(Received for publication, March 4, 1997, and in revised form, May 23, 1997)
From the Vascular Research Division, Department of
Pathology, Brigham and Women's Hospital, Harvard Medical School,
Boston, Massachusetts 02115 and the ¶ Bayer
Corporation, West Haven, Connecticut 06516
We have identified two compounds that inhibit the
expression of endothelial-leukocyte adhesion molecules intercellular
adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. These compounds act by inhibiting tumor necrosis factor--induced phosphorylation of I
B-
, resulting in decreased nuclear
factor-
B and decreased expression of adhesion molecules. The effects
on both I
B-
phosphorylation and surface expression of E-selectin were irreversible and occurred at an IC50 of
approximately 10 µM. These agents selectively and
irreversibly inhibited the tumor necrosis factor-
-inducible
phosphorylation of I
B-
without affecting the constitutive
I
B-
phosphorylation. Although these compounds exhibited other
activities, including stimulation of the stress-activated protein
kinases, p38 and JNK-1, and activation of tyrosine phosphorylation of a
130-140-kDa protein, these effects are probably distinct from the
effects on adhesion molecule expression since they were reversible. One
compound was evaluated in vivo and shown to be a potent
anti-inflammatory drug in two animal models of inflammation. The
compound reduced edema formation in a dose-dependent manner in the rat carrageenan paw edema assay and reduced paw swelling in a
rat adjuvant arthritis model. These studies suggest that inhibitors of
cytokine-inducible I
B
phosphorylation exert anti-inflammatory activity in vivo.
The adhesion of circulating leukocytes to vascular endothelium is
critical to inflammatory responses (reviewed in Refs. 1-3). Interaction of the selectin family of adhesion proteins and lectin counter-receptors is the predominant mechanism mediating initial adhesion between leukocytes and the vessel wall. The expression of
endothelial-leukocyte adhesion molecule-1 (E-selectin, CD62E), vascular
cell adhesion molecule-1
(VCAM-1,1 CD106), and
intercellular adhesion molecule-1 (ICAM-1, CD54) on the surface of
endothelial cells is elevated at sites of inflammation (2, 4).
Induction of these molecules by tumor necrosis factor- (TNF
) and
other inflammatory cytokines is regulated at the level of gene
transcription and requires binding of the transcription factor nuclear
factor-
B (NF-
B) to the regulatory regions within the promoters of
each of these genes (5-12).
The NF-B/Rel transcription factor family plays an important role in
cytokine-induced gene activation (13-15). The Rel family includes p50
(NFKB1), p52 (NFKB2), p65 (RelA), RelB, v-Rel, and c-Rel. In
endothelial cells, the p50·p65 heterodimer is the predominant species
that binds to
B consensus sequences in the VCAM-1, ICAM-1, and
E-selectin genes and activates gene transcription. NF-
B is located
in the cytoplasm of cells in an inactive form in association with the
inhibitor I
B-
. In response to TNF
stimulation, I
B-
is
phosphorylated on 2 serine residues (Ser-32 and Ser-36), ubiquitinated, and degraded by a proteosome-dependent pathway allowing
active NF-
B to translocate to the nucleus where it can activate gene expression (16-23). Many NF-
B-dependent genes including
the adhesion molecules and several cytokine genes are important
mediators of inflammation (reviewed in Ref. 24). A diverse range of
agents that block NF-
B signaling has been shown to decrease
expression of adhesion molecules (25-31). Recently, several clinically
important anti-inflammatory agents including glucocorticoids,
salicylates, and nitric oxide have been reported to inhibit
NF-
B-driven gene expression which may explain, at least in part, the
anti-inflammatory actions of these drugs (24-26, 31-34). Thus, novel
agents that block NF-
B/I
B-
signaling have the potential to
inhibit a wide range of inflammatory processes.
In this study, we identified two novel pharmacologic agents that
inhibit the TNF-induced surface expression of ICAM-1, VCAM-1, and
E-selectin in human endothelial cells. These compounds were examined
for their effects on cytokine-induced NF-
B/I
B-
signaling. Both
compounds decreased TNF
-induced nuclear translocation of NF-
B
through inhibition of the TNF
-induced phosphorylation of I
B-
.
