Báo cáo khoa học: FLIP and MAPK play crucial roles in the MLN51-mediated hyperproliferation of fibroblast-like synoviocytes in the pathogenesis of rheumatoid arthritis pdf

FLIP and MAPK play crucial roles in the MLN51-mediated
hyperproliferation of fibroblast-like synoviocytes in the
pathogenesis of rheumatoid arthritis
Ju-Eun Ha
1
, Young-Eun Choi
1
, Jinah Jang
2
, Cheol-Hee Yoon
1
, Ho-Youn Kim
3
and Yong-Soo Bae
1,2
1 Department of Biological Science, Sungkyunkwan University, Suwon, Gyeonggi-do, South Korea
2 Division of DC Immunotherapy, CreaGene Research Institute, Seongnam-shi, Gyeonggi-do, South Korea
3 Department of Medicine, Division of Rheumatology, Center for Rheumatoid Diseases and Rheumatism Research Center (RhRC), Catholic
Research Institutes of Medical Sciences, Catholic University of Korea, Seoul, South Korea
Rheumatoid arthritis (RA) is a chronic inflammatory
arthritis characterized by synovial hyperplasia with
local invasion of bone and cartilage. Accumulating evi-
dence suggests that RA fibroblast-like synoviocytes
(FLSs) possess unique characteristics in RA patho-
genesis [1]. FLSs play a key role in the development of
sustained inflammation and angiogenesis in arthritic
joints [2–4]. Several cytokines and RA factors existing
in the RA environment stimulate the overgrowth of
FLSs, leading to the aggravation of disease. Amongst
these factors, granulocyte–macrophage colony-stimu-
lating factor (GM-CSF) also plays an important role

in the pathogenesis of RA [5–7]. GM-CSF blockade
results in less severe disease and reduces cytokine levels
in tissue in vivo [8].
In RA synovium, FLSs express both tumor necrosis
factor-a (TNF-a) and Fas receptors, and their ligands
are detected in nearby macrophages or T cells [9,10].
However, previous studies have demonstrated that Fas
activation induces apoptosis in only a small proportion
of FLSs, because of their constitutive expression of the
FLICE-inhibitory protein (FLIP) [11]. FLIP expression
mediates the recruitment and activation of nuclear fac-
tor-jB kinase and several mitogen-activated protein
Keywords
FLICE-inhibitory protein; granulocyte–
macrophage colony-stimulating factor;
metastatic lymph node 51; mitogen-
activated protein kinase; rheumatoid arthritis
fibroblast-like synoviocyte
Correspondence
Y S. Bae, Department of Biological Science,
Sungkyunkwan University, 300 Chunchun-
dong, Jangan-gu, Suwon, Gyeonggi-do
440-746, South Korea
Fax: +82 31 290 7087
Tel: +82 31 290 5911
E-mail:
(Received 23 January 2008, revised 5 April
2008, accepted 9 May 2008)
doi:10.1111/j.1742-4658.2008.06500.x
One of the characteristic features of the pathogenesis of rheumatoid arth-

ritis is synovial hyperplasia. We have reported previously that metastatic
lymph node 51 (MLN51) and granulocyte–macrophage colony-stimulating
factor (GM-CSF) are involved in the proliferation of fibroblast-like synovi-
ocytes in the pathogenesis of rheumatoid arthritis. In this study, we have
found that: (1) GM-CSF-mediated MLN51 upregulation is attributable to
both transcriptional and post-translational control in rheumatoid arthritis
fibroblast-like synoviocytes; (2) p38 mitogen-activated protein kinase plays
a key role in the upregulation of MLN51; and (3) FLICE-inhibitory pro-
tein is upregulated downstream of MLN51 in response to GM-CSF, result-
ing in the proliferation of fibroblast-like synoviocytes. These results imply
that GM-CSF signaling activates mitogen-activated protein kinase,
followed by the upregulation of MLN51 and FLICE-inhibitory protein,
resulting in fibroblast-like synoviocyte hyperplasia in rheumatoid arthritis.
Abbreviations
DC, dendritic cell; ERK, extracellular signal-regulated kinase; FLIP, FLICE-inhibitory protein; FLS, fibroblast-like synoviocyte; JNK, c-Jun
N-terminal kinase; MAPK, mitogen-activated protein kinase; MLN51, metastatic lymph node 51; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide; OA, osteoarthritis; RA, rheumatoid arthritis; TNF-a, tumor necrosis factor-a.
3546 FEBS Journal 275 (2008) 3546–3555 ª 2008 The Authors Journal compilation ª 2008 FEBS
kinases (MAPKs), leading to cell survival, proliferation
or proinflammatory gene expression [12–14]. FLIP
expression is also upregulated during the differentia-
tion of monocytes into dendritic cells (DCs) [15]. In
addition, GM-CSF is a key component in the differen-
tiation of monocytes into DCs [16], and is generally
detected in RA synovial fluid [7,17]. Taken together,
these reports suggest that GM-CSF is probably associ-
ated with FLIP expression.
Our previous studies have demonstrated that active
RA FLSs express substantial amounts of metastatic
lymph node 51 (MLN51) in the presence of GM-

