Pulmonary and Critical Care Medicine, Stanford University Medical Center, Stanford, California 94305-5236
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ABSTRACT |
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Triptolide (PG490, 97% pure) is a diterpenoid
triepoxide with potent anti-inflammatory and immunosuppressive effects
in transformed human bronchial epithelial cells and T cells (Qiu D,
Zhao G, Aoki Y, Shi L, Uyei A, Nazarian S, Ng JC-H, and Kao PN.
J Biol Chem 274: 13443-13450, 1999). Triptolide,
with an IC50 of ~20-50 ng/ml, inhibits normal and
transformed human bronchial epithelial cell expression of interleukin
(IL)-6 and IL-8 stimulated by phorbol 12-myristate 13-acetate (PMA),
tumor necrosis factor-, or IL-1
. Nuclear runoff and luciferase
reporter gene assays demonstrate that triptolide inhibits IL-8
transcription. Triptolide also inhibits the transcriptional activation,
but not the DNA binding, of nuclear factor-
B. A cDNA
array and clustering algorithm analysis reveals that triptolide
inhibits expression of the PMA-induced genes tumor necrosis factor-
,
IL-8, macrophage inflammatory protein-2
, intercellular adhesion
molecule-1, integrin
6, vascular endothelial growth factor, granulocyte-macrophage colony-stimulating factor, GATA-3, fra-1, and NF45. Triptolide also inhibits constitutively expressed cell
cycle regulators and survival genes cyclins D1, B1, and A1, cdc-25,
bcl-x, and c-jun. Thus anti-inflammatory, antiproliferative, and proapoptotic properties of triptolide are associated with inhibition of nuclear factor-
B signaling and inhibition of genes known to regulate cell cycle progression and survival.
Tripterygium; interleukin-8; transcription; nuclear
factor-B; Atlas cDNA array; cyclin
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INTRODUCTION |
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BRONCHIAL EPITHELIAL CELL expression of inflammatory cytokines and growth factors contributes to the airway inflammation that is characteristic of asthma, chronic bronchitis, and bronchiectasis (reviewed in Ref. 21). Structural changes can develop in airways because of the chronic inflammation in cystic fibrosis or atypical mycobacterial pulmonary disease (11) or because of chronic allograft rejection after lung transplantation, which can manifest as obliterative bronchiolitis. Suppression of inappropriate airway inflammation by drugs delivered systemically or topically may provide therapeutic benefits in diseases such as asthma and chronic bronchitis and may help to reduce the airway destruction that occurs in bronchiectasis and obliterative bronchiolitis.
Extracts of the Chinese herb Tripterygium wilfordii exhibit potent immunosuppressive and anti-inflammatory properties and are effective traditional Chinese remedies for rheumatoid arthritis, systemic lupus erythematosis, and other autoimmune diseases. The active compounds in the extracts of Tripterygium are all structurally related to the diterpenoid triepoxide called triptolide. Triptolide possesses antileukemic activities (12) and inhibits proliferation of transformed cell lines (20, 24). A chloroform-methanol extract of Tripterygium, T2, inhibits expression of interleukin (IL)-2 and immunoglobulin by peripheral blood lymphocytes (22). A refined extract of Tripterygium, PG27, which contains PG490 (Pharmagenesis 490, 97% pure triptolide) as its active ingredient, is an effective immunosuppressant that prolongs heart and kidney allograft survival in rat transplantation models (Fidler J, unpublished data). In addition, PG27 effectively prevents graft versus host disease in a murine model of allogeneic bone marrow transplantation (8).
Qui et al. (18) recently described the potent inhibition
of T cell activation and IL-2 gene expression by triptolide.
Triptolide inhibits the transcriptional activation of the IL-2 gene by
inhibiting activation of the purine-box regulator of the nuclear factor
of activated T cells (NFAT) target DNA sequence in the IL-2 enhancer and by inhibiting nuclear factor-B (NF-
B) activation. Qui et al.
