Tài liệu Báo cáo khoa học: Molecular aspects of rheumatoid arthritis: role of transcription factors ppt

MINIREVIEW
Molecular aspects of rheumatoid arthritis: role of
transcription factors
Hiroshi Okamoto
1
, Thomas P. Cujec
2
, Hisashi Yamanaka
1
and Naoyuki Kamatani
1
1 Institute of Rheumatology, Tokyo Women’s Medical University, Japan
2 Ambrx, Inc. La Jolla, CA, USA
The central dogma of molecular biology is that DNA
produces RNA, which, in turn, produces protein. In
the process of transcription, RNA is produced from
the DNA and this conversion is an essential element in
gene expression. The central role of transcription in
the process of gene expression makes it an attractive
control process for regulating the expression of genes
in particular cell types or in response to a particular
signal, such as a cytokine. To study this control mech-
anism, the DNA sequences within individual genes
that are essential for basal or regulated gene expression
have been extensively studied. In most eukaryotic
genes a TATA box is found upstream of the site of
transcriptional initiation, although this element is lack-
ing in housekeeping genes and in some tissue-specific
genes. In the genes without a TATA box, a sequence
known as the initiator element, which is located over
the start site of transcription, appears to play a critical

role in determining the initiation point and acts as a
minimal promoter capable of producing basal levels of
transcription. In TATA-less promoters, the weak acti-
vity of the promoter is dramatically increased by other
elements located upstream of the proximal promoter
region. These elements are found in a wide variety of
Keywords
NFAT; NF-jB; rheumatoid arthritis;
transcription factors
Correspondence
H. Okamoto, Institute of Rheumatology,
Tokyo Women’s Medical University, 10-22
Kawada-cho, Shinjuku, Tokyo 162-0054,
Japan
Fax: +81 3 5269 1726
Tel: +81 3 5269 1725
E-mail:
(Received 14 March 2008, accepted 22 May
2008)
doi:10.1111/j.1742-4658.2008.06582.x
Rheumatoid arthritis is a multifactorial disease characterized by chronic
inflammation of the joints. Both genetic and environmental factors are
involved in the pathogenesis leading to joint destruction and ultimately dis-
ability. In the inflamed RA joint the synovium is highly infiltrated by
CD4
+
T cells, B cells and macrophages, and the intimal lining becomes
hyperplastic owing to the increased number of macrophage-like and fibro-
blast-like synoviocytes. This hyperplastic intimal synovial lining forms an
aggressive front, called pannus, which invades cartilage and bone struc-

tures, leading to the destruction and compromised function of affected
joints. This process is mediated by a number of cytokines (tumor
necrosis factor-a, interleukin-1, interleukin-6, interleukin-17 interferon-c,
etc.), chemokines (monocyte chemoattractant protein-1, monocyte chemo-
attractant protein-4 CCL18, etc.), cell adhesion molecules (intercellular
adhesion molecule-1, vascular cell adhesion molecule-1, etc.) and matrix
metalloproteinases. Expression of these molecules is controlled at the tran-
scription level and activation of a limited number of transcription factors is
involved in this process.
Abbreviations
AGE, advanced glycation end-product; AP-1, activator protein-1; FLIP, Fas-associated death domain-like interleukin 1b-converting enzyme-
inhibitory protein; FLS, fibroblast-like synoviocytes; GM-CSF, granulocyte–macrophage colony-stimulating factor; IKK, IjB kinase; IL,
interleukin; IjB, inhibitor of NF-jB proteins; MMP, matrix metalloproteinase; NFAT, nuclear factor for activation of T cells; NF-jB, nuclear
factor-jB; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response element; RA, rheumatoid arthritis;
RAGE, receptor for advanced glycation end-products; RANKL, receptor activation of NF-jB ligand; SAA, serum amyloid A; TNF-a, tumor
necrosis factor-a.
FEBS Journal 275 (2008) 4463–4470 ª 2008 The Authors Journal compilation ª 2008 FEBS 4463
genes and play a role in stimulating the constitutive
activity of promoters. In addition, the presence of spe-
cific DNA sequences that can bind particular proteins
will confer on a specific gene the ability to respond to
particular stimuli. Such binding proteins are transcrip-
tional factors. In this review, we focus on the role of
transcriptional factors on the pathology of rheumatoid
arthritis (RA).
Nuclear factor-jB
The nuclear factor-jB (NF-jB) proteins are a family
of ubiquitously expressed transcription factors that
play an essential role in most immune and inflamma-
tory responses. In mammals, the NF-jB family con-

