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B Activation and Gene Expression1
Cytokine Research Laboratory, Department of Molecular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B is a transcription factor activated in
response to a wide variety of inflammatory stimuli, including TNF, but
whether leflunomide blocks NF-B activation is not known. In the
present report we demonstrate that treatment of a human T cell line
(Jurkat) with leflunomide blocks TNF-mediated NF-B activation in a
dose- and time-dependent manner, with maximum inhibition at 5–10 µM.
Inhibition was not restricted to TNF-induced activation, because
leflunomide also inhibited NF-B activation induced by other
inflammatory agents, including phorbol ester, LPS,
H2O2, okadaic acid, and ceramide. Leflunomide
blocked the degradation of IB
and subsequent nuclear
translocation of the p65 subunit, steps essential for NF-B
activation. This correlated with inhibition of dual
specificity-mitogen-activated protein kinase kinase as well as an Src
protein tyrosine kinase, p56lck, by
leflunomide. Reducing agents did not reverse the effect of leflunomide.
Leflunomide also suppressed the TNF-activated NF-B-dependent
reporter gene expression. Our results thus indicate that leflunomide is
a potent inhibitor of NF-B activation induced by a wide variety of
inflammatory stimuli, and this provides the molecular basis for its
anti-inflammatory and immunosuppressive
effects. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
is a ubiquitous transcription factor that plays a critical role in
cells of the immune system. This transcription factor is sequestered in
its inactive state in the cytoplasm by a noncovalent association with
the inhibitory protein called
IB3 (for references,
see 1 . NF-B is activated by a wide variety of inflammatory
stimuli, including TNF, IL-1, okadaic acid, phorbol ester,
H2O2, ceramide, endotoxin, and 
-radiation.
Most of these agents induce the phosphorylation-dependent degradation
of IB, thus unmasking the nuclear localization signals on p65 and
activating NF-B. Indeed, IB-deficient mice exhibit
constitutive NF-B activation, severe runting, dermatitis, and
extensive granulopoiesis and die during the neonatal period 2 .
NF-B regulates the expression of many genes that play essential
roles in immune and inflammatory responses. It regulates the expression
of genes for various cytokines, the MHC, viral replication (e.g., type
I HIV), and cell proliferation 1, 2 . The inappropriate regulation of
NF-B and its dependent genes has been associated with various
pathological conditions, including septic shock, graft-vs-host
reaction, acute inflammatory conditions, acute phase response,
transplant rejection, autoimmune diseases, viral replication, radiation
damage, atherosclerosis, and cancer 1, 2 .
Agents that can block NF-B activation have potential for treatment
of the pathological situations indicated above. Leflunomide (HWA-486;
N-(4-trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide)
is one such agent, in that it exhibits antiinflammatory,
antiproliferative, and immunosuppressive effects through mechanisms
that are not fully understood 3, 4, 5, 6, 7 . HWA-486 is a prodrug that is
rapidly converted in the cell to an active metabolite,
N-(4-trifluoromethylphenyl-2,2-cyano-3-hydroxycrotoamide),
named A77 1726. The initial conversion involves the opening of the
isoxazole ring to produce A77 1726, which constitutes >95% of the
drug in the circulation. Early experiments suggest that A77 1726 blocks
T cell proliferation stimulated by anti-CD28 and PMA, anti-CD3,
and IL-2 5, 8 . It also prevents the proliferation of B cells and Ab
production by B cells 4 .
Since NF-B activation is critical for inflammation, and leflunomide
exhibits antiinflammatory and immunosuppressive effects, we
investigated whether it inhibits NF-B activation. Our results show
that the active form of leflunomide, A77 1726, is a potent inhibitor of
NF-B activation induced by TNF and other inflammatory agents.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leflunomide (A77 1726) was a gift from Dr. Robert R. Bartlett
(Hoechst, Weisbaden, Germany). A 5 mM solution of leflunomide was made
in water. Bacteria-derived human rTNF, purified to homogeneity with a
specific activity of 5 x 107 U/mg, was provided by
Genentech (South San Francisco, CA). Penicillin, streptomycin, RPMI
1640 medium, and FBS were obtained from Life Technologies (Grand
Island, NY). Tris, glycine, NaCl, SDS, PMA, and BSA were obtained from
Sigma (St. Louis, MO). The polyclonal Abs used were as follows:
anti-p65, against the epitope corresponding to amino acids mapping
within the amino-terminal domain of human NF-Bp65; anti-p50,
against a peptide 15 amino acids long mapping at the NLS region of
NF-B p50; anti-IB-, against amino acids 297–317 mapping
at the carboxyl terminus of IB-/MAD-3; and anti-c-Rel and
anti-cyclin D1 against amino acids 1–295, which represent
full-length cyclin D1 of human origin. All these Abs were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA).
Rat mdr1b promoter containing wild-type (MDR1bCAT) and mutated
(243RMICAT) NF-B binding sites and linked to the chloramphenicol
acetyltransferase (CAT) gene were supplied by Dr. M. Tien Kuo of the
University of Texas M. D. Anderson Cancer Center (Houston, TX).
The characterization of these plasmids has been described previously in
detail 9 .
