION, BUT IS NOT SUFFICIENT FOR E-SELECTIN EXPRESSION (*)

(Received for publication, January 24, 1996)

Atsushi Masamune (§) Yasuyuki Igarashi Sen-itiroh Hakomori (¶)

From the Biomembrane Institute, Seattle, Washington 98119 and the Department of Pathobiology, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recent studies indicate that sphingolipids mediate several cellular processes. We assessed roles of sphingolipids in the regulation of E-selectin expression in human umbilical vein endothelial cells. All exogenously-added sphingolipids (sphingosine, C(2)-ceramide, sphingosine 1-phosphate, and N,N-dimethylsphingosine) failed to induce E-selectin expression by themselves. C(2)-ceramide at 5 µM enhanced interleukin-1beta (IL-1beta)-induced E-selectin expression 2.7-fold, whereas other sphingolipids tested had no effects on this process. Sphingomyelinase, but not phospholipases A(2), C, or D, mimicked the enhancing effect of C(2)-ceramide. Northern blot analyses revealed that C(2)-ceramide and sphingomyelinase increased interleukin-1beta-induced E-selectin gene transcription levels. C(2)-ceramide and sphingomyelinase induced NF-kappaB activation by themselves and enhanced activation by IL-1beta, which is essential for E-selectin expression. Immunological analyses with anti-NF-kappaB antibodies showed that subunit composition of NF-kappaB activated by IL-1beta differs from that activated by C(2)-ceramide, suggesting that signaling pathways utilized by these stimuli may be different. Treatment with C(2)-ceramide or sphingomyelinase did not alter NF-ELAM1 specific binding activity. IL-1beta induced sphingomyelin hydrolysis to ceramide; intracellular ceramide level increased to 182% of control value at 30 min. Taken together, these findings suggest that (i) sphingomyelin hydrolysis to ceramide does not trigger, but rather enhances cytokine-induced E-selectin expression, in part through NF-kappaB; (ii) sphingomyelin hydrolysis to ceramide does not mediate all the effects of IL-1beta, although it may play important roles in IL-1beta signal transduction in human umbilical vein endothelial cells.


INTRODUCTION

Adhesion of blood leukocytes to endothelium is a critical early step of inflammatory and immune responses(1) . This step involves adhesion molecules expressed on the endothelial cell surface and their ligands on the leukocyte surface. The endothelial leukocyte adhesion molecule (E-selectin, or CD 62E) is a member of the selectin family of cell surface glycoproteins(2, 3) . Expression of E-selectin is both cell-type specific and stimulus specific; it is expressed exclusively on endothelial cells, in response to interleukin-1beta (IL-1), (^1)tumor necrosis factor-alpha (TNF), bacterial lipopolysaccharide, and phorbol myristate acetate(1, 2) . In cultured human umbilical vein endothelial cells (HUVEC), E-selectin is rapidly and transiently induced, with protein and mRNA peaking at about 4 h after cytokine treatment, and returning to near basal level by 24 h(2, 4) . Maximal transcriptional activity is observed within 1-2 h post-induction(4) . This tight regulation of gene activity presumably requires complex control mechanisms. E-selectin induction is transcriptionally mediated(5) . Roles of protein kinase C(6) , labile proteins(4) , nuclear factor kappaB (NF-kappaB)(5, 6) , and NF-ELAM1 (7) in E-selectin expression have been suggested, but are not fully understood. Better understanding of E-selectin expression control would provide further insights into inflammatory processes.

NF-kappaB is a member of the Rel family of transcriptional regulatory proteins; the family includes p50 (NF-kappaB1), p52 (NF-kappaB2), RelA (p65), c-Rel, RelB, and the Drosophila morphogen dorsal gene product(8, 9) . Phosphorylation and degradation of the inhibitory protein IkappaB, and subsequent dissociation of this protein from NF-kappaB are thought to be necessary for activation of NF-kappaB(10, 11, 12) . NF-kappaB is known to be involved in control of cytokine-induced expression of many immune and inflammatory-response genes(13) . In particular, NF-kappaB and IkappaB are an inducible regulatory system in endothelial activation(14) . Members of this family are capable of homo- and heterotypic dimerization through a 300-amino acid homologous domain(8, 9) . Both homo- and heterodimeric NF-kappaB complexes containing other Rel family members are able to influence gene transcription. The level of activation, and the type of NF-kappaB/Rel dimers activated, determine the extent and nature of the response induced(15, 16) .