sists of five members: RelA (p65), RelB, c-Rel, NF-
jB1 (p50 and its precursor p105) and NF-jB2 (p52
and its precursor p100). They form a variety of
homodimers and heterodimers, each of which activates
its own characteristic set of genes, and share a 300-
amino acid domain (designated the Rel homology
domain) that mediates their DNA binding, dimeriza-
tion and nuclear translocation [1–3]. Although, the
most prevalent activated form is the heterodimer RelA
(p65) and p50, different dimers can bind to the same
or distinct sites in NF-jB-dependent promoters, thus
regulating the transcription of response genes in a
cell-type and stimulus-type specific manner [4,5]. The
NF-jB proteins are retained in an inactive form in the
cytoplasm through their interaction with inhibitor of
NF-jB proteins (IjB). Cellular stimulation, by cyto-
kines such as tumor necrosis factor-a (TNF-a) and
interleukin (IL)-1b, activate the inhibitor of NF-jB
kinase [IjB kinase (IKK) complex] and then this com-
plex phosphorylates IjB, which leads to its ubiquitina-
tion and subsequent proteosomal degradation.
Degradation of IjB enables NF-jB to translocate to
the nucleus, leading to stimulation of the transcription
of genes containing the consensus jB sequence
5¢-GGGPuNNPyPyCC-3¢ (where Pu denotes a purine
and Py denotes a pyrimidine). The genes containing
the jB sequence include cytokine and chemokine genes
[TNF, IL-1, IL-2, IL-6, macrophage inflammatory pro-
tein-1b, macrophage inflammatory protein-2, regulated
on activation, normal, T-cell expressed, and secreted

(RANTES), etc.], adhesion molecule genes (E-selectin,
intercellular adhesion molecule-1, vascular cell adhe-
sion molecule-1, etc.), anti-apoptosis genes [XIAP,
c-IAPs, c-Fas-associated death domain-like interleukin
1b-converting enzyme-inhibitory protein (c-FLIP),
survivine, bcl-2, bcl-x
L
, etc.], NF-jB family genes
(p52 ⁄ p100, p50 ⁄ p105, c-Rel, IjBa, etc.), cell prolifera-
tion-associated genes [cyclin D1, c-Myc, bone morpho-
genetic protein-2 (BMP-2), etc.], viral genes [HIV-1,
simian immunodeficiency virus, Epstein–Barr virus,
etc.) and others [matrix metalloproteinases (MMPs),
vascular endothelial growth factor, inducible nitric
oxide synthase, cyclooxygenase-2, etc.). Some of these
genes have been reported to have important roles in
the pathogenesis of RA [1]. In addition, the NF-jB
family of genes has been reported to be highly
expressed and activated in RA-affected tissues, and
several interventions, such as dominant-negative IKK
and antisense NF-jB oligonucleotides, have effectively
prevented the expression of cytokines and the develop-
ment of arthritis in vitro and in animal models. Fur-
thermore, NF-jB has been reported to contribute to
the fierce proliferation of synovial cells. Several lines
of evidence suggest that RA synovial cells proliferate
as fiercely as tumor cells and that this aggressive pro-
liferation plays an important role in the pathogenesis
of RA. Synovial hyperproliferation has been reported
to be caused, at least in part, by impaired apoptosis of

