Departments of 1 Surgery and 2 Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130
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ABSTRACT |
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The objective of
this study was to assess the effects of two structurally distinct yet
selective proteasome inhibitors (PS-341 and lactacystin) on leukocyte
adhesion, endothelial cell adhesion molecule (ECAM) expression, and
nuclear factor-B (NF-
B) activation in tumor necrosis
factor (TNF)-
-stimulated human umbilical vein endothelial cells
(HUVEC) and the transformed, HUVEC-derived, ECV cell line. We found
that TNF (10 ng/ml) significantly enhanced U-937 and polymorphonuclear
neutrophil (PMN) adhesion to HUVEC but not to ECV; TNF also
significantly enhanced surface expression of vascular cell adhesion
molecule 1 and E-selectin (in HUVEC only), as well as intercellular
adhesion molecule 1 (ICAM-1; in HUVEC and ECV). Pretreatment of HUVEC
with lactacystin completely blocked TNF-stimulated PMN adhesion,
partially blocked U-937 adhesion, and completely blocked TNF-stimulated
ECAM expression. Lactacystin attenuated TNF-stimulated ICAM-1
expression in ECV. Pretreatment of HUVEC with PS-341 partially blocked
TNF-stimulated leukocyte adhesion and ECAM expression. These effects of
lactacystin and PS-341 were associated with inhibitory effects on
TNF-stimulated NF-
B activation in both HUVEC and ECV. Our results
demonstrate the importance of the 26S proteasome in TNF-induced
activation of NF-
B, ECAM expression, and leukocyte-endothelial
adhesive interactions in vitro.
inflammatory mediators; nuclear factor-B; adhesion molecules; leukocyte adhesion; U-937 cell line; neutrophils; human umbilical vein
endothelial cells; ECV cell line
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INTRODUCTION |
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A FUNDAMENTAL AND EARLY event in inflammation is
adhesion of leukocytes to the endothelium. This is mediated by binding
of leukocytes to endothelial cell adhesion molecules (ECAMs).
Inflammatory mediators such as tumor necrosis factor (TNF)-
stimulate transcription and subsequent cell surface expression of ECAMs
such as vascular cell adhesion molecule 1 (VCAM-1), intercellular
adhesion molecule 1 (ICAM-1), and E-selectin (20, 30). It has been
demonstrated that expression of ECAMs in response to TNF is controlled
by the activity of the transcription factor nuclear factor-
B
(NF-
B) (21).
Under basal conditions, NF-B is sequestered in an inactive form in
the cytoplasm by an inhibitory binding protein, I
B
. When
stimulated by inflammatory stress, I
B
undergoes postranslational modification (e.g., phosphorylation, polyubiquitination) that leads to
its degradation and dissociation from NF-
B (4, 19). The released
NF-
B is then translocated to the nucleus, where it activates
transcription of genes having NF-
B binding sites in their promoters.
Proteolytic degradation of IB
is thought to be mediated by the
26S proteasome complex and is required for activation of NF-
B (19).
Recently, it was shown that, in cells exposed to TNF-
, pretreatment
with peptide aldehydes, which inhibit chymotryptic activities of the
proteasome and possibly other proteases, prevents both activation of
NF-
B (19, 21) and increased ECAM expression and neutrophil adhesion
in endothelial cells (21). Thus it has been proposed that the latter
processes are mediated by proteasome activity. However, direct evidence
supporting this hypothesis has not yet been presented.
Although the peptide aldehydes are potent and relatively specific
proteasome inhibitors (19, 22), their Michaelis-Menten constants for
proteasome inhibition (5-12 nM) are similar to those for other
proteases, such as cathepsin B and calpain (10 nM) (19). Other,
nonproteasome inhibitors of cathepsins and calpain have no effect on
either NF-B precursor processing or NF-
B activation (19).
