From the Pulmonary and Critical Care Medicine, Stanford University Medical Center, Stanford, California 94305-5236
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ABSTRACT |
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PG490 (triptolide) is a diterpene triepoxide with
potent immunosuppressive and antiinflammatory properties. PG490
inhibits interleukin(IL)-2 expression by normal human peripheral blood lymphocytes stimulated with phorbol 12-myristate 13-acetate (PMA) and
antibody to CD3 (IC50 of 10 ng/ml), and with PMA and
ionomycin (Iono, IC50 of 40 ng/ml). In Jurkat T-cells,
PG490 inhibits PMA/Iono-stimulated IL-2 transcription. PG490 inhibits
the induction of DNA binding activity at the purine-box/antigen
receptor response element (ARRE)/nuclear factor of activated T-cells
(NF-AT) target sequence but not at the NF- Extracts of the Chinese herb Tripterygium Wilfordii hook have
potent antiinflammatory and immunosuppressive properties and have been
used successfully in traditional Chinese medicine for the treatment of
rheumatoid arthritis (1). One active component of Tripterygium extracts
is the diterpene triepoxide, triptolide, which possesses antileukemic
activities (2) and also inhibits proliferation of transformed cell
lines (3, 4). Kupchan and Shubert (5) described that triptolide
possesses a 9,11-epoxy-14 A refined extract of Tripterygium, PG27, which contains PG490
(triptolide) as its active compound, prolongs heart and kidney allograft survival in rat transplantation models and, furthermore, displays synergy with the immunosuppressant cyclosporin A
(CsA)1 in preventing cardiac
and renal allotransplant
rejection.2 The combination
of PG27 with CsA substantially prolongs hamster cardiac xenograft
survival in rat recipients and inhibits the production of serum
anti-hamster IgM and IgG xenoantibodies where single drug therapies are
ineffective.2 In addition, PG27 suppresses the development
of graft versus host disease associated with allogeneic bone
marrow transplantation.3 The
chloroform methanol extract of Triptyergium, T2 (6), has been studied
recently (7-9) and was shown to block mitogen-induced early cytokine
gene transcription in T-cells (10).
An early cytokine transcribed during T-cell activation is IL-2
(reviewed in Ref. 11). IL-2 transcription involves specific DNA binding
and transcriptional activation of a purine-box transcriptional regulator operative at the antigen receptor response element
(ARRE)/NF-AT target DNA sequence, and of NF- Cyclosporin A and FK506 interfere with the induction of
sequence-specific DNA binding activity at the purine-box/ARRE/NF-AT target DNA sequence (reviewed in Ref. 11). We previously purified to
homogeneity a CsA- and FK506-sensitive sequence-specific purine-box DNA
binding complex that contains NF45 and NF90 proteins (12, 13).
Recently, we showed that NF45 and NF90 associate tightly with the
catalytic subunit of DNA-dependent protein kinase and serve
to stabilize the association of the catalytic subunit of DNA-dependent protein kinase with DNA-targeting proteins,
Ku80 and Ku70 (14). The catalytic subunit of DNA-dependent
protein kinase and Ku have been shown to bind with sequence specificity to purine-rich target DNA sequences and to mediate sequence-specific transcriptional repression (15). We have recently shown that the
specific CsA-sensitive purine-box/ARRE DNA binding complex in human
bronchial epithelial cells involves NF45, NF90, Ku80, and Ku70
with no evidence for NF-ATp or NF-ATc proteins (16).
Activation of IL-2 transcription triggered through costimulatory
receptors such as CD28 involves NF- We investigated the immunosuppressive and antiinflammatory properties
of PG490 (pure triptolide) in human peripheral blood lymphocytes,
Jurkat T-cells, and human bronchial epithelial cells and show that the
mechanism of inhibition by PG490 differs fundamentally from that of CsA
and involves inhibition of transcriptional activation of the purine-box
regulator of the ARRE/NF-AT site and of NF- Source of Triptolide--
PG490 (triptolide, molecular weight
360) was obtained from Pharmagenesis (Palo Alto, CA). The material was
composed of white to off-white crystals, had a melting point of
226-240 °C, conformed to standard triptolide preparation by proton
nuclear magnetic resonance (2), and was 97% pure by reverse phase high
pressure liquid chromatography evaluation using
acetonitrile:methanol:water (18:9:73).4
Cell Culture and Stimulation Conditions--
Human peripheral
blood lymphocytes (PBL) were prepared by centrifugation on a gradient
of sodium diatrizoate/Ficoll (Sigma) of a buffy coat obtained from the
Stanford Hospital blood bank. Monocytes were depleted by adherence to
plastic culture dishes for 30 min at room temperature and then the PBLs
were stimulated for 12 h at a density of 1 × 107
cells/ml in RPMI 1640 supplemented with 10% fetal bovine serum. Jurkat
T-cells (clone E6-1) were obtained from American Type Culture Collection (Manassas, VA), and cultured in RPMI 1640 (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (BioWhittaker, Walkersville, MD). An SV-40 large T-antigen transformed human bronchial epithelial cell line 16HBE14o- (16HBE), which retains differentiated morphology and function of normal human airway epithelia (21), was cultured in
Eagle's minimum essential medium (BioWhittaker) supplemented with 10%
heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (BioWhittaker) as described (22). Human PBLs, Jurkat
T-cells, or monolayer 16HBE cells were stimulated for the indicated
times in culture media containing 20 ng/ml phorbol 12-myristate
13-acetate (PMA; Calbiochem), PMA + antibody to CD3 (clone HIT3a,
Pharmingen, San Diego, CA), PMA + antibody to CD28 (YTH913.12,
BIOSOURCE, Camarillo, CA), PMA + 2 µM ionomycin (Iono; Calbiochem), or 20 ng/ml TNF- RNA Extraction and Northern Analyses--
Total RNA was isolated
and analyzed by Northern hybridization as described (22). Complementary
DNA probes for human IL-2 (738 bp). IL-8 (289 bp), I Nuclear Extract Preparation and Electrophoretic Mobility Shift
Assays--
Jurkat T-cells were stimulated for 3 h, pelleted, and
washed, and cytosolic and nuclear extracts were prepared as described (12). Briefly, nuclear proteins were extracted from chromatin using 0.3 M (NH4)2SO4, and then
soluble nuclear proteins were precipitated using 1.5 M
(NH4)2SO4, followed by dialysis
into DNA binding buffer. Protein concentrations were determined by Bradford assay (Bio-Rad).