Cell line
For most studies an acute human T cell leukemia cell line (Jurkat) obtained from American Type Culture Collection (Manassas, VA) was used. Cells were routinely grown in RPMI 1640 medium supplemented with glutamine (2 mM), gentamicin (50 µg/ml), and FBS (10%). The cells were seeded at a density of 1 x 105 cells/ml in T25 flasks (Falcon 3013, Becton Dickinson Labware, Lincoln Park, NJ) containing 10 ml of medium and were grown at 37°C in an atmosphere of 95% air and 5% CO2. Cell cultures were split every 3 days.
Electrophoretic mobility shift assays (EMSAs)
Cells (2 x 106 cells/ml) were treated separately with different concentrations of activator at 37°C, and nuclear extracts were prepared as previously described 10 . Briefly, 2 x 106 cells were washed with cold PBS and suspended in 0.4 ml of lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.5 mg/ml benzamidine). Cells were lysed with 12.5 µl of 10% Nonidet P-40. The nuclear pellet was resuspended in 25 µl of ice-cold extraction buffer (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.5 mg/ml benzamidine). The supernatant, i.e., nuclear extract, was used immediately or was stored at -70°C for later use. The protein content was measured by the method of Bradford 11 .
EMSAs were performed by incubating 4 µg of protein of the nuclear
extract with 16 fmol of 32P end-labeled, 45-mer,
double-stranded NF-B oligonucleotide from the HIV-1 long terminal
repeat, 5'-TTGT TACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3' 12 ,
in the presence of 2–3 µg of poly(dI-dC) in a binding buffer (25 mM
HEPES (pH 7.9), 0.5 mM EDTA, 0.5 mM DTT, 1% Nonidet P-40, 5%
glycerol, and 50 mM NaCl) for 15 min at 37°C as described previously
13, 14 . The DNA-protein complex formed was separated from the
oligonucleotide on 7.5% native polyacrylamide gel using buffer
containing 50 mM Tris, 200 mM glycine (pH 8.5), and 1 mM EDTA 15 , and
then the gel was dried. An oligonucleotide with mutated binding sites
(5'-TTGTTACAACT CACTTTCCGCTGCRCACRRRCCAGGGAGGCGTGG-3') was used to
examine the specificity of binding of NF-B to the DNA. The
specificity of binding was also examined by competition with the
unlabeled oligonucleotide.
For supershift assays, nuclear extracts prepared from TNF-treated cells
were incubated with the Abs against either p50 or p65 of NF-B for 30
min at room temperature before the complex was analyzed by EMSA as
described previously in detail 16 . Abs against c-Rel B and cyclin D1
and preimmune serum were included as negative controls.
The EMSAs for AP-1 and Oct-1 were performed as described for NF-B
using 32P end-labeled double-stranded oligonucleotides.
Visualization and quantitation of radioactive bands were conducted by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software.
Western blotting for IB, p50, and p65
After the NF-B activation reaction described above,
postnuclear extracts were resolved on 10% SDS-polyacrylamide gels to
assay IB. To determine the p50 and p65 levels, nuclear and
postnuclear (cytoplasmic) extracts were resolved on 8% SDS-PAGE. After
the gels, the proteins were electrotransferred onto nitrocellulose
filters and probed with rabbit polyclonal Abs against IB, p50,
and p65, and bands were detected by chemiluminescence (ECL, Amersham,
Arlington Heights, IL) 17 . The bands were quantitated by personal
densitometer scan v1.30 using ImageQuant software version 3.3
(Molecular Dynamics).
Transient transfection and CAT assay
Jurkat cells were transiently transfected with -243RMICAT
(wild-type) and -243RMICAT-m (mutant) genes for 6 h by the
calcium phosphate method according to the instructions supplied by the
manufacturer (Life Technologies). After transfection, the cells were
incubated for 24 h at 37°C and then treated with leflunomide (10
µM) for 2 h before stimulation with 100 pM TNF for 1 h.
Then, the cells were washed with PBS and examined for CAT activity as
previously described 18 .
In vitro tyrosine kinase assay for p56lck
The in vitro kinase assay was performed using a modified method
as described previously 19 . Briefly, the Jurkat cells (5 x
106/ml) were treated with different concentrations of
leflunomide for 2 h and then stimulated with TNF (100 pM) for 15
min, cell extracts were prepared by lysing cells in buffer containing
20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2
µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml
benzamidine, 1 mM DTT, and 1 mM sodium o-vanadate (lysis
buffer). Cell extracts (400–500 µg/sample) were immunoprecipitated
with 0.3 µg anti-p56lck Ab (Santa
Cruz Biotechnology) after incubation for 60 min at 4°C. The immune
complex was collected by incubation with protein A/G-Sepharose beads
for 45 min at 4°C. The beads were washed four times with lysis buffer
(400 µl) and twice with the kinase buffer (400 µl; 20 mM HEPES (pH
7.4), 1 mM DTT, and 25 mM NaCl). Kinase assays were performed for 15
min at room temperature with 5 µg of histone 2B as a substrate in 50
mM HEPES (pH 7.4), 10 mM MgCl2, 10 mM MnCl2,
and 10 µCi of [32P] ATP. Reaction was stopped by
addition of 15 µl of SDS sample buffer (20% glycerol (v/v), 2% SDS,
62.5 mM Tris-Cl (pH 6.8), 5% 2-ME, and 0.0025% bromophenol blue),
boiled for 5 min, and subjected to SDS-PAGE (9 or 12%). After
electrophoresis, autophosphorylation of p56lck
and phosphorylation of exogenous histone 2B were analyzed by
visualization and quantitation of radioactive bands on a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA) using ImageQuant software.