synovial cells and deficient apoptosis of synovial cells
resulting from the upregulation of anti-apoptotic mole-
cules such as bcl-2 and FLIP [6,7]. Thus, NF-jB con-
tributes to the hyperproliferation of synovial cells in
RA by regulating the gene expression of FLIP and
bcl-2.
Nuclear factor-jB is activated by various inducers,
including cytokines (TNF-a, IL-1b, IL-2, IL-17, etc.),
mitogens [B-lymphocyte activating factor (BAFF),
CD40 ligand, etc.] and stess ⁄ cartinogens (ultraviolet
light, hypoxia, 4b-phorbol 12-myristate 13-acetate, etc.).
Another inducer is the serum amyloid A (SAA) protein,
an acute-phase protein produced by hepatocytes in
response to pro-inflammatory cytokines, and its expres-
sion is up-regulated during the course of the inflamma-
tory process [8]. Although a wealth of information
concerning the diagnosis and pathogenesis of AA amy-
loidosis has accumulated, the biological role(s) of SAA
in the pathogenesis of RA is still not fully understood.
Mullan et al. [9] reported that acute-phase SAA was
as effective at increasing the time-dependent and dose-
dependent expression of intercellular adhesion mole-
cule-1 and vascular cell adhesion molecule as IL-1b
and TNF-a, and that their expression was partially
mediated by NF-jB signaling. The accumulation of
advanced glycation end-products (AGEs), S100A12
and high-mobility-group-box chromosomal protein 1
(HMGB1) has been associated with joint inflammation
in RA. The receptor for these proteins, termed recep-
tor for AGEs (RAGE) has been reported to be highly

expressed in synovial tissue macrophages from RA
patients [10]. RAGE has also been reported to be a
receptor for the amyloidogenic form of SAA [11].
Role of transcription factors H. Okamoto et al.
4464 FEBS Journal 275 (2008) 4463–4470 ª 2008 The Authors Journal compilation ª 2008 FEBS
From these findings, we hypothesized that acute-phase
SAA could bind to RAGE on the surface of synovial
cells, thereby resulting in NF-jB signaling and the
active promotion of RA-mediated joint inflammation.
To study the biological implication of SAA expression
in RA joints, we further analyzed the in vitro effects of
SAA. We studied the effects of SAA on cytokine pro-
duction from fibroblast-like synoviocytes (FLS) and
found that SAA induced expression of the pro-inflam-
matory cytokines IL-6 and IL-8 in a dose-dependent
manner. Serum amyloid A stimulated the transcrip-
tional activation by NF-jB in a dose-dependent
manner in a reporter gene assay in 293T cells transfect-
ed with p4xjB-Luc plasmid. We studied the effects of
SAA on NF-jB activation and found that SAA
induced the degradation of IjBa as well as IL-1b
(10 ngÆmL
)1
). In order to study whether the effect of
SAA on NF-jB activation is mediated through the
binding of SAA to RAGE on synovial cells, we pre-
incubated SAA with various concentrations of soluble
recombinant RAGE protein before adding it to the
FLS. We observed a dose-dependent inhibition of
SAA-induced IjBa degradation. By immunofluores-

cent studies, we also found that SAA stimulation
promoted nuclear translocation of NF-jB, whereas
pre-incubation of SAA with RAGE inhibited nuclear
translocation [12]. These data suggested that SAA of
RA joints is actively involved in the pathogenesis of
RA through the SAA–RAGE–NF-jB signaling path-
way (Fig. 1).
We found that angiotensin II is also an inducer of
NF-jB activation in FLS. We have shown that angio-
tensin II activated NF-jB in synovial cells to induce
the monocyte chemoattractant protein-1 and that the
angiotensin receptor blocker inhibited this activation
[13].
It is noteworthy that some anti-RA drugs, including
corticosteroids, have been shown to block the NF-jB
activation cascade (Fig. 2). Among the drugs currently
used for the treatment of diseases other than RA, such
as diabetes, hyperlipidemia and hypertension, there are
some drugs that have the potential to inhibit NF-jB
activation. To seek other candidate compounds for use
in an anti-RA strategy, we studied several drugs that
have pleiotropic actions on the NF-jB activation cas-
cade. Ligands for peroxisome proliferator-activated
receptors (PPARs) are such examples. Peroxisome
proliferator-activated receptors are members of the
nuclear hormone receptor family, the largest family of
transcription factors [14]. Three distinct members of
the PPAR subfamily have been reported: a, d (also
called b, NUC-1) and c, all of them being activated by
naturally occurring fatty acids or fatty acid derivatives.

