вход по аккаунту


Differential mechanism of NF-╨Ю╤ФB inhibition by two glucocorticoid receptor modulators in rheumatoid arthritis synovial fibroblasts.

код для вставкиСкачать
Vol. 60, No. 11, November 2009, pp 3241–3250
DOI 10.1002/art.24963
© 2009, American College of Rheumatology
Differential Mechanism of NF-␬B Inhibition by
Two Glucocorticoid Receptor Modulators in
Rheumatoid Arthritis Synovial Fibroblasts
Valerie Gossye,1 Dirk Elewaut,2 Nadia Bougarne,1 Debby Bracke,1 Serge Van Calenbergh,1
Guy Haegeman,1 and Karolien De Bosscher1
addition, CpdA attenuated the TNF␣-induced nuclear
translocation and DNA binding of p65 in RA FLS, via
the attenuation of IKK phosphorylation and subsequent
I␬B␣ degradation. CpdA also displayed profound effects on TNF␣-induced MAPK activation. The effects of
CpdA on TNF␣-induced kinase activities occurred independently of the presence of GR. In sharp contrast, DEX
did not affect TNF␣-induced IKK phosphorylation,
I␬B␣ degradation, p65 nuclear translocation, or MAPK
activation in RA FLS.
Conclusion. DEX and CpdA display a dissimilar
molecular mechanism of interaction with the NF-␬B
activation pathway ex vivo. A dual pathway, partially
dependent and partially independent of GR (nongenomic), may explain the gene-inhibitory effects of
CpdA in RA FLS.
Objective. To investigate and compare the molecular mechanisms by which 2 glucocorticoid receptor
(GR)–activating compounds, dexamethasone (DEX)
and Compound A (CpdA), interfere with the NF-␬B
activation pathway in rheumatoid arthritis (RA) synovial cells.
Methods. Quantitative polymerase chain reaction
was performed to detect the tumor necrosis factor ␣
(TNF ␣ )–induced cytokine gene expression of
interleukin-1␤ (IL-1␤) and to investigate the effects of
DEX and CpdA in RA fibroblast-like synoviocytes (FLS)
transfected with small interfering RNA (siRNA) against
GR (siGR) compared with nontransfected cells. Immunofluorescence analysis was used to detect the subcellular distribution of NF-␬B (p65) under the various
treatment conditions, and active DNA-bound p65 was
measured using a TransAM assay and by chromatin
immunoprecipitation analysis of IL-1␤. Signaling pathways were studied via Western blotting of siGRtransfected cells, compared with nontransfected and
nontargeting siRNA–transfected control cells, to detect
the regulation of phospho-IKK, I␬B␣, phospho-p38,
phospho-ERK, and phospho-JNK.
Results. Both DEX and CpdA efficiently inhibited
IL-1␤ gene expression in a GR-dependent manner. In
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease that is characterized by the formation of an aggressive tumor-like pannus structure, which
invades and destroys the cartilage and bone. The pannus
is home to many cell types, including blood-borne cells
and mesenchymal cells (fibroblast-like synoviocytes
[FLS]) (1,2). It is now clear that FLS not only are
considered to be structural elements but also actively
contribute to the inflammatory and destructive processes in RA via the production of a battery of cytokines,
chemokines, and enzymes degrading the joint matrix (3).
Moreover, the invasive FLS display morphologic features that are characteristic of a transformed cell,
thereby supporting the hypothesis that the activated
phenotype of FLS is an intrinsic property of these cells
(4–8). Alternatively, it has been hypothesized that RA
FLS are bone marrow–derived mesenchymal cells that
are abundantly recruited to the RA synovium and are
arrested at various stages of differentiation in the inflamed environment. Aberrantly activated NF-␬B ap-
Dr. Gossye’s work was supported by the IWT-Vlaanderen.
Dr. De Bosscher’s work was supported by the FWO-Vlaanderen. Ms
Bougarne’s work was supported by the Interuniversitaire AttractiePolen (project IAP6).
Valerie Gossye, PhD, Nadia Bougarne, MSc, Debby Bracke,
PhD, Serge Van Calenbergh, PhD, Guy Haegeman, PhD, Karolien De
Bosscher, PhD: Ghent University, Ghent, Belgium; 2Dirk Elewaut,
MD, PhD: University Hospital Ghent, Ghent, Belgium.
Address correspondence and reprint requests to Karolien De
Bosscher, PhD, Laboratory for Eukaryotic Gene Expression and
Signal Transduction, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium.
Submitted for publication September 22, 2008; accepted in
revised form August 10, 2009.
pears to play a central role in the differentiation arrest of
these cells (9).
NF-␬B is a heterodimeric transcription factor,
usually composed of a p50 subunit and p65 subunit. A
vast number of studies have demonstrated the involvement of NF-␬B in the pathogenesis of RA, which is
attributed to the role of NF-␬B in the transcriptional
regulation of genes encoding different proinflammatory
mediators (10–15). Furthermore, by showing that activation of NF-␬B protects synovial cells against apoptosis, it has been hypothesized by Miagkov and colleagues
that this transcription factor might constitute the missing
link between inflammation and hyperplasia in the arthritic joint (16,17). In unstimulated cells, NF-␬B is kept
inactive in the cytoplasm by inhibitory proteins (I␬Bs).
Triggering of the cell with various extracellular stimuli,
including tumor necrosis factor ␣ (TNF␣), results in
activation of different signaling cascades, which converge on and activate the IKK complex. Subsequently,
I␬B␣ is phosphorylated and degraded, allowing NF-␬B
to translocate to the nucleus and to regulate the expression of its target genes (for review, see refs. 18–20).
The transcriptional activity of NF-␬B can be
further modulated in the nucleus by different kinases,
including the MAPKs (21,22). In this respect, previous
reports have described the importance of p38 and ERK
and the downstream nuclear kinase MSK-1 for the
phosphorylation of p65 at Ser276, thereby evoking fullblown NF-␬B activity (23–25). In this way, an additional
level of regulation is created, providing a basis for the
observed crosstalk with other signaling pathways (26).