(18) showed that triptolide inhibits T cell activation
through non-calcineurin-dependent mechanisms and that triptolide
inhibits T cell activation triggered through cyclosporin A
(CsA)-resistant signaling initiated at the costimulatory receptor CD28.
In Jurkat T cells and A549 cells (non-small cell lung cancer), our
laboratory demonstrated (13, 18) that triptolide inhibits
transcriptional activation, but not DNA binding, of NF-
B. These
results led us to propose that triptolide exerts its immunosuppressive
effects in the nucleus by inhibiting posttranslational modifications of transcription factors such as NF-
B that regulate transcriptional activation after specific binding to DNA (18).
NF-B is a pleiotropic transcriptional regulator that is intimately
involved in the regulation of many proinflammatory genes (reviewed in
Refs. 5 and 6). NF-
B activation can promote cell survival, and
NF-
B inhibition can promote apoptosis (7, 23). Through
inhibition of NF-
B transcriptional activation, triptolide sensitizes
tumor cells to tumor necrosis factor-
(TNF-
)-induced apoptosis of
tumor cells in vitro (13). We proposed that the mechanisms
of the synergy between triptolide and TNF receptor ligands in inducing
apoptosis of tumor cells includes inhibition by triptolide of the
survival signal conferred by NF-
B (13, 18).
In this study, we characterized the anti-inflammatory effects of
triptolide in normal (NHBE) and transformed (16HBE) human bronchial
epithelial cells. Triptolide potently inhibits the expression of
numerous genes including cytokines, chemokines, and adhesion molecules
associated with inflammation. The mechanisms of inhibition of
inflammatory gene expression by triptolide include inhibition of
transcriptional activation, but not DNA binding, of NF-B. Using cDNA
array hybridization, we identified bronchial epithelial cell genes
involved in the inflammatory response and cell survival that are
inhibited by triptolide.
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EXPERIMENTAL PROCEDURES |
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Source of triptolide. Triptolide (PG490; mol wt 360) was obtained from Pharmagenesis (Palo Alto, CA). Triptolide appeared as white to off-white crystals at room temperature, had a melting point of 226-240°C, conformed to standard triptolide preparation as assessed by proton nuclear magnetic resonance (12), and was 97% pure by reverse-phase HPLC evaluation with acetonitrile-methanol-water (18:9:73) (Fidler J and Jin RL, personal communication).
Cell culture and stimulation conditions.
16HBE (16HBE14o) cells are a HBE cell line that was
generated by transformation with SV40 large T antigen (9),
which retains the differentiated morphology and function of normal
human airway epithelia. 16HBE cells were cultured in Eagle's MEM
(BioWhittaker) supplemented with 10% heat-inactivated FBS, 100 U/ml of
penicillin, and 100 µg/ml of streptomycin (GIBCO BRL, Life
Technologies, Grand Island, NY). NHBE cells were purchased from
Clonetics and were cultured in bronchial epithelial cell growth medium
as recommended (Clonetics). Bronchial epithelial cells were stimulated
in culture for the times indicated in Figs. 1-4 in the
presence of phorbol 12-myristate 13-acetate (PMA; 20 ng/ml; Sigma),
TNF-
(20 ng/ml; BioSource), or IL-1
(20 ng/ml; BioSource) in the
absence and presence of triptolide or CsA (Sandoz).
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ELISA. IL-8 and IL-6 protein concentrations in epithelial cell supernatants (diluted 1:10) were measured by ELISAs according to the manufacturer's directions (Coulter).
RNA extraction and Northern analyses.
Total RNA was isolated and analyzed by Northern hybridization as
described (1, 18). cDNA probes for human IL-8 (289 bp) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1.1 kb; Clontech) were
labeled with [-32P]dCTP (Amersham) with the use of a
random hexamer labeling kit (Stratagene).
Nuclear runoff transcription.
For a detailed description, see Ausubel et al. (4).