Peroxisome proliferator-activated receptors heterodi-
merize with the retinoid X receptor and regulate tran-
scription of target genes through binding to specific
peroxisome proliferator response elements (PPREs),
which consist of a direct repeat of the nuclear receptor
hexameric DNA core recognition motif spaced by one
nucleotide. In addition to the regulation of gene tran-
scription via PPREs, PPARs modulate gene expression
in a DNA-binding-independent manner. Peroxisome
proliferator-activated receptor-a is highly expressed in
liver, heart, muscle, kidney and cells of the arterial
wall and it is activated by fibrate, fatty acids and eico-
sanoids. Peroxisome proliferator-activated receptor-a
ligands inhibit IL-1-induced production of IL-6 and
prostaglandin and inhibit the expression of cyclooxy-
genase-2 by negatively interfering with NF-jB tran-
scriptional activity. Peroxisome proliferator-activated
receptor-a ligands are thought to inhibit NF-jB activ-
ity by inducing IjBa, which, in turn, inhibits NF-jB
signaling. Peroxisome proliferator-activated receptor-c
is expressed at high levels in adipose tissue, is a critical
regulator of adipocyte differentiation and reportedly
plays a role in glucose homeostasis and insulin sensi-
tivity. In addition, PPAR-c has been suggested to be
an important immunomodulatory factor that is
expressed in cells of the immune system, specifically in
the spleen, monocytes, bone-marrow precursors and
helper T cells [15]. Peroxisome proliferator-activated
receptor-c ligands also reportedly inhibit disease pro-
gression of inflammatory bowel diseases, ischemic

heart diseases, experimental autoimmune encephalomy-
elitis and RA [16]. These PPAR-c ligands inhibit gene
expression by preventing the phosphorylation of IKK,
which, in turn, reduces the activity of the transcription
SAA
Chondrocytes
synovial cells
SAA
blood circulation
RAGE
P
P
Ubiquitination and
(Cytoplasm)
p65
Phosphorylation of
IκBα
degradation of IκBα
p50
IκBα
IκBα at Ser 32/36
p65
p50
p50
(Nucleus)
IL-6, IL-8, MMPs, etc.
Target gene
SAA
SAA
p65

Fig. 1. SAA in RA joints binds to RAGE on synovial cells and acti-
vates the NF-jB signaling pathway in these cells.
H. Okamoto et al. Role of transcription factors
FEBS Journal 275 (2008) 4463–4470 ª 2008 The Authors Journal compilation ª 2008 FEBS 4465
factor, NF-jB [17]. Taken together, these findings sug-
gest that PPAR-a and PPAR-c may negatively regu-
late the inflammatory processes in RA. To examine the
induction of IL-6, IL-8 and granulocyte–macrophage
colony-stimulating factor (GM-CSF), FLS obtained
from RA patients were stimulated with 10 ng ÆmL
)1
of
IL-1b. Interleukin-6, IL-8 and GM-CSF production
from FLS were suppressed in a dose-dependent man-
ner in the presence of PPAR-c ligands and a PPAR-a
ligand, fenofibrate. Neither PPAR-a nor PPAR-c
ligands inhibit basal level expression of these cyto-
kines, and these compounds are not toxic to FLS.
Next, we examined whether PPAR-a and PPAR-c
ligands inhibit nuclear translocation of NF-jBinan
immunohistochemical assay. As shown in Fig. 3A,
FLS were incubated in the presence of 10 ngÆmL
)1
of
IL-1b in order to stimulate NF-jB nuclear transloca-
tion. As expected, without IL-1b stimulation, NF-jB
remained localized in the cytoplasm. However, after
30 min of stimulation with IL-1b, NF-jB was mainly
localized in the nucleus. In the presence of 100 lm
pioglitazone, or fenofibrate, nuclear localization of