Ample amounts of evidence support the idea that
the antiinflammatory effects of glucocorticoids (GCs)
are a result of their interference with the NF-␬B signaling pathway (27,28). Indeed, GCs exert their effects via
the GC receptor (GR), which, in the absence of a ligand,
can be found in the cytoplasm of the cell in complex with
chaperone molecules. After diffusion through the cell
membrane, GCs bind to their cognate receptor, thereby
inducing its conformational change and spurring the
activated ligand–receptor complex to translocate to the
nucleus. In the nucleus, GR binds as a homodimer to
GC-responsive elements, leading to gene activation
(transactivation). Alternatively, the activated GR monomer also regulates gene expression in a negative way
(transrepression), mainly through negative interference
with the activity of proinflammatory transcription factors such as NF-␬B (29–31).
It is generally accepted that mainly the latter
characteristic of transrepression by GCs forms the basis
for their antiinflammatory potential, and thus explains
their success as drug targets (27,28). In addition, recent
evidence suggests that GR ligands can also induce
cellular responses that can occur in a time frame ranging
from a few seconds up to 1 hour (32,33). These rapid
effects cannot be explained by the well-known genomic
effects of GR ligands, which normally take a few hours
to days (32,33); yet, at the moment, it is not completely
understood which receptor or pathway is responsible for
mediating these so-called nongenomic effects (32).
We previously demonstrated that a small, nonsteroidal, plant-derived GR modulator, Compound A
(CpdA), stimulates the transrepression activity of GR,
but leaves the transactivation function of the receptor
unaffected (34,35). Consequently, it was observed that
the therapeutic potential of CpdA for the treatment of
collagen-induced arthritis in mice was not overshadowed
by diabetogenic effects (35). Subsequently, we were
interested in unraveling the precise molecular mechanisms by which CpdA interferes with the NF-␬B pathway
in primary FLS derived from patients with RA. As a
reference compound, we used dexamethasone (DEX), a
synthetic steroidal GR ligand.
Isolation and culture of cells. FLS were obtained from
patients with active RA, whose diagnosis met the revised
criteria of the American College of Rheumatology (formerly,
the American Rheumatism Association) (36). The study was
approved by the local ethics committee, and informed consent
was obtained from all patients. FLS were obtained by enzymatic digestion from RA synovial tissue, and the cells were
then cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal calf serum, 100 units/ml
penicillin, and 0.1 mg/ml streptomycin. To ascertain that
non-FLS cells were not contaminating the culture, FLS with a
passage number ranging from 4 to a maximum of 8 were used.
Cytokines and reagents. Recombinant murine TNF␣
was produced in our laboratory (29). DEX was purchased from
Sigma (St. Louis, MO). CpdA (Alexis Biochemicals, Lausen,
Switzerland) was synthesized as described previously (37).
Anti-p65 (C20) and anti-I␬B␣ (C21) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The
antibodies anti–phospho-p38 MAPK (Thr180/Tyr182), anti-p42/
p44 (anti-ERK), anti–phospho-p42/p44 (anti–p-ERK; Thr202/
Tyr204), anti–phospho-SAPK/JNK (Thr183/Tyr185), and anti–
phospho-IKK␣/␤ (Ser180/Ser181) were purchased from Cell
Signaling Technology (Beverly, MA). Anti– ␤ -catenin
(Ab2982) and antitubulin (clone B-5-1-2) were purchased from
Abcam (Cambridge, UK) and Sigma, respectively. Small interfering RNA (siRNA) against GR (siGR; siGENOME
SMARTpool human NR3C1) and nontargeting siRNA controls (siCtrl; siCONTROL Pool #1) were both purchased from
Dharmacon RNA Technologies (Lafayette, CO).
Quantitative polymerase chain reaction (PCR). FLS
were seeded in 6-well plates and serum-starved 24 hours prior
to induction. Total RNA was isolated using the RNeasy mini
kit (Qiagen, Chatsworth, CA), according to the manufacturer’s
instructions. The messenger RNA (mRNA) was reverse transcribed with the verso complementary DNA (cDNA) kit
(ABgene, Epsom, UK). The obtained cDNA was amplified in
a quantitative PCR reaction containing the iQ Custom SYBR
Green Supermix (Bio-Rad, Richmond, CA). Gene expression
values for the housekeeping gene hypoxanthine guanine phosphoribosyltransferase were used for normalization.
NF-␬B (p65) transcription factor assay. NF-␬B (p65)
activation was measured using the TransAM kit (Active Motif,
Carlsbad, CA). FLS were grown to subconfluence and serumstarved for 24 hours. After inductions, nuclear extracts were
prepared and used in the enzyme-linked immunosorbent assay
(ELISA)–based format, according to the manufacturer’s instructions.
Chromatin immunoprecipitation (ChIP) assay. FLS
were starved in 0% DMEM for 48 hours. After the appropriate
inductions, cells were subjected to a ChIP assay against NF-␬B
p65 or GR␣, which was performed using a method essentially
as described previously (38) except for the substitution of
sodium dodecyl sulfate with deoxycholate in all buffers. Quantitative PCR primers within the interleukin-1␤ (IL-1␤) promoter region near the NF-␬B site were as follows: forward
cycle values were analyzed using GENEX software (Bio-Rad).
The relative amount of the precipitated target sequence was
determined via normalization to the input values, i.e., the
purified total genomic DNA levels.
Indirect immunofluorescence analysis. FLS were
seeded on coverslips and grown in phenol red–free and
serum-free medium for 24 hours. Fixation, permeabilization,
and staining were performed as described previously (34).
Presence of p65 was visualized with a 1:200 dilution of anti-p65
antibody, followed by probing with a 1:800 dilution of Alexa Fluor
488–conjugated goat anti-rabbit IgG. Staining with 4⬘,6diamidino-2-phenylindole was used for visualization of the cell
nuclei. A Zeiss Axiovert 200M microscope was used for visualization of the images, and data were analyzed using Carl Zeiss
Axiovision software (Carl Zeiss Instruments, Jena, Germany).
Western blot analysis. FLS were seeded in 6-well
plates and serum-starved 24 hours prior to induction. Total
protein was extracted as described previously (39). The protein
concentration was determined according to the Bradford
method (40).
Small interfering RNA transfection into FLS. FLS
were transfected using Amaxa nucleofector technology (program number U-23; Amaxa, Gaithersburg, MD) in combination with the human dermal nucleofector kit. The protocol has
been previously described by Inoue et al (41).