However, it is possible that these other proteases (especially calpain)
might be involved in events required for ECAM surface mobilization and
leukocyte adhesion that are downstream from, or parallel to,
NF-
B-dependent ECAM gene expression. For example, known calpain
substrates include cytoskeletal proteins (actin-binding and
microtubule-associated proteins), adhesion molecules, and the Fos and
Jun components of transcription factor activating protein-1 (AP-1)
(25). Thus, in interpreting the results of studies using the peptide
aldehyde proteasome inhibitors, it is important to distinguish between
the effects of these compounds on proteasome activity and their
possible effects on other aspects of cell function that are likely also
important in ECAM surface expression and leukocyte adhesion. It remains
necessary to examine the specific role of the proteasome in mediating
the effect of TNF on ECAM expression and leukocyte adhesion.
Another, recently described, class of proteasome inhibitors is boronic acid peptides (1). These inhibitors are thought to form a more stable bond at the active site of the proteasome than the peptide aldehydes and are more potent and selective for proteasome inhibition (1). Moreover, whereas peptide aldehydes cannot be used in vivo due to their acute toxicity, peptide boronates have been used in animals (6). Use of peptide boronates in studies of ECAM expression and leukocyte adhesion have not been reported.
The most specific proteasome inhibitor described to date is
lactacystin, a Streptomyces metabolite
that is structurally distinct (10) from the peptide aldehyde and
peptide boronate proteasome inhibitors. Lactacystin is converted to a
-lactone intermediate (the active inhibitor), which binds
irreversibly to the subunit X of the proteasome and acylates the active
site NH2-terminal threonine (1,
8). It has been demonstrated using radiolabeled lactacystin that this
inhibitor binds only to the proteasome complex in cell extracts (8, 9).
Finally, it was shown that lactacystin selectively inhibits proteasome
activity, with no detectable effect on the activity of any other
protease, including cathepsin B and calpain (9). To date, lactacystin
remains the most specific proteasome inhibitor known (1, 9). Thus
lactacystin should be a useful tool in determining, unambiguously, the
role of the proteasome in mediating the effects of proinflammatory
stimuli such as TNF on ECAM expression and leukocyte-endothelial cell adhesion.
The objective of this study was to compare the effects of two
structurally unrelated yet selective proteasome inhibitors, lactacystin
and the tripeptide boronate PS-341, on TNF--stimulated NF-
B activation, surface expression of ECAMs (VCAM-1, ICAM-1, and
E-selectin), and endothelial adhesion of monocytes and
neutrophils. We examined these parameters in endothelial cells derived
from primary culture and in a transformed cell line (ECV) derived from human umbilical vein endothelial cells (HUVEC). Our results demonstrate the importance of the proteasome in mediating TNF-stimulated NF-
B activation, ECAM expression, and leukocyte adhesion.
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MATERIALS AND METHODS |
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Reagents
Antibodies to VCAM-1 (1.G11B1), ICAM-1 (15.2), and E-selectin (1.2B6) were purchased from Southern Biotechnology Associates (Birmingham, AL). Antibody to ICell Culture
HUVEC were isolated from human umbilical cords using a modification of the procedures described by Jaffe et al. (13). These primary cultures were seeded in T-25 flasks in endothelial growth medium (EGM) plus bovine pituitary brain extract (Clonetics, San Diego, CA). For experiments, they were trypsinized and passaged to either 24-well plates (monocyte adhesion and ECAM surface expression measurements) or 100-mm dishes (analysis of NF-Isolation of Neutrophils
Human neutrophilic polymorphonuclear leukocytes were isolated from venous blood of healthy adults using standard dextran sedimentation and gradient separation on Histopaque 1077 (Sigma) (12, 31). This procedure yields a polymorphonuclear leukocyte population that is 95-98% viable (by trypan blue exclusion) and 98% pure (by acetic acid-crystal violet staining).Monocyte and Neutrophil Adhesion Assay