NF-jB was inhibited. These results are consistent with
the PPAR-induced suppression of cytokine expression
described above and indicate that this suppression is
caused by the inhibition of NF-jB nuclear trans-
location in FLS. To investigate further the anti-NF-jB
effects of these compounds, we performed western
γ
Aspirin and glucocorticoids
I
κB
α
[Nature (2000) 403, 103–108]
[
J Clin Invest
(
1998
) 101 1163–1174]
IL-1 receptor
(Cytoplasm)
NIK
PPAR-
α ligands
[J Biol Chem
(2000)
275, 36703–36707]
PPAR-α ligands,
aspirin, salicylate,
sulindac
[Nature (1998)
396, 77–80,

Sulfasalazine
[
J Clin
Invest (
1998
) 101, 1163 1174]
NIK
IKK
Complex
β
α
IκBα
P
P
Ubiquitination and
degradation of IκBα
J Biol Chem
(1999)
274, 27307–27314]
p65
p50
IκBα
Phosphorylation of
IκBα at Ser 32/36
IκBα
p65
p50
PPAR-
α
I d ti f I B

(Nucleus)
p50
p65
Target gene
Co-activators
In
d
uction o
f
IκBα
TNF-
α, Cox-2, IL-1, IL-6, IL-8, MMPs, etc.
Glucocorticoids
[
Science
(1995)
270, 286–290, Science (1994) 265, 956–959, Science (1994) 270, 283–286]
[
Mol Cell Biol
(1995) 15, 943–953]
Fig. 2. NF-jB activation pathway and the
site of inhibition of various compounds.
PLC l d
Ca
2+
PLC coupled
receptor
CRAC
(cytoplasm)
Ca

2+
Calmodulin
NFAT
P
PLC-
IP
3
Ca
2+
Ca
2
+
Ca
2+
Ca
2+
C
2+
Ca
2+
Ca
2+
Ca
2+
Calcineurin A
ER
Ca
2
Ca
2+

Ca
2+
Ca
2+
Ca
2+
Ca
2+
Ca
2+
p38
MAP kinase
MEKK-1
CsA
FK-506
Cyclophilin
FKBP
(Nucleus)
NFAT
DYRK
Target ge ne
Co-activators
NFAT
IL-2, IL-4, IFN- , GM-CSF, CD40L, TNF-α,
αγ
etc.
Calcineurin B
Fig. 3. NFAT activation pathway and the
site of inhibition of immunosuppressive
drugs CD40 ligand (CD40L), dual-specificity

tyrosine-regulated kinase (DYRK), endo-
plasmic reticulum (ER), mitogen-activated
protein (MAP) kinase, MAPK kinase kinase-1
(MEKK-1).
Role of transcription factors H. Okamoto et al.
4466 FEBS Journal 275 (2008) 4463–4470 ª 2008 The Authors Journal compilation ª 2008 FEBS
blots to detect IjBa degradation by the IL-1b signal.
As demonstrated, the PPAR-c ligand (pioglitazone)
and fenofibrate inhibited the IL-1b-stimulated degrada-
tion of IjBa. Therefore, PPAR-a and PPAR-c ligands
induced NF-jB signaling in FLS, as illustrated in
Fig. 2. We tested the effect of PPAR-a and PPAR-c
ligands in vivo on the progression and severity of adju-
vant-induced arthritis in female Lewis rats and found
that pioglitazone and fenofibrate suppressed the pro-
gression of clinical arthritis compared with control rats
treated with NaCl ⁄ P
i
, as demonstrated by paw volume
and arthritis score. These data suggest that both
PPAR-a and PPAR-c ligands have anti-arthritis effects
in vivo [18]. Considering the wide array of events under
the control of NF-jB, including cytokine and cyclo-
oxygenase-2 expression, osteoclast differentiation and
apoptosis, and the impact of these events on the path-
ogenesis of RA, NF-jB is an efficient and feasible
therapeutic target for RA. Therapy with fenofibrate
may serve as a new anti-NF-jB strategy for the treat-
ment of RA. We have also shown, by case reports,
that fenofibrate is useful for the treatment of RA and