Compound 1 selectively inhibited the TNF
-inducible phosphorylation of I
B-
without affecting the constitutive I
B-
phosphorylation. To determine whether these agents may inhibit other
cellular phosphorylation events, we examined the effects of compound 1 on TNF
-induced activity of the stress-activated protein kinases, p38
and JNK-1. This agent increased the activity of p38 kinase and JNK-1
but had little or no effect on the activity of the MAP kinase, ERK-1. The agent was also examined for effects on protein tyrosine
phosphorylation since agents that block tyrosine phosphorylation have
been reported to inhibit NF-
B signaling and adhesion molecule
expression (29). Treatment of endothelial cells with the test compound
did not detectably inhibit protein tyrosine phosphorylation but rather resulted in an elevated level of a tyrosine-phosphorylated protein of
molecular mass 130-140 kDa. We evaluated compound 2 in two animal
models of inflammation. This agent reduced swelling in a
dose-dependent manner in both the rat carrageenan paw
edema assay and in a rat adjuvant arthritis model. Thus, we have
identified a novel class of anti-inflammatory agents that act by
selectively inhibiting the TNF
-induced phosphorylation of I
B
resulting in decreased expression of endothelial adhesion
molecules.
The structures of compounds 1 (BAY-117821;
(E)3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (CA
number, 195462-67-7)) and 2 (BAY 11-7083;
((E)3-[4-t-butylphenyl)-sulfonyl]-2-propenenitrile) are shown in Fig. 1. The compounds were prepared according to previously published procedures (35).
Cell Culture, Cytokine Treatment, and Toxicity Assay
Human
umbilical vein endothelial cells (HUVEC) were isolated and maintained
in culture using previously described procedures (36). For experiments
on cytokine induction, cells were exposed to recombinant human TNF
at a final concentration of 100 units/ml in complete media for the
times indicated. The proteosomal inhibitor carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG115) was prepared as a 40 mM stock solution in Me2SO and added to
complete medium to a final concentration of 40 µM. Cell
toxicity was assessed by morphology and by 3-(4,5-dimethyl
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (37).
Cell surface binding
assays were performed at 4 °C on viable human umbilical vein
endothelial cell monolayers in microtiter plates, using saturating
concentrations of monoclonal antibody supernatants and a secondary
fluorescent-conjugated F(ab)2 goat anti-murine IgG (Caltag
Labs, San Francisco, CA) as previously detailed (38). Antibodies to
E-selectin (H4/18), VCAM-1 (E1/6), and ICAM-1 (Hu5/3) culture
supernatants were kindly provided by Dr. Michael A. Gimbrone, Jr.
Fluorescence intensities were determined using an automated microtiter
plate reader (Pandex, Baxter Healthcare Corp.).
The effects of
compounds 1 and 2 on interleukin-6 (IL-6) and interleukin-8 (IL-8)
production were evaluated on HUVEC that were grown to confluence on
96-well microtiter plates. The cells were preincubated with the drugs
at concentrations of 0, 1, 5, 10, or 25 µM and then
incubated with TNF (10 units/ml) and drug for 16 h. The culture
supernatants were removed and assayed for IL-6 and IL-8 content using
enzyme-linked immunoassay kits from R & D Systems (Minneapolis,
MN).
Nuclear extracts were
prepared from test or control HUVEC in the presence of 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1.5 µg/ml pepstatin A, 40 µM ALLN (calpain inhibitor 1), 1 mM sodium
orthovanadate, and 1 mM sodium fluoride as described
previously (10). Oligonucleotides were gel-purified, annealed, and
end-labeled with [-32P]dCTP (50 µCi; specific
activity of 3000 Ci/mmol, NEN Life Science Products) and the Klenow
fragment of Escherichia coli DNA polymerase I. Binding
reactions were performed in the presence of 10 mM Tris, pH
7.5, 1 mM dithiothreitol, 1 mM EDTA, 5%
glycerol, and 1 µg of poly(dI·dC) and electrophoresis was carried
out as described previously (39). The following oligonucleotides were
utilized: VCAM-
B (vNF-WT), 5
CTGGGTTTCCCCTTGAAGGGATTTCCCTC and the
complementary strand. Protein DNA complexes were resolved on 4%
polyacrylamide gels.