Transcription factor DNA binding activities in Jurkat T-cell nuclear
extracts were assayed using electrophoretic mobility shift assays
(EMSA). 10 µg of nuclear proteins were incubated for 30 min at
25 °C in 20 µl of binding buffer (25 mM HEPES, pH 7.6, 0.1 mM EDTA, 10% glycerol, 50 mM KCl, 0.05 mM dithiothreitol) containing 1-2 µg of poly(dI-dC) and
2.5 pg of 32P-labeled oligonucleotide probe (approximately
1 × 105 cpm). The sequences of oligonucleotide
probes used were agctAAAGAGGGACTTTCCCTAAA for the immunoglobulin Plasmids, Transfections, and Luciferase Reporter Gene
Assays--
Luciferase reporter gene constructs were under the control
of the IL-2 enhancer (nucleotides
Jurkat T-cells were transfected by electroporation with reporter and
expression plasmids as described (13, 26). T-cells were stimulated for
6-12 h (24 h after electroporation in the case of transient
transfections) and then cells were washed in phosphate-buffered saline
and pelleted by centrifugation. Cell pellets were resuspended in 50 µl of lysis buffer (1% Triton X-100, 0.1 mM HEPES, pH
7.6, 1 mM dithiothreitol, and 2 mM EDTA, pH
8.0) for 10 min at 4 °C, then the cell lysates were centrifuged at 13,000 rpm for 10 min. The supernatants were collected as whole cell
extracts, and the Bradford reagent (Bio-Rad) was used to measure
protein concentration. 20 µg of protein was mixed with 200 µl of
luciferase reaction mixtures (1 mg/ml bovine serum albumin, 5 mM ATP, pH 7.6, 25 mM glycylglycine, and 15 mM MgSO4) and 100 µl of 1 mM
D-luciferin (Analytical Luminescence Laboratory, San Diego,
CA). Triplicate determinations of luminescence were each read for
20 s using a MonolightTM 2010 luminometer (Analytical
Luminescence Laboratory), and were measured in relative light units
(RLU). In transient transfection experiments that incorporated the pEF
Renilla luciferase normalizing plasmid, 20-µl aliquots of whole cell
extracts were analyzed sequentially for firefly and Renilla luciferase
activities using a dual luciferase assay kit (Promega), and the ratio
of firefly to Renilla RLU was taken to represent the normalized
(firefly) luciferase activity.
Western Immunoblotting--
Cytosolic extracts (10 µg of
protein) were fractionated by SDS-polyacrylamide gel electrophoresis
(8% separating gel) and transferred to nitrocellulose membranes
(Schleicher and Schuell). I Data and Statistical Analysis--
Significance of the
differences between the experimental conditions were determined by
paired two sample Student's t test (Microsoft EXCEL). The
data presented are the means ± S.D.
PG490 Inhibits IL-2 Expression by Normal Human Peripheral Blood
Lymphocytes--
PG490 potently inhibits IL-2 expression by human PBLs
stimulated by PMA + anti-CD3 (PMA/ PG490 Inhibits Jurkat T-Cell Cytokine and Cytokine Regulator Gene
Expression Differently Than CsA--
Similar to the result in human
PBLs, we show that PG490 potently inhibits Jurkat T-cell expression of
IL-2 (Fig. 1B, panel a). Stimulation with
PMA/Iono bypasses membrane signaling events of T-cell activation (27)
and strongly induces IL-2 protein and mRNA expression (Fig.
1B, panels a and b, lane
6). PG490 at 20 ng/ml (56 nM) causes over 80%
inhibition of PMA/Iono-stimulated IL-2 protein and mRNA expression
(Fig. 1B, panel a and b, lane 7 versus lane 6). PG490 at 200 ng/ml more completely
suppresses Jurkat T-cell IL-2 protein and mRNA expression
stimulated by PMA and by PMA/Iono than 1,000 ng/ml of CsA (Fig.
1B, panels a and b, lanes 3 and 5 versus lane 2, and lanes 8 and
10 versus lane 6).
Using Northern hybridization analysis, we show that PG490 has distinct
effects on the mRNA expression of cytokine regulators I PG490 Inhibits IL-2 Gene Transcription through Mechanisms Different
Than CsA--
We next demonstrated that PG490 inhibits activation of
an IL-2 luciferase reporter gene transfected transiently and stably into Jurkat T-cells (Fig. 2), and this
result implies that PG490 inhibits IL-2 expression at the level of
transcriptional activation of the IL-2 gene.
We used the IL-2 luciferase assay to test the ability of PG490 to
inhibit the CsA-resistant pathway of T-cell activation achieved by
stimulation with PMA in combination with anti-CD28 monoclonal antibody
(18). We stimulated Jurkat T-cells that stably express the IL-2
luciferase reporter gene with either PMA + anti-CD28 antibody
(PMA/
To explore further the mechanistic differences between PG490 and CsA
and FK506 inhibition of IL-2 transcription, we tested the effects of
calcineurin overexpression on sensitivity to inhibition by these
immunosuppressant drugs (Fig. 2B) Overexpression of
calcineurin in Jurkat T-cells confers relative resistance to the
inhibitory effects of CsA and FK506 upon transcriptional activation of
the IL-2 gene (29, 30). We transiently cotransfected Jurkat T-cells with the IL-2 luciferase reporter plasmid together with either an empty
expression vector or with expression vectors encoding calcineurin A and
B subunits (Fig. 2B). The transfected T-cells were
stimulated with PMA/Iono in the presence of increasing doses of PG490
(Fig. 2B, left panel), CsA (Fig. 2B,
center panel), or FK506 (Fig. 2B, right
panel). There is no shift in the dose-inhibition curve of IL-2
transcription by PG490 conferred by overexpression of calcineurin (Fig.