U-937 cells and freshly isolated neutrophils were washed twice with labeling medium (RPMI 1640 plus 1% FBS) and then incubated for 1 h (37°C; 5% CO2) with 51CrO4 (sodium salt; DuPont NEN, Boston, MA; 3-5 µCi/5 × 107 cells; 2-ml incubation volume). Labeled leukocytes were washed four times with labeling medium and then resuspended in fresh labeling medium at 2 × 107 cells/ml. After control or experimental treatments, endothelial cell monolayers in 24-well plates were washed twice with labeling medium, and then 450 µl of labeling medium were added to each well. For static adhesion assays, 50 µl of labeled monocyte or neutrophil suspension (1 × 106 cells) were added to each well of endothelial cells (2:1 ratio of leukocytes to endothelial cells), and the plates were gently agitated and placed in a cell culture incubator for 30 min. At the end of the incubation period, the medium from each well was aspirated and saved for radioactive counting. The monolayer was gently washed three times with cold PBS; collected washes were combined with medium and counted, yielding a measure of nonadherent leukocytes. Preliminary experiments indicated that three washes of the monolayer decreased control or basal adhesion by 50-100% (compared with a single wash), while effecting a minimal decrease (<2%) in TNF-stimulated adhesion. After the final wash, monolayers were lysed for 1 h with 1 M NaOH; counting of the lysate [in counts/min (cpm)] yielded a measure of adherent leukocytes. Adhesion was expressed as the percent of total collected radioactivity present in the lysate: %adhesion = [cpm in lysate / (cpm in lysate + cpm in supernatant and washes)] × 100.Effect of TNF on Leukocyte Adhesion to Endothelial Monolayers
Stock solutions of PS-341 and lactacystin were prepared in DMSO immediately before use. TNF (human recombinant; Calbiochem, La Jolla, CA) was dissolved in Hanks' buffered saline solution (HBSS) with 0.5% BSA and stored in small aliquots atEffect of TNF on Endothelial Expression of ECAMs
Surface expression of VCAM-1, E-selectin, and ICAM-1 was assayed using the method of Khan et al. (15). Endothelial cells were grown in 24-well culture plates. After exposure to TNF with or without pretreatment with PS-341 or lactacystin, wells were gently washed once with PBS and then incubated with antibodies to VCAM-1, E-selectin, or ICAM-1 (1.G11B1, 1.2B6, or 15.2, respectively; Southern Biotechnology Associates) diluted 1:400 in PBS plus 5% FCS at 37°C for 30 min. Wells were washed twice with PBS and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates) in PBS plus 5% FCS at 37°C for 30 min. Wells were washed four times with PBS and then incubated with 0.003% hydrogen peroxide plus 0.1 mg/ml 3,3',5,5'-tetramethylbenzidine (Sigma) for 30 min in the dark. The color reaction was stopped by addition of 75 µl of 8 N H2SO4, and the samples were transferred to 96-well plates. Plates were read on a microplate reader at 450 nm, blanking on wells stained with only secondary antibody. For a given experiment, each treatment was performed in triplicate.Activation of NF-B
Isolation of nuclear extracts. Methods used were a modification of Schreiber et al. (26). HUVEC and ECV were incubated for 4 h with either vehicle or TNF (10 ng/ml), after pretreatment for 1 h with or without PS-341 (0.01, 0.1, 1.0, or 10 µM) or lactacystin (0.01, 0.1, 1.0, 10, 20, or 50 µM). Incubation medium was aspirated, and the cells were scraped from the plate in PBS. Cells were pelleted (1,500 rpm, 5 min) at 4°C, the PBS was decanted, and the pellets were resuspended in 0.4 ml of buffer A [in mM: 10 HEPES (pH 7.9), 10 KCl, 0.1 EDTA, 0.1 EGTA, 1 dithiothreitol (DTT), and 0.5 phenylmethylsulfonyl fluoride (PMSF), with 1 µg/ml leupeptin and 1 µg/ml aprotinin] by gentle pipetting through a large-bore pipette tip. The cell suspension was allowed to swell on ice for 15 min, after which 25 µl of 10% NP-40 were added and the suspension was vortexed for 10 s. Homogenates were centrifuged at 10,000 g for 30 s at 25°C; the nuclear pellet was washed once with and resuspended in 50 µl of buffer C [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin]. The resuspended nuclear fraction was sonicated on ice and then centrifuged at 12,000 g for 1 min at 4°C. The supernatant from this spin (nuclear extract) and the cytosolic extract were assayed for protein concentration using the Bio-Rad DC protein assay (Hercules, CA) and then immediately used for gel shift assay.