Sphingoglycolipids play important roles in cell-to-cell interaction, modulation of cell growth, and differentiation(17) . They modulate transmembrane signaling through interaction with tyrosine kinases associated with growth factor receptors, and protein kinase C(17, 18) . It has become increasingly evident that the signal-modulatory effect of sphingoglycolipids resides not only in the glycosylated structures but also in their backbone structures and metabolites such as ceramide(19, 20) , sphingosine (Sph)(21, 22) , sphingosine 1-phosphate (Sph-1-P)(23, 24) , and N,N-dimethylsphingosine (DMS)(18) . Biological functions of these sphingolipids have been extensively studied, and they have been implicated as important signaling molecules in regulation of numerous cellular functions. For example, TNF and IL-1 activate sphingomyelinases (SMase), resulting in sphingomyelin hydrolysis and generation of ceramide. Conversely, ceramide appears to mediate effects of these agonists on cell growth, differentiation, and apoptosis in several cell lines(19, 20) . Ceramide is also reported to enhance IL-1-induced prostaglandin E(2) secretion in human fibroblasts(25) , and interleukin-2 secretion in lymphocytes(26) , suggesting that ceramide may regulate immune function and inflammatory responses.

In this study, we investigated the roles of sphingolipids in regulation of IL-1-induced E-selectin expression in HUVEC. N-Acetylsphingosine (C(2)-ceramide (C(2)-cer), a cell-permeable ceramide analogue) and bacterial SMase enhanced IL-1-induced E-selectin expression, but did not induce E-selectin expression in the absence of IL-1. Hydrolysis of sphingomyelin to ceramide was stimulated by IL-1 in HUVEC. Our results suggest that IL-1-induced sphingomyelin hydrolysis and generation of ceramide do not trigger, but rather regulate E-selectin expression in HUVEC. Possible mechanisms consistent with these findings are discussed.


EXPERIMENTAL PROCEDURES

Materials

Sph, Sph-1-P, and DMS were prepared as described previously(27, 28, 29) , dissolved, and stocked at 2 mM ethanol/water (50:50, v/v). C(2)-cer was from Biomol (Plymouth Meeting, PA). Ceramide (Type III), sphingomyelin (chicken egg yolk), SMase (Staphylococcus aureus), phospholipase (PL) A(2) (bee venom), phosphatidylinositol-specific PLC (Bacillus cereus), PLD (cabbage), paraformaldehyde, glycine, bovine serum albumin, sodium azide, mouse IgG, tricyclodecan-9-yl xanthogenate (D609), ammonium chloride, monensin, phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, pepstatin A, Nonidet P-40, glycerol, and gelatin were from Sigma. sn-1,2-Diacylglycerol kinase from Escherichia coli and dithiothreitol were purchased from Calbiochem (San Diego, CA). Poly(dIbulletdC)-poly(dIbulletdC) was from Pharmacia (Piscataway, NJ). [-P]ATP, [alpha-P]dCTP, and [^3H]choline chloride were purchased from DuPont NEN. Recombinant IL-1 was from Boehringer Mannheim. Oligonucleotide probe containing the NF-ELAM1 binding site (5`-GATCTTCTGACATCATTGTAAT-3`) (30) and NF-kappaB from the E-selectin promoter (5`-GCCATTGGGGATTTCCTCTTTACTGG-3`) (6) were synthesized by Life Technologies Inc. (Gaithersburg, MD). Oligonucleotide probe for the AP-2 consensus sequence was from Promega (Madison, WI). Rabbit polyclonal antibodies against human p50, p65, and c-Rel were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-human E-selectin monoclonal antibody 3B7 (5 mg of IgG/ml of culture supernatants) (31) was kindly provided by Otsuka Pharmaceutical Co. (Tokyo, Japan).

Cell Treatment

HUVEC and their optimized culture medium containing endothelial cell growth factor, and fetal calf serum, were purchased from Clonetics (San Diego, CA). Optimized medium contains 2% fetal calf serum. For some experiments, serum-free medium from the same company was used. HUVEC were maintained according to the manufacturer's instruction and cells between passages three and five were used for all experiments. HUVEC were grown on 0.2% gelatin-coated 96-well flat-bottom plates (Costar, Cambridge, MA) for enzyme-linked immunosorbent assay (ELISA), or on tissue culture dishes (Corning, Corning, NY) for other experiments. On the day of experiment, HUVEC monolayer was refed with fresh serum-free medium supplemented with 1% fetal calf serum and treated with experimental reagents for 30 min at 37 °C except as specifically indicated. IL-1 was subsequently added to culture medium to a final concentration of 60 IU/ml, and incubation was continued. For permeabilization experiments, cells were treated with Trans-Port transient cell permeabilization kit (Life Technologies) according to the manufacturer's protocol.

Enzyme-linked Immunosorbent Assay

ELISA was performed essentially as described previously (32) following 4 h incubation with or without IL-1. Briefly, cells were washed and fixed with 1% paraformaldehyde for 30 min. Following washes with 20 mM glycine, cells were blocked with bovine serum albumin and 0.1% sodium azide for 1 h. Cells were incubated with 100 µl of 3B7 or with 5 µg/ml mouse IgG as a control for 1 h. After washes, anti-mouse IgG antibody conjugated with peroxidase (Southern Biotechnology Association, Birmingham, AL) was added and incubated for 1 h. Soluble substrate 1,2-phenylenediamine (Dako, Glostrup, Denmark) and hydrogen peroxide (final concentration 0.03%) were added according to the manufacturer's instruction. E-selectin expression was determined by the difference in absorbance at wavelength 490 versus 630 nm.