autoimmune hepatitis [19,20].
Besides its involvement in immunoregulation, NF-
jB has also been reported to be associated with the
inhibition of programmed cell death and has an impor-
tant role in the development and homeostasis of the
immune, hepatic and nervous systems. The embryonic
lethality of RelA-deficient mice was one of the first
indications that NF-jB contributes a crucial anti-
apoptotic effect during normal development and this
embryonic death was attributed to extensive apoptosis
of developing hepatocytes. A similar phenotype is seen
in mice lacking both copies of IKK, lacking IKK
along with IKK, or lacking the IKK regulator NEMO.
In addition, the initial cloning of the NF-jB p50 ⁄ p105
subunit cDNA revealed homology to the cellular
homolog (c-Rel) of the oncoprotein (v-Rel) from the
avian reticuloendotheliosis virus, suggesting a potential
link between NF-jB and oncogenesis. In fact, struc-
tural alteration of the NF-jB p52 ⁄ p100 subunit
encoded by the NFKB2 gene has been reported in cer-
tain T-cell lymphomas, chronic lymphocytic leukemias,
myelomas and B-cell lymphomas. Amplification of the
c-rel gene has also been reported in several types of
B-cell lymphoma. In addition to these genetic observa-
tions, several lines of evidence have demonstrated
that NF-jB transcription targets are linked to pro-
mote the oncogenic phenotype. For example, NF-jB
can promote retinoblastoma hyperphosphorylation by
binding and activating the cyclin D1 promoter, result-
ing in progression into the S phase of the cell cycle,

and IKK has been proposed to play a role in cyclin
D1 transcription through a T-cell factor site in the
promoter. Nuclear factor-jB has also been reported to
potentiate cancer cell growth by the NF-jB-associated
upregulation of hypoxia-inducible factor-1 and its reg-
ulation of c-myc transcription. Resistance to apoptosis
is a common feature of cancer cells and is associated
with the increased expression of anti-apoptotic factors,
such as Bcl-2 or Bcl-x
L
. Nuclear factor-jB directly reg-
ulates a potent anti-apoptotic pathway, and genes
regulated by NF-jB that suppress apoptosis, such as
Bcl-2 and Bcl-x
L
, are often expressed in human can-
cers. Given the strong association between NF-jB and
the regulation of apoptosis, many studies suggest that
NF-jB controls the anti-apoptotic mechanisms associ-
ated with oncogenesis, and extensive evidence demon-
strates that compounds which block NF-jB activation
can serve as an anticancer strategy [21]. In the pathol-
ogy of RA, it is widely accepted that the progressive
destruction of articular cartilage is reliant on the evo-
lution of hyperplastic synovial tissue, and that hyper-
plasia of FLS is dependent on dysregulated
proliferation and apoptosis [22]. Methotrexate, which
is a well-known antitumor agent, is now widely
accepted as a standard therapeutic strategy for RA,
and the mechanism of action of methotrexate is