Briefly, 16HBE cells (seven 100-mm petri dishes/condition) were
either not stimulated, stimulated for 6 h with 20 ng/ml of
IL-1, or stimulated for 6 h with IL-1
in the presence of 200 ng/ml of triptolide. Cells were rinsed with PBS, scraped with a rubber policeman, and lysed two times in 4 ml of Nonidet P-40 lysis
buffer A; then nuclei were resuspended in glycerol storage
buffer and frozen in liquid nitrogen. Runoff transcription was
performed by incubating thawed nuclei with nucleotides plus 5 µl of
10 mCi/ml of [
-32P]UTP at 30°C for 30 min. Template
DNA was digested with RNase-free DNase I, RNA was extracted with
phenol-chloroform-isoamyl alcohol, precipitated with trichloracetic
acid-sodium pyrophosphate onto a 0.45-µm nitrocellulose filter
(Schleicher and Schuell), and washed. The RNA was eluted from the
filter, reextracted with phenol-chloroform, and precipitated with 3 M
sodium acetate and ethanol. The precipitated RNA was resuspended, and
an aliquot was used for determination of radioactivity by liquid
scintillation counting. The radiolabeled RNA (1 × 106
counts/min in 1 ml for each condition) was hybridized to the IL-8 cDNA
[PCR 2.1 vector containing 289-bp IL-8 cDNA (2)] that
was immobilized on nitrocellulose membrane at 65°C for 36 h,
then the membrane was washed with 2× saline-sodium citrate, blotted
dry, and exposed to the phosphorimager screen.
Plasmids, transfections, and luciferase reporter gene assays.
Luciferase reporter gene constructs were under the control of the IL-8
enhancer [IL-8-luciferase, nucleotides 1481 to +44 of the IL-8
enhancer (2)], the IL-8
B site (TGGAATTTCCT,
IL-8
B-luciferase), or the immunoglobulin
light chain NF-
B
sequence monomer (GGGACTTTCCT) in the context of the minimal IL-8
promoter (nucleotides
45 to +44 of the human IL-8 promoter Ig
NF-
B-luciferase). These plasmids also contained a neomycin
resistance gene under control of the constitutively active SV40
promoter. 16HBE cells were transfected with plasmids with the use of
LipofectAMINE (GIBCO BRL), and cell lines that stably expressed these
luciferase reporter constructs were selected in the presence of G-418
(1.5 mg/ml). These 16HBE/IL-8-luciferase, IL-8
B-luciferase, or
Ig-NF-
B-luciferase cell lines were not stimulated or were stimulated
for 6 h in the absence and presence of triptolide or CsA, and then
20 µg of whole cell extract proteins were assayed for firefly
luciferase activity as described (3).
Nuclear extract preparation and electrophoretic mobility shift
assays.
16HBE cells were stimulated for 3 h, pelleted, and washed, and
then cytoplasmic and nuclear extracts were prepared as described (2). NF-B DNA binding activities in the 16HBE cell
nuclear extracts were assayed by incubating 10 µg of nuclear proteins for 30 min at 4°C in 20 µl of binding buffer (25 mM HEPES, pH 7.6, 0.1 mM EDTA, 10% glycerol, 50 mM KCl, and 0.05 mM dithiothreitol) containing 1-2 µg of poly(dI-dC) and 2.5 pg of
32P-labeled oligonucleotide probe (~1 × 105 counts/min). For supershift assays, 10 µg of nuclear
proteins were incubated with 2 µl of anti-NF-
B p65 IgG (Santa Cruz
Biotechnology, Santa Cruz, CA) for 30 min at 25°C, radiolabeled probe
and poly(dI-dC) were added, and the incubation was continued for an
additional 30 min at 25°C. The sequence of oligonucleotide probe used
was agctAAAGAGGGACTTTCCCTAAA for the immunoglobulin
light chain NF-
B site (5). The lowercase letters indicate
overhanging bases used for incorporation of [
-32P]dCTP
and nonradioactive dATP, dGTP, and dTTP with Klenow DNA polymerase.