2B, left panel). In contrast, overexpression of
calcineurin shifts the IC50 for CsA inhibition of IL-2
transcription from approximately 2 to 10 ng/ml (Fig. 2B,
center panel), and shifts the IC50 for FK506
inhibition of IL-2 transcription from approximately 0.2 to 1 ng/ml
(Fig. 2B, right panel). These results demonstrate
that PG490 inhibits IL-2 transcription through mechanisms distinct from
CsA and FK506, and which probably do not involve calcineurin.
PG490 Inhibits Transcriptional Activation and Also DNA Binding of
the Regulator of the Purine-box/ARRE/NF-AT Target Site in the IL-2
Enhancer--
After demonstrating that PG490 inhibits IL-2 expression
at the level of transcription, we next investigated the effects of PG490 on the induction of DNA binding activity and transcriptional activation of specific factors controlling the purine-box/ARRE/NF-AT and NF- PG490 Inhibits Transcriptional Activation and not DNA Binding of
NF-
At the level of NF-
Despite the development of a strong NF-
Although PMA stimulation alone activates NF-
PG490 nearly completely inhibits NF- PG490 Inhibits Activation through NF- PG490 Inhibits IL-8 Expression in 16HBE Human Bronchial Epithelial
Cells--
NF- PG490, which is pure triptolide, inhibits T-cell activation and
early cytokine gene transcription in T-cells and epithelial cells
through mechanisms that are different from CsA and FK506. PG490
inhibits transcriptional activation at the purine-box/ARRE/NF-AT and
NF- In previous studies of the T2 ethyl acetate extract of Tripterygium,
Tao et al. (9) inferred that the immunosuppressive properties of T2 are due to triptolide and tripdiolide and showed that
T2 completely suppressed mitogen-stimulated IL-2 and interferon- In our experiments, we studied PG490, which is 97% pure triptolide, in
contrast to the T2 ethyl acetate extract that contains approximately
5% triptolide. We found that PG490 potently inhibits IL-2 protein and
mRNA expression, and this inhibition occurs at the level of IL-2
transcription, and our results are consistent with the characterization
of T2 (10). Using an in vivo assay, we found that
overexpression of calcineurin in Jurkat T-cells does not alter
sensitivity to inhibition by PG490 while conferring a 5-fold relative
resistance to the inhibitory effects of both CsA and FK506. This result
establishes that PG490 inhibits IL-2 transcription through mechanisms
distinct from CsA and FK506 and which likely do not involve
calcineurin. In addition, we show that PG490 but not CsA inhibits IL-2
transcription stimulated by PMA and monoclonal antibody YTH913.12 to
the costimulatory receptor, CD28. In contrast to our results, Tao
et al. (10) found that a maximal dose of T2 (2 µg/ml) was
unable to inhibit IL-2 secretion by purified T-cells stimulated with
PMA + monoclonal antibody 9.3 to CD28. We believe that this difference
in results can most likely be attributed to our use of pure triptolide
compared with the use of the T2 mixture (10), which contains only a
fraction of triptolide. The other compounds present in T2 may be
physiologically inactive, in which case their presence might simply
limit the dose availablity of triptolide; alternatively, other
compounds present in T2 might modulate the immunosuppressive activities of T2 so that it becomes ineffective in comparison to pure triptolide in inhibiting T-cell activation through costimulatory receptors.
The principal regulators of IL-2 transcriptional activation operate at
the purine-box/ARRE/NF-AT and NF- NF- We extended our analysis of PG490 inhibition of NF- From our results, we have developed a model for the potential
mechanisms of PG490 inhibition of transcriptional activation at the
NF-B site. PG490 can
completely inhibit transcriptional activation at the
purine-box/ARRE/NF-AT and NF-
B target DNA sequences triggered by all
stimuli examined (PMA, PMA/Iono, tumor necrosis factor-
). PG490 also
inhibits PMA-stimulated activation of a chimeric transcription factor
in which the C-terminal TA1 transactivation domain of NF-
B p65 is
fused to the DNA binding domain of GAL4. In 16HBE human bronchial
epithelial cells, IL-8 expression is regulated predominantly by
NF-
B, and PG490 but not cyclosporin A can completely inhibit expression of IL-8. The mechanism of PG490 inhibition of cytokine gene
expression differs from cyclosporin A and involves nuclear inhibition
of transcriptional activation of NF-
B and the purine-box regulator
operating at the ARRE/NF-AT site at a step after specific DNA binding.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxy system, which is important for
biologic activity, and proposed that this system may be involved in
selective alkylation by nucleophilic groups such as thiols present in
key target enzymes involved in growth regulation.
B, AP-1, and Oct-1
(reviewed in Ref. 11). The T-cell immunosuppressants, CsA and FK506,
inhibit transcription of the IL-2 gene through mechanisms that may
involve the serine/threonine protein phosphatase, calcineurin (reviewed in Ref. 11).
B (17) and is largely resistant
to inhibition by CsA (17, 18). The activation of transcription by
NF-
B involves stimulation-induced degradation of I
B, which serves
to release NF-
B p65 for translocation from the cytoplasm into the
nucleus. In the nucleus, p65 binds as a heterodimer to its target DNA
sequence and then interacts with transcriptional regulatory components
to signal initiation of transcription by RNA polymerase II (reviewed in
Ref. 19). NF-
B signaling is regulated by phosphorylation in the
cytoplasm at the level of I
B in the nucleus at the level of specific
DNA binding and also at the level of transcriptional activation
(reviewed in Ref. 20).
B at a step after
specific binding to DNA.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(BIOSOURCE) in the presence of PG490, CsA
(Sandoz), or FK506 (Fujisawa).
B
(883 bp),
NF90 (2,008 bp), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
0.6-kilobase XbaI-HindIII fragment of cDNA) were labeled with [
-32P]dCTP using a random hexamer
labeling kit (Stratagene).
light chain NF-
B site (19), and aagaAAGGAGGAAAAACTGTTTCATA (
259 to
284 in the human IL-2 enhancer) for the
purine-box/NF-AT site (11). Probes were labeled by filling in
overhanging ends (identified by lowercase letters) using Klenow DNA
polymerase (New England Biolabs) and [
-32P]dCTP
(Amersham Pharmacia Biotech), and nonradioactive dGTP, dATP, and dTTP.