Electrophoretic mobility shift assay.
NF-B consensus oligonucleotide 5'-AGTTGAGGGGACTTTCCCAGGC
(Promega, Madison, WI) was end labeled with
[32P]ATP using T4
polynucleotide kinase (Promega gel shift assay system) according to
manufacturer's instructions. Nuclear extracts (20 µg protein) were
preincubated for 10 min at 25°C in a solution containing (in mM) 1 MgCl2, 2.5 EDTA, 2.5 DTT, 250 NaCl, and 50 Tris · HCl (pH 7.5), with 0.25 µg/µl
poly(dI-dC) in 20% glycerol. Labeled oligonucleotide (0.07 pmol) was
then added (total assay volume 20 µl), and binding reactions were
incubated for 30 min at 25°C. Reactions were stopped by addition of
4 µl of 10× gel loading buffer [250 mM
Tris · HCl (pH 7.4), 0.2% bromphenol blue, 0.2%
xylene cyanol, and 40% glycerol]. Specificity of binding was
verified by including a 100-fold molar excess of unlabeled oligonucleotide in some reactions. Reaction mixtures were applied to a
nondenaturing, 4% polyacrylamide gel and electrophoresed at 125 V
(constant voltage) for 3 h. Gels were fixed in 10% acetic acid-40%
methanol for 15 min, washed in distilled water, dried, and then exposed
to X-ray film (Kodak X-OMAT) for 4-16 h at
70°C. Activation of NF-
B (relative to non-TNF-treated control) was determined by performing densitometric analysis (ImageQuant software, Molecular Dynamics, Sunnyvale, CA) on shifted bands from scanned autoradiographs.
Cytosolic IB
in TNF-Stimulated
HUVEC
Statistical Analysis
Data were analyzed by one-way ANOVA with multiple comparisons, using Bonferroni's method (18). Means were considered significant when P < 0.05. ![]() |
RESULTS |
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Leukocyte Adhesion to Endothelial Cells
We examined the effects of preincubation of HUVEC monolayers with PS-341 or lactacystin on both basal and TNF-stimulated adhesion of U-937 cells and neutrophils (Fig. 1). Neither PS-341 nor lactacystin (incubated with HUVEC for 5 h) affected basal adhesion of monocytes and neutrophils to HUVEC. Exposure to TNF (10 ng/ml) for 4 h stimulated U-937 and neutrophil adhesion to HUVEC by ~10-fold and 3- to 10-fold, respectively. Preincubation of HUVEC with either PS-341 or lactacystin for 1 h before exposure to TNF significantly attenuated leukocyte adhesion. Lactacystin completely blocked TNF-stimulated neutrophil adhesion and decreased TNF-stimulated U-937 adhesion by 62% (Fig. 1). PS-341 (10 µM) decreased TNF-stimulated neutrophil and U-937 adhesion to HUVEC by 67 and 49%, respectively. A higher dose of PS-341 (50 µM) was also tested; this dose had no further effect on either monocyte or neutrophil adhesion to HUVEC compared with 10 µM (data not shown).
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We were unable to demonstrate increased adhesion of either U-937 cells or neutrophils to ECV monolayers in response to TNF at doses up to 20 ng/ml (data not shown).