Protein-DNA complexes were resolved from free probe with 4%
nondenaturing polyacrylamide gels in 0.5× Tris-borate-EDTA (pH 8.3)
and visualized by fluorography.
Western immunoblotting.
The expression of IB-
protein in 15 µg of
SDS-PAGE-fractionated cytoplasmic extracts of 16HBE cells was
determined by immunoblotting with a primary rabbit polyclonal IgG
antibody to I
B-
(1:1,000 dilution; Santa Cruz Biotechnology),
followed by a horseradish peroxidase-conjugated secondary antibody
(1:3,000 dilution) and detection with enhanced chemiluminescence (Amersham).
cDNA array analysis. The effects of triptolide on the expression of multiple genes in 16HBE cells were analyzed with the Atlas array (Clontech) of 588 immobilized cDNAs selected for their relevance to growth control, stress responses, gene regulation, and intercellular signaling. 16HBE cells were grown to 90% confluence on 100-mm petri dishes and were either not stimulated, not stimulated and treated with triptolide (200 ng/ml) for 6 h, stimulated for 6 h with PMA (20 ng/ml), or stimulated for 6 h with PMA in the presence of triptolide (200 ng/ml). Total RNA was then isolated with RNA-STAT-60 (Tel-Test), and poly(A)+ mRNA was purified from 250 µg of total RNA with the use of an Oligotex mRNA mini kit (QIAGEN). The poly(A)+ mRNA (2 µg) from the PMA and PMA-triptolide conditions was then used as a template for reverse transcription and 32P radiolabeling (Amersham) to generate a pool of first-strand cDNA probes according to the manufacturer's directions (Clontech). The Atlas arrays were incubated with the radiolabeled cDNA probes, and specific binding was detected by Southern hybridization according to the manufacturer's directions (final wash with 0.1× saline-sodium citrate-0.5% SDS at 68°C; Clontech). Specific hybridization was quantitated and analyzed with a phosphorimager with data analysis software (Cyclone, Packard Instruments). Further information about the 588 genes immobilized on the Atlas array is available at the manufacturer's web site.
Cluster analysis.
Expression-level data generated by the phosphorimager were stored
in a table where the rows represented individual cDNA clones in
different experimental states and the columns represented one experimental condition each. With the expression levels of the housekeeping gene -tubulin, the raw expression levels of all the
genes were adjusted between the four arrays to eliminate differences resulting from overall (background) exposure variations (Microsoft EXCEL). The adjusted expression levels were then log transformed in
base 2 and median centered within each experimental condition. Average-linkage hierarchical clustering was performed with the program
Cluster, and the results were analyzed with TreeView (10). The computational methods are discussed in further detail in the Cluster manual (Eisen M;
http://rana.stanford.edu/software/). The color representation
encodes the expression level as red with upregulation and green with
downregulation. The color intensity increases with deviation from the
median, with desaturated (black) areas representing no change from the
median. Clusters of coregulated genes that reveal biologically
interesting consequences of cell treatments with triptolide were
selected for presentation as image files.
Data and statistical analyses. Significance of the differences between the experimental conditions was determined by paired two-sample Student's t-test (Microsoft EXCEL). The data are presented as the means ± SD, and the lowest dose of triptolide that produced a significant change from the stimulated condition is identified in Figs. 1-4.
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RESULTS |
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We demonstrated that triptolide inhibits bronchial epithelial cell
IL-8 and IL-6 protein secretion triggered by diverse stimuli in NHBE
and 16HBE cells. The inhibition of IL-8 mRNA expression occurs at the
level of transcription. Furthermore, triptolide inhibits NF- B
transcriptional activation at a novel step after the induction of
nuclear DNA binding activity.
Triptolide inhibited inflammatory cytokine gene expression by
stimulated HBE cells.
16HBE cells stimulated for 12 h with PMA, TNF-, or IL-1
markedly increased secretion of IL-8 and IL-6 protein (Fig.
1, A and D).
