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B, Activator Protein-1, c-Jun N-Terminal Protein Kinase, and Apoptosis1
Cytokine Research Laboratory, Department of Bioimmunotherapy, 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 activation by suppressing I-B
(inhibitory subunit of
NF-B) degradation. We in this study show that leflunomide also
blocks NF-B reporter gene expression induced by TNFR1,
TNFR-associated factor 2, and NF-B-inducing kinase (NIK), but not
that activated by the p65 subunit of NF-B, suggesting that
leflunomide acts downstream of NIK. Leflunomide suppressed TNF-induced
phosphorylation of I-B, as well as activation of I-B
kinase-
located downstream to NIK. Leflunomide also inhibited
TNF-induced activation of AP-1 and the c-Jun N-terminal protein kinase
activation. TNF-mediated cytotoxicity and caspase-induced
poly(ADP-ribose) polymerase cleavage were also completely abrogated by
treatment of Jurkat T cells with leflunomide. Leflunomide suppressed
TNF-induced reactive oxygen intermediate generation and lipid
peroxidation, which may explain most of its effects on TNF signaling.
The suppressive effects of leflunomide on TNF signaling were completely
reversible by uridine, indicating a critical role for pyrimidine
biosynthesis in TNF-mediated cellular responses. Overall, our results
suggest that suppression of TNF signaling is one of the possible
mechanisms for inhibitory activity of leflunomide against rheumatoid
arthritis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation has
been suggested (11). Recent studies have begun to clarify its mechanism of action. Leflunomide is a potent inhibitor of dihydroorotate dehydrogenase (DHODH),3 a rate-limiting enzyme in the biosynthetic pathway of pyrimidines (12, 13, 14, 15, 16). In vitro the Ki of inhibition of DHODH by leflunomide ranges from 179 nM to 2.7 µM (13, 14). Its ability to suppress proliferation of T and B cells (2, 3, 4) has been suggested to be due to inhibition of DHODH (12, 13, 14, 15), a pathway critical for the proliferation of these cells. The reversal of antiproliferative effects of leflunomide by uridine further suggests the critical role of DHODH (12, 15).
Leflunomide can also inhibit several protein tyrosine kinases, including those of src family (p59fyn and p56lck) (6, 12), the Janus kinase (JAK) family (JAK1 and JAK3) (10), and epidermal growth factor receptor kinase (17). The concentration of leflunomide required to inhibit these protein tyrosine kinases is 10–500 times higher than that required to inhibit DHODH. Inhibition of tyrosine phosphorylation of JAK3 and STAT6 by leflunomide has been implicated in inhibition of IgG1 secretion (18). While leflunomide suppresses proliferation of cells by inhibiting DHODH, protein tyrosine kinase inhibition was implicated in its ability to suppress autoimmune and lymphoproliferative disorders (16).
Leflunomide has been recently approved for treatment of rheumatoid
arthritis (RA) (19), but how its antiarthritis effects are
mediated is not known. Some recent studies suggest TNF may be the
mediator. TNF has been implicated in causing RA (20), and
agents that can down-regulate TNF-mediated cellular responses, such as
TNF-soluble receptors or Ab against TNF, have been approved for
treatment of RA (21, 22). We have previously shown
that leflunomide blocks TNF-mediated NF-B activation by suppressing
I-B degradation (11). We now further extend these
studies to the effects of leflunomide on TNF signaling. In our study,
leflunomide blocked TNF-induced NF-B activation, I-B
(inhibitory subunit of NF-B) phosphorylation, I-B kinase (IKK)
activation, and activation of AP-1, c-Jun N-terminal protein kinase,
and suppressed TNF-induced apoptosis. Leflunomide also suppressed
TNF-induced reactive oxygen intermediate (ROI) generation and lipid
peroxidation. We also demonstrate that the suppressive effects of
leflunomide on TNF signaling can be reversed by uridine, suggesting
an essential role for pyrimidine biosynthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leflunomide (A77 1726), a generous gift from Dr. Robert R.