CSF, and the upregulation of MLN51 is associated
with the hyperproliferation of FLSs [7]. MLN51 was
first identified in breast cancer cells. Later, it was
reported that MLN51 is associated with the exon
junction complex in the nucleus and remains stably
associated with mRNA in the cytoplasm [18,19]. A
recent study has found that MLN51 is essential for
the formation of stress granules occurring in malig-
nant tumors [20].
In the present study, we have investigated the mech-
anism underlying the GM-CSF-mediated and MLN51-
associated hyperproliferation of FLSs using an RA
FLS cell line, MH7A [21]. We found that GM-CSF
upregulates MLN51 expression through the activation
of MAPK, followed by the induction of FLIP expres-
sion. The present data strongly suggest that MLN51
and MLN51-induced FLIP play critical roles in FLS
hyperplasia under GM-CSF conditions by facilitating
cell proliferation and blocking apoptosis in the patho-
genesis of RA.
Results
GM-CSF induces the proliferation of MH7A cells
and the expression of MLN51
Our recent study has shown that GM-CSF is
involved in the proliferation of primary RA FLSs
[7]. In the present study, we investigated cell proli-
feration and MLN51 expression using an RA FLS
cell line, MH7A, in the presence of GM-CSF. As
shown in Fig. 1A, MH7A cell proliferation was
enhanced by GM-CSF treatment in a dose-dependent

manner at concentrations up to 100 ngÆmL
)1
, but
0 10 50 100 300 500 (ng·mL
–1
)
MLN51
0 10 50 100 300 500 (ng·mL
–1
)
MLN51
GM-CSF
GM-CSF
0 0.25 0.5 1 1.5 2 6 12 24 (h)
0 0.25 0.5 1 1.5 2 6 12 24 (h)
MLN51
MLN51
GM-CSF (ng·mL
–1
)
0.0
0.5
1.0
1.5
2.0
2.5
A
B
C
0 10 100 1000

Number of cells (×10
4
)
*
WB
RT-PCR
hMLN51
β
-actin
β
-actin
β
-actin
β
-actin
β
-actin
0 0.5 1 3 6 12
0 0.5 1 3 6 12 (h)
RA FLS (#1) RA FLS (#2)
GM-CSF
Fig. 1. GM-CSF induces FLS proliferation
and MLN51 expression. (A) MH7A cells
were incubated with GM-CSF at various
concentrations for 24 h. Cell proliferation
was assessed by MTT assay. The results
are expressed as the mean ± standard devi-
ation in triplicate. *P < 0.01. (B) Dose kinet-
ics (left) and time kinetics (right) of GM-CSF
effects on MLN51 expression in MH7A