Effect of TNF on Endothelial Expression of ECAMs
We next examined the effects of the two proteasome inhibitors on basal and TNF-stimulated ECAM surface expression in HUVEC. TNF enhanced surface expression of VCAM-1, ICAM-1, and E-selectin (Fig. 2). Lactacystin had no effect on basal ECAM expression but completely blocked the increases in ECAM surface expression produced by TNF. Although PS-341 significantly attenuated surface expression of all ECAMs examined (VCAM-1, 69% decrease; ICAM-1, 48% decrease; E-selectin, 41% decrease), all three ECAMs remained significantly elevated compared with basal expression. Higher doses of PS-341 (50 or 100 µM) had no further inhibitory effect on TNF-stimulated ECAM expression (data not shown).
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We then examined the effects of TNF with or without lactacystin on ECAM
expression in the ECV cells. Basal expression of VCAM-1 and E-selectin
could not be demonstrated, and TNF did not significantly stimulate
surface expression of these ECAMs after either 4 or 16 h of exposure to
TNF (data not shown). Basal expression of ICAM-1 was comparable to or
slightly higher than that in HUVEC (Fig.
3). Incubation with TNF for 4 h produced a
significant, 45% increase in surface ICAM-1 expression (considerably
lower than the 300-700% increase produced in HUVEC; Fig. 2).
Preincubation with lactacystin (50 µM) attenuated the TNF-elicited
increase in ICAM-1 surface expression by 60%. Similar increases in
ICAM-1 expression were observed after 16-h incubation of ECV with TNF, but lactacystin did not inhibit this increase.
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Activation of NF-B
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Degradation of Cytosolic IB
in HUVEC
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DISCUSSION |
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The recent availability of specific proteasome inhibitors now allows
unequivocal examination of the role of proteasome activity in mediating
diverse cellular functions. In the present work, we examined the effect
of two such inhibitors, the peptide boronate PS-341 and the
structurally distinct bacterial metabolite lactacystin, on several
measures of inflammatory activation of endothelial cells. Both
compounds significantly attenuated TNF-stimulated leukocyte adhesion
and ECAM surface expression in HUVEC. These effects were correlated
with inhibition of NF-B activation as measured by electrophoretic
mobility shift assay. Our results unequivocally support the hypothesis
that proteasome-dependent processes, including activation of NF-
B,
are necessary for TNF-elicited increases in both ECAM expression and
leukocyte adhesion in endothelial cells.
In the experiments examining leukocyte adhesion to HUVEC, our results clearly demonstrate that lactacystin is more effective at blocking TNF-elicited adhesion of both monocytes and neutrophils. The relatively incomplete blockade of both TNF-stimulated monocyte and neutrophil adhesion in HUVEC by PS-341 (49 and 67% inhibition, respectively) compared with lactacystin (62 and 100% inhibition, respectively) was directly correlated to similar differences in effect on TNF-stimulated ECAM surface expression between these two inhibitors: whereas lactacystin virtually completely prevented TNF-elicited upregulation of VCAM-1 (92% inhibition), ICAM-1 (84% inhibition), and E-selectin (97% inhibition), the inhibitory effect of PS-341 on TNF-elicited stimulation of these ECAMs was less marked (VCAM-1, 69% inhibition; ICAM-1, 48% inhibition; E-selectin, 41% inhibition). Because VCAM-1, ICAM-1, and E-selectin could play a role in mediating monocyte adhesion to endothelial cells and because ICAM-1 and E-selectin are probably involved in adhesion of neutrophils, it is reasonable to conclude that differences between lactacystin and PS-341 in their effects on ECAM surface expression probably account for the relative effects of the two inhibitors on leukocyte adhesion.