Radiolabeling of DNAs

E-selectin cDNA (R& Systems, San Diego, CA) and beta-actin cDNA generated by polymerase chain reaction (33) were radiolabeled with [alpha-P]dCTP using Ready-To-Go DNA labeling kit (Pharmacia). Double-stranded oligonucleotide probes were labeled with [-P]ATP by incubation with T4 polynucleotide kinase (Promega) at 37 °C for 10 min. The labeled probes were separated from unincorporated nucleotides using Quick Spin G-50 or G-25 column (Boehringer Mannheim).

RNA Isolation and Northern Analysis

Following 4 h incubation, total RNA was isolated from HUVEC by the method of Chomczynski and Sacchi(34) . Total RNA (10 µg/lane) was separated on a 1% agarose denaturing gel containing 2.2 M formaldehyde, transferred to Hybond-N nylon filters (Amersham), and hybridized to radiolabeled E-selectin cDNA for 16 h at 42 °C. Filters were washed twice in 2 times SSC, 0.1% SDS for 30 min at 42 °C followed by a wash in 0.2 times SSC, 0.1% SDS for 1 h at 42 °C. Washed filters were subjected to autoradiography overnight at -80 °C. As a control, stripped filters were rehybridized with human beta-actin cDNA probes.

Nuclear Extract Preparation

Nuclear extracts were prepared from approximately 1 times 10^7 cells using the method of Dignam et al.(35) and Osborn et al.(36) with slight modification. Following 1 h incubation, cells were harvested and washed twice with ice-cold phosphate-buffered saline. Cells were lysed in 400 µl of buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 2 mM MgCl(2), 0.5 mM dithiothreitol, 1 mM PMSF, 5 µg/ml aprotinin, 5 µg/ml pepstatin A, and 5 µg/ml leupeptin) containing 0.1% Nonidet P-40 for 15 min on ice, vortexed vigorously for 15 s, and centrifuged at 14,000 rpm for 30 s. The pelleted nuclei were resuspended in 40 µl of buffer C (20 mM Hepes, pH 7.9, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl(2), 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF, 5 µg/ml aprotinin, and 5 µg/ml leupeptin). After 30 min on ice, lysates were centrifuged at 14,000 rpm for 10 min. Supernatants containing the nuclear proteins were diluted with 20 µl of modified buffer D (20 mM Hepes, pH 7.9, 20% (v/v) glycerol, 0.05 M KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM PMSF) and stored at -80 °C until use. Protein concentration was determined using Bio-Rad protein assay (Bio-Rad).

Electrophoretic Mobility Shift Assay

Nuclear extracts (2-3 µg) were incubated in a binding reaction buffer (10 mM Tris-Cl, pH 7.5, 1 mM MgCl(2), 50 mM NaCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF, 4% (v/v) glycerol, 0.1 mg/ml poly(dIbulletdC)-poly(dIbulletdC)) with labeled oligonucleotide probe for 20 min at 22 °C. They were electrophoresed through 6% polyacrylamide gel in 0.5 times TBE buffer (44 mM Tris, 44 mM boric acid, 1 mM EDTA). Gels were dried and autoradiographed at -80 °C for 4 h to overnight. 100-fold molar excess of unlabeled oligonucleotide was incubated with nuclear extracts for 10 min at 22 °C prior to addition of radiolabeled probe in competition experiments. For super-shift analyses, nuclear extracts were incubated for 1 h at 4 °C with rabbit polyclonal antibodies against the various subunits of NF-kappaB complexes prior to incubation with labeled probe. Anti-p50, anti-p65, or anti-c-Rel antibodies were added to the reaction mixture, at various concentrations (0, 2, 4, or 8 µl/sample), to ascertain saturation.

Sphingomyelin Measurement

At the time of plating, [^3H]choline chloride (specific activity, 81.0 Ci/mmol) was added to medium to a final concentration of 1 µCi/ml. Following 72 h labeling, cells were washed three times with phosphate-buffered saline followed by addition of serum-free fresh medium, and incubated for 3 h at 37 °C. Cells were treated with IL-1 (60 IU/ml) for the indicated times. Lipids were extracted by the method of Bligh and Dyer(37) , applied to Silica Gel 60 high performance thin layer chromatography plates (Merck, Darmstadt, Germany), and developed in chloroform:methanol:acetic acid:water (50:30:8:5, v/v). Sphingomyelin spots were visualized by fluorography, scraped, and quantitated by scintillation spectrometry. In parallel experiments, sphingomyelin spots were scraped, lipids were eluted from silica gel in chloroform:methanol (2:1, v/v), and sphingomyelin was quantitated based on phosphate content as described previously (38) .