TNF-
and IL-1
are physiological stimuli for epithelial cell
inflammation, whereas PMA represents a nonphysiological, supranormal
stimulus. We included PMA in our experimental protocol to discern
whether the novel drug triptolide is capable of inhibiting epithelial
inflammation triggered by supranormal stimuli. The induced expression
of IL-8 after stimulation with PMA, TNF-
, or IL-1
involved
increased expression of IL-8 mRNA (Fig. 1, B and
C), suggesting that IL-8 induction involves increased gene transcription and/or mRNA stability. We show that triptolide treatment caused dose-dependent inhibition of PMA-, TNF-
-, and
IL-1
-stimulated IL-8 and IL-6 protein secretion, with an
IC50 of ~20 ng/ml (Fig. 1, A and
D). Triptolide potently inhibited stimulated IL-8 mRNA expression while causing no significant change in GAPDH mRNA
expression, suggesting that the inhibitory effect of triptolide on IL-8
and IL-6 expression is not caused by a global inhibitory effect on transcription. In contrast to these inhibitory effects demonstrated for
triptolide, the immunosuppressant CsA, which blocks calcium-dependent cytokine activation in lymphocytes, produces no significant inhibition of stimulated IL-8 or IL-6 expression in 16HBE cells (Fig. 1, lanes 6 vs. 2, 12 vs. 8,
and 18 vs. 14).
Triptolide inhibits transcriptional activation of the IL-8 gene.
We performed a nuclear runoff transcription assay to demonstrate
that IL-1-stimulation of 16HBE cells results in a 1.6-fold increase
in transcription of the endogenous IL-8 gene (Fig.
2A). Importantly, this
increase was completely blocked when 16HBE cells were stimulated with
IL-1
in the presence of 200 ng/ml of triptolide (Fig.
2A). This result indicates that triptolide inhibits IL-8 gene expression at the level of transcription.
Triptolide inhibits NF-B transcriptional activation after
induction of specific DNA binding.
The IL-8 NF-
B DNA target sequence may be bound by multiple
nuclear factors and mediates complex regulation likely to involve both
derepression and stimulation-dependent activation (14, 15,
17). To simplify our analysis of the effects of triptolide on
NF-
B signaling, we chose to study DNA binding and transcriptional activation regulated by a consensus NF-
B sequence derived from the
immunoglobulin
light chain enhancer. In stimulated,
triptolide-treated 16HBE cells, we correlated 1) cytoplasmic
expression of the NF-
B anchoring protein I
B-
, 2)
nuclear induction of NF-
B DNA binding activity, and 3)
transcriptional activation of a consensus NF-
B-luciferase reporter
gene (Fig. 3). Cytoplasmic
I
B-
protein expression decreases markedly in 16HBE cells treated
with triptolide (Fig. 3A, lanes 3-5 vs.
1). Specific DNA binding activities of NF-
B in nuclear extracts and its modulation by triptolide were characterized with the
use of electrophoretic mobility shift assays. Using the consensus NF-
B oligonucleotide probe, we observed a single DNA binding complex
(Fig. 3B). With the IL-8 NF-
B sequence as an
oligonucleotide probe, we observed multiple specific DNA binding
complexes (Aoki Y and Kao PN, unpublished observations), which serve to
complicate the analysis of the effects of triptolide on NF-
B
signaling. 16HBE cell stimulation with PMA, TNF-
, or IL-1
enhanced nuclear NF-
B DNA binding (Fig. 3B, lanes
2 vs. 1, 14 vs. 13, and
26 vs. 25). Triptolide treatment produced further
increases in nuclear NF-
B DNA binding (Fig. 3B,
lanes 3-5 vs. 2, 15-17 vs.
14, and 27-29 vs. 26). Triptolide
increased nuclear NF-
B DNA binding in proportion to its inhibition
of cytoplasmic I
B-
expression (Fig. 3B; compare with
Fig. 3A). A specific antibody against the NF-
B p65
subunit effectively supershifted the NF-
B DNA binding complex,
thereby establishing the specificity of this DNA binding complex (Fig.