Bartlett (Hoechst AG, Weisbaden, Germany), was made up as 5 mM solution
in water. Penicillin, streptomycin, RPMI 1640 medium, and FCS were
obtained from Life Technologies (Grand Island, NY). Glycine, NaCl, and
BSA were obtained from Sigma (St. Louis, MO). Bacteria-derived human
rTNF, purified to homogeneity with a sp. act. of 5 x
107 U/mg, was kindly provided by Genentech (South
San Francisco, CA). Abs against I-B, JNK1, cyclin D1, c-Jun,
c-Fos, and p50 were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). Poly(ADP-ribose) polymerase (PARP) Ab was purchased from
PharMingen (San Diego, CA). Phospho-I-B
(Ser32) Ab was purchased from New England BioLabs
(Beverly, MA). Anti-IKK or anti-IKK Abs were kindly provided
by Imgenex (San Diego, CA). Expression plasmids encoding FLAG-tagged
NF-B-inducing kinase (NIK) (23) were kindly provided by
Dr. David Wallach (Weizmann Institute of Science, Rehovot, Israel). The
expression plasmid encoding myc-tagged TNFR-associated
factor 2 (TRAF2) has been previously described (24).
Cell lines
HeLa (human epithelial cells) and T-Jurkat (T cells) were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml).
Isolation of PBLs
Freshly drawn human blood was incubated with 2.5% gelatin in saline (1:1 ratio) for 30 min at 37°C. The supernatant was layered on Histopaque 1077 (from Sigma) and centrifuged at 1500 rpm for 30 min at room temperature. The cells were then collected from the top layer of Histopaque, diluted with Dulbecco’s PBS, and centrifuged at 2000 rpm for 10 min. To get rid of mixed reticulocytes, pellet was suspended in 0.2% NaCl for 1 min, immediately diluted with equal volume of 1.6% NaCl, and centrifuged at 1000 rpm. To remove macrophages by adherence, the pellet was suspended in RPMI 1640 medium supplemented with 10% FBS and cultured for 2 h at 37°C, CO2 incubator in a petri dish. Then the lymphocytes were harvested from the medium by centrifugation at 1000 rpm.
NF-B activation assays
To determine NF-B activation, EMSA were conducted essentially
as described (25). Briefly, nuclear extracts prepared from
TNF-treated cells (2 x 106/ml) were
incubated with 32P end-labeled 45-mer
double-stranded NF-B oligonucleotide (4 µg protein with 16 fmol
DNA) from the HIV long terminal repeat,
5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3'
(underline indicates NF-B binding sites) for 15 min at 37°C,
and the DNA-protein complex formed was separated from free
oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded
mutated oligonucleotide,
5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-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. The dried gels were visualized, and
radioactive bands were quantitated by a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA) using ImageQuant software.
NF-B-dependent reporter gene transcription
The effect of leflunomide on TNF-, TRAF-2-, NIK-, and p65
(transactivation subunit of NF-B)-induced NF-B-dependent reporter
gene transcription was measured as previously described
(24). Briefly, HeLa cells (0.1 x
106 cells/well) were plated in six-well plates,
pretreated with 10 µM leflunomide for 2 h, and then transfected
by the calcium phosphate method with medium (1 ml) containing plasmid
DNAs for TRAF2, NIK, or p65 (1 µg each) along with 0.5 µg NF-B
promoter DNA linked to heat-stable secretory alkaline phosphatase
(SEAP) gene. The total final amount of DNA was maintained at 3 µg by
the addition of the control plasmid pCMVFLAG1 DNA.
To examine TNF-induced reporter gene expression, we transfected cells
with the SEAP expression plasmid for 10 h before treating them
with TNF (1 nM). Treatment with leflunomide was continued during the
transfection reaction. Twenty-four hours after transfection, cell
culture-conditioned medium was harvested and analyzed (25 µl) for
alkaline phosphatase activity essentially as described by the protocol
of Clontech (Palo Alto, CA). The activity of SEAP was assayed on a
96-well fluorescent plate reader (Fluoroscan II; Labsystems, Chicago,
IL) with excitation set at 360 nm and emission at 460 nm. This reporter
system was specific because TNF-induced NF-B SEAP activity was
inhibited by overexpression of either an I-B mutant lacking
Ser32/36, a kinase-inactive NIK, or a
dominant-negative TRAF2 mutant (24).