Ceramide Measurement

Following treatment of cells with IL-1, lipids were extracted and incubated with diacylglycerol kinase as described previously(39, 40) . Major lipid products of the phosphorylation reaction were phosphatidic acid (from diacylglycerol) and ceramide 1-phosphate (from ceramide). They were completely resolved by thin-layer chromatography using chloroform:acetone:methanol:acetic acid:water (10:4:2:2:1, v/v) as solvent, and visualized by autoradiography. Ceramide 1-phosphate spots were identified by comparison with standard prepared as described previously(41) , scraped, and quantitated by scintillation counting. Intracellular ceramide levels were measured following bacterial SMase treatment in a similar manner.

Statistics

Differences between experimental groups were evaluated by the two-tailed unpaired Student's t test. A p value less than 0.05 was considered statistically significant.


RESULTS

C(2)-ceramide and Sphingomyelinase Enhance IL-1-induced E-selectin Expression

We first examined the effects of sphingolipids on E-selectin expression in HUVEC by ELISA. Without cytokine stimulation, no E-selectin expression was detected. None of the sphingolipids tested (Sph, C(2)-cer, DMS, Sph-1-P) induced E-selectin expression at concentrations between 0.001-10 µM in the absence of IL-1 (Table 1). We examined the expression of E-selectin as long as 24 h after sphingolipids treatment, but no expression was observed (data not shown). Treatment of permeabilized cells with sphingolipids up to 10 µM did not result in E-selectin expression. Stimulation of HUVEC with IL-1 (60 IU/ml) for 4 h induced E-selectin expression (2) . IL-1-induced E-selectin expression was enhanced by C(2)-cer treatment (Table 1). This effect was dose-dependent and maximal (2.7-fold increase) at 5 µM (Fig. 1A). Another permeable ceramide analogue, C(6)-ceramide, enhanced the E-selectin expression to a smaller extent. Other sphingolipids tested showed no stimulatory or inhibitory effects at concentrations up to 10 µM (Table 1). In these experiments, sphingolipids were added to culture medium using ethanol as a vehicle, and the final concentration of ethanol (0.25%) did not affect viability or morphology of endothelial cells. Viability under these conditions (10 µM sphingolipid in 0.25% ethanol) was >95% as determined by trypan blue exclusion assay. However, when HUVEC were treated with sphingolipids at concentrations above 10 µM, cells became round-shaped and detached from the plate during incubation. Sphingosine and DMS, known as potent protein kinase C inhibitors, showed no inhibitory effects on E-selectin expression. This is consistent with previous findings that several protein kinase C inhibitors failed to diminish IL-1-induced E-selectin expression (42) .




Figure 1: Effects of C(2)-cer (A) and SMase (B) on IL-1-induced E-selectin expression. HUVEC were treated with IL-1 (60 IU/ml) and increasing concentrations of C(2)-cer or SMase (B). After 4 h, E-selectin expression was measured by ELISA as described under ``Materials and Methods.'' The data represent mean values ± S.D. from four independent experiments.



SMase hydrolyzes sphingomyelin, and generates ceramide and phosphorylcholine(19, 20) . To determine whether endogenous ceramide generated by SMase mimics the enhancing effect of C(2)-cer on E-selectin expression, we treated cells with bacterial SMase, alone, or in combination with IL-1, and measured E-selectin expression. SMase treatment enhanced IL-1-induced E-selectin expression in a dose-dependent manner (Fig. 1B). Maximal stimulation, achieved at 100 milliunits/ml SMase, represented a 2.2-fold increase compared with stimulation by IL-1 alone. Treatment of cells with SMase alone at concentrations from 1 times 10 to 1000 milliunits/ml had no effect on E-selectin expression. It seems unlikely that failure of E-selectin induction is due to an insufficient amount of ceramide generated by SMase treatment, because SMase at 200 milliunits/ml resulted in an approximately 5.2-fold increase in ceramide content within 15 min. SMase treatment of permeabilized HUVEC did not result in E-selectin expression over the same range of concentration. PLA(2), C, or D, with or without IL-1 had no effect, suggesting that the effect of SMase is specific (Table 1). Exogenous addition of phosphorylcholine had no effect. Taken together, these results suggest that ceramide or its metabolites enhance IL-1-induced E-selectin expression.

``Acidic Sphingomyelinase Pathway'' Inhibitors Do Not Attenuate IL-1-induced E-selectin Expression

There are at least two pools of ceramide within cells: plasma membrane ceramide generated by neutral SMase, and internal ceramide generated by acidic SMase(43) . Internal ceramide is reported to play a central role in activation of NF-kappaB in Jurkat and U-937 cells(43, 44) . Acidic SMase is induced by phosphatidylcholine (PC)-specific PLC following TNF stimulation. To elucidate possible involvement of the PC-specific PLC-acidic SMase pathway in E-selectin expression, we examined effects of putative inhibitors of this pathway. We first employed a rather specific inhibitor of PC-PLC, D609(44) . Pretreatment with D609 up to 25 µg/ml did not attenuate IL-1-induced E-selectin expression. Higher concentrations of D609 resulted in cell morphological changes. Other inhibitors of the pathway, monensin (up to 25 mM) and ammonium chloride (up to 5 µg/ml), did not inhibit E-selectin expression. These agents raise the pH in endolysosomal compartments and are expected to inhibit the sequential activation of endosomal acidic SMase, leaving the membrane-associated neutral SMase unaffected(43) . It seems unlikely, therefore, that the ``PC-PL C-acidic SMase pathway'' is involved in E-selectin expression.