Protein-DNA complexes were resolved from free probe using 4%
nondenaturing polyacrylamide gels in 0.5× Tris borate EDTA (pH 8.3)
and visualized by fluorography.
326 to +48, pCLN15deltaCX, prepared by D. Durand, Stanford University), or three copies of the
purine-box/NF-AT regulatory sequence sequence (
285 to
255 of the
human IL-2 enhancer) in the context of the minimal IL-2 promoter, or
the immunoglobulin
light chain NF-
B sequence monomer in the
context of the minimal IL-8 promoter (
45 to +40 of the human IL-8
promoter). These plasmids also contain a neomycin resistance gene under
control of the constitutively active SV40 promoter, and this allows
G418 antibiotic (Life Technologies, Inc.) selection of cell lines that
stably express the luciferase reporter constructs. The GAL4-luciferase
reporter contains five copies of the GAL4 target DNA sequence upstream
of the minimal IL-2 promoter and was prepared by J. Riegel at Stanford
University. For normalizing the transient transfection assays, plasmid
pEF Renilla luciferase was generated by cloning the elongation factor 1
promoter (23) into the pRL null vector (Promega) between the
EcoRI and HindIII sites. Expression plasmids pEF
CNA and pEF CNB contain the elongation factor 1
promoter (23)
upstream of rat calcineurin A and calcineurin B cDNAs (24).
Expression plasmids GAL4p65TA1 and GAL4p65{TA1+TA2} utilize the
Rous sarcoma virus promoter to drive expression of a chimeric protein
with the GAL4 DNA binding domain (amino acids 1-147) fused to the p65 transactivating domains and were generated by Schmitz and Baeuerle (25)
and obtained through A. Baldwin (University of North Carolina, Chapel Hill).
B
was detected using rabbit polyclonal
IgG primary antibody (Santa Cruz Biotechnology) at 1:500 dilution for
2 h at 37 °C and horseradish peroxidase-conjugated goat
anti-rabbit secondary antibody at 1:3,000 dilution for 1 h at room
temperature. Detection was with enhanced chemiluminsescence, according
to the manufacturer's directions (Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CD3, Fig.
1A, rows 5-8
versus row 4), and by PMA + ionomycin (Fig.
1A, rows 15-17 versus row 14). The
IC50 for PG490 inhibition of PMA/
CD3-stimulated IL-2
expression is approximately 10 ng/ml (28 nM). PG490 at 200 ng/ml (560 nM) causes more complete inhibition of
PMA/
CD3-stimulated IL-2 expression than CsA at 1,000 ng/ml (832 nM, Fig. 1A, row 7 versus
row 11). Ionomycin stimulation alone causes minimal
induction of IL-2 expression, which is inhibited by the lowest dose of
4 ng/ml PG490 (Fig. 1A, rows 12 and
13). The IC50 for PG490 inhibition of
PMA/Iono-stimulated IL-2 expression is approximately 40 ng/ml (112 nM).
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Fig. 1.
PG490 inhibition of T-cell expression of
IL-2. A, PG490 inhibits IL-2 expression by normal human
peripheral blood lymphocytes (PBL) stimulated with PMA,
CD3, and with P/I. B, PG490 inhibits Jurkat T-cell
expression of IL-2 protein (panel a), IL-2 mRNA
(panel b) and I
B
mRNA (panel c), and
increases expression of NF90 mRNA (panel d) with no
changes of GAPDH mRNA (panel e). Normal human peripheral
blood lymphocytes or Jurkat T-cells were stimulated for 12 h with
PMA (P) (20 ng/ml), anti-CD3 (
CD3, clone HIT3a, 10 µg/ml in solution, Pharmingen), ionomycin (I) (2 µM), P/
CD3, or P/I in the presence of the indicated
concentrations of PG490 or CsA. Cell supernatants were assayed for IL-2
by ELISA (Immunotech, Westbrook, ME), and Jurkat total cellular RNA was
fractionated and analyzed by Northern hybridization. The PBL-IL-2-ELISA
data represent the means ± S.D. from four independent
experiments; statistically significant inhibitions compared with no
drug treatment are indicated (*p < 0.05;
**p < 0.01).
B
and
NF90 (Fig. 1B, panels c and d). We
observe constitutive expression of I
B
mRNA (28) (Fig.
1B, panel c, lane 1), and this
expression is further stimulated by PMA and PMA/Iono (Fig. 1B, panel c, lanes 2 and
6). PG490 potently inhibits I
B
mRNA expression
(Fig. 1B, panel c, lanes 3,
4, and 7-9), whereas 1,000 ng/ml CsA
shows only limited inhibition (Fig. 1B, panel c,
lanes 5 and 10). In contrast, mRNA expression
for NF90, a transcriptional regulator of IL-2 gene expression (13), is
significantly induced in the presence of 200 and 1,000 ng/ml PG490
(Fig. 1B, panel d, lanes 3 and
4 versus lane 2 and lanes 8 and 9 versus lane 6). Finally, the levels of
GAPDH mRNA are unaltered in response to stimulation and drug
treatment (Fig. 1B, panel e). Our observations that PG490 causes more complete inhibition of IL-2 expression than CsA
and that PG490 but not CsA inhibits I
B
mRNA expression and
enhances NF90 mRNA expression establish that PG490 modulates gene
expression through distinctly different mechanisms than CsA.
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Fig. 2.
PG490 inhibits IL-2 transcriptional
activation through mechanisms different from CsA. A,
PG490 but not CsA inhibits T-cell activation stimulated through
costimulatory receptor CD28. Jurkat T-cells that stably
express an IL-2 luciferase reporter plasmid were stimulated for 15 h with either P/I or P+ CD28 monoclonal antibody (YTH913.12,
BIOSOURCE) then whole cell extracts were assayed
for luciferase activity. The mean luciferase activity in nonstimulated
cells was ~2,000 RLU, in P/I-stimulated cells was ~76,000 RLU, and
in P/
CD28-stimulated cells was ~3,400 RLU. For each independent
experiment, the P/I-stimulated and the P/
CD28-stimulated luciferase
values with zero drug present were set as 100% (these values are not
plotted because the x axis is on a log scale). The data
shown are the means ± S.D. from three independent experiments.