Although lactacystin completely prevented the upregulation of surface
expression of ICAM-1, VCAM-1, and E-selectin and polymorphonuclear neutrophil adhesion in HUVEC, it did not completely block the increase
in monocyte adhesion (62% inhibition). A higher dose of lactacystin
(50 µM) had no further effect; these results suggest the existence of
TNF-stimulated adhesive determinants, other than those assayed in the
present study, that mediate a component of TNF-stimulated U-937
adhesion to endothelial cells and are not influenced by proteasome
inhibition. Other molecular determinants for monocyte-endothelial
adhesion have recently been described (3, 16); however, it is unknown
whether cytokine-elicited stimulation of their surface expression
depends on NF-B activation or proteasome activity.
Immunoblot analysis of cytosolic IB
supports the role of the
proteasome in activating NF-
B in HUVEC. TNF elicited a decrease in
cytosolic levels of I
B
; this was blocked by preincubation with
both lactacystin and PS-341; importantly, inhibition of degradation of
I
B
was associated with a relative accumulation of phosphorylated forms of this protein similar to those reported previously (4, 19, 21),
indicating that neither lactacystin nor PS-341 affects these earlier
steps in I
B
processing. The other major finding was the
appearance of smaller molecular weight immunoreactive bands in extracts
from HUVEC that had been pretreated with proteasome inhibitor for 1 h
and then treated with TNF. These bands were not detectable in control
or TNF-treated HUVEC without proteasome inhibitor
pretreatment. These observations suggest that prolonged inhibition of the proteasome has no effect on eventual degradation of
excess accumulated I
B
by nonproteasomal cellular proteases (11),
which under conditions of normal proteasome function would not
encounter a sufficient level of accumulated I
B
substrate to
produce detectable smaller molecular weight proteolytic products. Examples of proteasome-independent NF-
B activation have been reported previously (2, 24); our findings in HUVEC are consistent with
such activity, and this latter finding could explain the apparent
discrepancy between the effects of proteasome inhibitors on ECAM
expression and apparent NF-
B activation (see below).
Our findings confirm those of previous workers (20) that only partial
inhibition of NF-B activation (as assessed by gel shift assay)
produces a greater inhibition of ECAM expression. The
reasons for this finding are not clear but have been previously considered (20). One possibility may involve other aspects of cytokine-stimulated activation of NF-
B besides nuclear translocation and DNA binding, such as phosphorylation of p65 (17). Alternatively, the apparent discrepancy between the level of NF-
B activation and
the extent of ECAM expression may reflect amplification in the effects
of relatively small changes in the levels of active, nuclear NF-
B on
the transcription of NF-
B-dependent genes (i.e., such genes may
require only a threshold, submaximal level of NF-
B activation to be
transcriptionally active) (1, 20), especially since NF-
B works with
other transcription factors to induce ECAM expression. Our Western blot
data on cytosolic levels of I
B
suggest another explanation,
namely, that the ability of NF-
B to activate genes with
B
promoters may depend on whether its presence in the nucleus is
dependent on proteasome activity.
The differences in effects of PS-341 and lactacystin on ECAM surface
expression in HUVEC did not precisely correlate with differences in the
extent of attenuation of NF-B activation by these two inhibitors;
indeed, the 40% decrease in TNF-elicited NF-
B activation by
lactacystin was associated with almost complete inhibition of ECAM
expression, whereas PS-341 produced a slightly greater suppression of
NF-
B activation yet attenuated TNF-stimulated increases in ECAM
surface expression by only 40-70%. The reasons for these
differences are not clear. All available evidence indicates that both
PS-341 and lactacystin are specific in their proteasome-inhibitory activity (1). Thus one explanation for the above differences may be
that TNF-stimulated increases in ECAM surface expression depend on
additional, as yet uncharacterized, cellular functions of the
proteasome besides activation of NF-
B, functions that may
vary in their susceptibility to inhibition by lactacystin and PS-341.
Our Western blot analysis of cytosolic levels of I
B
in the
presence or absence of proteasome inhibition is consistent with this hypothesis.