C(2)-ceramide and Sphingomyelinase Increase the Level of E-selectin mRNA Induced by IL-1

We next studied effects of C(2)-cer and SMase on E-selectin gene transcription. E-selectin mRNA level is maximal at 4 h post-cytokine stimulation (2) . We determined E-selectin-specific mRNA levels 4 h post-induction by Northern blot analyses. HUVEC treated with IL-1 contained substantial amounts of E-selectin specific transcripts corresponding to 3.9 kilobases(2) , while unstimulated endothelial cells contained no detectable E-selectin mRNA (Fig. 2). C(2)-cer and SMase, but not Sph, increased the amount of E-selectin transcripts produced by IL-1. In Northern blot analyses of mRNA from HUVEC treated with sphingolipids or phospholipases in the absence of IL-1, no E-selectin transcripts were detected (data not shown). These results were roughly consistent with results from ELISA, and suggest that ceramide may regulate the steady-state level of E-selectin mRNA.


Figure 2: Induction of E-selectin gene transcription. HUVEC were incubated in medium alone (C), medium containing IL-1 (60 IU/ml), IL-1 with C(2)-cer (5 µM), IL-1 with Sph (5 µM), or IL-1 with SMase (100 milliunits/ml). Total RNA was isolated and Northern analyses were performed as described under ``Materials and Methods.'' Results shown in figure are representative of four similar experiments.



Activation of NF-kappaB by C(2)-ceramide Differs from That by IL-1 in Terms of Subunit Composition

E-selectin gene transcription is mediated in part by cytokine-induced activation of NF-kappaB and its subsequent binding to E-selectin promoter DNA(5) . We examined the effects of sphingolipids on NF-kappaB binding activity by EMSA. Treatment of HUVEC with IL-1 resulted in two NF-kappaB specific protein-DNA complexes (Fig. 3A, arrows). Specific NF-kappaB binding complexes were revealed by addition of a 100-fold molar excess of unlabeled (cold) NF-kappaB or AP-2 oligonucleotides in competition analyses (data not shown). Treatment with C(2)-cer induced formation of one NF-kappaB specific complex, which comigrated with upper complex as observed with nuclear extracts from IL-1-treated cells (Fig. 3A, lane 3). Sph also induced formation of one DNA-protein complex (Fig. 3A, lane 4). The binding activity was weaker than that induced by C(2)-cer. Whether Sph activates NF-kappaB directly or through its metabolites (e.g. ceramide) is now being investigated. Neither DMS nor Sph-1-P induced NF-kappaB specific binding activity. We performed EMSA with nuclear extracts treated with PLA(2), C, D, or SMase. Only SMase induced NF-kappaB specific binding activity (Fig. 3B).


Figure 3: Effects of IL-1, sphingolipids (A), and phospholipases (B) on NF-kappaB specific binding activity. Nuclear proteins were prepared as described under ``Materials and Methods'' from HUVEC treated with IL-1 (60 IU/ml), sphingolipids (5 µM), or phospholipases (100 milliunits/ml). Arrows labeled NF-kappaB denote specific inducible complexes competitive with double-stranded NF-kappaB oligonucleotide.



Because NF-kappaB specific complexes induced by C(2)-cer appeared quantitatively different from those induced by IL-1, we examined subunit composition of these complexes by supershift analyses. Anti-p50, anti-p65, or anti-c-Rel polyclonal antibody were added prior to addition of radiolabeled NF-kappaB probe. Preincubation of nuclear extracts from IL-1-treated HUVEC with anti-p50 antibody completely abolished lower complex formation, reduced upper complex abundance in a dose-dependent manner, and generated a further gel retardation (super-shift) (Fig. 4A, lanes 2 and 3). Eight µl of anti-p50 antibody failed to completely inhibit NF-kappaB specific upper complex formation (data not shown), suggesting that the complex may contain non-p50 dimer. Preincubation with anti-p65 antibody abolished upper complex formation and generated a super-shift, but had no effect on lower complex (Fig. 4A, lanes 4 and 5). The complexes were unaffected by anti-c-Rel antibody, suggesting that c-Rel is not a component (Fig. 4A, lanes 6 and 7). These results indicate that IL-1 activates p50/p65 heterodimer (upper complex) and p50 homodimer or p50/other subunit heterodimer (lower complex). Super-shift analyses of nuclear extracts from HUVEC treated with C(2)-cer revealed that subunit composition of the complex induced by C(2)-cer was identical to that of the upper one in IL-1-treated cells; it consisted predominantly of p50/p65 heterodimer (Fig. 4B). Higher concentration of C(2)-cer did not induce lower complex formation (data not shown). These results suggest that NF-kappaB activation by C(2)-cer may differ from that by IL-1 in terms of subunit composition.