MAPK assay
Leflunomide-pretreated Jurkat cells were stimulated with different concentrations of TNF. After incubation for 30 min at 37°C, cells were washed with Dulbecco’s PBS and then extracted with lysis buffer containing 20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, 1 mM DTT, and 1 mM sodium o-vanadate. The protein concentration in the supernatant was determined and then resolved with 50 µg of protein/lane on 10% SDS-PAGE. After the electrophoresis, the proteins were electrotransferred to nitrocellulose filters, probed with the phospho-specific anti-p44/42 MAPK (Thr202/Tyr204) Ab (New England Biolabs, Beverley, MA) raised in rabbits (1/3000 dilution). Then the membrane was incubated with peroxidase-conjugated anti-rabbit IgG (1/3000 dilution), and bands were detected by chemiluminescence (ECL, Amersham).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B by different
inflammatory stimuli. We used an acute leukemia T cell line (Jurkat)
for these studies because leflunomide is known to suppress T
cell-mediated responses. The time of incubation and the concentration
of drug used in our studies had no effect on cell viability (data not
shown). Leflunomide (HWA-486; or
N-(4-trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide)
is a prodrug that is rapidly converted in the cell to an active
metabolite,
N-(4-trifluoromethylphenyl-2,2-cyano-3-hydroxycrotoamide),
named A77 1726 (see Fig. 1
). The initial
conversion involves the opening of the isoxazole ring to produce A77
1726, which constitutes >95% of the drug in the circulation. In the
present studies we used the A77 1726 compound, which does not require
conversion to be active.
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B activation
Jurkat cells were preincubated for 2 h with different
concentrations of leflunomide and then treated with TNF (100 pM) for 30
min at 37°C. Nuclear extracts were prepared and assayed for NF-B
activation by EMSA. The results presented in Fig. 2A indicate that 5–10 µM
leflunomide inhibited most of the TNF-induced NF-B activation
and that leflunomide by itself did not activate NF-B. To
determine the effect of leflunomide on the kinetics of NF-B
activation by TNF, both untreated and leflunomide-pretreated cells were
incubated with TNF for different times. The activation of NF-B was
detected with increases in incubation time as shown by the increase in
band intensity in untreated cells. The leflunomide-pretreated cells
showed a dramatic decrease in activation of NF-B even after up to 60
min of TNF stimulation (Fig. 2B).
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B within 5 min and that
this induction is higher in its intensity than that obtained with cells
using 100-fold lower concentrations of TNF for longer times 20 . To
determine the effect of leflunomide on NF-B activation at higher
concentrations, both untreated and leflunomide-pretreated cells were
incubated with various concentrations of TNF (0–10,000 pM) for 30 min,
and then NF-B was assayed by EMSA (Fig. 2
C). Although the
activation of NF-B by 10 nM TNF was strong, leflunomide almost
completely inhibited it as efficiently as it did at 0.01 nM. These
results show that leflunomide is a very potent inhibitor of TNF-induced
NF-B activation.
We next tested the length of leflunomide incubation on NF-B
activation by TNF. For this, cells were incubated with leflunomide for
120, 60, and 30 min before the addition of TNF; at the same time as the
addition of TNF; or 5, 15, and 30 min after the addition of TNF. The
cells were treated with TNF for 30 min. Only when the cells were
pretreated for 120 min with leflunomide was NF-B activation almost
completely inhibited, and the inhibition decreased gradually with
decreased preincubation time. Cotreatment or posttreatment with
leflunomide was not effective in the inhibition of NF-B activation
by TNF (Fig. 2D).
We also investigated whether the effect of leflunomide on the
TNF-dependent NF-B activation was reversible. To determine this,
Jurkat cells were treated with 10 µM leflunomide for 2 h, then
the drug was removed, and the cells were incubated for different times
in the drug-free medium before checking for TNF-induced NF-B
activation. As shown in Fig. 2E, suppression of NF-B
activation is reversible; it remained suppressed up to 3 h after
the removal of the drug and then reversed at 6 h. These results
show that the effects of leflunomide are reversible.
Activated NF-B inhibited by leflunomide consists of p50 and p65
subunits
Various combinations of Rel/NF-B proteins can constitute an
active NF-B heterodimer that binds to specific sequences in DNA. To
show that the retarded band visualized by EMSA in TNF-treated cells was
indeed NF-B, we incubated nuclear extract from TNF-activated
cells with Abs to either p50 (NF-BI) or p65 (Rel A) subunits and
then conducted EMSA. Abs to either subunit of NF-B shifted the band
to a higher m.w. (Fig. 3A),
thus suggesting that the TNF-activated complex consists of p50 and p65
subunits. When a higher concentration of the Ab or the combination of
p50 and p65 Abs were used, a complete supershift of the band was
observed (data not shown). Neither preimmune serum nor irrelevant Abs
such as anti-c-Rel or anti-cyclin DI had any effect on the
mobility of NF-B. This is consistent with a recent report from our
laboratory 21 .