Following experimental treatment of HUVEC,
cytosolic and nuclear protein extracts were prepared, subjected to
electrophoresis on 10% SDS-polyacrylamide gels, and transferred to
nitrocellulose in 25 mM Tris, 192 mM glycine,
5% methanol at 100 V for 1 h as described previously (10, 27).
Anti-IB
and anti-p38 antisera were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA) and used at dilutions of 1:1000.
Rabbit antisera directed against the phosphorylated p38 (Tyr-182) were
obtained from New England Biolabs (Beverly, MA) and used at a 1:1000
dilution. Mouse anti-phosphotyrosine antibodies were obtained from
Upstate Biotechnology Inc. (Lake Placid, NY) and used at 1:1000
dilution. Immunoreactive proteins were detected by enhanced
chemiluminescent protocol (Amersham Corp.) using 1:10,000 horseradish
peroxidase-linked donkey anti-rabbit or sheep anti-mouse secondary
antiserum. Blots were exposed to film for 1-15 min and then
developed.
Extracts were prepared from
control and TNF-treated HUVEC. Cells were solubilized with Triton
lysis buffer (TLB, 20 mM Tris, pH 7.4, 1% Triton X-100,
10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM
-glycerophosphate, 1 mM sodium
orthovanadate, 2 mM pyrophosphate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin). Extracts were
centrifuged at 14,000 × g for 15 min at 4 °C. The
JNK, p38, or ERK protein kinases were immunoprecipitated by incubation
for 1 h at 4 °C with specific rabbit polyclonal antibodies
bound to protein-A Sepharose (Pharmacia Biotech Inc.). The rabbit
polyclonal JNK-1 and p38 antibodies have been described (40). The
immunoprecipitates were washed twice with TLB and twice with kinase
buffer (20 mM Hepes, pH 7.4, 20 mM
-glycerophosphate, 20 mM MgCl2, 2 mM dithiothreitol, 0.1 mM sodium
orthovanadate). The kinase assays were initiated by the addition of 1 µg of substrate protein and 50 µM
[
-32P]ATP) (10 Ci/mmol) in a final volume of 25 µl.
The reactions were terminated after 15 min at 30 °C by addition of
Laemmli sample buffer. Control experiments demonstrated that the
phosphorylation reaction was linear with time for at least 30 min under
these conditions. The phosphorylation of the substrate proteins was examined by SDS-polyacrylamide gel electrophoresis followed by autoradiography.
In gel kinase assay for the proteins
that phosphorylate IB-
was carried out according to the method of
Hibi et al. (41) and as detailed below. Whole cell extracts
were prepared from HUVEC treated with TNF
(100 units/ml) for 15 min
in the presence or absence of compound 1 (20 µM,
pretreatment for 1 h) as indicated. Proteins were separated on a
10% SDS gel containing 0.5 mg/ml HIS-I
B-
. Gels were washed two
times in 20% propanol, 50 mM Hepes, pH 7.6, for 30 min and
two times in buffer A (50 mM Hepes, pH 7.6, 5 mM 2-mercaptoethanol) for 30 min, followed by a 1-h
incubation with buffer A containing 6 M urea, 1 h each
in 3, 1.5, and 0.75 M urea in buffer A and 0.05% Tween 20 and 1 h in buffer A with 0.05% Tween 20. The kinase assay was
carried out for 1 h at 30 °C in the presence of 50 µM ATP, 5 µCi/ml [32P]ATP, 20 mM Hepes, pH 7.6, 20 mM MgCl2, 20 mM
-glycerophosphate, 20 mM
p-nitrophenyl phosphate, 1 mM sodium vanadate, 2 mM dithiothreitol. The gel was washed with 5%
trichloroacetic acid and 1% sodium pyrophosphate, dried, and exposed
to film. A separate gel with no HIS-I
B-
was assayed as a
control.
Male Harlan Sprague Dawley rats 150-175 g were used. A 1% suspension of carrageenan (Marine Colloids, Springfield, MA) in distilled water was administered to rats as 0.1 ml subplantar injection into the footpad of the right hind paw as described previously (42). One h prior to injection rats were treated intraperitoneally with vehicle (polyethylglycol 400 diluted 1:5 in 5% bovine serum albumin/H2O) or a fine suspension of compound 2 (1, 5, or 50 mg/kg) in vehicle. A positive control group was also included in which rats were pretreated with 20 mg/kg ibuprofen. Four hours after carrageenan administration, the volume of the injected paw was measured by means of a water displacement plethysmograph. Edema volumes were determined as the difference between the paw volumes of each rat at time 0 and 4 h. Each group contained five animals. Data were analyzed by a one-way analysis of variance and, if indicated, differences between groups analyzed by Bonferroni's modified t test. A p < 0.05 was considered significant.