3B, lanes 7-12, 19-24, and
31-36, NF-
B*).
cDNA array analysis of triptolide effects on 16HBE cell gene expression. We extended our analysis of the effects of triptolide on the expression of multiple genes in 16HBE cells using an array of immobilized cDNAs (Atlas array, Clontech). This Atlas array contains 588 genes selected for their relevance to growth control, stress responses, gene regulation, and signaling. 16HBE cells were either not stimulated, not stimulated and treated with triptolide (200 ng/ml), stimulated for 6 h with PMA (20 ng/ml), or stimulated for 6 h with PMA in the presence of triptolide (200 ng/ml). Poly(A)+ mRNA was isolated, radiolabeled by reverse transcription, and used to probe the Atlas cDNA arrays by Southern hybridization and phosphorimager analysis. These cell culture conditions were selected to produce supramaximal stimulation of bronchial epithelial cells and to determine the effectiveness of triptolide (applied at 4-10 times the IC50 for inhibition of 16HBE expression of IL-8) in inhibiting inflammatory gene expression.
The analysis of changes in gene expression across the four different conditions was performed by applying a clustering algorithm (10). This method is useful for extracting information that identifies genes that are similarly regulated during cellular perturbations. The phosphorimager data of specific Southern blot hybridization intensities was median centered and converted to logarithmic scale. Then the genes with expression above the median are presented as increased intensity of red color, and genes with expression below the median are presented as an increasing intensity of green color (Fig. 4). Genes expressed at the median level appear black. We present selected strips of the cluster analysis containing regions of genes with interesting modulation by triptolide (Fig. 4). Genes with low expression in nonstimulated 16HBE cells that are induced by PMA and suppressed by triptolide are clustered as shown (Fig. 4A; green, green, red, green). Notable among these genes are cytokines, chemokines, adhesion molecules, and growth factors such as TNF- ![]() |
DISCUSSION |
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We present the first characterization of the novel anti-inflammatory mechanisms of triptolide in HBE cells. Triptolide effectively inhibits the expression of numerous genes involved in the bronchial epithelial cell inflammatory and proliferative responses.
The expression of IL-8 in bronchial epithelial cells is regulated both
at the level of transcription and by posttranscriptional mRNA
stabilization (19). We demonstrated that triptolide
potently inhibited epithelial cell expression of IL-8 mRNA and protein. Triptolide inhibited IL-8 expression at the level of transcription, as
we demonstrated using both an IL-8 nuclear run-on assay and IL-8
luciferase reporter gene experiments. Within the IL-8 enhancer, the
IL-8 NF-B target sequence contributes importantly to activation in
bronchial epithelial cells (2, 19). The inhibitory effects of triptolide on the 1.4-kb IL-8 enhancer are preserved on an IL-8
NF-
B luciferase reporter (spanning nucleotides
82 to +44 of the
IL-8 promoter sequence).
We performed a more detailed characterization of the effects of
triptolide on DNA binding and transcriptional activation regulated by
the consensus NF-B target DNA sequence. Triptolide enhances the
nuclear DNA binding activity of NF-
B in stimulated epithelial cells,
likely through inhibition of I
B-
expression, which serves to
release more p65 for translocation from the cytoplasm into the nucleus
(18). Remarkably, triptolide inhibits NF-
B
transcriptional activation even in the presence of enhanced nuclear
NF-
B DNA binding activity as Qui et al. (18) showed
previously in Jurkat T cells. This important result establishes that
triptolide inhibits NF-
B transcriptional activation at a novel
nuclear site in the signaling pathway. No other inhibitor is known to
operate so distally in the NF-
B signaling pathway. Transcriptional
regulation by NF-
B in the nucleus may involve coactivators, such
as p300 or pCAF, that modulate chromatin conformation, in addition to
interactions with the RNA polymerase II holoenzyme. The inhibitory
effects of triptolide that we have observed on transiently and stably transfected reporter genes controlled by NF-
B imply that
triptolide inhibition does not require chromatin conformation
changes. At present, we do not know the precise mechanisms of
transcriptional inhibition by triptolide in the nucleus.