I-B phosphorylation
To determine the effect of leflunomide on I-B
phosphorylation, cytoplasmic extracts were prepared from cells (2
x 106/ml) treated with leflunomide (10 µM) for
2 h, then with N-acetyl leucyl leucyl nonleucinal
(ALLN) (100 µg/ml) for an additional 1 h, and then with TNF (1
nM) for 15 min, resolved on 10% SDS-PAGE, and then analyzed by Western
blot using Abs against either I-B or phosphorylated I-B
(26). After electrophoresis, the proteins were detected by
chemiluminescence (ECL; Amersham, Arlington Heights, IL).
I-B kinase assay
The IKK assays were performed as described (27).
Briefly, IKK signalosomes were precipitated by treating 300 µg
cytoplasmic extracts with 1µg anti-IKK Ab (IMG-136) overnight
at 4°C, followed by treatment with 20 µl protein A/G-Sepharose
(Pierce, Rockford, IL). After 2 h, the beads were washed three
times with lysis buffer and three times with the kinase assay buffer,
and then resuspended in 20 µl of kinase assay mixture containing 50
mM HEPES (pH 7.4), 20 mM MgCl2, 2 mM DTT, 20
µCi 
-ATP, 10 mM unlabeled ATP, and 2 µg of substrate
GST-I-B 1–54(1–54). After incubation at 30°C for 30 min, the
reaction was terminated by boiling with 5 µl of 5x SDS sample buffer
for 5 min. Finally, the protein was resolved on 10% polyacrylamide gel
under reducing conditions, the gel was dried, and the radioactive bands
were visualized by PhosphorImager. To determine the total amounts of
IKK and IKK in each sample, 60 µg of the cytoplasmic protein
was resolved on 7.5% acrylamide gels, electrotransferred to a
nitrocellulose membrane, blocked the membrane with 5% nonfat milk
protein for 1 h, and then incubated with either anti-IKK or
anti-IKK (IMG-129) Abs (at 1/500 dilution) for 1 h. The
membrane was then washed and reacted with HRP-conjugated secondary
anti-mouse IgG Ab, and finally detected by chemiluminescence (ECL;
Amersham).
AP-1 activation assay
The activation of AP-1 was determined as described (28). Briefly, 6 µg of nuclear extract prepared as indicated above was incubated with 16 fmol of the 32P end-labeled AP-1 consensus oligonucleotide 5'-CGCTTGATGACTCAGCCGGAA-3' (bold indicates AP-1 binding site) for 15 min at 37°C and analyzed by using 6% native polyacrylamide gel. The specificity of binding was examined by supershift with anti-c-fos and anti-c-jun Abs and by competition with unlabeled oligonucleotide. Visualization and quantitation of radioactive bands were done as indicated above.
c-Jun kinase assay
The c-Jun kinase assay was performed by a modified method, as
described earlier (29). Briefly, after treatment of cells
with TNF for 10 min, cell extracts were prepared, immunoprecipitated
with anti-JNK Ab, collected the immune complexes by incubation with
protein A/G-Sepharose beads, and performed the in vitro kinase assays
using GST-Jun 1–79(1–79) as a substrate and
[-32P]ATP. Reactions were stopped by the
addition of SDS sample buffer and subjected the samples to SDS-PAGE.
GST-Jun 1–79(1–79) was visualized by staining with Coomassie blue, and the
dried gel was analyzed by a PhosphorImager (Molecular Dynamics).
Cytotoxicity assay
TNF-induced cytotoxicity was measured by the MTT assay (28). Briefly, 5000 cells/well were incubated in the presence or absence of the indicated test sample in a final volume of 0.1 ml for 72 h at 37°C. Thereafter, 0.025 ml of MTT solution (5 mg/ml in PBS) was added to each well. After a 2-h incubation at 37°C, 0.1 ml of the extraction buffer (20% SDS, 50% dimethyl formamide) was added. After an overnight incubation at 37°C, the OD at 590 nm were measured using a 96-well multiscanner autoreader (Dynatech MR 5000), with the extraction buffer as a blank.