Adjuvant ArthritisInbred, male Lewis rats 8-10 weeks of age weighing 250-275 g were obtained from Charles River (Wilmington, MA). Five animals per group were used, and the animals were allowed to feed ad libitum on laboratory rat chow and water. Heat-inactivated Mycobacterium butyricum (Difco) was suspended at 10 mg/ml in mineral oil (Purepac Lubinol, Purepac Pharmaceuticals, Elizabeth, NJ) and administered as 0.1-ml injection (1 mg/animal) at the base of the tail. Paw volumes were determined by a water displacement plethysmograph as described above. Volumes were determined on the indicated dates and values compared with initial time 0 measurements. Vehicle (0.5% methyl cellulose) or drug (compound 2 or dexamethasone at the indicated concentrations) was administered once a day as an intraperitoneal injection (200 µl). Data from these studies are expressed as the mean difference in foot pad volume. At day 20 animals were sacrificed by CO2 inhalation.
Two structurally related compounds,
compound 1 and compound 2 (Fig. 1), were
identified as inhibitors of cytokine-induced surface expression of
ICAM-1 as measured by fluorescence immunoassay as described by
Gerritsen et al. (42). Drug effects on TNF-induced surface expression of E-selectin, VCAM-1, and ICAM-1 were determined by
fluorescence immunoassay as described under "Experimental
Procedures." Compound 1 and compound 2 inhibited the surface
expression of all three adhesion molecules with IC50 values
in the range of 5-10 µM (Fig.
2). To determine whether the effects of
the test compounds were reversible, we compared E-selectin levels in
cells stimulated with TNF
in the presence of compound 1 (10 µM) with the levels obtained when the cells were
pretreated with compound 1 followed by a 1 h "washout" period
and then a 3-h stimulation with TNF
in the absence of test drug.
Treatment of HUVEC with 10 µM compound inhibited
E-selectin expression by 57% and a similar level of inhibition was
seen when the drug was "washed out" prior to TNF
treatment (Fig.
3). Thus, compound 1 irreversibly
inhibits surface expression of E-selectin. Other effects of compound 1 were reversible (see below) suggesting that the inability to reverse the inhibition of TNF
-induced E-selectin expression was not due to
retention of the compound by the cell. There was no detectable cytotoxicity as measured by MTT assay even after 16 h treatment of
cells with this dose (10 µM) of the test compound
(IC50 in the MTT assay ranged from 25-38
µM). Thus, it is likely that the drug irreversibly
modifies a cellular target.
We also determined whether the test compounds could inhibit E-selectin
surface expression when added after initiation of TNF treatment.
Maximal inhibition occurred when the test compound was included from
the start of the TNF
induction period, and no significant inhibition
was observed when the test compound was added after 1 h (Fig. 3).
These results are consistent with the drug acting rapidly and
irreversibly within the 1st h of the TNF
induction.
Additionally, we determined whether the test compounds could inhibit
cytokine production, as well as expression of leukocyte adhesion
molecules. Compounds 1 and 2 also reduced TNF-induced IL-6 and IL-8
production in a dose-dependent manner. This inhibition was
greater than 50% at 10 µM and virtually complete at 25 µM (data not shown).
The TNF-induced expression
of adhesion molecules E-selectin, VCAM-1, and ICAM-1 requires the
transcription factor NF-
B (5-12). Therefore, we evaluated the test
compounds for effects on nuclear translocation of NF-
B. We carried
out electrophoretic mobility shift assay to determine the levels of
NF-
B in nuclear extracts from HUVEC treated with TNF
in the
presence of compound 1 or compound 2. As previously observed,
TNF
-induced nuclear translocation of NF-
B occurs within 15 min in
the absence of test compound (Fig.