B, PG490 inhibition of T-cell activation is unaffected by
overexpression of calcineurin. Jurkat T-cells were transiently
transfected with an IL-2 luciferase reporter construct, together with
either empty expression vector (filled squares), or with
expression vectors directing synthesis of calcineurin A and calcineurin
B (empty squares). 20 h following transfection, cells
were stimulated with P/I for 6 h in the presence of PG490, CsA, or
FK506 at the indicated doses then whole cell extracts were prepared for
analysis of luciferase activity. The mean luciferase activity for the
nonstimulated condition was 300 RLU and for the P/I-stimulated
condition was ~300,000 RLU. For each experiment, the P/I-stimulated
value with zero drug present was set at 100% (this point is not
plotted because the x axis is on a log scale). The data
shown are the means ± S.D. from three independent experiments.
Statistically significant differences in IL-2 transcription at the same
doses of CsA or FK506 following transfection of calcineurin expression
plasmids are indicated (*p < 0.05; **p < 0.01).
CD28), or with PMA/Iono, each in the presence of PG490 or CsA
(Fig. 2A). For T-cells stimulated with PMA/Iono (Fig. 2A, left panel), the IC50 is
approximately 20 ng/ml (56 nM) for PG490 and approximately
3 ng/ml (2.5 nM) for CsA inhibition of IL-2 transcription.
In contrast, for T-cells stimulated with PMA/
CD28 (Fig.
2B, right panel), the IC50 is
approximately 50 ng/ml for PG490 inhibition of IL-2 transcription, and
CsA is completely ineffective in inhibiting this pathway of
stimulation. At the two lower doses of PG490 but not of CsA, we
observed modest increases in IL-2 luciferase activity before inhibition
occurs at the higher doses. These results demonstrate that although CsA
is more potent than PG490 in inhibiting PMA/Iono-stimulated IL-2
luciferase activity, PG490 is capable of inhibiting T-cell activation
triggered through a costimulatory receptor, a situation in which CsA is ineffective.
B target sequences in the IL-2 enhancer (reviewed in Ref. 11). We used EMSA to analyze the effects of PG490 on the
PMA/Iono-induced Jurkat T-cell purine-box/ARRE/NF-AT DNA binding
activity (Fig. 3A). PG490
inhibits the induction of the purine-box/ARRE·EMSA complex with an
IC50 slightly below 200 ng/ml (Fig. 3A,
lane 4 versus lane 2). At 1,000 ng/ml
PG490, the PMA/Iono-induced purine-box/ARRE·EMSA complex is
undetectable (Fig. 3A, lane 5) and is therefore
weaker than the EMSA complex constitutively present in nonstimulated cells (Fig. 3A, lane 1), and weaker than the
complex induced in the presence of 1,000 ng/ml CsA (Fig. 3A,
lane 6). PG490 is more potent in inhibiting
PMA/Iono-stimulated transcriptional activation of a purine-box/NF-AT
luciferase reporter gene with an IC50 of approximately 20 ng/ml (Fig. 3B, lane 3 versus lane
2). Taken together, these results demonstrate that PG490
inhibits both transcriptional activation and also DNA binding of the
regulator operating at the purine-box/ARRE/NF-AT target sequence and
that the predominant site of signaling inhibition is at the level of
transcription after specific binding to DNA.
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Fig. 3.
PG490 inhibits purine-box/NF-AT
transcriptional activation and induced purine-box/ARRE/NF-AT DNA
binding. A, EMSA analysis showing inhibition of induced
purine-box/ARRE/NF-AT DNA binding activity by PG490. Jurkat T-cells
were stimulated for 3 h with P/I in the presence of the indicated
concentrations of PG490 then nuclear extracts were prepared, and DNA
binding activities were analyzed by EMSA. B, inhibition of
purine-box/NF-AT transcriptional activation by PG490. Jurkat T-cells
that stably express a purine-box/NF-AT luciferase reporter construct
were stimulated for 6 h with P/I in the presence of the indicated
doses of PG490. The mean luciferase activity in nonstimulated cells was
550 RLU and in P/I-stimulated cells was ~1,100,000 RLU. The data
shown are the means ± S.D. from three independent experiments.
Statistically significant inhibitions compared with no drug treatment
are indicated (*p < 0.05; **p < 0.01). Pu, purine.
B--
We investigated the effects of PG490 on NF-
B signaling
in Jurkat T-cells (Fig. 4). We show
constitutive expression of the NF-
B p65 anchoring protein, I
B
,
in the cytoplasm of nonstimulated Jurkat T-cells (Fig. 4A,
lane 1), and I
B
expression decreases following
stimulation with PMA (Fig. 4A, lane 2 versus lane 1). PG490 at 200 and 1,000 ng/ml causes nearly
complete inhibition of I
B
protein expression in PMA-stimulated
T-cells (Fig. 4A, lanes 4 and 5), and
this result correlates with PG490 inhibition of I
B
mRNA
expression (Fig. 1B, panel c, lanes 3 and 4).
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Fig. 4.
PG490 inhibits NF- B
transcriptional activation with no inhibition of induced
NF-
B DNA binding. A, Western
immunoblot of cytoplasmic I
B
expression following PMA stimulation
and effects of PG490. B, EMSA analysis of nuclear NF-
B
DNA binding activity following PMA stimulation and effects of PG490.
Jurkat T-cells were stimulated with PMA (20 ng/ml) for 3 h in the
presence of the indicated concentrations of PG490 or CsA and then
cytoplasmic and nuclear extracts were prepared and used for Western and
EMSA analyses. Supershift analyses were performed by the addition of 2 µl of IgG against NF-
B p65 (sc-109, Santa Cruz Biotechnology).