Immunoblot analysis of PARP degradation
TNF-induced apoptosis was examined by proteolytic cleavage of PARP (30). Briefly, cells (2 x 106/ml) were treated with different concentrations of leflunomide at 37°C for 2 h and then stimulated with 1 nM TNF with cycloheximide (2 µg/ml) for 2 h at 37°C. The cells were then washed and extracted by treatment for 30 min on ice with 0.05 ml 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 vanadate. The lysate was centrifuged, and the supernatant was collected. Cell extract protein (50 µg) was resolved on 7.5% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, blotted with mouse anti-PARP Ab, and then detected by chemiluminescence (ECL; Amersham). Apoptosis was represented by the cleavage of 116-kDa PARP into a 85-kDa product.
Determination of lipid peroxidation
TNF-induced lipid peroxidation was determined by detection of thiobarbituric acid-reactive malondialdehyde (MDA), an end product of the peroxidation of polyunsaturated fatty acids and related esters, as described (26). Results were normalized with the amount of MDA equivalents/mg of protein and expressed as a percentage of thiobarbituric acid-reactive substances above control values. Untreated cells showed 0.571 ± 0.126 nmol of MDA equivalents/mg of protein.
Measurement of ROI
The production of ROI upon treatment of cells with TNF was determined by flow cytometry, as described (26).
Radiolabeling of TNF and receptor-binding assay
Human TNF was iodinated with [125I]Na by the Iodogen method, purified, and examined for cell surface receptors, as described previously (30).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
activation in this cell (11). The concentration of
leflunomide and its time of exposure had no effect on the viability of
these cells (data not shown).
Leflunomide represses TNF-induced NF-B-dependent reporter gene
expression
Previously, we have shown that leflunomide blocks
TNF-induced NF-B activation, I-B degradation, and
NF-B-dependent reporter gene expression (11).
TNF-induced NF-B activation is mediated through sequential
interaction of the TNFR (TNFR1) with TNFR-associated death domain,
TRAF2, NIK, and IKK-, resulting in phosphorylation of I-Ba
(31, 32). To delineate the site of action of leflunomide
in the TNF-signaling pathway leading to NF-B activation, HeLa cells
were transfected with TNFR1, TRAF2, NIK, and p65 plasmids, and then
NF-B-dependent SEAP expression monitored in leflunomide-untreated
and -treated cells. Due to higher transfection efficiency, HeLa cells
were used. As shown in Fig. 1
, TNFR1,
TRAF2, NIK, and p65 plasmids induced gene expression, and leflunomide
suppressed TNFR1-, TRAF2-, and NIK-induced expression, but had little
effect on p65-induced NF-B reporter expression. RANK, another
NF-B-inducing receptor, which is a member of the TNFR1 family, was
minimally affected by leflunomide, indicating the specificity.
Specificity of the assay results is also indicated by suppression of
TNF-induced NF-B reporter activity by the dominant-negative
I-B plasmid. Thus, leflunomide must act at a step downstream from
IKK-. Because NIK is known to activate IKK-, which in turn
phosphorylates I-B, it appears that leflunomide blocked the
activity of IKK-, a kinase that phosphorylates I-B
directly.
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B
To determine whether inhibition of TNF-induced I-B
degradation by leflunomide was due to suppression of I-B
phosphorylation, cells were treated with the proteosome inhibitor ALLN
(33) for 1 h, and then the hyperphosphorylated form
of I-B was assayed by Western blot using Abs that detect only the
serine-phosphorylated form of I-B. The results clearly show that
TNF induced the phosphorylation of I-B and leflunomide suppressed
it (Fig. 2A, upper
panel). The hyperphosphorylated form of I-B also appeared as
a slow-migrating band on SDS-PAGE (Fig. 2A, lower
panel). The lack of a slow-migrating band in leflunomide-treated
cells indicates that leflunomide blocked TNF-induced I-B
phosphorylation.
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activation
Because TNF-induced phosphorylation of I-B is mediated
through IKK-, these results suggest that leflunomide must inhibit
IKK- activation. Therefore, we investigated the effect of
leflunomide on TNF-induced IKK- activation. As shown in Fig. 2
B (upper panel), in the immune complex kinase
assays, TNF activated IKK- in a time-dependent manner and
leflunomide treatment completely suppressed the activation. Under these
conditions, leflunomide had no effect on the IKK- (middle
panel) and IKK- (lower panel) protein
levels.