4A, lane 2 and Fig. 4B,
lane 2). At 20 µM, both test compounds completely
inhibited nuclear NF-
B (Fig. 4A, lane 4, Fig. 4B, lane 3). A lower dose of compound 1 (10 µM) also
reduced nuclear NF-
B (Fig. 4A, lane 3).
Inhibition of TNF
The TNF-induced
regulation of NF-
B involves the phosphorylation, ubiquitination, and
degradation of the cytoplasmic inhibitor, I
B-
(16-23). In
endothelial cells, the phosphorylation and degradation of I
B-
have been shown to occur within 15 min of TNF
treatment allowing
NF-
B to translocate to the nucleus where it can activate gene
expression (8). To determine whether the test compounds may affect
TNF
-inducible phosphorylation and/or degradation of I
B-
, we
examined the levels of I
B-
in the cytoplasm of endothelial cells
pretreated with increasing concentrations of test compounds and then
stimulated with TNF
for 15 min. The results of Western blot analysis
of endothelial cytoplasmic extracts using I
B
-specific antisera
are shown in Fig. 5. A 37-kDa protein was
detected in cytoplasmic extracts from unstimulated cells (Fig. 5,
lane 1). Treatment of HUVEC with TNF
led to a rapid loss
of I
B-
from the cytoplasm (Fig. 5, lane 2). Both test
compounds stabilized I
B-
in a dose-dependent manner
with an IC50 value of approximately 10 µM
(Fig. 5, lanes 3-5). There was a clear correlation between the concentration of drug that stabilized I
B-
, the concentration that inhibited nuclear levels of NF-
B, and the concentration that
inhibited adhesion molecule expression. The levels of p38 were not
significantly affected by the test compounds (Fig. 5) suggesting that
these agents did not result in nonspecific effects on protein
stability. In addition, these concentrations of test compounds did not
affect protein synthesis as assessed by [3H]leucine
incorporation into 5% trichloroacetic acid-precipitable protein.
The effects of test compounds on levels of IB-
protein could be
due to an inhibition of I
B-
phosphorylation or a block of
degradation. To determine whether these agents affect I
B-
phosphorylation, we examined the I
B-
levels in cells treated with
both compound 1 and the proteosome inhibitor
carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG115) (Fig. 5, lanes
6 and 7). As previously reported (19, 22, 27), the
proteosome inhibitor blocks the degradation of I
B-
, allowing
visualization of the phosphorylated form of I
B-
, detected as a
slower migrating band (Fig. 5, lane 7,
I
B-
-P). The faster migrating form present in
unstimulated cells is basally phosphorylated I
B
(18, 19). The
I
B-
protein present in TNF
-stimulated cells treated with
compound 1 appears as a single faster migrating band corresponding to
basally phosphorylated I
B-
(Fig. 5, lane 5).
Stimulation of cells with TNF
in the presence of both compound 1 and
the proteosome inhibitor resulted in stabilization of the basally
phosphorylated form of I
B-
(Fig. 5, lane 6). Little or
no inducibly phosphorylated I
B-
protein was detected (Fig. 5,
lanes 6 and 7,
I
B-
-P), suggesting that compound
1 inhibits TNF
-inducible phosphorylation of I
B-
. Similar results were observed with compound 2 (data not shown). The I
B-
which has not undergone inducible phosphorylation is not targeted for
degradation (19, 43). Thus, in contrast to the proteosome inhibitors
that block degradation of the phosphorylated I
B-
, compounds 1 and
2 inhibit inducible phosphorylation of I
B-
. Compounds 1 and 2 may
inhibit a TNF
-inducible kinase and/or activate a cellular
phosphatase activity.
Because the effects of these agents on adhesion molecule expression
were found to be irreversible (Fig. 3), we tested whether the
stabilization of IB-
was similarly irreversible. The levels of
cytoplasmic I
B-
were examined in HUVEC pretreated with compound 1 for 1 h and then incubated with media alone for 1 h prior to treatment with TNF
in the absence of drug. The wash out of this compound had little or no effect on the levels of I
B-
(Fig. 6, lanes 6 and 7).