cells. MH7A cells were treated with various
concentrations of GM-CSF for 1 h for dose
kinetics, and with 100 ngÆmL
)1
of GM-CSF
for different periods of time for time kinet-
ics. MLN51 expression was then assessed
by western blot (WB) analysis and RT-PCR
at the protein (top) and mRNA (bottom)
levels. (C) Time kinetics of GM-CSF effects
on MLN51 expression in primary RA FLSs.
Primary RA FLS cells were treated with
100 ngÆmL
)1
of GM-CSF for the periods of
time shown in the figure. MLN51 expres-
sion was then assessed by RT-PCR.
J E. Ha et al. FLIP and MAPK in MLN51-mediated FLS hyperproliferation
FEBS Journal 275 (2008) 3546–3555 ª 2008 The Authors Journal compilation ª 2008 FEBS 3547
not at extreme concentrations such as 500 ngÆmL
)1
(Fig. 1A).
We have reported previously that MLN51 is upregu-
lated in the FLSs of RA patients, and is enhanced
6 days after GM-CSF treatment [7]. In the present
experiments, we assessed the dose and time kinetics of
the effects of GM-CSF on MLN51 expression in MH7A
cells. MLN51 was fully induced at the protein level fol-
lowing treatment with GM-CSF at a concentration of
50–100 ngÆmL

)1
(left panel in Fig. 1B) for 1–2 h (right
panel in Fig. 1B). However, GM-CSF treatment did not
affect the mRNA level of MLN51 over the entire range
of kinetics in MH7A cells (Fig. 1B). These data suggest
that GM-CSF-mediated MLN51 upregulation in
MH7A cells is likely to depend on translational or post-
translational control. However, in the case of primary
RA FLSs, MLN51 expression was induced at both
mRNA and protein levels within 12 h following GM-
CSF treatment (Fig. 1C), suggesting that GM-CSF-
mediated MLN51 upregulation in primary RA FLSs is,
at least in part, dependent on transcriptional control.
GM-CSF-mediated MLN51 upregulation is
attributable to both transcriptional and
post-translational control in RA FLSs
As a next step, we investigated the control mecha-
nism underlying the expression of MLN51 following
GM-CSF treatment. When MH7A cells were pretreat-
ed with a-amanitin or cycloheximide, the mRNA and
protein levels, respectively, of MLN51 were signifi-
cantly decreased compared with those of untreated
cells (Fig. 2A,B). However, pretreatment with a-amani-
tin (Fig. 2A) or cycloheximide (Fig. 2B) did not affect
the GM-CSF-mediated upregulation of MLN51 in
MH7A cells, suggesting that the GM-CSF-mediated
upregulation of MLN51 in MH7A cells is not depen-
dent on transcriptional or translational control. In
contrast, GM-CSF-mediated MLN51 upregulation was
completely obliterated and significant amounts of

MLN51 were detected even in the absence of GM-CSF
in MH7A cells following pretreatment with MG-132, a
proteasome inhibitor (Fig. 2C). In order to confirm
this result, we treated the MH7A cells with MG-132 in
the presence of GM-CSF, and measured MLN51 pro-
tein expression at different time points. In accordance
with the results shown in Fig. 2C, once cells had been
pretreated with MG-132, significant amounts of
MLN51 were detected from the beginning, and were
maintained for longer than 4 h without any additional
effects of GM-CSF (Fig. 2D). However, in the case of
RA FLSs, MG-132 pretreatment showed additive
effects on the GM-CSF-mediated upregulation of
MLN51 expression at the protein level (Fig. 2E). These
findings suggest that GM-CSF-mediated MLN51
upregulation in primary RA FLSs is, to some extent,
MG-132
0 0.5 1 4 0 0.5 1 4 (h)
MLN51
GM-CSF
WB
MLN51
GM-CSF
A B
C
E
D
MLN51
β
-actin

β
-actin
β
-actin
β
-actin
β
-actin
β
-actin
β
-actin
β
-actin
– + – +
α
-amanitin
WB
RT-PCR
CHX
GM-CSF
– + – +
MLN51
MLN51
WB
RT - PCR
MG-132
MLN51
MLN51
GM-CSF

– + – +
WB
RT-PCR
GM-CSF – + – +
MG132
Relative band intensity
GM-CSF – + – +
MLN51
MG132
WB
RA-FLS
0
5
10
15
20
25
30
Fig. 2. GM-CSF-induced MLN51 expression
acts as a post-transcriptional regulator rather
than a transcriptional or translational control.
MLN51 expressed in MH7A cells was
assessed in the presence or absence of
10 lgÆmL
)1
of a-amanitin for 20 h (A),
25 lgÆmL
)1
of cycloheximide (CHX) for 20 h
(B) and 20 l