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Whether leflunomide affects DNA binding of other transcription
factors was investigated. Leflunomide had no effect on the Oct-1
transcription factor (Fig. 3B, left panel). DNA
binding of AP-1 transcription factors 22 , however, was down-modulated
only at the highest concentration (25 µM) of leflunomide. Up to a
10-µM concentration of leflunomide, which inhibits most of the
NF-B activation, had no effect on the AP-1 transcription factors
(Fig. 3B, right panel).
Leflunomide does not interfere with the DNA-binding ability of
NF-B proteins
It has been shown that both TPCK, a serine protease inhibitor, and
herbimycin A, a protein tyrosine kinase inhibitor, down-regulate
NF-B activation by chemical modification of the NF-B subunits,
thus preventing its binding to DNA 23, 24 . To determine whether
leflunomide also directly modifies NF-B proteins, we incubated
cytoplasmic extracts from untreated cells and those treated with
deoxycholate (DOC; 0.8%) for 15 min at room temperature or incubated
nuclear extracts from TNF-triggered cells and then treated them with
various concentrations of leflunomide. Then DNA binding activity was
detected using EMSA. The DOC treatment has been shown to dissociate the
IB subunit, thus releasing NF-B for binding to the DNA. Our
results in Fig. 3, C and D, show that leflunomide
did not modify the DNA-binding ability of NF-B proteins prepared by
treatment with either DOC or TNF. Therefore, leflunomide inhibits
NF-B activation through a mechanism different from that of TPCK or
herbimycin A.
Leflunomide blocks phorbol ester-, LPS-, okadaic acid-, ceramide-,
and H2O2-mediated activation of NF-B
Besides TNF, NF-B is also activated by phorbol ester,
H2O2, LPS, okadaic acid, and ceramide 25 .
However, the signal transduction pathways induced by these agents
differ. We therefore examined the effect of leflunomide on the
activation of transcription factor by these various agents. The results
shown in Fig. 4 indicate that leflunomide
completely blocked the activation of NF-B induced by all five
agents. These results suggest that leflunomide may act at a step where
all these agents converge in the signal transduction pathway leading to
NF-B activation.
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B activation by leflunomide is not cell type
specific
All the effects of leflunomide described above were found using
Jurkat T cells. Whether leflunomide affects other cell types was also
investigated. We examined the ability of leflunomide to block
TNF-induced NF-B activation in myeloid (U-937), epithelial (HeLa),
and glioma (H4) cells. The results of these experiments (Fig. 5) indicate that leflunomide inhibited
NF-B in all three cell types. Almost complete inhibition was
observed with epithelial and glioma cells, and partial inhibition was
found with myeloid cells, thus suggesting that this effect of
leflunomide is not cell type specific. The NF-B binding in all cells
was abrogated by a 25-fold molar excess of unlabeled oligonucleotide.
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It has been shown that while agents such as TPCK, which modify the
sulfhydryl group in NF-B, inhibit NF-B activation, this
inhibition can be reversed by DTT 23 . To determine whether the
inhibitory effect of leflunomide on NF-B could be reversed by this
reducing agent, Jurkat cells were treated with leflunomide in the
presence or the absence of DTT and then examined for the activation of
NF-B by TNF. DTT did not reverse the inhibition caused by
leflunomide (data not shown), suggesting that leflunomide did not
suppress NF-B activation by blocking sulfhydryl groups.
Leflunomide inhibits TNF-dependent degradation of IB and
hence nuclear translocation of the p65 subunit
The translocation of NF-B to the nucleus is preceded by the
phosphorylation and proteolytic degradation of IB 26 . To
determine whether the inhibitory action of leflunomide was due to its
effect on IB degradation, the cytoplasmic levels of IB
protein were examined by Western blot analysis. An IB decrease
appeared within 5 min of TNF treatment of Jurkat cells, and it
completely disappeared by 15 min. The IB was fully resynthesized
by 60 min, as indicated by the reappearance of the band (Fig. 6A). The presence of
leflunomide completely blocked the TNF-induced disappearance of the
band. Thus, these results strongly suggest that leflunomide blocks
TNF-mediated degradation of IB.
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B activation also requires nuclear translocation of the
p65 subunit of NF-B, we measured the level of p65 in the cytoplasm
and in the nucleus. As expected, upon TNF treatment the level of p65
declined in the cytoplasm with a concurrent increase in the nucleus
(Fig. 6B). Treatment of the cells with leflunomide abolished
the TNF-dependent change in the nuclear and cytoplasmic p65 pools.
These results show that leflunomide inhibits the TNF-induced
translocation of p65 to the nucleus, which is consistent with the
inhibition of TNF-dependent degradation of IB. Besides p65, the
effect of leflunomide was also examined on the cytoplasmic and nuclear
pools of other members of the Rel family of proteins. The results shown
in Fig. 6C indicate that neither TNF by itself nor in
combination with leflunomide had any effect on the level of p50. Leflunomide blocks the TNF-induced p56lck and MAPKK activation
Previously it has been shown that leflunomide is a potent
inhibitor of p56lck, a member of the Src family
of protein tyrosine kinases 8, 19 . There is also recent report that
shows that pervanadate-induced NF-B activation requires the
activation of p56lck 27 . Whether TNF activates
p56lck and if this activation is blocked by
leflunomide are not known. The results shown in Fig. 7A indicate that TNF activates
p56lck in a dose-dependent manner in Jurkat
cells and that this activation is blocked quite effectively by
leflunomide (Fig. 7B). The inhibitory effect could be noted
with as little as 1 µM leflunomide.