Leflunomide inhibits TNF-induced NF-B activation in normal
cells
All the experiments described above were performed with the Jurkat
cell lines. Previously, we have shown that leflunomide also blocks
TNF-induced NF-B activation in U-937 cells (myeloid) and epithelial
(HeLa) and glioma (H4) cells (11). Whether leflunomide
also affects NF-B in normal cells was examined. As shown in Fig. 2C, TNF activated NF-B by 4-fold in normal human PBLs and
the pretreatment with leflunomide abolished the activation in a
dose-dependent manner. These results suggest that the suppressive
effect of leflunomide is not restricted to tumor cells.
Leflunomide inhibits TNF-induced AP-1 activation
Previously, we have shown that leflunomide has no effect on the
constitutive levels of AP-1 (11). TNF is also one of the
most potent activators of AP-1 (34). TNF induced AP-1
expression by 5-fold in Jurkat cells at 1 nM concentration (Fig. 3A). The activation of AP-1
was completely inhibited by leflunomide in a concentration-dependent
manner, with maximum suppression occurring at 5 µM (Fig. 3A). Supershift analysis with specific Abs against c-fos and
c-jun indicates that TNF-induced AP-1 consisted of fos and jun (Fig. 3B). The lack of supershift by unrelated Abs and
disappearance of the AP-1 band by competition with unlabeled
oligonucleotide indicate the inhibition was specific (Fig. 3B).
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Previously, we have shown that leflunomide blocks TNF-induced
mitogen-activated protein/extracellular signal-related kinase kinase
activation (11). JNK, the downstream kinase, is known to
be required for the activation of AP-1 (35, 36, 37). Whether
JNK is also modulated by leflunomide was examined. Jurkat cells were
pretreated with different concentrations of leflunomide for 2 h
and then stimulated with TNF (1 nM) for 10 min; activation of JNK was
then measured. TNF activated JNK by about 13-fold, an activation that
gradually decreased with increasing concentrations of leflunomide. A 10
µM concentration of leflunomide inhibited most of the JNK induced by
TNF (Fig. 3C). Thus, it is possible that leflunomide blocks
TNF-induced AP-1 activation through suppression of JNK activation.
Leflunomide blocks TNF-induced cytotoxicity and caspase activation
Among the cytokines, TNF is one of the most potent inducers of
apoptosis (for references, see Ref. 38). Whether
leflunomide modulates TNF-induced apoptosis was also investigated.
Jurkat cells were treated with various concentrations of leflunomide
either in the absence or presence of TNF and then examined for
cytotoxicity by the MTT method (Fig. 4A). TNF was cytotoxic to
Jurkat cells, and leflunomide abolished TNF-induced cytotoxicity in a
dose-dependent manner, reaching complete inhibition at 10 µM
leflunomide. We also examined the cytotoxic effect of various
concentrations of TNF either in the absence or presence of leflunomide
(Fig. 4B). The cytotoxic effects of TNF in Jurkat cells were
dose dependent, with almost 60% killing occurring at 0.5 nM
concentration of the cytokine. This cytotoxicity was nearly completely
inhibited at all TNF doses by treatment of cells with 10 µM
leflunomide.
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C, TNF
induced complete cleavage of PARP, and this cleavage was inhibited in a
dose-dependent manner by treatment of cells with leflunomide, with
maximum effect at 10 µM concentration. Thus, leflunomide also blocked
TNF-induced apoptosis. Leflunomide blocks TNF-induced ROI generation and lipid peroxidation
Previous reports have shown that TNF activates NF-B, AP-1, JNK,
and apoptosis through generation of ROI (27, 39, 40, 41).
Whether leflunomide suppresses TNF signaling through suppression of ROI
generation was examined by flow cytometry. As shown in Fig. 5A, TNF induced ROI generation
in a time-dependent manner, and this was suppressed on pretreatment of
cells with leflunomide. Because lipid peroxidation has also been
implicated in TNF signaling (42), we also examined the
effect of leflunomide on TNF-induced lipid peroxidation. Results in
Fig. 5B show that TNF induced lipid peroxidation in Jurkat
cells, and it was completely suppressed by leflunomide. Thus, it is
quite likely that leflunomide blocks TNF signaling through suppression
of ROI generation and lipid peroxidation. Although earliest time we
examined ROI generation is at 1 h, whereas NF-B and AP-1
activation can be seen at 15 min, it is possible that low levels of ROI
generated at early times are either not detectable or other mechanisms
are involved in TNF signaling.