M MG-132 for 6 h (C). The
expression of MLN51 was assessed by
RT-PCR and western blot (WB) analysis. (D)
The expression of MLN51 was assessed via
WB analysis after treatment with GM-CSF
for the indicated periods of time in the pres-
ence or absence of MG-132. (E) MLN51
expressed in primary RA FLSs was
assessed in the presence or absence of
20 l
M MG-132 for 6 h. The expression of
MLN51 was assessed by WB analysis (left).
The histogram (right) represents the relative
band intensity of the WB data, assessed by
IMAGE J software ( />FLIP and MAPK in MLN51-mediated FLS hyperproliferation J E. Ha et al.
3548 FEBS Journal 275 (2008) 3546–3555 ª 2008 The Authors Journal compilation ª 2008 FEBS
dependent on both post-translational and transcrip-
tional control, probably by blocking of the proteasome
degradation pathway.
GM-CSF-induced MLN51 is involved in the
hyperproliferation and anti-apoptosis of MH7A
cells via the upregulation of FLIP
Next, we investigated the contribution of MLN51 to
MH7A cell proliferation. When MLN51 was knocked
down by transfection of siRNA (si-MLN51), cell pro-
liferation was completely abrogated, whereas the over-
expression of MLN51 enhanced MH7A cell
proliferation (Fig. 3A). These results strongly suggest
that MLN51 plays a critical role in the proliferation of
MH7A cells.

In order to determine whether MLN51 is involved
in the anti-apoptosis as well as cell proliferation of
MH7A in response to GM-CSF, we examined the
apoptosis of normal and MLN51-knockdown MH7A
cells in the presence or absence of GM-CSF. As shown
in Fig. 3B, cell apoptosis was substantially increased
by MLN51-knockdown, and the increased apoptosis
was not attenuated by additional GM-CSF treatment.
These data suggest that MLN51 is also involved in
anti-apoptosis.
Amongst the several anti-apoptotic molecules, FLIP
mRNA was markedly enhanced by MLN51 over-
expression in MH7A cells (Fig. 3C). Once treated with
GM-CSF, FLIP mRNA was also increased, together
with MLN51 mRNA, in primary RA FLSs, but was
undetectable in osteoarthritis (OA) FLSs even in the
presence of GM-CSF (Fig. 3D). Transient expression
of MLN51 induced the expression of FLIP (Fig. 3E),
whereas MLN51-knockdown attenuated FLIP expres-
sion in MH7A cells regardless of GM-CSF treatment
(Fig. 3F). These results indicate that MLN51 causes
the upregulation of FLIP expression, followed by the
blocking of FLS apoptosis.
FLIP upregulated by MLN51 plays a crucial role in
the anti-apoptosis of MH7A cells
We examined whether FLIP was involved in the anti-
apoptosis of MH7A cells. As shown in other cells [22],
FLIP-knockdown (si-FLIP) completely abrogated
MH7A cell proliferation, even in the presence of GM-
CSF, when compared with control cells (si-con)

(Fig. 4A). In addition, FLIP-knockdown markedly
increased the cell apoptosis of MH7A, and the apopto-
tic ratio was not attenuated by GM-CSF treatment
(Fig. 4B). These data suggest that FLIP plays an
important role in GM-CSF-mediated cell proliferation
and anti-apoptosis. In contrast, FLIP-knockdown did
not show any discernible effects on the expression of
MLN51 (Fig. 4C), implying that FLIP works down-
stream of MLN51 in the GM-CSF-mediated signaling
pathway to FLS proliferation.
MAPK functions in the upregulation of MLN51
under GM-CSF conditions
Activation of the GM-CSF receptor leads to the acti-
vation of multiple cytoplasmic signaling molecules,
including MAPK. The MAPKs are key regulators of
cytokine and metalloproteinase production, and thus
may be targeted in RA. It has been reported previ-
ously that all three MAPK families, extracellular
signal-regulated kinase (ERK), c-Jun N-terminal
kinase (JNK) and p38, are expressed in rheumatoid
synovial tissue, and also play a key role in RA FLS
activation [23]. We investigated whether or not GM-
CSF activates MAPK in MH7A cells. Following the
addition of GM-CSF to cultures of MH7A cells,
JNK and p38 were dramatically phosphorylated
within approximately 1 h, whereas ERK phosphoryla-
tion was slightly enhanced during the same period of
time (Fig. 5A). In good accordance with the data
shown in Fig. 5A, pretreatment of MH7A cells with
SB203580 (p38 inhibitor) completely abrogated the