Statistical analysis. Comparisons between groups
were performed by one-way analysis of variance. P values less
than 0.05 were considered statistically significant.
Effect of CpdA and DEX on NF-␬B nuclear
translocation, activation, and DNA binding. To establish
the effect of CpdA on NF-␬B–driven gene expression,
Figure 1. Down-regulation of NF-␬B–driven interleukin-1␤ (IL-1␤)
cytokine gene expression by Compound A (CpdA) and dexamethasone
(DEX) in rheumatoid arthritis fibroblast-like synoviocytes (FLS). The
FLS were pretreated with DEX (1 ␮M), CpdA (10 ␮M), or ethanol
(EtOH) control solvent for 1 hour, after which tumor necrosis factor ␣
(TNF␣) (2,000 IU/ml) was added to the cells for the remaining 6
hours. To determine the regulation of gene expression, total RNA was
isolated from the cells, but only the mRNA was reverse transcribed
and subjected to quantitative polymerase chain reaction with primers
targeting the IL-1␤ transcript. Bars show the mean and SD results
from triplicate experiments, normalized to the expression values for
the housekeeping gene hypoxanthine guanine phosphoribosyltransferase. ⴱⴱ ⫽ P ⬍ 0.01 versus TNF␣-stimulated controls, by one-way
analysis of variance.
we first performed quantitative PCR analysis for the
detection of proinflammatory cytokine gene transcription, using IL-1␤ as an example of a typical NF-␬B–
regulated gene (16,42). Stimulation of the FLS with
TNF␣ for 6 hours resulted in a significant increase in
IL-1␤ transcription. Pretreatment of the FLS with CpdA
(10 ␮M) repressed the TNF␣-induced IL-1␤ transcription to a similar extent as that after pretreatment with
DEX (1 ␮M) (Figure 1).
To study the impact of both compounds on the
NF-␬B activation pathway, we performed immunofluorescence analysis for localization of the p65 subunit of
NF-␬B. In the presence of TNF␣ (30-minute period of
induction), p65 was predominantly localized in the nucleus of the cell. Treatment with DEX did not change
the subcellular distribution of p65 after stimulation with
TNF␣ (Figure 2A). In sharp contrast, after stimulation
of the FLS with TNF␣ followed by treatment with
CpdA, p65 was either detected mainly in the cytoplasm
of the cell or equally distributed over both subcellular
compartments (Figure 2A).
To determine whether the hampered cytoplasmic-to-nuclear translocation after CpdA treatment
was correlated with the amount of active p65, we per-
Figure 2. Attenuation of the nuclear translocation, DNA binding, and activation of p65 by CpdA, and stimulation of glucocorticoid receptor (GR)
recruitment onto the IL-1␤ promoter in rheumatoid arthritis FLS. Cells were pretreated with DEX (1 ␮M), CpdA (10 ␮M), or EtOH control solvent
for 1 hour, followed by the addition of TNF␣ (2,000 IU/ml). A, After pretreatment, TNF␣ was added for 30 minutes and localization of p65 was
determined by immunofluorescence analysis (top), and 100 randomly chosen cells were counted and categorized into 3 groups according to the
subcellular distribution of p65, i.e., mainly cytoplasmic, mainly nuclear, or equally distributed (nuclear/cytoplasmic) (bottom). B, After pretreatment,
TNF␣ was added for 1 hour or 2 hours, as indicated. Binding of active nuclear p65 to an oligonucleotide was determined via the TransAM (p65)
assay. The amount of active p65 was normalized to the levels in control solvent cultures. ⴱⴱ ⫽ P ⬍ 0.01 versus TNF␣-stimulated controls, by one-way
analysis of variance. C, Following serum starvation for 48 hours, FLS were incubated with control solvent or TNF␣ (2,000 IU/ml) for 30 minutes,
with or without pretreatment for 1 hour with CpdA (10 ␮M) or DEX (1 ␮M). Crosslinked and sonicated cell lysates were subjected to chromatin
immunoprecipitation against p65 or GR␣; IgG was used as the control. Quantitative polymerase chain reaction was performed to assay recruitment
at the IL-1␤ gene promoter. ⴱ ⫽ P ⬍ 0.05; ⴱⴱⴱ ⫽ P ⬍ 0.001, by one-way analysis of variance. Bars in B and C show the mean and SD results from
triplicate experiments. DAPI ⫽ 4⬘,6-diamidino-2-phenylindole; NS ⫽ not significant (see Figure 1 for other definitions).
formed a TransAM assay to detect active DNA-bound
p65 (Figure 2B). After the appropriate inductions, nuclear proteins were extracted from the FLS and analyzed
for binding of active p65 to a target oligonucleotide in
the ELISA-based format. Stimulation of the cells with
TNF␣ for 1 hour or 2 hours resulted in a significant
increase in binding of active p65. Although treatment of
TNF␣-stimulated FLS with DEX resulted in a small, but
significant, decrease in active DNA-bound p65 after 1
hour of TNF␣ stimulation, this effect of DEX was
completely abolished when TNF␣ remained on the cells
for 2 hours. The effect of CpdA on the amount of active
p65 DNA binding was more pronounced than the effect
of DEX. Although there was a significant CpdAmediated decrease in the amount of p65 DNA binding
after 1 hour of stimulation with TNF␣, the effect of
CpdA was even more pronounced after 2 hours of TNF␣
stimulation, as was exemplified by a partial, yet significant, inhibition of p65 DNA binding (Figure 2B).
In addition, recruitment of the p65 subunit of
NF-␬B or of the GR␣ isoform onto the IL-1␤ promoter
was analyzed via a ChIP assay, after treatment of the
FLS for 30 minutes with TNF␣ in the absence or
presence of DEX or CpdA. TNF␣ was able to induce an
enhanced promoter recruitment of p65, and pretreatment with DEX or CpdA resulted in decreased p65
DNA binding at the IL-1␤ promoter (Figure 2C), which
is in accordance with the results obtained in the
TransAM assay after 1 hour of stimulation with TNF␣.
Importantly, both DEX and CpdA were able to stimulate promoter accumulation of GR␣, strongly suggesting
the importance of the GR in the transrepression mechanism of both compounds (Figure 2C).