In the transformed, HUVEC-derived cell line ECV, basal surface
expression of VCAM-1 and E-selectin was undetectable by our assay
system, and TNF did not elicit an increase in surface expression of
these proteins. Similarly, we were unable to detect a significant increase in either polymorphonuclear neutrophils or U-937 adhesion to
ECV treated with TNF. In contrast to VCAM-1 and E-selectin, basal
expression of ICAM-1 in ECV was similar to that found in HUVEC, and TNF
produced a significant increase in ICAM-1 surface expression, which was
partially blocked by lactacystin. It should be noted that the
TNF-elicited upregulation of ICAM-1 in ECV was markedly blunted (58%
increase above basal) compared with this response in HUVEC
(300-700% increase above basal). In HUVEC, lactacystin completely
blocked TNF-stimulated increases in ICAM-1 expression, but in ECV, in
contrast, lactacystin only partially (60%) blocked the TNF-stimulated
increase in ICAM-1 expression. The reason for this is not clear, but
one possibility is that other pathways for activation of ICAM-1
expression [e.g., AP-1-dependent mechanisms (23)] may play
a more important role in the control of ICAM expression during
proteasome blockade in ECV than they do in HUVEC. The reason for
lactacystin not blocking increases in ICAM-1 expression in response to
16 h of exposure to TNF is not certain. This observation might suggest
proteasome-independent processes playing a role in long-term ICAM-1
expression, but a simpler and more likely explanation is the relatively
short half life of the biologically active intermediate in vivo
metabolite of lactacystin (the -lactone) (8).
Our findings in ECV are essentially in agreement with previous
conclusions (27) that ECV are a poor model for examining the effects of
proinflammatory stimuli on ECAM expression. Our results extend these
conclusions by suggesting where the relative defect in ECV may lie.
Recently, Cobb et al. (5) demonstrated marked induction of VCAM-1 and
ICAM-1 promoter-luciferase reporter genes by cytokines in ECV cells
transfected with such constructs. The results of Cobb et al. (5)
combined with our findings that TNF stimulates NF-B activation in
ECV that is similar to that in HUVEC yet does not induce
NF-
B-dependent ECAM expression make it seem likely that the defect
in ECAM expression in ECV lies between NF-
B binding to promoter
sites and activation of transcription of certain NF-
B-dependent
ECAMs. Experiments addressing this issue are needed, because the
precise molecular defect in ECV remains unknown.
The effects of lactacystin and PS-341 on ECAM surface expression in
HUVEC indicate that proteasome activity is both necessary and
sufficient to elicit increases in surface expression of ICAM-1, VCAM-1,
and E-selectin in response to stimulation by TNF. These events were
correlated with lactacystin-induced inhibition of NF-B activation,
but, in view of the difference in the extent of inhibition of ECAMs by
lactacystin and PS-341, it seems unlikely that the mechanism for
inhibition of TNF-stimulated ECAM expression by lactacystin is solely
the prevention of NF-
B activation. Rather, our data suggest that the
proteasome may play additional roles in mediating the effects of TNF on
adhesion molecule expression. Thus whether the effects of lactacystin
are entirely mediated by direct effects of proteasome inhibition on
availability of NF-
B for binding to ECAM NF-
B promoter sites or
whether inhibition of other proteasome-dependent processes such as
nitric oxide production (6, 7, 14) might play a contributory role will
require further investigation.
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ACKNOWLEDGEMENTS |
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We thank Laura L. Coe for isolating and culturing the HUVEC used in these studies.
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FOOTNOTES |
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This work was supported by funds from the Louisiana State University Medical Center Dept. of Surgery and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52148 (T. J. Kalogeris) and DK-43785 (to M. B. Grisham).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. B. Grisham, Dept. of Molecular and Cellular Physiology, 1501 Kings Hwy., Shreveport, LA 71130 (E-mail: mgrish{at}lsumc.edu).
Received 6 July 1998; accepted in final form 6 January 1999.
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