Figure 4: Super-shift analyses of NF-kappaB specific activity induced by IL-1 (A) or C(2)-cer (B). Nuclear extracts were incubated with polyclonal antibodies against various subunits of NF-kappaB complexes prior to addition of labeled oligonucleotide probes as described under ``Material and Methods.'' Anti-p50, anti-p65, or anti-c-Rel antibodies were added at various concentrations (0, 2, and 4 µl/sample) as indicated.



C(2)-ceramide and Sphingomyelinase Enhance IL-1-induced NF-kappaB Activation, but Do Not Alter NF-ELAM1 Binding Activity

Because C(2)-cer and SMase enhanced IL-1-induced E-selectin expression, we also studied their effects on the cytokine-induced binding activities of NF-ELAM1 and NF-kappaB. NF-ELAM1 does not have enhancer activity on its own, but up-regulates NF-kappaB activation(7) . NF-ELAM1 may therefore be a control element in the mechanism by which cytokines induce E-selectin transcription(7) . EMSA of unstimulated cells with NF-ELAM1 probe revealed several shifted bands (Fig. 5A). Competition analyses with unlabeled NF-ELAM1 or AP-2 oligonucleotide showed that most of the shifted bands are specific for NF-ELAM1. Treatment of cells with C(2)-cer or SMase, alone or in combination with IL-1, had no effect on its constitutive binding activities of NF-ELAM1 (Fig. 5A). In contrast, IL-1-induced NF-kappaB specific binding activity was enhanced by C(2)-cer and SMase (Fig. 5B). It seems likely that C(2)-cer and SMase enhance E-selectin expression not through altered NF-ELAM1 binding activity, but in part through enhanced NF-kappaB activation.


Figure 5: EMSA of NF-ELAM1 specific (A) and NF-kappaB specific (B) binding activities induced by IL-1 and/or C(2)-cer or SMase. HUVEC were treated with C(2)-cer (5 µM) or SMase (100 milliunits/ml), alone or in combination with IL-1 (60 IU/ml). Nuclear extracts were prepared and EMSA were performed as described under ``Materials and Methods.'' Arrow denotes nonspecific bands.



IL-1 Stimulates Sphingomyelin Hydrolysis to Ceramide

IL-1 has been shown to induce hydrolysis of sphingomyelin to ceramide in human dermal fibroblasts (25) and murine thymoma EL4 cells(26) . These reports, along with our observation that exogenous C(2)-cer and SMase enhanced IL-1-induced E-selectin expression, prompted us to examine the effect of IL-1 on intracellular sphingomyelin and ceramide levels. HUVEC were labeled with [^3H]choline for 72 h to ensure equilibrium labeling of sphingomyelin, and stimulated with IL-1. Under these conditions, IL-1 induced time-dependent sphingomyelin hydrolysis (Fig. 6A). IL-1 induced a detectable reduction in sphingomyelin content within 15 min, from a baseline of 1488 ± 70 pmol to 1357 ± 35 pmol/10^6 cells (p < 0.05). After 30 min of IL-1 stimulation, the time for maximal sphingomyelin breakdown, sphingomyelin level was reduced to 82% of control value, from 1488 to 1229 ± 33 pmol/10^6 cells (p < 0.01), a decrease of 259 pmol/10^6 cells. Sphingomyelin returned to the basal level after 1 h. No significant changes in phosphatidylcholine labeling were detected over the same interval.


Figure 6: Effects of IL-1 on sphingomyelin hydrolysis to ceramide. HUVEC labeled with [^3H]choline (A) or unlabeled HUVEC (B) were treated with IL-1 (60 IU/ml). Then sphingomyelin (A) and ceramide (B) levels at the indicated time points were determined as described under ``Materials and Methods.'' The results represent mean values ± S.D. from five independent experiments.



Conversely, IL-1 induced a statistically significant increase in ceramide content (Fig. 6B). Ceramide level increased from 268 ± 16 to 326 ± 22 pmol/10^6 cells (p < 0.05) in 15 min. After 30 min, ceramide level increased maximally to 183% of control value, i.e. to 492 ± 63 pmol, an increase of 224 pmol/10^6 cells (p < 0.01). The mass of ceramide generated by 30 min following IL-1 treatment was roughly consistent with the mass of hydrolyzed sphingomyelin. Given these results, we postulate that IL-1 activates a SMase, and that activation of this pathway is a significant signal transduction event mediating at least some of the actions of IL-1 in vivo.