Effect of CpdA and DEX on the upstream pathways leading to NF-␬B activation. To investigate the
molecular basis of the altered subcellular localization
and DNA binding of p65 after CpdA treatment, we
assessed the I␬B␣ protein levels in the FLS (Figure 3A).
After stimulation of the FLS with TNF␣ for 15 minutes,
I␬B␣ protein expression completely disappeared, in
contrast to the findings with the ethanol control solvent
(compare lane 1 with lane 4 in Figure 3A). Resynthesis
of the I␬B␣ protein could be observed after TNF␣
stimulation for 60 minutes (compare lane 4 with lane 10
in Figure 3A). These results confirm the known mechanism of NF-␬B–driven resynthesis of I␬B␣.
Following treatment of the cells with DEX and
stimulation with TNF␣, a regulation pattern similar to
that found in untreated cells stimulated with TNF␣
alone was observed; I␬B␣ protein was completely degraded and only reappeared after 1 hour of TNF␣
stimulation (compare lane 4 with lane 5 and lane 10 with
lane 11 in Figure 3A). Remarkably, in the RA FLS,
TNF␣ stimulation in combination with CpdA treatment
Figure 3. Attenuation of NF-␬B activation by CpdA via the inhibition
of I␬B␣ degradation, in a manner independent of the glucocorticoid
receptor (GR), in rheumatoid arthritis FLS. Cells were treated for 1
hour with DEX (1 ␮M), CpdA (10 ␮M), or EtOH control solvent, after
which a time kinetics experiment was started. TNF␣ (2,000 IU/ml) was
added and left on the FLS for the indicated time periods (15, 30, or 60
minutes). A, Western blot analysis with I␬B␣ and phospho-IKK
antibodies was performed on total extracts. Detection of a nonspecific
(NS) band or of ERK was used as a loading control. B, Before starting
the inductions, cells were treated for 1 hour with cycloheximide
(CHX), or its solvent (DMSO), at a concentration of 20 ng/ml.
Western blot analysis with an I␬B␣ antibody was performed on total
extracts. The detection of ␤-catenin (␤-cat) protein levels was used for
verifying CHX functionality. Equal loading was determined by the
detection of ERK or tubulin (tub). C, At 30 hours before starting the
inductions, FLS were transfected with control small interfering RNA
(siCtrl) or small interfering RNA against GR (siGR), as indicated.
Western blot analysis with I␬B␣, phospho-IKK, and GR antibodies
was performed on total extracts; p65 was used as a loading control. See
Figure 1 for other definitions.
did not lead to lower I␬B␣ levels (compare lane 4 with
lane 6 in Figure 3A).
Since the concentration of I␬B␣ in the cells after
TNF␣ stimulation is the result of an equilibrium between cyclic events of I␬B␣ degradation and I␬B␣
resynthesis after binding of NF-␬B to the I␬B␣ promoter
(43), we next determined whether these high levels of
I␬B␣ protein observed in the FLS after combined TNF␣
stimulation and CpdA treatment could result from an
inhibition of I␬B␣ degradation. CpdA, in contrast to the
effects of DEX, indeed hampered the TNF␣-induced
phosphorylation of IKK␣/␤ (compare lane 5 with lane 6
in Figure 3A).
To exclude the possibility that a fast resynthesis
of I␬B␣ might also be partially responsible for the effect
of CpdA on I␬B␣ protein levels, we pretreated the cells
with cycloheximide (CHX) for 1 hour, before stimulation with TNF␣ and treatment with DEX or CpdA. The
functionality of CHX was confirmed through the detection of ␤-catenin protein, which is known for its quick
turnover rate (44). Higher levels of ␤-catenin were
apparent in control-treated samples in comparison with
CHX-treated samples, thereby showing that new protein
synthesis was efficiently blocked (compare lane 13 with
lane 14 in Figure 3B). Even in the absence of new
protein synthesis, I␬B␣ protein levels were much higher
in the CpdA-treated TNF␣-stimulated FLS compared
with the DEX-treated TNF␣-stimulated FLS (compare
lanes 10 and 11 with lane 12 in Figure 3B). Treatment of
the FLS with CpdA and stimulation with TNF␣ resulted
in protein levels similar to the levels observed in the
basal state (compare lane 1 with lane 6 in Figure 3B).
Since the effects of CpdA on the I␬B␣ protein
levels occurred rapidly and in the absence of new protein
synthesis, we investigated whether these effects were
GR-dependent. Consequently, the FLS were transfected
with siGR or with nontargeting siCtrl (Figure 3C).
Expression of the GR was indeed efficiently silenced in
the siGR-transfected cells (compare lanes 1–6 with lanes
7–12 in Figure 3C). In parallel, we measured the levels
of phosphorylated IKK␣/␤ and found that in siGRtransfected cells, the same pattern of regulation could be
observed as that in siCtrl-transfected cells (compare
lanes 1–6 with lanes 7–12 in Figure 3C). Treatment of
the cells with CpdA hampered the TNF␣-induced phosphorylation of IKK␣/␤ in both siGR- and siCtrltransfected cells (compare lane 4 with lane 6 and lane 10
with lane 12 in Figure 3C), an effect that could not be
observed after DEX treatment (compare lane 4 with
lane 5 and lane 10 with lane 11 in Figure 3C).
Effect of CpdA and DEX on MAPK activation.
MAPK pathways are considered important for finetuning the activity of NF-␬B in the nucleus. Therefore,
we investigated the effect of DEX and CpdA on activation of the MAPKs p38, ERK, and JNK (Figure 4A).
Stimulation of the FLS with TNF␣ for 15 minutes
resulted in a significant increase in the amount of
phosphorylated p38, ERK, and JNK (compare lane 1
with lane 4 in Figure 4A). Interestingly, in RA FLS,
treatment with CpdA repressed the TNF␣-induced
phosphorylation of p38, ERK, and JNK almost completely, which is in contrast to the effects of DEX
(compare lane 5 with lane 6 in Figure 4A).