thought to be its inhibitory effects on the hyperplasia
of synovial tissue. Therefore, any compound that could
inhibit the fierce hyperplasia of synovial cells has
potential as a promising anti-RA strategy. Ligands for
PPAR-c have been reported to inhibit arthritis in ani-
mal models through the activation of synoviocyte
apoptosis [16]. The anti-arthritis effects of ligands for
PPAR-a and PPAR-c might be caused by their pro-
apoptotic effects through the inhibition of NF-jB sig-
naling. Besides anti-NF-jB compounds, some com-
pounds that possess anti-proliferation effects in
synoviocytes have been reported as potential candi-
dates in anti-RA therapeutic strategies. Lipophilic sta-
tins, such as fluvastatin, have been reported to induce
apoptosis in RA synoviocytes and have potential as
novel therapeutic agents for RA [23]. In addition, a cy-
clin-dependent kinase inhibitor, p16INK4a, has been
shown to suppress synovial cell proliferation, resulting
in inhibition of RA pathology in an animal model [24].
Vitamin K2 (menaquinone-4, MK-4) has been reported
to induce apoptosis in hepatocellular carcinoma, leuke-
mia and MDS cell lines. Thus, we investigated the
effect of MK-4 on the proliferation of rheumatoid
synovial cells and the development of arthritis in a col-
lagen-induced rat model. Our results indicated that
MK-4 inhibited the proliferation of cultured synovial
fibroblasts and the development of collagen-induced
arthritis in a dose-dependent manner. We concluded
H. Okamoto et al. Role of transcription factors
FEBS Journal 275 (2008) 4463–4470 ª 2008 The Authors Journal compilation ª 2008 FEBS 4467

that MK-4 may represent a new agent for the treat-
ment of RA in a combination therapy with other dis-
ease-modifying antirheumatic drugs [25,26].
Nuclear factor for activation of T cells Ca
2+
is a sig-
naling molecule that functions in a great variety of
organs and cells. One of the roles of Ca
2+
is to regulate
calcineurin, which in turn dephosphorylates and
induces the nuclear localization of the cytoplasmic
components of nuclear factor for activation of T cells
(NFAT) transcription complexes. In the nucleus,
NFAT transcription complexes assemble on target
DNA to activate the expression of genes such as IL-2,
IL-3, GM-CSF, IL-4, IL-5, IL-13, IFN-c, TNF-a,
CD40 ligand and Fas ligand, etc. (Fig. 3). Ligand bind-
ing of various receptors results in the activation of
phospholipase C (PLC), the release of inositol 1,4,5-tri-
phosphate (IP
3
), and a transient release of Ca
2+
from
intracellular stores through IP
3
receptors. This initial
release of Ca
2+

is not sufficient to activate NFAT tar-
get genes, and an influx of Ca
2+
through Ca
(2+)
-
release-activated Ca
(2+)
(CRAC) channels is required
[27]. Pharmacologic inhibitors of NFAT translocation,
such as tacrolimus (FK506) and cyclosporine A, are
administered to patients as part of the transplant ther-
apy because of their ability to prevent an immune
response to transplanted tissue. These compounds bind
to two different intracellular proteins, namely FK506-
binding protein (FKBP) and cyclophilin, and the
drug–protein complex then binds to the interface of the
calcineurin A ⁄ B complex and blocks its phosphatase
activity by preventing substrate access. Initial evidence
showing the importance of NFATs in the pathogenesis
of RA is the clinical observation that treatment with
cyclosporine A is effective in otherwise refractory RA.
Furthermore, tacrolimus (FK506) is now widely used
as a treatment of RA [28]. Experimental evidence has
shown that NFATs (NFAT1–5) are expressed in the
RA synovium and that NFAT1 knockout mice and
NFAT1 ⁄ 4 double- knockout mice developed an asym-
metric oligoarthritis [29]. Nuclear factor for activation
of T cells has been shown to have roles in the bone
destruction of RA. Bone destruction has been shown to