C, inhibition of PMA-stimulated NF-
B transcriptional
activation by PG490. The mean luciferase activity per 20 µg of
protein in NS cells was 34,500 RLU and ~1,320,000 RLU in
PMA-stimulated cells. D, EMSA analysis of nuclear NF-
B
DNA binding activity following P/I stimulation and effects of PG490.
The nuclear extracts analyzed are the same as in Fig. 3A and
were prepared from Jurkat T-cells stimulated for 3 h with
PMA/Iono. E, inhibition of PMA/Iono-stimulated NF-
B
transcriptional activation by PG490. The mean luciferase activity per
20 µg of protein in NS cells was 60,595 RLU and ~2,439,652 RLU in
PMA/Iono-stimulated cells. F, inhibition of
TNF-
-stimulated NF-
B transcriptional activation by PG490. The
mean luciferase activity per 20 µg of protein in NS cells was 82,147 RLU and ~351,202 RLU in TNF-
-stimulated cells. For the
NF-
B-luciferase experiments in C, E, and
F, Jurkat T-cells that stably express an NF-
B luciferase
reporter construct were stimulated for 6 h either with PMA (20 ng/ml), P/I (2 µM), or TNF-
(20 ng/ml) in the presence
of the indicated doses of PG490 or CsA. The data shown are the
means ± S.D. from three independent experiments. Statistically
significant inhibitions compared with no drug treatment are indicated
(*p < 0.05; **p < 0.01).
B DNA binding, stimulation of Jurkat T-cells with
PMA induces the appearance of a new band (NF-
B complex, Fig.
4B, lanes 2-6). The presence of the NF-
B p65
subunit within this complex is established by the quantitative
supershift of the inducible complex with an antibody to p65 (Fig.
4B, lanes 7-12). PG490 at 20 ng/ml causes no
significant effect on the NF-
B·EMSA complex (Fig. 4B,
lane 3 versus lane 2), and PG490 at
200 and 1,000 ng/ml causes a significant increase in the strength of
the NF-
B complex (Fig. 4B, lanes 4 and
5 versus lanes 2 and 10,
lane 11 versus lane 8). We propose that the
increase in NF-
B DNA binding activity which we observe at 200 and
1,000 ng/ml PG490 occurs as a consequence of PG490 inhibition of
I
B
expression, which likely allows increased nuclear
translocation of p65.
B DNA binding complex in the
nucleus of T-cells stimulated with PMA in the presence of 200 ng/ml
PG490 (Fig. 4B, lane 4), we observe nearly 90%
inhibition of transcriptional activation of an NF-
B luciferase
reporter gene (Fig. 4C, lane 4). CsA at 1,000 ng/ml shows no significant inhibition of PMA-stimulated NF-
B
transcription (Fig. 4C, lane 6), which is
expected, because CsA acts predominantly to inhibit calcium-mediated
activation pathways. The inhibitory effects of PG490 on
NF-
B-mediated transcriptional activation were identical using stably
or transiently transfected cells, or when using a consensus
immunoglobulin
light chain NF-
B binding site monomer in the
context of the minimal IL-8 promoter, or the IL-2 NF-
B site trimer
in the context of the minimal IL-2 promoter (data not shown).
B DNA binding and
transcription (Fig. 4, B and C), substantial
expression of IL-2 mRNA and protein requires the additional calcium
mobilization achieved following ligation of the T-cell receptor (Fig.
1A) or with ionophore (Fig. 1, A and
B). We, therefore, also examined the effects of PG490 upon
NF-
B signaling triggered with PMA + ionomycin (Fig. 4, D
and E). Similar to the results observed with PMA
stimulation, PMA/Iono stimulation causes the induction of specific
nuclear NF-
B DNA binding activity (Fig. 4D, lanes
2 and 8), which increases when cells are stimulated in
the presence of PG490 (Fig. 4D, lanes 3-5 and
9-11). Again, a specific antibody to NF-
B p65
causes a quantitative supershift of the inducible DNA binding complex
(Fig. 4D, lanes 7-12). Cells stimulated with PMA/Iono in the presence of CsA show much less induction of NF-
B DNA
binding activity than cells stimulated in the presence of PG490 (Fig.
4D, lane 6 versus lanes 3-5),
underscoring the different mechanisms of action of these
immunosuppressant drugs. Despite the development of strong NF-
B DNA
binding activity, PG490 causes near complete inhibition of
PMA/Iono-stimulated NF-
B transcriptional activation with an
IC50 of approximately 50 ng/ml (Fig. 4E,
lanes 3-5). Transcriptional activation of NF-
B
stimulated by TNF-
is also inhibited by PG490, with an
IC50 of approximately 50 ng/ml (Fig. 4F,
rows 3-6).
B-mediated transcription
triggered by all stimuli tested (PMA, PMA/Iono, TNF-
, and IL-1
data not shown). Taken together, our results demonstrate that PG490
inhibits NF-
B transcriptional activation at a step after specific
binding to DNA.
B Transactivation Domains
TA1 and TA2--
Because PG490 inhibits NF-
B transcriptional
activation without inhibiting nuclear NF-
B DNA binding activity
(Fig. 4, B-E), we next investigated whether
PG490 can inhibit transcriptional activation through a chimeric
transcription factor in which the transactivating domain of p65 is
fused to the yeast GAL4 DNA binding domain. Schmitz and Baeuerle (25)
identified that the C-terminal portion of NF-
B p65 contains two
transactivation domains, TA1 and TA2, and showed that chimeric fusion
proteins with the TA1 (p65 amino acids 522-551) or TA1 + TA2 (p65
amino acids 286-521) domains fused to the DNA binding domain of GAL4
(amino acids 1-147) confer PMA-inducible transcriptional activation
onto a GAL4 reporter gene (25, 31). We transiently transfected Jurkat
T-cells with a GAL4 response element-luciferase reporter construct,
together with expression constructs encoding either the GAL4 DNA
binding domain alone (GAL4DB), GAL4DB-p65TA1, or GAL4DB p65(TA1+ TA2). After recovery from the transfection, the cells were stimulated with
PMA, in the presence of increasing doses of PG490. The expression construct for GAL4DB alone shows no transactivation of the GAL4 response element (GAL4RE, data not shown), demonstrating that Jurkat
T-cells contain no endogenous activators of the GAL4RE reporter. There
is constitutive transcription by the chimeric transcription factor,
GAL4DB-p65 TA1, and this transcription is enhanced approximately
1.6-fold following stimulation with PMA, and the PMA enhancement of
transcription is inhibited by PG490 (Fig.