effects of GM-CSF on the upregulation of MLN51,
whereas pretreatment with SP600125 (JNK inhibitor)
or PD98059 (ERK inhibitor) partially or barely atten-
uated the effects of GM-CSF on the expression of
MLN51 and FLIP, respectively (Fig. 5B). These data
indicate that MAPKs, particularly p38 and partly
JNK, but not ERK, play an important role in the
upregulation of MLN51 and FLS overgrowth
upstream of MLN51 under the GM-CSF signaling
pathway, as summarized in Fig. 6.
Discussion
Inflammatory cell infiltration and the expansion of an
aggressive FLS population in the synovial membrane
are the pathological hallmarks of RA [1,24]. A number
of growth factors and cytokines have been described in
association with the proliferative response of FLSs,
including transforming growth factor-b, platelet-
derived growth factor, fibroblast growth factor, inter-
leukin-1b, TNF-a and interleukin-6. However, these
factors are not sufficient to cover the active prolifera-
tion capacity of RA FLSs, thus indicating that other
factors must be involved in this proliferation. In our
previous studies, we have determined that GM-CSF in
the synovial fluid plays an important role in the hyper-
J E. Ha et al. FLIP and MAPK in MLN51-mediated FLS hyperproliferation
FEBS Journal 275 (2008) 3546–3555 ª 2008 The Authors Journal compilation ª 2008 FEBS 3549
proliferation of RA FLSs through the upregulation of
MLN51 [7].
In the present study, we have investigated the
mechanism underlying the GM-CSF-mediated and

MLN51-associated hyperproliferation of FLSs using
an RA FLS cell line, MH7A. As shown previously
with primary RA FLSs, GM-CSF treatment
increased the number of MH7A cells in culture, as
well as the expression of MLN51 in these cells
(Fig. 1).
MLN51
Bcl2
c-IAP
x-IAP
NF
K
B (p65)
NF
K
B (p50)
FLIP
Control
pcDNA3.1
pcDNA-MLN51
GM-CSF – + – +
si-con si-MLN51
MLN51
FLIP
MLN51
FLIP
si-
con
pcDNA3.1
si

-
MLN51
pcDNA-MLN51
*
*
*
2.5
AB
CD
EF
2.0
1.5
1.0
0.5
0.0
Cell number (×10
4
)
pcDNA3.1 -hMLN51
Untreated
GM-CSF
MLN51
β-actin
MLN51 -siRNA
con -siRNA
β
-actin
β
-actin
β

-actin
β
-actin
11.05
si-con
25.81
si-MLN51
+
+
GM-CSF
Annexin V
26.52
si-MLN51
hMLN51
FLIP
GM-CSF
– + – + – +
RA FLS (#1)
RA FLS (#2)
OA FLS (#1)
Fig. 3. MLN51 induces FLIP expression in the GM-CSF-mediated proliferation of FLSs. (A) MH7A cells were transfected with 200 pmol siR-
NA against MLN51 (si-MLN51) or with 3 lg of pcDNA3.1-MLN51 plasmids using Lipofectamine 2000. One day later, the cells were stimu-
lated with GM-CSF for 24 h or were left unstimulated. Cell numbers were assessed by MTT assay, and expressed as the mean ± standard
deviation in triplicate. *P < 0.01. (B) MH7A cells transfected with 200 pmol of MLN51 siRNA (si-MLN51) or non-targeting siRNA (si-con)
were stimulated with 100 ngÆmL
)1
of GM-CSF for 24 h, or were left unstimulated. Apoptosis of each sample was assessed by flow cytome-
try after Annexin V–FITC staining. (C) MH7A cells were transfected with 1 lg of mock vector or pcDNA3.1-MLN51 plasmids. The levels of
several anti-apoptotic gene mRNAs were assessed by semi-quantitative RT-PCR with specific PCR primer sets. (D) Primary RA and OA FLS
samples were cultured for 6 h in the presence or absence of GM-CSF. The mRNAs of MLN51 and FLIP were assessed by semi-quantitative