Thus, this drug appears to irreversibly stabilize I
B-
. Treatment
of cells with the test compound 15 min after TNF
induction and
immediately prior to preparation of extracts did not result in
stabilization of I
B-
(Fig. 6, lane 8). Clearly, the
drug blocks TNF
-induced phosphorylation and degradation of I
B-
in intact cells and not during extract preparation. The effects on both
I
B-
phosphorylation and surface expression of E-selectin were
rapid, irreversible, and occurred at an IC50 of approximately 10 µM. This suggests that the inhibition of
I
B-
phosphorylation and degradation causes the decrease in
NF-
B, resulting in decreased transcription and surface expression of the adhesion molecules.
Effect on Constitutive Phosphorylation of I
The
IB-
protein is regulated by cytokine-inducible phosphorylation on
Ser-32 and Ser-36 (16-23). In addition, the C-terminal sequences of
I
B-
contain a consensus sequence for casein kinase II that may be
important for basal phosphorylation of the I
B-
protein (19,
44-46). To determine whether compound 1 selectively inhibited
TNF
-inducible phosphorylation of I
B-
or might also inhibit the
activity of a constitutive I
B-
kinase that basally phosphorylates
I
B-
, we carried out an in gel kinase assay for proteins that
phosphorylate I
B-
(Fig. 7). Two
proteins of molecular mass of approximately 36 to 41 kDa were observed
in whole cell extracts. The activities of these kinases were unaffected
by 15 min treatment with TNF
or by compound 1 (20 µM).
The molecular weights of these kinases correspond to those expected for
the catalytic subunits of casein kinase II; however, Bennett et
al. (47) have recently described I
B-
kinases of similar
molecular weight in endothelial cells that are distinct from casein
kinase II. Our results suggest that compound 1 had no effect on the
activity of these I
B-
kinases. Thus, compound 1 selectively
inhibits the TNF
-inducible phosphorylation of I
B-
without
affecting the constitutive I
B-
phosphorylation.
Activation of Stress-activated Protein Kinases, p38 and JNK-1, with No Effect on ERK-1
The treatment of endothelial cells with TNF
induces multiple signaling events that might be affected by the test
compounds. It has been shown that TNF
treatment stimulates the
stress-activated protein kinase cascade (40, 41, 48-53). The JNK-1 and
p38 kinases are activated by dual phosphorylation of threonine and
tyrosine. To determine whether compound 1 selectively inhibits
I
B-
phosphorylation without affecting other TNF
-induced
phosphorylation events, we examined the effects of compound 1 on MAP
kinase activity. We carried out Western blot analysis with antisera
specific for the phosphorylated form of p38 to determine whether this
agent affected the phosphorylation of the p38 kinase. Results are shown
in the bottom panel of Fig. 5. There was a small increase in
phosphorylation of p38 with TNF
alone and a marked increase in
phosphorylation of p38 in HUVEC-treated with TNF
in the presence of
20 µM compound 1 (Fig. 5, lanes 2 and
5). In addition, there was a slight but detectable increase
in p38 phosphorylation in HUVEC treated with compound 1 alone (data not
shown). The level of p38 phosphorylation in cells treated with both
compound 1 and TNF
was higher than that seen in with TNF
alone or
compound 1 alone (Fig. 5, lanes 2 and 5). The
total levels of p38 in these cells did not change. These results
suggest that compound 1 stimulates TNF
-induced phosphorylation of
p38 and that compound 1 does not globally inhibit all TNF
-induced
phosphorylation. In contrast to the effects on I
B-
and the
effects on adhesion molecule expression, the observed activation of p38
phosphorylation was reversible with 1 h treatment in the absence
of drug (data not shown). Thus, the increase in p38 phosphorylation did
not correlate with the inhibition of adhesion molecule expression.
The effect of this compound on MAP kinase signaling was also measured
by immunoprecipitation kinase assays for activity of p38, JNK-1, or
ERK-1. The results are shown in Fig. 8.