5, panel a). We observed a
greater signal with a similar pattern of regulation using the chimeric
transcription factor GAL4DB p65(TA1 + TA2); the constitutive
transcription is enhanced approximately 2.3-fold following stimulation
with PMA, and the PMA enhancement of transcription is inhibited by
PG490 (Fig. 5, panel b). Notably, PG490, but not CsA,
inhibits transcription through the C-terminal transactivation domains
TA1 and TA1 + TA2 of p65 (Fig. 5, panels a and
b). The IC50 for PG490 inhibition of the
chimeric transcription factors is approximately 50 ng/ml, similar to
the IC50 for PG490 inhibition of endogenous NF-
B
activity (Fig. 4, C, E, and F).
View larger version (25K):
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Fig. 5.
PG490 inhibits activation through chimeric
transcription factors
GAL4DB-NF- BTA1 and
GAL4DB-NF-
B{TA1+TA2}. Jurkat T-cells were transiently
transfected with 2 µg of GAL4RE luciferase reporter plasmid and 4 µg of expression plasmids for either GAL4DB-p65 TA1 or
GAL4DB-p65{TA1+TA2}. In panel a, cells were also
cotransfected with 1 µg of pEF Renilla luciferase plasmid to
normalize for transfection efficiency. 20 h after transfection,
cells were stimulated for 6 h with PMA then whole cells extracts
were assayed for luciferase activity (panel b); in
panel a, both firefly and Renilla luciferase were assayed
using a dual luciferase assay kit (Promega). In panel a, the
mean normalized luciferase activity in NS cells was 6,600 RLU and
~9,600 RLU in PMA-stimulated cells; in panel b, the mean
luciferase activity per 20 µg of protein in NS cells was 258,000 RLU
and ~600,000 RLU in PMA-stimulated cells. The data shown represent
the means ± S.D. from five (panel a) and three
(panel b) independent experiments. Statistically significant
inhibitions of the PMA-stimulated condition by PG490 are indicated
(*p < 0.05; **p < 0.01).
B is important for transcriptional activation of
cytokine genes such as IL-8 in nonlymphoid cells including epithelial and endothelial cells (32). We therefore examined the effects of PG490
on expression of IL-8 protein and mRNA in 16HBE transformed human
bronchial epithelial cells (Fig. 6).
PG490 at 20 ng/ml inhibits PMA-stimulated IL-8 protein and mRNA
expression more effectively than CsA at 1,000 ng/ml (Fig. 6,
panels a and b, lane 3 versus lane 5), and PG490 at 200 and 1,000 ng/ml inhibits
PMA/Iono-stimulated IL-8 expression more effectively than 1,000 ng/ml
CsA (Fig. 6, lanes 8 and 9 versus
lane 10). These results demonstrate that PG490 inhibits
inflammatory cytokine gene expression in epithelial cells, as well as
in lymphoid cells, and the mechanism of inhibition is distinctly
different from CsA and probably involves transcriptional inhibition of
NF-
B.
View larger version (49K):
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Fig. 6.
PG490 inhibits bronchial epithelial cell IL-8
expression. 16HBE cells were treated for 6 h with the
indicated stimulants and immunomodulating drugs then secreted IL-8 was
analyzed by ELISA (Immunotech), and total RNA was prepared and analyzed
for IL-8 and GAPDH mRNA expression by Northern hybridization. The
ELISA data are the means ± S.D. from four independent
experiments; statistically significant inhibitions by PG490 or CsA are
indicated (*p < 0.001).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B target sequences after specific binding to DNA.
expression in T-cells but showed only limited inhibition of IL-6, IL-2
receptor, and transferrin receptor expression (10). IL-2 expression was
inhibited by T2 at the level of transcription, as revealed by an IL-2
reporter gene assay in Jurkat T-cells (10). The in vitro
phosphatase activity of calcineurin was not significantly inhibited by
immunosuppressive doses of T2, triptolide, or tripdiolide (10).
B target DNA sequences (11). We
demonstrate that PG490 inhibits purine-box/ARRE/NF-AT signaling both at
the level of transcriptional activation and also at the level of
specific binding to DNA. The IC50 for PG490 inhibition of
PMA + ionomycin(P/I)-stimulated purine-box/NF-AT transcriptional
activation is ~20 ng/ml, whereas the IC50 for PG490
inhibition of the induced specific purine-box/ARRE/NF-AT DNA binding
complex is ~200 ng/ml. From this result, we conclude that PG490
predominantly inhibits purine-box/NF-AT signaling at the level of
transcriptional activation after specific binding to DNA.
B signaling involves stimulation-induced degradation of
cytoplasmic I
B (19, 33), releasing p65 for translocation from the
cytoplasm into the nucleus, where p65 interacts with p50 and binds
specifically to the NF-
B target DNA sequence. Following specific
binding to DNA, transcriptional activation of NF-
B is regulated
through specific phosphorylation of p65 at several distinct sites
(34-37). We found that PG490 potently inhibits the expression of
I
B
mRNA and protein, and these decreases in I
B
protein likely serve to increase the amount of p65 released for translocation into the nucleus and specific binding to DNA. Although PG490 causes an
increase in nuclear NF-
B DNA binding activity, PG490 inhibits NF-
B transcriptional activation at a step after specific binding to
DNA. The I
B
enhancer contains at least two potential binding sites for NF-
B (38). We hypothesize that PG490 inhibits I
B
mRNA and protein expression through transcriptional inhibition of
the I
B
gene, probably at these NF-
B target sites.