In addition, we also investigated the effect of GR
knockdown on the TNF␣-induced MAPK activation
status in the absence or presence of DEX and CpdA
(Figure 4B), in the same experiment as depicted in
Figure 3C. Intriguingly, similar to the effects on IKK␣/␤
phosphorylation, treatment of the cells with CpdA could
also block TNF␣-induced MAPK activation, in both
Figure 5. Functional necessity of the glucocorticoid receptor (GR)
for the antiinflammatory effects of CpdA. Rheumatoid arthritis FLS
were transfected with control small interfering RNA (siCtrl) or small
interfering RNA against GR (siGR). At 30 hours thereafter, cells were
treated with EtOH control solvent, DEX (1 ␮M), or CpdA (10 ␮M) for
1 hour, after which TNF␣ (2,000 IU/ml) was added for 6 hours. Total
RNA was isolated and subjected to reverse transcription–polymerase
chain reaction (PCR). A, Silencing of the GR was monitored by
quantitative PCR analysis. The GR expression levels in siCtrltransfected cells were set at 100%, and values in siGR-transfected cells
were determined as the percent silencing relative to siCtrl-transfected
samples. B, The amount of cDNA for the proinflammatory cytokine
IL-1␤ after siCtrl or siGR transfection was measured by quantitative
PCR. Expression levels in each treatment group were normalized to
the values in the TNF␣-stimulated control samples. Bars show the
mean and SD results from triplicate experiments. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽
P ⬍ 0.01, by one-way analysis of variance. NS ⫽ not significant (see
Figure 1 for other definitions).
Figure 4. Attenuation of MAPK activation by CpdA, in a glucocorticoid receptor (GR)–independent manner. A, Cells were treated for 1
hour with DEX (1 ␮M), CpdA (10 ␮M), or EtOH control solvent, after
which a time kinetics experiment was started. TNF␣ (2,000 IU/ml) was
added and left on the FLS for the indicated time periods (15, 30, or 60
minutes). Western blot analysis with phosphorylation-specific antibodies that detect phosphorylated p38, ERK, and JNK was performed on
total extracts. A nonspecific (NS) band was used as a loading control.
B, At 30 hours before starting the inductions, FLS were transfected
with control small interfering RNA (siCtrl) or small interfering RNA
against GR (siGR), as indicated. Western blot analysis with phosphop38, phospho-ERK, and phospho-JNK antibodies was performed on
total extracts. Detection of p65 was used as a loading control. MAPK
blots were obtained during the same experiment as shown in Figure
3C. See Figure 1 for other definitions.
siCtrl-transfected and siGR-transfected cells (Figure
Antiinflammatory, GR-dependent effect of CpdA.
We previously demonstrated that the gene-repressive
effect of CpdA depends on the presence of functional
GR. To underscore the functional necessity of GR in the
antiinflammatory action of CpdA in FLS, the FLS were
transfected with siGR or with siCtrl (Figure 5). Downregulation of the receptor was monitored with quantitative PCR. A silencing efficiency of 80–90% was achieved
with siGR (Figure 5A). In parallel, we measured the
mRNA levels of TNF␣-induced IL-1␤ gene expression
after treatment with CpdA and DEX in siRNAtransfected FLS. In the siCtrl-transfected FLS, the
TNF␣-induced increase in IL-1␤ levels was efficiently
down-regulated by both DEX and CpdA, but in siGRtransfected FLS, both DEX and CpdA failed to exert an
efficient repressive effect on this proinflammatory cytokine.
Since it has been repeatedly reported that GCs
exert their beneficial effects, at least in part, via interference with the NF-␬B activation pathway (27–31), a
pathway that has been firmly linked to the pathogenesis
of RA (9–17), we set out to unravel how the dissociative
CpdA imposes its negative effect on the NF-␬B activation pathway in human FLS. The results described in
previous reports and in the present study clearly indicate
that CpdA down-regulates the TNF␣-induced and NF␬B-driven transcriptional expression of matrix metalloproteinase 1 (MMP-1), MMP-3, TNF␣ (35), IL-6, IL-8,
monocyte chemotactic protein 1 (45), and IL-1␤ (Figure
1) to a similar extent as that after treatment with DEX
in the ex vivo cell system. Despite the fact that the
degree of response may vary when primary material
isolated from different patients is studied, our findings
confirm that both compounds can efficiently downregulate the expression of TNF␣-induced IL-1␤ expression in RA FLS.
Although the amount of IL-1␤ in FLS culture
supernatants was too low for detection with ELISA, it is
interesting to note that this cytokine is transcriptionally
regulated by TNF␣ in these cells, and thus can be used
for studying the NF-␬B activation pathway in the cells
(46,47). Indeed, even though the IL-1␤ promoter contains response elements for a variety of transcription
factors, thereby allowing its regulation by different signaling pathways (48), the central role of the NF-␬B
pathway in IL-1␤ gene expression has repeatedly been
recognized (42,49). Moreover, in FLS, it has been reported that TNF␣-induced IL-1␤ gene expression can be
ablated by expression of the super-repressor I␬B␣ (16).
These findings are supported by the results presented
herein, which show that after stimulation of FLS with
TNF␣, an increased amount of p65 was recruited onto
the promoter of IL-1␤ (Figure 2C).
We therefore determined the functional necessity of activated GR for the repression of inflammatory
cytokine production by both DEX and CpdA, and used
IL-1␤ as a paradigm. Our results underscore that knockdown of GR abrogates the repressive effects of both
compounds on IL-1␤ expression (Figure 5). However,
the molecular mechanisms by which DEX and CpdA
affect the NF-␬B activation pathway differ drastically.
Although DEX exerted a slight, but significant, effect on
active DNA-bound p65 after 30 minutes or 1 hour of
TNF␣ stimulation, as determined by TransAM assay
(Figure 2B) and ChIP analysis (Figure 2C), we could not
observe such an effect after 2 hours of TNF␣ administration (Figure 2B). These discrepancies regarding the
degree of GC-mediated inhibition of NF-␬B binding at
different time points, and yet occurring in the same cell,
are likely to depend on the repressive mechanisms that
prevail in any given situation at any given time (for
review, see ref. 50).