RT-PCR with specific PCR primer sets. (E) MH7A cells transfected with 1 lg of pcDNA3.1-MLN51 or 200 pmol MLN51 siRNA were har-
vested at 24 h post-transfection, and subjected to western blot analysis for the expression of MLN51and FLIP. (F) MH7A cells transfected
with control (si-con) or MLN51 (si-MLN51) siRNAs were treated with 100 ngÆmL
)1
of GM-CSF, or were left untreated. The cells were har-
vested and assessed by western blot analysis for the expression of MLN51and FLIP.
FLIP and MAPK in MLN51-mediated FLS hyperproliferation J E. Ha et al.
3550 FEBS Journal 275 (2008) 3546–3555 ª 2008 The Authors Journal compilation ª 2008 FEBS
In our previous paper [7], we examined the mRNA
and protein levels of MLN51 6 days after GM-CSF
treatment of a culture of RA FLSs. In the present
study, however, MLN51 expression was examined
within 24 h after GM-CSF treatment in MH7A cells
at both mRNA and protein levels. In the case of
MH7A cells, MLN51 was constitutively expressed at
the mRNA level under normal conditions, and was
not changed by GM-CSF treatment over 24 h. In con-
trast, the protein level of MLN51 was low in the
untreated control, but rapidly increased over 1–2 h
following GM-CSF treatment, and the enhanced level
lasted longer than 24 h. When the cells were pretreated
with MG-132, the protein level of MLN51 was as high
as that of GM-CSF-treated cells, even in the absence
of GM-CSF, suggesting that GM-CSF-mediated
MLN51 upregulation in MH7A cells is probably post-
translational.
However, when examined in primary RA FLSs over
12 h, both mRNA and protein levels of MLN51 were
enhanced at 3–12 h following GM-CSF treatment
(Fig. 1C). The pretreatment of RA FLSs with MG-132

showed an additional increment in the protein level of
GM-CSF-induced MLN51 (Fig. 2E). These data indi-
cate that GM-CSF not only induces the expression of
MLN51, but also blocks the proteasome-mediated
degradation of MLN51 in RA FLSs by an un-
known mechanism. In other words, GM-CSF-mediated
MLN51 upregulation is attributable to both transcrip-
tional and post-translational control in RA FLSs.
The overgrowth of RA FLSs may result from unbal-
anced proliferation and apoptosis, and both processes
have been detected on tissue sections of rheumatoid
synovium [25,26]. In the present study, the MLN51-
knockdown and ectopic expression of MLN51
(Fig. 3A,B) experiments have shown that MLN51 plays
an important role in GM-CSF-mediated MH7A cell
proliferation. In order to determine whether MLN51 is
also involved in anti-apoptosis, we investigated the
anti-apoptotic molecules, and found that FLIP expres-
sion was upregulated by MLN51 (Fig. 3C,D). MLN51
is a subunit of the exon junction complex, which is
involved in post-splicing events, such as mRNA export,
nonsense-mediated mRNA decay and translation
[27–29]. Taken together, these findings allow us to
assume that MLN51 may facilitate the export of FLIP
mRNA from the nucleus, or stabilize FLIP mRNA in
the cytoplasm, followed by the blocking of cell apopto-
sis, and therefore involvement in FLS overgrowth.
Apoptosis stimulators, such as TNF-a and FasL, nor-
mally induce cell apoptosis. In RA FLSs, however,
activation of the TNF-a receptor or Fas receptor