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B-inducing kinase in TNF-induced NF-B activation has been
implicated 28, 29 . Whether leflunomide inhibits TNF-induced NF-B
activation by blocking this pathway was also investigated. For this,
Jurkat cells were pretreated with leflunomide and then activated with
TNF; cytoplasmic extracts were prepared and then assayed for activation
of MEK by Western blot analysis. The results presented in Fig. 7C show that TNF activated MEK in a dose-dependent manner.
Treatment of cells with leflunomide completely suppressed the
TNF-induced activation of MEK.
Leflunomide represses the MDR-NF-B-CAT gene expression
To determine the effect of leflunomide on TNF-induced
NF-B-dependent gene expression, the promoter of the rat
mdr1b gene containing the NF-B binding site linked to the
CAT reporter gene was used. Jurkat cells were transiently transfected
with the CAT reporter construct and then stimulated with TNF in either
the presence or the absence of leflunomide. Almost threefold increase
in CAT activity was obtained upon stimulation with TNF (Fig. 8). However, TNF-induced CAT activity was
reduced significantly when the cells transfected with the wild-type
NF-B sequence were pretreated with leflunomide for 2 h before
TNF treatment. Transfection with the MDR gene containing mutated
NF-B binding site did not result in induction of CAT by TNF. These
results demonstrate that leflunomide represses gene expression induced
by TNF.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation.
Inhibition was not restricted to TNF, as NF-B activation induced by
other inflammatory agents was also blocked. The inhibitory effect of
leflunomide was not cell type specific, as NF-B activation was
inhibited in myeloid and epithelial cells as well as in T cells.
Leflunomide blocked the phosphorylation and degradation of IB and
subsequent nuclear translocation of p65 subunit, steps essential for
NF-B activation. Leflunomide also blocked the activation of a
tyrosine kinase p56lck and of a dual specificity
kinase MEK. Leflunomide completely suppressed the TNF-induced gene
expression dependent on NF-B activation. Thus, these results may
help to explain why leflunomide is a potent immunosuppressive and
anti-inflammatory agent.
How leflunomide inhibited NF-B activation is not clear at present.
Since NF-B activation induced by highly diverse stimuli, including
TNF, H2O2, LPS, PMA, okadaic acid, and
ceramide, was inhibited, leflunomide must block activation at a step
common to all these activators. In response to most of these stimuli,
IB undergoes phosphorylation at serines 32 and 36 by activation
of kinase complex consisting of I- and
I-ß (for references, see Refs. 30 and 31), which leads
to ubiquitination at lysines 21 and 22 and then degradation 1, 2 .
Since leflunomide blocked this entire cascade, it must act upstream of
IB phosphorylation. Previously, it has been reported that
leflunomide can inhibit several protein tyrosine kinases, including
those of the Src family (p59fyn and
p56lck) 8, 19 , the Janus kinase family (JAK1
and JAK3) 32 , and epidermal growth factor receptor kinase 33 . More
recent studies have shown that inhibition of tyrosine phosphorylation
of JAK3 and STAT6 by leflunomide causes inhibition of IgG1 secretion
34 . The concentration of leflunomide required to inhibit these PTKs,
however, is much higher than that used in our studies. For instance, in
vitro leflunomide blocks the autophosphorylation and histone 2B
phosphorylation by p59fyn with
IC50 values of 125–175 µM and 22–40
µM, respectively 8 . Similarly, p56lck is
inhibited with IC50 values of 160 and 65 µM for
autophosphorylation and histone, respectively 8, 19 . A complete
inhibition of NF-B activation, however, occurs at 5–10 µM
leflunomide. We found that this concentration of leflunomide was
sufficient to inhibit TNF-induced activation of
p56lck. This is the first demonstration that TNF
can activate p56lck. The role of
p56lck in pervanadate-induced NF-B
activation has been reported 27 . Our results show that
p56lck may also play a role in the pathway
leading to NF-B activation by TNF and perhaps other agents.
Our results indicate that leflunomide inhibits NF-B activation not
only in T cells but also in myeloid and epithelial cells. The
p56lck is a T cell-specific protein tyrosine
kinase. Thus, how leflunomide inhibits NF-B activation in other cell
types is not clear. It is possible that there are other kinases that
are more ubiquitously expressed, and their inhibition by leflunomide
blocks NF-B activation. MEKK1 is one such kinase; it is expressed in
all different cell types and has been shown to play a role in NF-B
activation 28 . The target of this kinase is MEK, a dual-specific
kinase, the activation of which leads to phosphorylation of MAPK at
threonine and tyrosine residues. Another structural homologue of MEKK1,
NF-B-inducing kinase, has been identified, which is involved in
TNF-induced NF-B activation 29 . We found that leflunomide also
completely suppressed the TNF-induced activation of MEK. Therefore, it
is possible that leflunomide blocks NF-B activation in all different
cell types by suppression of the MAPK pathway activated by TNF (see
Fig. 7C).
