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

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-

(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

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-

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

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

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

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+

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

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

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+

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

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

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