The lack of effect on DNA binding of p65 in the
nucleus of FLS after 2 hours of TNF␣ stimulation
(Figure 2B) is in concordance with our observations
further upstream in the NF-␬B activation pathway,
showing that treatment of the cells with DEX did not
hamper the TNF␣-induced phosphorylation of IKK and
the subsequent degradation of I␬B␣ (Figure 3A). Our
results are consistent with previous findings reported by
Han and colleagues (47), who also failed to observe an
effect of DEX on the I␬B␣ expression levels and on
NF-␬B translocation and who subsequently concluded
that steroids act independently of NF-␬B to reduce
inflammatory cytokine production in FLS. However, it is
important to note that in recent years, evidence has
accumulated to show that I␬B␣ up-regulation does not
constitute an exclusive mechanism to explain the crosscoupling between GR and NF-␬B (27,28). It was shown,
for example, that blockage of novel protein synthesis still
allowed for an efficient DEX-mediated cytokine gene
repression at the transcriptional level in a murine fibroblast cell line (29). As such, it may be more likely to
assume that also in FLS, a nuclear interference between
steroid-activated GR and NF-␬B may exist, which may,
but does not necessarily have to, preclude the DNA
binding of p65. Indeed, depending on the time point
studied, we observed that treatment of the FLS with
DEX could result in a decrease in p65 DNA binding
(Figures 2B and C), concomitant with the absence of a
cytoplasmic effect, namely, an effect on I␬B␣ protein
levels, and with the absence of a p65 redistribution from
the nucleus to the cytoplasm.
Interestingly, we found that the interaction of
CpdA with the NF-␬B pathway in FLS differed drastically from the pattern observed after DEX treatment. In
sharp contrast to the effects of DEX, we observed that
CpdA could attenuate cytoplasmic-to-nuclear translocation of p65. This observation is also reflected in the
amount of activated p65 that could be found in the
nucleus of the RA FLS (Figure 2A) and bound on DNA
after CpdA treatment (Figures 2B and C). Indeed, both
the TransAM assay (Figure 2B) and the ChIP analysis
(Figure 2C) consistently showed a lower level of NF-␬B
DNA binding, regardless of the duration of TNF␣
We investigated the mechanism of CpdAmediated p65 cytoplasmic retention in more depth,
which revealed that CpdA interferes with the NF-␬B
pathway in the cytoplasm of the cell. We showed that
CpdA attenuates IKK phosphorylation, and thereby
hampers I␬B␣ degradation (Figure 3A). This attenuation of degradation seems to be the only reason for the
observed higher levels of I␬B␣, since blocking of new
protein synthesis did not lower the I␬B␣ levels after
CpdA treatment (Figure 3B). As such, it is quite unlikely
that a rapid resynthesis of I␬B␣ is responsible for the
higher I␬B␣ levels observed.
Intriguingly, this rapid effect of CpdA on the
phosphorylation of IKK seems to occur largely independent of the presence of the GR (Figure 3C). However,
the above finding does not exclude an additional crosscoupling between CpdA-activated GR and NF-␬B in the
nucleus of FLS. This would be in agreement with
previous results showing that CpdA, similar to GCs,
interferes with the transactivation potential of a nuclear
Gal4-p65 fusion protein, thereby ruling out the absolute
necessity for cytoplasmic events in the negative interference between GR and NF-␬B in L929sA cell lines (34).
In line with this reasoning, we found that the generepressive effect of CpdA on TNF␣-induced IL-1␤
expression was negatively affected when the levels of GR
were knocked down (Figure 5). Moreover, consistent
with a definite role for the GR in the gene-repressive
effect of CpdA, we found an increased GR recruitment
onto the TNF␣-stimulated IL-1␤ promoter in the presence of CpdA (Figure 2C). Our results thus indicate that
although the DEX-activated GR may partially interfere
with p65 recruitment at the IL-1␤ promoter, without a
subsequent redistribution of p65 into the cytosol, CpdA
may also interfere with p65 recruitment via an additional
partial interference with the upstream signaling pathways, resulting in a more cytoplasmic phenotype of p65
(Figure 2).
In addition, we observed that the 3 MAPK pathways, i.e., p38, JNK, and ERK, are efficiently activated
by TNF␣ in FLS. Although it has been described that
GCs interfere with MAPK signaling pathways, thereby
hampering full-blown NF-␬B activity, this effect seems
to be cell type–dependent (31), and we could not
observe such an effect in RA FLS. In contrast to what
has been described by Toh and coworkers (51), who
demonstrated a clear suppressive effect of DEX on the
amounts of phosphorylated p38, JNK, and ERK, our
data showed an almost negligible response to DEX on
the level of inhibition of MAPK phosphorylation in FLS
(Figure 4A). The differential results in these studies of
FLS may be due to the fact that we pretreated the cells
with DEX for 1 hour, whereas in the former study (51),
a 24-hour pretreatment was applied, a strategy that does
not exclude a role for secondary effects.
Notwithstanding these differences, investigation
of the effect of CpdA on the MAPK pathways in FLS
revealed that CpdA treatment resulted in a strikingly
more efficient down-regulation of MAPK phosphorylation, and thus of MAPK activation, in comparison with
the effects of DEX (Figure 4B). The observation that
both DEX and CpdA suppress IL-1␤ cytokine mRNA
expression equally well would suggest either that blockage of MAPK is not a determining factor for mediating
efficient cytokine gene repression or, alternatively, that
DEX could target still other transcription factors or
cofactors present in the IL-1␤ promoter enhanceosome.
Similar to the inhibition of IKK (Figure 3C), we found
that the CpdA-mediated inhibition of activated MAPK
also stayed invariable following knockdown of GR (Figure 4B), suggesting a dual mechanism of CpdA for
interfering with the pathways leading toward NF-␬B.
It has been observed, even in the same cell type,
that different mechanisms can contribute to the GCmediated inhibition of NF-␬B at different time points
(50). However, which mechanism predominates when a
longer time period is assessed seems to be highly cell
type– and promoter-specific. In this respect, results from
our ChIP assay (Figure 2C) and siRNA analyses (Figure
5) suggest that for the repression of IL-1␤, observed
over a time span of 6 hours, the genomic effects of the
GR, i.e., its interference with proinflammatory transcription factors, such as NF-␬B, in the nucleus of the
cell do seem to outweigh the rapid, GR-independent
effects observed herein.
The present report thus describes 2 compounds
with completely different structures, both of which exert
their antiinflammatory effects in FLS via the same
receptor, namely, the GR. However, in sharp contrast to
the classic steroid DEX, CpdA is able to additionally
interfere with the NF-␬B activation pathway ex vivo in
the cytoplasm of RA FLS. A dual pathway, partially
dependent and partially independent of the GR, may
therefore explain the gene-inhibitory effects of CpdA in
We are grateful to Dr. Firestein and his collaborators
for helping us with the FLS transfection protocol. We also
thank Dr. M. Van Gele and Prof. Dr. J. Lambert from the
Department of Dermatology (University Hospital Ghent) for
placing their Amaxa nucleofector at our disposition.