Several reports indicate that leflunomide can block the proliferation
of T and B cells 4, 5, 6 and inhibit dihydroorotate dehydrogenase
(DHODH) 19, 35, 36, 37 , a rate-limiting enzyme in the biosynthesis
pathway of pyrimidine, a pathway critical for the proliferation of
these cells 4, 5, 6 . The cell proliferation block was due to the
inhibition of DHODH by leflunomide, given that the antiproliferative
effects could be reversed by addition of uridine 19, 37 . In vitro the
Ki of inhibition of DHODH by leflunomide
ranges from 179 nM to 2.7 µM 35, 36 , which is 10–500 times lower
than that for PTK. While leflunomide suppresses the proliferation of
cells by inhibiting DHODH, PTK inhibition was implicated in its ability
to suppress autoimmune and lymphoproliferative disorders 38 . It is
unlikely that inhibition of NF-B activation by leflunomide is due to
inhibition of DHODH, because NF-B activation occurs very rapidly at
a low concentration of leflunomide and does not require any new protein
synthesis 1, 2 .
Identifying how leflunomide blocks the activation of NF-B requires
an understanding of the mechanism by which various inducers activate
this important transcription factor. TNF is one of the most potent
activators of NF-B, but the mechanism by which the activation occurs
is not understood. Roles for ceramide, superoxide radicals, proteases,
protein serine kinases, and protein tyrosine phosphatases upstream of
IB phosphorylation have been suggested 1, 15, 39, 40, 41 . Whether
these signals are generated by TNF sequentially or independently of
each other, however, is not known. Such NF-B inducers as phorbol
ester, H2O2, and TNF are known to produce
reactive oxygen intermediates (ROI). The inhibitors of
mitochondrial electron transport have been shown to impair the
TNF-induced activation of NF-B 42 , thus also suggesting a role for
ROI. Therefore, it is possible that leflunomide blocks NF-B
activation by quenching ROI production. However, there is no published
report to suggest that leflunomide has antioxidant properties.
Our results indicate that leflunomide also blocks NF-B-dependent
gene expression. Transcription of a number of genes, including those
involved in inflammation, transplant rejection, tumor promotion, tumor
metastasis, cell proliferation, and autoimmunity, requires NF-B
activation. It is quite likely that the roles of leflunomide in the
suppression of transplant rejection 43, 44, 45 , adjuvant arthritis 3 ,
proliferation of B and T cells and smooth muscle cells 8, 32, 43 , and
IL-2R expression 8 are due to the inhibition of NF-B activation.
Overall, we conclude that because of its very low pharmacological
toxicity 46 and its ability to modulate activation of NF-B by
various agents, leflunomide has a high potential for use as an
immunosuppressive and growth modulatory agent.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Cytokine Research Laboratory, Department of Molecular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. E-mail address:
3 Abbreviations used in this paper: IB, inhibitor of nuclear factor-B; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; MAPK, mitogen-activated protein kinase; AP-1, activating protein-1; TPCK, L-p-tosylamino-2-phenylethyl chloromethyl ketone; DOC, deoxycholate; MEKK, mitogen-activated protein kinase kinase kinase; PTK, protein tyrosine kinase; DHODH, dihydroorotate dehydrogenase; ROI, reactive oxygen intermediates.
Received for publication May 12, 1998. Accepted for publication October 29, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B/IB family: intimate tales of association and dissociation. Genes Dev. 9:2723.B. Annu. Rev. Cell Biol. 10:405.
B mediated induction of mdrIb expression by insulin in rat hepatoma cells. J. Biol. Chem. 272:15174.B-like motifs and of constitutive and inducible forms of NF-B. Mol. Cell. Biol. 10:1498.B. J. Biol. Chem. 270:10631.B activation induced by tumor necrosis factor requires membrane-associated components: comparison with pathway activated by ceramide. J. Biol. Chem. 269:25369.B causes resistance to apoptosis in human cutaneous T cell lymphoma HuT-78 cells: autocrine role of TNF and reactive oxygen intermediates. J. Biol. Chem. 273:14008.B is not sufficient for its dissociation from NF-B and is inhibited by protease inhibitors. Proc. Natl. Acad. Sci. USA 91:11884.B activation in T lymphocytes: evidence for covalent modification of the p50 subunit. J. Biol. Chem. 270:28557.B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J. 12:2005.[Medline]
B: a lesson in family values. Cell 80:529.[Medline]
B- activates NF-B without proteolytic degradation of IB-. Cell 86:787.[Medline]
B kinase complex by MEKK1, a kinase of the JNK pathway. Cell 88:213.[Medline]
B induction by TNF, CD95 and IL-1. Nature 385:540.[Medline]
B kinase complex. Science 278:818.B activation: the IB kinase revealed?. Cell 90:299.