0.0
0.5
1.0
1.5
2.0
si-con
A
B
C
si-FLIP
Untreated
GM-CSF
Cell number (×10
4
)
*
si-con
si-FLIP
+ GM-CSF
si-FLIP
Annexin V
46.92
14.06
44.57
si-con si-FLIP
GM-CSF
–+ –+
MLN51
β
-actin

FLIP
Fig. 4. MAPK and FLIP are involved in the GM-CSF-mediated FLS
proliferation mechanism upstream and downstream of MLN51,
respectively. (A) MH7A cells transfected with 100 pmol of FLIP
(si-FLIP) or control (si-con) siRNAs were stimulated with
100 ngÆmL
)1
of GM-CSF 24 h post-transfection, or were left
unstimulated. At 24 h after treatment, cell numbers were assessed
by MTT assay. The results are expressed as the mean ± standard
deviation in triplicate. *P < 0.01. (B) Control (si-con) or FLIP si-RNA-
transfected (si-FLIP) cells were treated with 100 ngÆmL
)1
of
GM-CSF for 24 h, or were left untreated. They were assessed for
cell apoptosis by flow cytometry after Annexin V–FITC staining. (C)
Control (si-con) or FLIP si-RNA-transfected (si-FLIP) cells were trea-
ted with 100 ngÆmL
)1
of GM-CSF for 24 h, or were left untreated.
FLIP and MLN51 expression was assessed in the cells by western
blot analysis.
J E. Ha et al. FLIP and MAPK in MLN51-mediated FLS hyperproliferation
FEBS Journal 275 (2008) 3546–3555 ª 2008 The Authors Journal compilation ª 2008 FEBS 3551
induces NF-jB translocation, which leads to increased
FLIP expression [30]. This NF-jB loop may protect
RA FLSs from TNF-a ⁄ FasL-mediated cell death,
resulting in FLS overgrowth. However, we found that
MLN51 induced FLIP expression in the absence of
TNF-a or FasL stimulation. These data suggest that

RA FLSs are resistant to cell apoptosis via, at least
in part, MLN51-mediated FLIP upregulation under
GM-CSF conditions.
The activation of MAPK is almost exclusively found
in synovial tissue from RA patients. This activation is
induced by inflammatory cytokines [23]. Amongst
these proinflammatory cytokines, GM-CSF induces
phosphorylation of Ser345 in the MAPK consensus
sequence [31].
We have found that GM-CSF induces the phos-
phorylation of p38 and JNK predominantly
(Fig. 5A), and that a p38 inhibitor (SB203580) com-
pletely abrogates the GM-CSF-mediated upregulation
of MLN51 and FLIP in MH7A cells (Fig. 5B).
Although ERK (p42 ⁄ 44) is constitutively activated in
MH7A cells (Fig. 5A), as reported previously [21],
ERK inhibitor (PD98059) does not affect MLN51
and FLIP induction in MH7A cells (Fig. 5B), indi-
cating that ERK is unlikely to be involved in the
GM-CSF-mediated induction of MLN51 and FLIP
in RA FLSs. These data suggest that MAPK, partic-
ularly p38, is activated by GM-CSF, and plays an
important role in the post-translational modification
of MLN51, resulting in the protection of MLN51
from ubiquitin-mediated degradation.
In summary, in RA FLSs: (1) GM-CSF signaling
activates p38 MAPK; (2) this is followed by MLN51
upregulation via both transcriptional and post-transla-
tional control; (3) FLIP expression is induced; and (4)
this results in the anti-apoptotic proliferation of FLS,