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. De Bosscher had full access to all
of the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Gossye, Elewaut, Haegeman, De Bosscher.
Acquisition of data. Gossye, Bougarne, Bracke.
Analysis and interpretation of data. Gossye, Elewaut, Bougarne,
Bracke, Van Calenbergh, De Bosscher.
1. Knedla A, Neumann E, Muller-Ladner U. Developments in the
synovial biology field 2006. Arthritis Res Ther 2007;9:209–16.
2. Smolen JS, Steiner G. Therapeutic strategies for rheumatoid
arthritis. Nat Rev Drug Discov 2003;2:473–88.
3. Muller-Ladner U, Ospelt C, Gay S, Distler O, Pap T. Cells of the
synovium in rheumatoid arthritis. Synovial fibroblasts. Arthritis
Res Ther 2007;9:223–33.
4. Muller-Ladner U, Kriegsmann J, Franklin BN, Matsumoto S,
Geiler T, Gay RE, et al. Synovial fibroblasts of patients with
rheumatoid arthritis attach to and invade normal human cartilage
when engrafted into SCID mice. Am J Pathol 1996;149:1607–15.
5. Smith TJ. Insights into the role of fibroblasts in human autoimmune diseases. Clin Exp Immunol 2005;141:388–97.
6. Abeles AM, Pillinger MH. The role of the synovial fibroblast in
rheumatoid arthritis: cartilage destruction and the regulation of
matrix metalloproteinases. Bull NYU Hosp Jt Dis 2006;64:20–4.
7. Karouzakis E, Neidhart M, Gay RE, Gay S. Molecular and cellular
basis of rheumatoid joint destruction. Immunol Lett 2006;106:
8. Mor A, Abramson SB, Pillinger MH. The fibroblast-like synovial
cell in rheumatoid arthritis: a key player in inflammation and joint
destruction. Clin Immunol 2005;115:118–28.
9. Li X, Makarov SS. An essential role of NF-␬B in the “tumor-like”
phenotype of arthritic synoviocytes. Proc Natl Acad Sci U S A
10. Asahara H, Asanuma M, Ogawa N, Nishibayashi S, Inoue H. High
DNA-binding activity of transcription factor NF-␬B in synovial
membranes of patients with rheumatoid arthritis. Biochem Mol
Biol Int 1995;37:827–32.
11. Vincenti MP, Coon CI, Brinckerhoff CE. Nuclear factor ␬B/p50
activates an element in the distal matrix metalloproteinase 1
promoter in interleukin-1␤–stimulated synovial fibroblasts. Arthritis Rheum 1998;41:1987–94.
12. Miyazawa K, Mori A, Yamamoto K, Okudaira H. Transcriptional
roles of CCAAT/enhancer binding protein-␤, nuclear factor-␬B,
and C-promoter binding factor 1 in interleukin (IL)-1␤-induced
IL-6 synthesis by human rheumatoid fibroblast-like synoviocytes.
J Biol Chem 1998;273:7620–7.
13. Miyazawa K, Mori A, Yamamoto K, Okudaira H. Constitutive
transcription of the human interleukin-6 gene by rheumatoid
synoviocytes: spontaneous activation of NF-␬B and CBF1. Am J
Pathol 1998;152:793–803.
14. Han Z, Boyle DL, Manning AM, Firestein GS. AP-1 and NF-␬B
regulation in rheumatoid arthritis and murine collagen-induced
arthritis. Autoimmunity 1998;28:197–208.
15. Firestein GS. NF-␬B: holy grail for rheumatoid arthritis? [editorial]. Arthritis Rheum 2004;50:2381–6.
16. Miagkov AV, Kovalenko DV, Brown CE, Didsbury JR, Cogswell
JP, Stimpson SA, et al. NF-␬B activation provides the potential
link between inflammation and hyperplasia in the arthritic joint.
Proc Natl Acad Sci U S A 1998;95:13859–64.
17. Makarov SS. NF-␬B in rheumatoid arthritis: a pivotal regulator of
inflammation, hyperplasia, and tissue destruction. Arthritis Res
18. Ghosh S, Karin M. Missing pieces in the NF-␬B puzzle. Cell
2002;109 Suppl:S81–96.
19. Hayden MS, Ghosh S. Signaling to NF-␬B. Genes Dev 2004;18:
20. Perkins ND, Gilmore TD. Good cop, bad cop: the different faces
of NF-␬B. Cell Death Differ 2006;13:759–72.
21. Campbell KJ, Perkins ND. Post-translational modification of
RelA(p65) NF-␬B. VI. Biochem Soc Trans 2004;32:1087–9.
22. Vermeulen L, Vanden Berghe W, Haegeman G. Regulation of
NF-␬B transcriptional activity. Cancer Treat Res 2006;130:89–102.
23. Vanden Berghe W, Plaisance S, Boone E, De Bosscher K, Schmitz
ML, Fiers W, et al. p38 and extracellular signal-regulated kinase
mitogen-activated protein kinase pathways are required for nuclear factor-␬B p65 transactivation mediated by tumor necrosis
factor. J Biol Chem 1998;273:3285–90.
24. Vermeulen L, De Wilde G, Van Damme P, Vanden Berghe W,
Haegeman G. Transcriptional activation of the NF-␬B p65 subunit
by mitogen- and stress-activated protein kinase-1 (MSK1). EMBO
J 2003;22:1313–24.
25. Jamaluddin M, Wang S, Boldogh I, Tian B, Brasier AR. TNF-␣induced NF-␬B/RelA Ser(276) phosphorylation and enhanceosome formation is mediated by an ROS-dependent PKAc pathway. Cell Signal 2007;19:1419–33.
26. Baud V, Karin M. Signal transduction by tumor necrosis factor and
its relatives. Trends Cell Biol 2001;11:372–7.