B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell 71:765.[Medline]
activates the sphingomyelin signal transduction pathway in a cell-free system. Science 255:1715.. Proc. Natl. Acad. Sci. USA 88:10009.This article has been cited by other articles:
|
|
 |
|
  H. Wang, T. Cui, F. Hou, Z. Ni, X. Chen, F. Lu, F. Xu, X. Yu, F. Zhang, X. Zhao, et al. Induction treatment of proliferative lupus nephritis with leflunomide combined with prednisone: a prospective multi-centre observational study Lupus, July 1, 2008; 17(7): 638 - 644. [Abstract] [PDF]
|
|
|
|
 |
|
 N. Leca, K. A. Muczynski, J. A. Jefferson, I. H. de Boer, J. Kowalewska, E. A. Kendrick, R. Pichler, and C. L. Davis Higher Levels of Leflunomide Are Associated with Hemolysis and Are not Superior to Lower Levels for BK Virus Clearance in Renal Transplant Patients Clin. J. Am. Soc. Nephrol., May 1, 2008; 3(3): 829 - 835. [Abstract] [Full Text] [PDF]
|
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|
 |
|
 V S Sangle, S R Sangle, and D P D'Cruz Leflunomide as a remission-maintaining therapy in difficult-to-treat dermatomyositis Ann Rheum Dis, May 1, 2008; 67(5): 723 - 723. [Full Text] [PDF]
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|
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|
I Tchetverikov, M C Kraan, B van El, R Hanemaaijer, J DeGroot, and T W J Huizinga Leflunomide and methotrexate reduce levels of activated matrix metalloproteinases in complexes with {alpha}2 macroglobulin in serum of rheumatoid arthritis patients Ann Rheum Dis, January 1, 2008; 67(1): 128 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. F. Si, J. Li, X. W. Lu, and Y. Jin Suppressive Effects of Leflunomide on Leptin-Induced Collagen I Production Involved in Hepatic Stellate Cell Proliferation Experimental Biology and Medicine, March 1, 2007; 232(3): 427 - 436. [Abstract] [Full Text] [PDF]
|
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 |
|
 E. Kreijveld, H. J. P. M. Koenen, L. B. Hilbrands, H. J. P. van Hooff, and I. Joosten The immunosuppressive drug FK778 induces regulatory activity in stimulated human CD4+CD25- T cells Blood, January 1, 2007; 109(1): 244 - 252. [Abstract] [Full Text] [PDF]
|
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|
 |
|
 D. Uygur, H. Aytan, S. Zergeroglu, and S. Batioglu Leflunomide--an Immunomodulator--Induces Regression of Endometrial Explants in a Rat Model of Endometriosis Reproductive Sciences, July 1, 2006; 13(5): 378 - 383. [Abstract] [PDF]
|
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|
|
|
|
M Cutolo, S Capellino, P Montagna, A Sulli, B Seriolo, and B Villaggio Anti-inflammatory effects of leflunomide in combination with methotrexate on co-culture of T lymphocytes and synovial macrophages from rheumatoid arthritis patients Ann Rheum Dis, June 1, 2006; 65(6): 728 - 735. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Migita, T. Miyashita, Y. Maeda, M. Nakamura, H. Yatsuhashi, H. Ishibashi, and K. Eguchi An active metabolite of leflunomide, A77 1726, inhibits the production of serum amyloid A protein in human hepatocytes Rheumatology, April 1, 2005; 44(4): 443 - 448. [Abstract] [Full Text] [PDF]
|
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|
|
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J Grisar, M Aringer, M D Koller, G H Stummvoll, D Eselbock, B Zwolfer, C W Steiner, B Zierhut, L Wagner, P Pietschmann, et al. Leflunomide inhibits transendothelial migration of peripheral blood mononuclear cells Ann Rheum Dis, December 1, 2004; 63(12): 1632 - 1637. [Abstract] [Full Text] [PDF]
|
|
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|
 |
|
 T. Korn, T. Magnus, K. Toyka, and S. Jung Modulation of effector cell functions in experimental autoimmune encephalomyelitis by leflunomide-- mechanisms independent of pyrimidine depletion J. Leukoc. Biol., November 1, 2004; 76(5): 950 - 960. [Abstract] [Full Text] [PDF]
|
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|
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 |
|
 U. Mahlknecht, J. Will, A. Varin, D. Hoelzer, and G. Herbein Histone Deacetylase 3, a Class I Histone Deacetylase, Suppresses MAPK11-Mediated Activating Transcription Factor-2 Activation and Represses TNF Gene Expression J. Immunol., September 15, 2004; 173(6): 3979 - 3990. [Abstract] [Full Text] [PDF]
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|
M C Kraan, T J M Smeets, M J van Loon, F C Breedveld, B A C Dijkmans, and P P Tak Differential effects of leflunomide and methotrexate on cytokine production in rheumatoid arthritis Ann Rheum Dis, September 1, 2004; 63(9): 1056 - 1061. [Abstract] [Full Text] [PDF]
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D Hammaker, S Sweeney, and G S Firestein Signal transduction networks in rheumatoid arthritis Ann Rheum Dis, November 1, 2003; 62(90002): ii86 - 89. [Abstract] [Full Text] [PDF]
|
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|
 |
|
 Z. H. Mnjoyan, R. Dutta, D. Zhang, B.-B. Teng, and K. Fujise Paradoxical Upregulation of Tumor Suppressor Protein p53 in Serum-Stimulated Vascular Smooth Muscle Cells: A Novel Negative-Feedback Regulatory Mechanism Circulation, July 29, 2003; 108(4): 464 - 471. [Abstract] [Full Text] [PDF]