DISCUSSION

We studied the effects of sphingolipids on E-selectin expression in order to elucidate the molecular mechanisms of this phenomenon. None of the sphingolipids tested induced E-selectin expression by themselves, but C(2)-cer enhanced IL-1-induced E-selectin expression (Fig. 1A, Table 1). Structurally related compounds including Sph, Sph-1-P, and DMS had no effect. SMase, which hydrolyzes sphingomyelin and generates ceramide, mimicked the effect of C(2)-cer; it did not induce E-selectin expression in the absence of IL-1, but enhanced IL-1-induced expression. Because SMase treatment (200 milliunits/ml) generated an amount of ceramide nearly three times greater than that from IL-1 treatment, it seems unlikely that failure of E-selectin induction by C(2)-cer and SMase is due to dosage problems. In contrast, PLA(2), C, and D did not enhance IL-1-induced expression. Hence, enhancement of IL-1-induced E-selectin expression appears specific for ceramide.

Recent studies have indicated that hydrolysis of sphingomyelin to ceramide, termed the ``sphingomyelin cycle'' (19) or ``sphingomyelin pathway''(20) , mediates many biological effects of TNF and IL-1. Involvement of sphingomyelin hydrolysis in cellular processes has been described in hematopoietic cells such as HL-60 and U-937 cells(19, 20) . Treatment of cells with cell-permeable ceramide analogues or SMase induces cell proliferation, differentiation, and apoptosis. Extensive studies have revealed that cytokines may utilize this pathway for signal transduction in other cells(19, 20) . It has been proposed that TNF and IL-1 activate SMase to generate ceramide, which in turn activates NF-kappaB and transduces signals to the nucleus(19, 20) . Our present results suggest that C(2)-cer and SMase induce NF-kappaB specific binding activity, which is known to be essential for E-selectin expression in HUVEC(5) . This is consistent with previous findings in HL-60 leukemia cells(45) , Jurkat cells(44) , and U-937 cells(46) . We now show that, in spite of their ability to activate NF-kappaB, neither C(2)-cer nor SMase induce E-selectin expression. Therefore, NF-kappaB activation by ceramide may not trigger E-selectin expression.

Although it has been reported that NF-kappaB activation is not sufficient for E-selectin expression(4, 5) , failure of E-selectin induction by ceramide raises the possibility that signaling pathways leading to NF-kappaB utilized by ceramide differ from those utilized by cytokines. To explore this possibility, we examined subunit composition of NF-kappaB by super-shift analysis. NF-kappaB activation by C(2)-cer is not identical to that by IL-1 in terms of subunit composition; C(2)-cer activates p50/p65 heterodimer, whereas IL-1 can activate p50 homodimer or p50/other subunit heterodimer as well as p50/p65 (Fig. 4). Furthermore, NF-kappaB binding activity induced by IL-1 is greater than that induced by C(2)-cer at 5 µM. Higher concentrations of C(2)-cer did not induce stronger NF-kappaB binding activity nor lower complex formation (data not shown). These results are important because they suggest that the signal pathway leading to NF-kappaB activation by IL-1 is different from that by ceramide. Alternatively, additional pathway(s) may be necessary for IL-1-induced activation. For example, it has been reported that in SW 480 cells, inhibition of ceramide pathway has no effect on the ability of TNF to activate NF-kappaB(47) . In U-937 cells, NF-kappaB activation by TNF was shown to be tyrosine kinase-dependent, while that by ceramide was tyrosine kinase-independent(48) . Furthermore, interferon- causes ceramide production, but has no effect on NF-kappaB activation(49) . Collectively, these findings suggest that ceramide may not mediate all the signals necessary for NF-kappaB activation by cytokines.

It has been shown that plasma membrane ceramide generated by neutral SMase activates mitogen-activated protein kinases, while internal ceramide generated by acidic SMase activates NF-kappaB translocation (43) . Acidic SMase is activated by phosphatidylcholine-specific PLC following TNF stimulation in Jurkat and U-937 cells, resulting in NF-kappaB activation(44) . Exogenous addition of SMase and ceramide may preferentially activate neutral, but not acidic SMase, explaining the poor NF-kappaB translocation(44) . To rule out the possibility that failure of E-selectin induction by ceramide is due to poor accessibility in intact cells, HUVEC were permeabilized and treated with C(2)-cer and SMase. Under these conditions, both agents failed to induce E-selectin expression, suggesting that internal ceramide is not sufficient for this purpose. We also assessed involvement of the acidic SMase pathway in IL-1-induced E-selectin expression using inhibitors of the pathway. Neither D609, ammonium chloride, nor monensin inhibited the expression. At this point, it seems unlikely that internal ceramide generated by PC-PLC specific acidic SMase plays a central role in IL-1-induced E-selectin expression in HUVEC.

In primary cultured cells such as HUVEC, both free-radical dependent oxidation and protein phosphorylation are required for release of NF-kappaB from IkappaB-alpha(50) . The effect of ceramide on NF-kappaB might therefore include stimulation of protein kinases or production of oxygen radicals. A number of potential targets for ceramide have been identified, including a novel membrane-associated protein kinase termed ceramide-activated protein kinase(51) , protein phosphatase(52, 53) , and mitogen-activated protein kinase(54) . None of these has been shown to be directly involved in NF-kappaB activation. Machleidt et al.(46) showed that serine/threonine protease inhibitors abolished degradation of IkappaB-alpha by ceramide, suggesting involvement of serine-like protease in ceramide-mediated NF-kappaB activation. It was reported recently that protein kinase C- may be an immediate target of ceramide and induce translocation of NF-kappaB to the nucleus in U-937 cells(55) . Involvement of Raf was also suggested(56) , but obviously needs to be investigated further.