27. De Bosscher K, Vanden Berghe W, Haegeman G. Nuclear receptors and NF-␬B. Oncogene 2006;51:6868–86.
28. Gossye V, Haegeman G, De Bosscher K. Therapeutic implications
of the nuclear factor-␬B/nuclear receptor cross-talk. Front Biosci
29. De Bosscher K, Schmitz ML, Vanden Berghe W, Plaisance S, Fiers
W, Haegeman G. Glucocorticoid-mediated repression of nuclear
factor-␬B-dependent transcription involves direct interference
with transactivation. Proc Natl Acad Sci U S A 1997;94:13504–9.
30. Nissen RM, Yamamoto KR. The glucocorticoid receptor inhibits
NF␬B by interfering with serine-2 phosphorylation of the RNA
polymerase II carboxy-terminal domain. Genes Dev 2000;14:
31. De Bosscher K, Vanden Berghe W, Haegeman G. The interplay
between the glucocorticoid receptor and nuclear factor-␬B or
activator protein-1: molecular mechanisms for gene repression.
Endocr Rev 2003;24:488–522.
32. Lowenberg M, Stahn C, Hommes DW, Buttgereit F. Novel
insights into mechanisms of glucocorticoid action and the development of new glucocorticoid receptor ligands. Steroids 2008;73:
33. Lowenberg M, Verhaar AP, van den Brink GR, Hommes DW.
Glucocorticoid signaling: a nongenomic mechanism for T-cell
immunosuppression. Trends Mol Med 2007;13:158–63.
34. De Bosscher K, Vanden Berghe W, Beck IM, Van Molle W,
Hennuyer N, Hapgood J, et al. A fully dissociated compound of
plant origin for inflammatory gene repression. Proc Natl Acad Sci
U S A 2005;102:15827–32.
35. Dewint P, Gossye V, De Bosscher K, Vanden Berghe W, Van
Beneden K, Deforce D, et al. A plant-derived ligand favoring
monomeric glucocorticoid receptor conformation with impaired
transactivation potential attenuates collagen-induced arthritis.
J Immunol 2008;180:2608–15.
Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF,
Cooper NS, et al. The American Rheumatism Association 1987
revised criteria for the classification of rheumatoid arthritis.
Arthritis Rheum 1988;31:315–24.
Louw A, Swart P, de Kock SS, van der Merwe KJ. Mechanism for
the stabilization in vivo of the aziridine precursor –(4acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride by
serum proteins. Biochem Pharmacol 1997;53:189–97.
Clayton AL, Rose S, Barratt MJ, Mahadevan LC. Phosphoacetylation of histone H3 on c-fos- and c-jun-associated nucleosomes
upon gene activation. EMBO J 2000;19:3714–26.
Vanden Berghe W, Francesconi E, De Bosscher K, Resche-Rigon M,
Haegeman G. Dissociated glucocorticoids with anti-inflammatory
potential repress interleukin-6 gene expression by a nuclear factor␬B-dependent mechanism. Mol Pharmacol 1999;56:797–806.
Bradford MM. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 1976;72:248–54.
Inoue T, Hammaker D, Boyle DL, Firestein GS. Regulation of
JNK by MKK-7 in fibroblast-like synoviocytes. Arthritis Rheum
Cogswell JP, Godlevski MM, Wisely GB, Clay WC, Leesnitzer
LM, Ways JP, et al. NF-␬B regulates IL-1␤ transcription through
a consensus NF-␬B binding site and a nonconsensus CRE-like site.
J Immunol 1994;153:712–23.
Natoli G, Chiocca S. Nuclear ubiquitin ligases, NF-␬B degradation, and the control of inflammation. Sci Signal 2008;1:pe1.
Gherzi R, Trabucchi M, Ponassi M, Ruggiero T, Corte G, Moroni
C, et al. The RNA-binding protein KSRP promotes decay of
␤-catenin mRNA and is inactivated by PI3K-AKT signaling. PLoS
Biol 2006;5:e5.
Gossye V, Elewaut D, Van Beneden K, Dewint P, Haegeman G,
De Bosscher K. A plant-derived glucocorticoid receptor modulator attenuates inflammation without provoking ligand-induced
resistance. Ann Rheum Dis 2009. In press.
Sugiyama E, Kuroda A, Taki H, Ikemoto M, Hori T, Yamashita N,
et al. Interleukin 10 cooperates with interleukin 4 to suppress
inflammatory cytokine production by freshly prepared adherent
rheumatoid synovial cells. J Rheumatol 1995;22:2020–6.
Han CW, Choi JH, Kim JM, Kim WY, Lee KY, Oh GT.
Glucocorticoid-mediated repression of inflammatory cytokine
production in fibroblast-like rheumatoid synoviocytes is independent of nuclear factor-␬B activation induced by tumour necrosis
factor ␣. Rheumatology (Oxford) 2001;40:267–73.
Zhang Y, Saccani S, Shin H, Nikolajczyk BS. Dynamic protein
associations define two phases of IL-1␤ transcriptional activation.
J Immunol 2008;181:503–12.
Goto M, Katayama KI, Shirakawa F, Tanaka I. Involvement of
NF-␬B p50/p65 heterodimer in activation of the human prointerleukin-1␤ gene at two subregions of the upstream enhancer
element. Cytokine 1999;11:16–28.
Newton R, Holden NS. Separating transrepression and transactivation: a distressing divorce for the glucocorticoid receptor? Mol
Pharmacol 2007;72:799–809.
Toh ML, Yang Y, Leech M, Santos L, Morand EF. Expression of
mitogen-activated protein kinase phosphatase 1, a negative regulator of the mitogen-activated protein kinases, in rheumatoid
arthritis: up-regulation by interleukin-1␤ and glucocorticoids. Arthritis Rheum 2004;50:3118–28.
DOI 10.1002/art.27206
American College of Rheumatology Office Has Moved
American College of Rheumatology members and others who correspond with the College should be aware
that, as of November 1, 2009, the ACR office is at a new location. Please send all correspondence to
American College of Rheumatology, 2200 Lake Boulevard NE, Atlanta, GA 30319-5312. The ACR telephone
and main fax numbers have not changed (phone 404-633-3777; fax 404-633-1870).
Без категории
Размер файла
325 Кб
two, inhibition, glucocorticoid, mechanism, differential, arthritis, modulators, synovial, receptov, rheumatoid, fibroblasts
Пожаловаться на содержимое документа