B transcriptional
activation by demonstrating that PG490 inhibits the PMA-stimulated
activation of chimeric transcription factors containing the NF-
B p65
TA1 and TA2 transactivation domains. PG490 inhibits PMA-stimulated
transcriptional activation through the TA1 domain, which is highly
conserved between humans, mice, and Xenopus, and extends
from amino acid 521 to the C terminus at amino acid 551 in humans. This
result implies that a target of PG490 inhibition resides within these
31 amino acids. Within TA1 there are 7 serines, which are predicted to
align on one side of an
helix and form a polar region believed to
act similar to acidic transcriptional activators (39). These serine
residues are potential phosphorylation sites for a p65 regulatory
kinase stimulated by PMA. Ostrowski et al. (40) identified a
serine kinase activity that existed in the nucleus and that copurified
with p65 through two sequential ion-exchange purifications. TNF-
stimulates phosphorylation of p65 on Ser-529 within the TA1 domain, and
this phosphorylation contributes to the regulation of transcriptional
activation (37). Kinases that have been shown to be involved in the
regulation of NF-
B transcriptional activation include protein kinase
A (34), p38 mitogen-activated protein kinase (35), and casein kinase II
(36), although none of these kinases have been demonstrated to
phosphorylate p65 within the TA1 domain. Nuclear coactivator proteins,
cAMP response element-binding protein and p300, have been shown to
interact with the TA1 + TA2 domains of p65 (41) and contribute to
NF-
B transcriptional activation (41, 42).
B and purine-box/NF-AT sites (Fig.
7). We present the NF-
B heterodimer
interacting with a consensus NF-
B target regulatory sequence, and
the purine-box transcriptional regulator interacting with the
purine-box/ARRE/NF-AT target sequence in the IL-2 enhancer. Following
stimulation of cells with PMA, PMA + ionomycin, TNF, or other
NF-
B-activating stimuli, there is induced degradation of I
B,
releasing p65 for translocation from the cytoplasm into the nucleus.
Once in the nucleus, p65 combines with p50, and the p50-p65 heterodimer
binds specifically to the NF-
B target DNA sequence. We postulate
that PG490 inhibition of NF-
B and purine-box/NF-AT transcriptional
activation after specific DNA binding involves inhibition of regulated
phosphorylation of the transactivation domains (TA) of each
transcriptional regulator by a nuclear kinase, which we designate the
TA kinase (Fig. 7). Because PG490 at higher doses also acts to inhibit
the induced DNA binding activity of the purine-box regulator binding to
the ARRE/NF-AT target sequence, we hypothesize that there is a
PG490-sensitive nuclear phosphorylation step that regulates specific
DNA binding of the purine-box regulator (Fig. 7). The effects of PG490
in inhibiting transcriptional activation in the nucleus after specific
binding of NF-
B to DNA might also involve interference with
recruitment of coactivator proteins cAMP response element-binding
protein /p300 or inhibition of interactions between p65 and RNA
polymerase II (Fig. 7).
View larger version (26K):
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Fig. 7.
Schematic showing postulated mechanisms of
PG490 inhibition of transcriptional activation at the
NF- B and purine-box/ARRE/NF-AT target DNA
sequences. T-cells stimulated at the antigen receptor and through
costimulatory receptors initiate signaling pathways that lead to
transcriptional activation at the purine-box/ARRE/NF-AT and NF-
B
target sequences in the IL-2 enhancer. The consensus NF-
B target DNA
sequence from the mouse Ig
light chain is shown. PG490 inhibits
NF-
B transcriptional activation at a step after nuclear
translocation of p65 and specific binding of the p50-p65 heterodimer to
DNA. PG490 also inhibits purine-box/NF-AT transcriptional activation
more potently than it inhibits induced purine-box/ARRE/NF-AT DNA
binding activity. A nuclear kinase that regulates transactivation of
NF-
B and the purine-box regulator (TA kinase), which is a potential
target of PG490 inhibition, is shown. Other potential sites of PG490
inhibition of transcriptional activation in the nucleus include
recruitment of transcriptional coactivators cAMP response
element-binding protein or p300 to p65 or interactions between p65 and
the purine-box regulator and RNA polymerase II.
Our identification that PG490 effectively inhibits T-cell activation
and IL-2 gene expression triggered through pathways that are resistant
to CsA suggest therapeutic utility for PG490 as an immunosuppressant
capable of treating chronic allograft rejection and also graft
versus host disease. Furthermore, signaling through members
of the TNF receptor superfamily serves to simultaneously activate
pathways of inflammation involving NF-B and apoptosis involving
caspases. The balance of survival over death can be influenced by the
degree of activation of NF-
B (43, 44). The antiinflammatory effects
of PG490 involve inhibition of NF-
B transcriptional activation after
specific binding to DNA, and this inhibition of NF-
B activation
probably contributes to the pro-apoptotic effects observed with PG490
(45, 46).
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ACKNOWLEDGEMENTS |
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We thank Ed Lennox, Glenn D. Rosen, and T. W. Wiedmann for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R01-AI39624 and K04-01147 (to P. N. K.), and gifts from the Donald E. and Delia B. Baxter Foundation and Pharmagenesis.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 650-725-0570;
Fax: 650-725-5489; E-mail: peterkao{at}leland.stanford.edu.
2 J. Fidler, Pharmagenesis, Palo Alto, CA, unpublished data.
3 N. Chao, Duke University, and J. Fidler, Pharmagenesis, Palo Alto, CA, unpublished data.
4 J. Fidler and R.-L. Jin, Pharmagenesis, private communication.
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ABBREVIATIONS |
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The abbreviations used are: CsA, cyclosporin A; ARRE, antigen receptor response element; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde phosphate dehydrogenase; IL, interleukin; Iono, ionomycin; NF-AT, nuclear factor of activated T-cells; TNF, tumor necrosis factor; PG490, Pharmagenesis 490, 97% pure triptolide; PMA, phorbol 12-myristate 13-acetate; PBL, peripheral blood lymphocytes; bp, base pair(s); RLU, relative light units; IFN, interferon; P/I, PMA + ionomycin; ELISA, enzyme-linked immunosorbent assay; NS, nonstimulated.
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REFERENCES |
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