The mechanism by which C(2)-cer and SMase enhance IL-1-induced E-selectin expression remains to be elucidated. We examined their effects on NF-kappaB and NF-ELAM1 binding activities, which are known to cooperatively mediate cytokine-induced E-selectin expression (Fig. 5). EMSA revealed that C(2)-cer and SMase enhance NF-kappaB activation by IL-1. Similarly, C(2)-cer was reported to potentiate activation of NF-kappaB in response to TNF in HL-60 cells(57) . Positive feedback regulation of NF-kappaB activation may explain, at least in part, the enhancement of E-selectin expression by ceramide. Treatment with C(2)-cer or SMase had no effect on NF-ELAM1-specific binding activity. It seems unlikely that ceramide enhances IL-1-induced E-selectin expression by altering NF-ELAM1 binding activity. It is quite possible that ceramide acts not by altering binding of proteins, but by phosphorylating the proteins already bound to NF-ELAM1 element. Ceramide is reported to activate ceramide-activated protein kinase(51) , mitogen-activated protein kinases(58) , and stress-activated protein kinases(59) . Activation of such pathways may explain the augmentation of E-selectin expression by ceramide. Further studies to elucidate the mechanism are obviously needed.

We have demonstrated that IL-1 stimulates sphingomyelin hydrolysis to ceramide in HUVEC (Fig. 6). Although this is not direct evidence that ceramide is involved in the signaling pathways initiated by IL-1, the close relationships between IL-1, ceramide, and E-selectin expression suggest possible involvement of ceramide in IL-1 signal transduction. The time course is consistent with a proposed role for ceramide in regulating the effects of IL-1 which lead to E-selectin expression. Significant hydrolysis of sphingomyelin was observed by 15 min, accompanied by increased levels of intracellular ceramide. Maximal effect occurred within 30 min. The time course of sphingomyelin response to IL-1 was similar to that seen in HL-60 cells in response to TNF(60) . The increase of ceramide level was slightly delayed compared with that in EL-4 cells, in which sphingomyelin hydrolysis was observed as early as 30 s post-IL-1 treatment(26) . In human fibroblasts, IL-1-induced sphingomyelin hydrolysis occurs by 2 h, and is accompanied by increased levels of ceramide between 2 and 4 h. Ceramide levels remained elevated for at least 24 h. These differences in time course may reflect biological roles of ceramide; if it mediates very early responses, prompt activation of the pathway would be necessary. Prolonged duration of sphingomyelin turnover may be better suited for mediating long-term cellular responses such as differentiation and proliferation. In HUVEC, NF-kappaB activation was not observed until around 15 min and maximal transcriptional activity occurred around 1 h following cytokine treatment(5, 6) . The delayed increase of ceramide level and prompt return to basal level may be appropriate for modulation of E-selectin expression through NF-kappaB.

In summary, we have shown that sphingomyelin hydrolysis and the concomitant generation of ceramide may not trigger, but rather regulate IL-1-induced E-selectin expression in HUVEC. Ceramide may exhibit these effects at least in part through enhancing NF-kappaB activation by IL-1. To our knowledge, this is the first demonstration of IL-1-induced sphingomyelin hydrolysis to ceramide and its involvement in adhesion molecule expression in HUVEC. E-selectin expression is a key event of many pathological states including inflammation. Elucidation of the factors involved in the regulation of its expression can be expected to provide better understanding and rational approaches for control of inflammatory processes.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Outstanding Investigator Grant CA-42505 (to S. H.) and by funds from the Biomembrane Institute, in part under research contracts with Otsuka Pharmaceutical Co. and Seikagaku Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Visiting scientist from the Third Department of Internal Medicine, Tohoku University School of Medicine, Sendai 980-77 Japan.

To whom correspondence should be addressed: The Biomembrane Institute and University of Washington, 201 Elliott Ave., West #305, Seattle, WA 98119-4237. Tel.: 206-285-4209; Fax: 206-281-9893.

(^1)
The abbreviations used are: IL-1, interleukin-1beta; C(2)-cer, C(2)-ceramide; DMS, dimethylsphingosine; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; HUVEC, human umbilical vein endothelial cell(s); NF-kappaB, nuclear factor kappaB; PC, phosphatidylcholine; PL, phospholipase(s); PMSF, phenylmethylsulfonyl fluoride; SMase, sphingomyelinase; Sph, sphingosine; Sph-1-P, sphingosine 1-phosphate; TNF, tumor necrosis factor-alpha.


ACKNOWLEDGEMENTS

We thank Stephen Anderson, Ph.D., for scientific editing of the manuscript, Jumi Sakurai for figure preparation, and Yasue Shuto for encouragement throughout the study.


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