The level of ERK-1 activity detected in cells treated with TNF in
the presence of compound 1 (20 µM) was similar to the
ERK-1 activity in cells treated with TNF
alone suggesting that
compound 1 does not affect ERK-1 kinase activity (Fig. 8, lanes
2 and 3). The JNK-1 and p38 kinase activities were
induced with 15-min TNF
treatment (Fig. 8, lane 2). The
level of JNK-1 activity was somewhat enhanced in cells treated with
TNF
in the presence of compound 1 (Fig. 8, lanes 2 and
3). Notably, there was a significant increase in the
activity of the p38 kinase activity in cells treated with TNF
in the
presence of 20 µM compound 1 (Fig. 8, lanes 2 and 3). This suggests that compound 1 stimulates the
TNF
-induced p38 kinase and JNK-1 kinase activities with no
detectable effect on ERK-1 kinase activity. This compound may activate
signaling events upstream of p38 and JNK-1 that are distinct from the
pathway activating ERK-1 (54, 55). Alternatively, the compound may inhibit a dual specificity phosphatase such as M3/6 which selectively regulates p38 and JNK-1 but not ERK-1 (56). Taken together, our Western
blot and immunoprecipitation kinase assays suggest that the compound
does not act as a global inhibitor of TNF
-induced phosphorylation
events but selectively inhibits phosphorylation of I
B-
.
Effects on Tyrosine Phosphorylation
Protein tyrosine kinase
inhibitors have been reported to block NF-B/I
B-
signaling and
inhibit adhesion molecule expression (29). To determine whether the
selected agents may act as protein tyrosine kinase inhibitors, we
assayed the tyrosine-phosphorylated proteins in whole cell extracts
from endothelial cells treated with TNF
or TNF
and compound 1. Extracts were analyzed by Western blot with anti-phosphotyrosine
antisera as described under "Experimental Procedures." Multiple
bands at a variety of molecular weights were reactive with the
anti-phosphotyrosine antisera. There was not a general reduction in the
pattern or intensity of the bands in compound 1-treated cells. Indeed,
cells treated with TNF
and compound 1 showed a dramatic increase in
a tyrosine-phosphorylated protein with a molecular mass of 130-140 kDa
(Fig. 9). There was some increase in this
tyrosine-phosphorylated protein observed with compound 1 alone, and
further analysis of nuclear and cytoplasmic extracts was done to
determine that this protein was predominantly present in the cytoplasm
(data not shown). The effect of the drug on tyrosine phosphorylation
was reversible and thus probably distinct from the irreversible effects
on I
B-
phosphorylation and adhesion molecule expression. Our data
suggest that compound 1 does not act as a global protein tyrosine
kinase inhibitor and may in fact activate some specific tyrosine
phosphorylation.
Anti-inflammatory Actions of Compound 2
Compound 2 was
evaluated in two in vivo models of inflammation. As shown in
Fig. 10, compound 2 demonstrated a
dose-dependent reduction in swelling in the rat carrageenan
paw model. Compound 2 was also evaluated in established rat adjuvant
arthritis (Fig. 11). In the
vehicle-treated control group, the mean volume of both hind paws
increased by 0.39 ± 0.15 ml. Compound 2, given intraperitoneally at 20 mg/kg, but not at 5 mg/kg, significantly reduced the mean paw
edema of the rats, to levels similar to those observed with the
positive control, dexamethasone, at 1 mg/kg intraperitoneally. Thus,
this compound acted as an anti-inflammatory agent in both the rat
carrageenan paw and the rat adjuvant arthritis model.
We have identified two structurally related compounds that inhibit
the expression of ICAM-1, VCAM-1, and E-selectin in human endothelial
cells. These compounds act by selectively inhibiting TNF-induced
phosphorylation of I
B-
, resulting in decreased nuclear NF-
B
and decreased expression of adhesion molecules. These compounds
selectively inhibited the TNF
-inducible phosphorylation of I
B-
without affecting the constitutive I
B-
phosphorylation. Although
these agents were shown to exhibit reversible effects on other cellular
phosphorylation events including activation of the stress-activated
protein kinases, p38 and JNK-1, and activation of protein tyrosine
phosphorylation, it is likely that these effects are distinct from the
effects on adhesion molecule expression which were irreversible. One of
these agents, compound 2, was tested in vivo in two animal
models of inflammation. The compound reduced swelling in both the rat
carrageenan paw edema assay and in a rat adjuvant arthritis model.
These studies suggest that novel pharmacologic agents that inhibit
cytokine-inducible phosphorylation of I
B
can act as
anti-inflammatory agents.