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The antiproliferative effects of leflunomide on B and T cells can
be reversed by uridine, suggesting the critical role of DHODH
(12, 15). To determine whether uridine reverses the
suppression of TNF-induced NF-B activation in Jurkat cells, we
pretreated cells with leflunomide in the presence of various
concentrations of uridine and then treated them with TNF. As shown in
Fig. 6A, TNF-induced NF-B
activation was suppressed by leflunomide, and uridine reversed the
suppression in a dose-dependent manner. Uridine or leflunomide by
themselves did not activate NF-B, and uridine alone had minimal
effect on TNF-mediated NF-B activation. Thus, leflunomide mediated
its effects on TNF-induced NF-B activation by interfering with the
pyrimidine biosynthesis pathway. We also examined the effect of uridine
on the leflunomide-induced suppression of TNF-mediated cytotoxicity. As
shown in Fig. 6B, 50 µM uridine completely reversed the
effects of leflunomide on the cytotoxicity caused by TNF. Thus,
pyrimidine biosynthesis plays a critical role in the TNF signaling.
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Because leflunomide blocked a variety of signals activated by TNF, it is possible that leflunomide down-regulated TNFR. Therefore, we examined the effect of leflunomide on TNFR by receptor-binding assays. TNF bound to Jurkat cells, and this binding was completely unaffected by pretreatment of cells with leflunomide (data not shown), suggesting that the effects of leflunomide were not due to down-regulation of TNFR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation, I-B
phosphorylation, and activation of IKK, JNK, and AP-1, and suppressed
TNF-induced apoptosis. None of these effects were mediated through the
down-regulation of TNFR. Leflunomide suppressed TNF-induced ROI
generation and lipid peroxidation. The suppressive effects of
leflunomide on TNF signaling could be reversed by uridine.
There are several ways to explain how leflunomide might inhibit
TNF-induced NF-B activation. One is by suppressing TNF-induced
phosphorylation of I-B. The phosphorylation of I-B is
regulated by a large number of kinases, including IKK-, IKK-,
IKK-, NIK, TGF--activated kinase-1, AKT, and mitogen-activated
protein/extracellular signal-related kinase kinase kinase 1 (MEKK1),
MEKK2, and MEKK3 (43). However, only IKK- mediates
TNF-induced phosphorylation of I-B at positions 32 and 36. Thus,
leflunomide may suppress NF-B activation through suppression of
IKK-. That leflunomide blocked TNFR1-, TRAF2-, and NIK-induced
NF-B-mediated reporter gene expression, but not that activated by
p65 also suggests that the site of action of leflunomide is downstream
from NIK. The latter is known to activate IKK-, thus leading to
I-B phosphorylation.
Our results indicate that leflunomide also blocked TNF-induced JNK and
AP-1 activation. TRAF2, which is known to bind to TNFR through
TNFR-associated death domain, is also required for AP-1 and JNK
activation (31). The suppression of TRAF2 activity may
explain how leflunomide inhibits NF-B, AP-1, and JNK. While MEKK1,
MEKK2, and MEKK3 have been implicated in NF-B activation, MEKK4
activates JNK (44). There are some reports that indicate
that AKT and NIK activate IKK-, whereas MEKK1 and atypical PKC
activate IKK- (for references, see Ref. 45). Thus, it
is possible that leflunomide inhibited I-B phosphorylation by
inhibiting the activity of IKK-.