be caused by an abnormal activation of the immune
system in RA. Osteoclasts are cells of monocyte ⁄ mac-
rophage origin and are the key players in the control of
bone metabolism. Receptor activation of NF-jB ligand
(RANKL) induces osteoclast differentiation in the pres-
ence of the macrophage colony-stimulating factor.
RANKL activates the TNF receptor-associated factor
6, c-Fos, and calcium signaling pathways, all of
which are indispensable for the induction and activa-
tion of NFAT1. NFAT1 is the master transcription fac-
tor for osteoclast differentiation and regulates many
osteoclast-specific genes. Therefore, NFAT plays
important roles not only in inflammation but also in
osteoclast differentiation, resulting in the bone destruc-
tion associated with RA pathology.
Activator protein-1
The activator protein-1 (AP-1) transcription factor is
composed of members of the Fos, Jun and activating
transcription factor families of proteins. While the Fos
proteins (Fos, FosB, Fra-1 and Fra-2) can only hetero-
dimerize with members of the Jun family, the Jun pro-
teins (Jun, JunB and JunD) can both homodimerize
and heterodimerize with Fos members to form trans-
criptionally active complexes. Activator protein-1 trans-
duces extracellular signals to immune cells, resulting in
changes in the expression of specific target genes with
an AP-1 binding site(s) in their promoter ⁄ enhancer
regions. Activator protein-1 can affect the severity of
inflammation through several mechanisms, such as (a)
activation of cytokine production in co-operation with

transcription factors of the NFAT family, (b) regula-
tion of naive T-cell differentiation into T helper-1 or T
helper-2 cells or (c) interaction and trans-repression of
the glucocorticoid receptor. Most cytokine genes are
regulated by a transcription factor complex consisting
of AP-1 and NFAT, and their co-operation is essential
in most of these genes. NFAT and AP-1 have been
shown to form highly stable ternary complexes on com-
posite DNA-binding sites. As mentioned above, NFAT
is a prerequisite for the differentiation of osteoclasts.
Fos ⁄ AP-1 is also required for integration of the
RANKL and macrophage colony-stimulating factor
signals in osteoclast differentiation. Other than osteo-
clasts, the expression of MMPs contributes to the bone
destruction associated with RA. Expression of MMPs
is proposed to be regulated by AP-1, and the upregula-
tion of MMPs in the RA synovium correlates with
increased DNA-binding activity of AP-1 and increased
expression of Fos ⁄ Jun [30]. Thus, AP-1 proteins have
significant pathological roles in RA.
Other transcription factors
Other transcription factors implicated in the patho-
genesis of RA are the signal transducer and activator
of transcription (STAT) family of proteins, interferon
regulatory factors (IRFs), Forkhead (Fox) family pro-
teins, T-box transcription factor 21 (TBX21) ⁄ T-box
expressed in T cells (T-bet), the CCAAT-enhancer-
binding protein family and the Ets transcription factor
family [31]. Extensive genetic studies of RA have
revealed an association between RA and single nucleo-

Role of transcription factors H. Okamoto et al.
4468 FEBS Journal 275 (2008) 4463–4470 ª 2008 The Authors Journal compilation ª 2008 FEBS
tide polymorphisms in the Runt-related transcription
factor 1 (Runx1)-binding site of the SLC22A4 gene, in
the major histocompatibility complex class II trans-
activator (MHC2TA) gene, and in the STAT4 gene
[32–35].
Concluding remarks
Transcription factors play critical roles in the function
of immune effector cells, including cytokine ⁄ chemokine
expression and also in the control of synovial cell apop-
tosis. These cells have prerequisite roles in the pathogen-
esis of RA. Growing experimental evidence emphasizes
the importance of the NF-jB, NFAT and AP-1 tran-
scription factors in RA, and therefore signaling cascades
of these transcription factors are feasible targets for a
comprehensive anti-RA strategy. New therapeutic strat-
egies must be targeted at modulating transcription fac-
tor activity, such as control of their synthesis or activity,
including the inhibition of protein–protein interactions
in the activating signaling cascade of the transcription
factor of interest. Specific inhibitors have already been
reported, for example a small-molecule inhibitor of
NFAT, decoy oligonucleotides for NF-jB and interfer-
ing RNAs targeting components of the STAT pathway
[36–38]. As most of the transcription factors involved in
RA have pleiotropic roles in other biological processes,
inhibition of these transcription factors might invite
unexpected side effects in vivo. Co-operative contribu-
tion of both clinical studies and molecular biological

studies is required for the development of optimal thera-
peutic strategies against RA.
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