The precise molecular target for these agents is not yet clear. While
these drugs were shown to inhibit IB-
phosphorylation, this may
be the result of direct inhibition of a TNF
-inducible I
B-
kinase or due to inhibition of a signaling event upstream of the
I
B-
kinase. Alternatively, the regulation of I
B-
phosphorylation involves cellular phosphatase activities that may be
activated by these drugs (19). Once the TNF
-inducible I
B-
kinase(s) and regulatory phosphatase(s) are identified, it will be
interesting to determine if these molecules are the direct target of
compound 1 or 2. It has been observed that upstream activators of the
MAP kinase pathway can induce NF-
B/I
B-
signaling, suggesting
that the MAP kinase and NF-
B cascades share some common
intermediates (57-61). Since TNF
signaling of MAP kinases was not
inhibited by compound 1, the target for this drug is likely to be
downstream of the events that are common to both NF-
B and MAP kinase
signaling pathways. Recent reports have suggested that the TNF
signaling of NF-
B may occur by a ceramide-dependent
mechanism, whereas the TNF
signaling of p38 and JNK-1 kinases in
endothelial cells may be ceramide-independent (62). Therefore,
ceramide-dependent protein kinases (63) and/or
ceramide-dependent protein phosphatases (64) may be potential
targets of the drug action.
In endothelial cells, cytokines increase superoxide anion production
(30, 65), and reactive oxygen intermediates may act as an important
regulator of NF-B (reviewed in Ref. 66). A number of antioxidants
have been reported to inhibit cytokine-induced I
B-
phosphorylation, nuclear translocation of NF-
B, and
NF-
B-dependent transcription of VCAM-1 (24, 30, 67-70).
However, it has been suggested that the expression of ICAM-1 and
E-selectin may be less affected by antioxidants (30, 70, 71). The
potential of the novel compounds described in this study to act as
antioxidants by inhibiting the generation of reactive oxygen
intermediates or by scavenging free radicals has not been
evaluated.
Protein tyrosine kinase inhibitors have been shown to block
phosphorylation of IB-
and adhesion molecule expression. Recent reports suggest that reactive oxygen intermediates activate NF-
B by
a tyrosine kinase-dependent mechanism (29, 72). It is
possible that the compounds described in this study could act to
inhibit a specific protein tyrosine kinase that is upstream of the
I
B-
kinase; however, it does not appear that these compounds act
as globally active tyrosine kinase inhibitors since we observed an increase in phosphotyrosine activity as measured by Western blot with
anti-phosphotyrosine antibodies. In addition, these compounds activated
tyrosine phosphorylation of p38 as measured by Western blot with
phosphospecific antisera and stimulated p38 and JNK-1 activities that
are up-regulated by tyrosine phosphorylation (40, 48, 49). The
mechanism by which test compounds may stimulate tyrosine
phosphorylation has not been determined; however, it is possible that
these compounds may inhibit some protein tyrosine phosphatase activity.
Protein tyrosine phosphatase inhibitors have been reported to inhibit
NF-
B signaling (73, 74); however, the mechanism for this inhibition
is unclear. Serine protease inhibitors have also been reported to
inhibit NF-
B signaling and decrease adhesion molecule expression
(28). We have not tested our novel compounds for specific effects on
serine protease activity.
The anti-inflammatory effects of the test compounds in two animal
models are striking and are consistent with the action of other
pharmacologic agents that inhibit adhesion molecule expression and
leukocyte recruitment (75). Since these novel compounds inhibit NF-B
signaling, they would be expected to affect
B-dependent expression of many other genes including IL-1, IL-6, tissue factor, and
TNF
in lymphoid cells, monocytes, and endothelial cells (reviewed in
Ref. 24). Thus, the observed anti-inflammatory action probably reflects
not just inhibition of adhesion molecules but also effects on many
other important mediators of inflammation in a variety of cell types.
Understanding the mechanism by which these agents disrupt the
NF-
B/I
B regulatory pathway will be useful in identifying novel
anti-inflammatory agents that are both highly specific and effective.
Such drugs may be useful as therapeutic agents in disorders involving
up-regulation of endothelial adhesion molecules including ischemia,
reperfusion injury, asthma, transplantation, inflammatory bowel
disease, rheumatoid arthritis, and atherosclerosis.
We thank Kay Case, Margaret A. Read, Cathy Bull, Carol Perry, Chien-Ping Shen, Stacie Phan, Gwenda Ligon, and William Carley for their expert technical assistance, as well as Dr. Roger Davis for the p38 antibody used in the immunoprecipitation kinase assays.