(Received for publication, January 24, 1996)
From the
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-ceramide, sphingosine 1-phosphate, and N,N-dimethylsphingosine) failed to induce E-selectin
expression by themselves. C
-ceramide at 5 µM enhanced interleukin-1
(IL-1
)-induced E-selectin
expression 2.7-fold, whereas other sphingolipids tested had no effects
on this process. Sphingomyelinase, but not phospholipases
A
, C, or D, mimicked the enhancing effect of
C
-ceramide. Northern blot analyses revealed that
C
-ceramide and sphingomyelinase increased
interleukin-1
-induced E-selectin gene transcription levels.
C
-ceramide and sphingomyelinase induced NF-
B
activation by themselves and enhanced activation by IL-1
, which is
essential for E-selectin expression. Immunological analyses with
anti-NF-
B antibodies showed that subunit composition of NF-
B
activated by IL-1
differs from that activated by
C
-ceramide, suggesting that signaling pathways utilized by
these stimuli may be different. Treatment with C
-ceramide
or sphingomyelinase did not alter NF-ELAM1 specific binding activity.
IL-1
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-
B; (ii) sphingomyelin
hydrolysis to ceramide does not mediate all the effects of IL-1
,
although it may play important roles in IL-1
signal transduction
in human umbilical vein endothelial cells.
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-1 (IL-1), (
)tumor necrosis factor-
(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
B (NF-
B)(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-B is a member of the Rel family of transcriptional
regulatory proteins; the family includes p50 (NF-
B1), p52
(NF-
B2), RelA (p65), c-Rel, RelB, and the Drosophila morphogen dorsal gene product(8, 9) .
Phosphorylation and degradation of the inhibitory protein I
B, and
subsequent dissociation of this protein from NF-
B are thought to
be necessary for activation of
NF-
B(10, 11, 12) . NF-
B is known to
be involved in control of cytokine-induced expression of many immune
and inflammatory-response genes(13) . In particular, NF-
B
and I
B 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-
B
complexes containing other Rel family members are able to influence
gene transcription. The level of activation, and the type of
NF-
B/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 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-ceramide
(C
-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.
Figure 1:
Effects of C-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
-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-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
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
, 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.
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-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.
Figure 3:
Effects of IL-1, sphingolipids (A), and phospholipases (B) on NF-B 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-
B denote specific
inducible complexes competitive with double-stranded NF-
B
oligonucleotide.
Because NF-B
specific complexes induced by C
-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-
B 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-
B 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
-cer revealed that subunit composition of the complex
induced by C
-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
-cer
did not induce lower complex formation (data not shown). These results
suggest that NF-
B activation by C
-cer may differ from
that by IL-1 in terms of subunit composition.
Figure 4:
Super-shift analyses of NF-B specific
activity induced by IL-1 (A) or C
-cer (B). Nuclear extracts were incubated with polyclonal
antibodies against various subunits of NF-
B 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.
Figure 5:
EMSA of NF-ELAM1 specific (A) and
NF-B specific (B) binding activities induced by IL-1
and/or C
-cer or SMase. HUVEC were treated with
C
-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.
Figure 6:
Effects of IL-1 on sphingomyelin
hydrolysis to ceramide. HUVEC labeled with
[H]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 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
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.
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-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
-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
-cer and SMase is due to dosage problems. In contrast,
PLA
, 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-B
and transduces signals to the nucleus(19, 20) . Our
present results suggest that C
-cer and SMase induce
NF-
B 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-
B, neither C
-cer
nor SMase induce E-selectin expression. Therefore, NF-
B activation
by ceramide may not trigger E-selectin expression.
Although it has
been reported that NF-B 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-
B utilized by ceramide differ from those utilized by
cytokines. To explore this possibility, we examined subunit composition
of NF-
B by super-shift analysis. NF-
B activation by
C
-cer is not identical to that by IL-1 in terms of subunit
composition; C
-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-
B binding
activity induced by IL-1 is greater than that induced by
C
-cer at 5 µM. Higher concentrations of
C
-cer did not induce stronger NF-
B binding activity
nor lower complex formation (data not shown). These results are
important because they suggest that the signal pathway leading to
NF-
B 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-
B(47) . In U-937 cells, NF-
B 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-
B activation(49) . Collectively, these findings suggest
that ceramide may not mediate all the signals necessary for NF-
B
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-B translocation (43) . Acidic SMase is activated by
phosphatidylcholine-specific PLC following TNF stimulation in Jurkat
and U-937 cells, resulting in NF-
B activation(44) .
Exogenous addition of SMase and ceramide may preferentially activate
neutral, but not acidic SMase, explaining the poor NF-
B
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
-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-B from
I
B-
(50) . The effect of ceramide on NF-
B 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-
B activation. Machleidt et al.(46) showed that serine/threonine protease inhibitors
abolished degradation of I
B-
by ceramide, suggesting
involvement of serine-like protease in ceramide-mediated NF-
B
activation. It was reported recently that protein kinase C-
may be
an immediate target of ceramide and induce translocation of NF-
B
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-cer and SMase enhance
IL-1-induced E-selectin expression remains to be elucidated. We
examined their effects on NF-
B and NF-ELAM1 binding activities,
which are known to cooperatively mediate cytokine-induced E-selectin
expression (Fig. 5). EMSA revealed that C
-cer and
SMase enhance NF-
B activation by IL-1. Similarly,
C
-cer was reported to potentiate activation of NF-
B in
response to TNF in HL-60 cells(57) . Positive feedback
regulation of NF-
B activation may explain, at least in part, the
enhancement of E-selectin expression by ceramide. Treatment with
C
-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-B 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-
B.
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-B 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.