|
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|
M Cutolo, A Sulli, P Ghiorzo, C Pizzorni, C Craviotto, and B Villaggio Anti-inflammatory effects of leflunomide on cultured synovial macrophages from patients with rheumatoid arthritis Ann Rheum Dis, April 1, 2003; 62(4): 297 - 302. [Abstract] [Full Text] [PDF]
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 |
|
 A. Varin, S. K. Manna, V. Quivy, A.-Z. Decrion, C. Van Lint, G. Herbein, and B. B. Aggarwal Exogenous Nef Protein Activates NF-kappa B, AP-1, and c-Jun N-Terminal Kinase and Stimulates HIV Transcription in Promonocytic Cells. ROLE IN AIDS PATHOGENESIS J. Biol. Chem., January 17, 2003; 278(4): 2219 - 2227. [Abstract] [Full Text] [PDF]
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D. Burger, N. Begue-Pastor, S. Benavent, L. Gruaz, M.-T. Kaufmann, R. Chicheportiche, and J.-M. Dayer The active metabolite of leflunomide, A77 1726, inhibits the production of prostaglandin E2, matrix metalloproteinase 1 and interleukin 6 in human fibroblast-like synoviocytes Rheumatology, January 1, 2003; 42(1): 89 - 96. [Abstract] [Full Text] [PDF]
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P. Dimitrova, A. Skapenko, M. L. Herrmann, R. Schleyerbach, J. R. Kalden, and H. Schulze-Koops Restriction of De Novo Pyrimidine Biosynthesis Inhibits Th1 Cell Activation and Promotes Th2 Cell Differentiation J. Immunol., September 15, 2002; 169(6): 3392 - 3399. [Abstract] [Full Text] [PDF]
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D L Scott, J S Smolen, J R Kalden, L B A van de Putte, A Larsen, T K Kvien, M Schattenkirchner, P Nash, C Oed, and I Loew-Friedrich Treatment of active rheumatoid arthritis with leflunomide: two year follow up of a double blind, placebo controlled trial versus sulfasalazine Ann Rheum Dis, October 1, 2001; 60(10): 913 - 923. [Abstract] [Full Text] [PDF]
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 T. Korn, K. Toyka, H.-P. Hartung, and S. Jung Suppression of experimental autoimmune neuritis by leflunomide Brain, September 1, 2001; 124(9): 1791 - 1802. [Abstract] [Full Text] [PDF]
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S. Majumdar and B. B. Aggarwal Methotrexate Suppresses NF-{kappa}B Activation Through Inhibition of I{kappa}B{alpha} Phosphorylation and Degradation J. Immunol., September 1, 2001; 167(5): 2911 - 2920. [Abstract] [Full Text] [PDF]
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S. K. Manna, A. Mukhopadhyay, and B. B. Aggarwal Leflunomide Suppresses TNF-Induced Cellular Responses: Effects on NF-{kappa}B, Activator Protein-1, c-Jun N-Terminal Protein Kinase, and Apoptosis J. Immunol., November 15, 2000; 165(10): 5962 - 5969. [Abstract] [Full Text] [PDF]
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S. K. Manna, A. Mukhopadhyay, and B. B. Aggarwal IFN-{alpha} Suppresses Activation of Nuclear Transcription Factors NF-{kappa}B and Activator Protein 1 and Potentiates TNF-Induced Apoptosis J. Immunol., November 1, 2000; 165(9): 4927 - 4934. [Abstract] [Full Text] [PDF]
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F C Breedveld and J-M Dayer Leflunomide: mode of action in the treatment of rheumatoid arthritis Ann Rheum Dis, November 1, 2000; 59(11): 841 - 849. [Abstract] [Full Text]
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 H. C. Nousari and G. J. Anhalt Bullous Pemphigoid Treated With Leflunomide: A Novel Immunomodulatory Agent Arch Dermatol, October 1, 2000; 136(10): 1204 - 1205. [Full Text] [PDF]
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 S. Pampfer, S. Cordi, S. Cikos, B. Picry, I. Vanderheyden, and R. D. Hertogh Activation of Nuclear Factor {kappa}B and Induction of Apoptosis by Tumor Necrosis Factor-{alpha} in the Mouse Uterine Epithelial WEG-1 Cell Line Biol Reprod, September 1, 2000; 63(3): 879 - 886. [Abstract] [Full Text]
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J. S. Smolen, W. B. Graninger, and P. Emery Leflunomide, a new disease-modifying anti-rheumatic drug and the never ending rheumatoid arthritis story Rheumatology, July 1, 2000; 39(7): 689 - 692. [Full Text] [PDF]
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S. K. Manna and B. B. Aggarwal Vesnarinone Suppresses TNF-Induced Activation of NF-{kappa}B, c-Jun Kinase, and Apoptosis J. Immunol., June 1, 2000; 164(11): 5815 - 5825. [Abstract] [Full Text] [PDF]
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S. K. Manna and B. B. Aggarwal Differential Requirement for p56lck in HIV-tat Versus TNF-Induced Cellular Responses: Effects on NF-{kappa}B, Activator Protein-1, c-Jun N-Terminal Kinase, and Apoptosis J. Immunol., May 15, 2000; 164(10): 5156 - 5166. [Abstract] [Full Text] [PDF]
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S. K. Manna, N. K. Sah, and B. B. Aggarwal Protein Tyrosine Kinase p56lck Is Required for Ceramide-induced but Not Tumor Necrosis Factor-induced Activation of NF-kappa B, AP-1, JNK, and Apoptosis J. Biol. Chem., April 28, 2000; 275(18): 13297 - 13306. [Abstract] [Full Text] [PDF]
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