Our results also indicate that leflunomide is a potent inhibitor of
AP-1. This is not too surprising because most agents that activate
NF-B also activate AP-1. Activation of AP-1 requires JNK, another
kinase of the mitogen-activated protein kinase family, so it is
possible that AP-1 is suppressed through inhibition of JNK. Recent
studies from our laboratory showed that overexpression of cells with
either superoxide dismutase (27) or with
-glutamylcysteine synthetase, a rate-limiting enzyme in the
glutathione biosynthesis pathway (40), blocked both
NF-B and AP-1 activation induced by TNF, indicating a similar
mechanism of activation of both transcription factors. These results
also suggest that leflunomide may suppress these factors by regulating
the redox status of the cells. We did indeed find that leflunomide
blocks TNF-induced ROI production and lipid peroxidation.
TNF-induced cyotoxicity and caspase activation were also blocked by
leflunomide. Because NF-B activation has been shown to play an
antiapoptotic role (59), the suppression of apoptosis by
leflunomide may seem paradoxical. The overexpression of the antioxidant
enzymes manganous superoxide dismutase or -glutamylcysteine
synthetase has also has been shown to suppress TNF-induced apoptosis
and NF-B (27, 40), suggesting that the mechanisms of
activation of apoptosis and NF-B are very similar. Our discovery
that leflunomide blocks TNF-induced ROI generation and lipid
peroxidation may explain the mechanism by which leflunomide exerts some
of its effects.
The suppressive effects of leflunomide on TNF signaling were reversible by uridine. These results are consistent with previous reports (12, 15). Leflunomide is a potent inhibitor of DHODH, a rate-limiting enzyme in the biosynthesis pathway of pyrimidine (12, 13, 14, 15). In vitro the Ki of inhibition of DHODH by leflunomide ranges from 179 nM to 2.7 µM (13, 14). The ability of leflunomide to suppress proliferation of T and B cells (2, 3, 4) has been suggested to be due to inhibition of DHODH (12, 13, 14, 15), a pathway critical for the proliferation of these cells. Our results are the first to demonstrate the critical role of this pathway in TNF signaling.
We found that leflunomide blocked NF-B-dependent reporter gene
expression. Several genes that are involved in RA are regulated by
NF-B (19). These include inflammatory cytokines,
cyclooxygenase-2, metalloproteinases, urinary plasminogen activator, NO
synthase, and cell surface adhesion molecules (47, 48, 49, 50, 51, 52).
Thus, it is possible that leflunomide mediates its effects against RA
through suppression of NF-B-regulated genes. Indeed, the modulation
by leflunomide of cyclooxygenase-2, NO synthase, inflammatory
cytokines, and their receptors has recently been reported
(53, 54, 55). Because NF-B-regulated gene products have
also been implicated in tumorigenesis, leflunomide may prove useful in
suppressing tumorigenesis (56). Indeed, in vivo antitumor
activity against C6 glioma has been recently assigned to leflunomide
(57).
Adenoviral I-B, an NF-B inhibitor, has been used for the
treatment of RA and tumorigenesis (58, 59). Because
leflunomide lacks delivery problems, the suppression of NF-B
activation by leflunomide is preferable. Leflunomide is also preferable
over soluble TNFR or anti-TNF Abs, because formation of Abs against
either of the proteins can have serious side effects.
Adenovirus-enforced overexpression of mitochondrial superoxide
dismutase has also been used as gene therapy for ischemia/reperfusion
injury of the liver through the down-regulation of NF-B and AP-1
activation (60). Our results indicate that suppressive
effects of leflunomide on NF-B and AP-1 activation and on other
TNF-mediated cellular responses may have protective effects on liver
and against cardiovascular diseases.
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Acknowledgments |
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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 Bioimmunotherapy, Box 143, 1515 Holcombe Boulevard, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030.
3 Abbreviations used in this paper: DHODH, dihydroorotate dehydrogenase; ALLN, N-acetyl leucyl leucyl nonleucinal; I-B, inhibitory subunit of NF-B; IKK, I-B kinase; JAK, Janus kinase; JNK, c-jun N-terminal protein kinase; MDA, malondialdehyde; MEKK, mitogen-activated protein/extracellular signal-related kinase kinase kinase; NIK, NF-B-inducing kinase; PARP, poly(ADP-ribose) polymerase; RA, rheumatoid arthritis; ROI, reactive oxygen intermediate; SEAP, secretory alkaline phosphatase; TRAF, TNFR-associated factor.
Received for publication April 7, 2000. Accepted for publication August 21, 2000.
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