When we compare the IL-8 NF-B luciferase experiments with the
consensus NF-
B luciferase experiments, we note that 20 ng/ml of
triptolide produces a significant enhancement of IL-8 NF-
B luciferase activity but not of consensus NF-
B luciferase activity. We never observed superinduction of the entire IL-8 enhancer luciferase reporter or enhancement of IL-8 mRNA or protein expression at any dose
of triptolide. Thus superinduction of the IL-8 NF-
B luciferase
reporter activity at 20 ng/ml of triptolide likely arises from the
complex regulation that occurs at this nonconsensus NF-
B regulatory
sequence. The IL-8 NF-
B sequence bears similarity to the
purine-rich/NFAT target DNA sequence in the human IL-2 enhancer
(3) and has been identified as a genetic end target of the
inhibitory effects of FK506 in Jurkat T cells (16). We characterized a CsA- and FK506-sensitive purine-box transcriptional regulator that is present in 16HBE cells (3) and have
characterized its specific binding to an IL-8 NF-
B oligonucleotide
probe (Aoki Y and Kao PN, unpublished observations). In Jurkat T cells,
triptolide inhibits activation of the purine-box regulator at doses
lower than those necessary for inhibition of NF-
B (18).
We propose that in 16HBE cells, 20 ng/ml of triptolide primarily
inhibits the purine-box regulator, thereby derepressing the IL-8
NF-
B site and superinducing transcriptional activation.
The cDNA array analysis revealed that triptolide exerts broad
inhibitory effects on the expression of genes associated with the
cellular inflammatory response. Among the important genes of
inflammation inhibited by triptolide are TNF-, IL-8 and MIP-2
, and ICAM-1. The transcriptional regulation of TNF-
, IL-8, ICAM-1, and numerous other genes associated with inflammation involves regulation by NF-
B (reviewed in Ref. 5). Based on our demonstration that triptolide inhibits NF-
B signaling and transcriptional
activation in the nucleus, we propose that triptolide inhibition of
cytokine, chemokine, and integrin gene expression operates in part
through inhibition of NF-
B transcriptional activation.
The cDNA array analysis also revealed that triptolide inhibits
expression of the genes involved in cell cycle progression and cell
survival. Triptolide inhibition of cyclin expression would serve to
inhibit cell cycle progression and cell growth, and triptolide
inhibition of bcl-x expression would promote apoptosis. In combination
with ligands of the TNF receptor family, triptolide promotes apoptosis
of tumor cells (13). The mechanisms underlying triptolide-induced apoptosis may involve inhibition of a survival signal conferred by NF-B signaling as well as inhibition of survival signals associated with bcl-x expression. Furthermore, triptolide likely inhibits cell cycle progression through inhibition of the expression of cyclins D1, B1, and A1.
In summary, triptolide is a novel drug with potent anti-inflammatory
and antiproliferative effects on epithelial cells. Some of the
mechanisms of triptolide involve transcriptional inhibition of NF-B
signaling in the nucleus at a novel, distal site in the signaling
pathway. Triptolide may be of benefit in diseases characterized by
inappropriate bronchial epithelial inflammation such as the allograft
rejection that follows organ transplantation and other conditions that
involve airway and parenchymal lung inflammation.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Allergy and Infectious Diseases Grant R01-AI-39624 and National Heart, Lung, and Blood Institute Grant R01-HL-62588 and by gifts from the Donald E. and Delia B. Baxter Foundation and Pharmagenesis.
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FOOTNOTES |
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G. Zhao was supported by the Ho-Ho-Li fellowship and a Dean's Postdoctoral Fellowship from Stanford University.
Address for reprint requests and other correspondence: P. N. Kao, Pulmonary and Critical Care Medicine, Stanford Univ. Medical Center, Stanford, CA 94305-5236 (E-mail: peterkao{at}stanford.edu).
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.
Received 18 August 1999; accepted in final form 11 May 2000.
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