contributing to the pathogenesis of RA (Fig. 6).
MLN51 and MLN51-induced FLIP are believed to
play important roles in FLS hyperplasia by participat-
ing in FLS proliferation and anti-apoptosis in RA
pathogenesis. Thus, MLN51 and FLIP are attractive
targets for the development of new RA therapeutics.
Experimental procedures
Isolation and establishment of RA FLSs from RA
patients
Fibroblast-like synoviocyte samples were obtained from
synovectomized tissue of RA and OA patients undergoing
Fig. 6. Summary diagram showing the role of MLN51 in the
GM-CSF-mediated proliferation of RA FLSs via MAPK activation
and induction of FLIP expression.
A
B
Fig. 5. MLN51 expression via MAPK activation by GM-CSF. (A)
MH7A cells were treated with 100 ngÆmL
)1
of GM-CSF for the indi-
cated periods of time. Cells were assessed by western blotting for
the expression of three different MAPKs and their phospho-forms.
(B) MH7A cells were pre-incubated with 20 l
M SP600125, 50 lM
PD98059 and 20 lM SB203580 for 1 h, and were then cultured in
the presence or absence of GM-CSF (100 ngÆmL
)1
) for an additional
1 h. MLN51 and FLIP expression in each sample was assayed by
western blot analysis. DMSO, dimethylsulfoxide.

FLIP and MAPK in MLN51-mediated FLS hyperproliferation J E. Ha et al.
3552 FEBS Journal 275 (2008) 3546–3555 ª 2008 The Authors Journal compilation ª 2008 FEBS
joint replacement surgery at Kangnam St Mary Hospital,
Catholic University of Korea, Seoul, South Korea. Institu-
tional Review Board (IRB) approval and patient informed
consent from each enrolled participant were obtained. RA
and OA FLS cells were prepared as described previously
[7].
Cell line, chemicals and antibodies
A human synovial cell line (MH7A), which was prepared
from FLSs isolated from the knee joint of an RA patient,
was obtained from Riken Cell Bank, Tsukuba, Ibaraki,
Japan. MH7A cells were maintained in RPMI1640
(HyClone, Logan, UT, USA) supplemented with 10% fetal
bovine serum (Gibco, Grand Island, NY, USA)
and 100 lgÆmL
)1
each of penicillin and streptomycin.
GM-CSF (LG Life Science, Seoul, Korea), cycloheximide
(Calbiochem, San Diego, CA, USA), a-amanitin (Sigma,
St Louis, MO, USA), MAPK inhibitors SP600125,
PD98059, SB203580 (Calbiochem) and MG-132 (AG Scien-
tific Inc., San Diego, CA, USA) were used in the present
experiments. FLIP antibodies were purchased from Santa
Cruz Co. (Santa Cruz, CA, USA). SAPT ⁄ JNK, phospho-
SAPT ⁄ JNK, ERK, phospho-ERK, p38 and phospho-p38
antibodies were purchased from Cell Signaling Inc. (Dan-
vers, MA, USA). Anti-b-actin (Sigma), anti-rabbit and
anti-mouse IgG-HRP (Sigma) and Annexin V–fluorescein
isothiocyanate (Becton Dickinson, Mountain View, CA,

USA) IgG were also used in this study.
Recombinant plasmids
MLN51-expressing plasmids (pcDNA3.1-MLN51 and
pET28-MLN51) were prepared by cloning full-length
hMLN51 cDNA into pcDNA3.1 (Invitrogen, San Diego,
CA, USA) at the EcoRI ⁄ XhoI site and partial hMLN51
cDNA into the pET28a(+) vector (Novagen, Madison,
WI, USA) at the HindIII ⁄ XhoI site, respectively. Full-length
and partial cDNAs of hMLN51 [18] were prepared by
RT-PCR amplification of MH7A mRNA using appropriate
primer pairs for cDNAs of hMLN51: full-length, 5¢-TATG
AATTCGTTCTCCGTAAGATGGCGGAC-3¢ and 5¢-TA
TCTCGAGTTAACTGGAACCCCTGCTTACAA-3¢; par-
tial length, 5¢-ATCAAGCTTTGGTGCGTAAGGAGCT
GAC-3¢ and 5¢-ATACTCGAGCTTAGCAGCTGGAGTC
GTTT-3¢.
Preparation of recombinant MLN51 protein and
antiserum
Recombinant BL21(DE3) cells that had been transformed
by pET28a(+)-MLN51 were cultured in 2· yeast extract
and tryptophan medium. Recombinant proteins were
purified using a nickel nitrilotriacetic acid-conjugated
bead column. MLN51 antiserum was prepared by immu-
nizing New Zealand white rabbits three times at 3-week
intervals with recombinant hMLN51 proteins emulsified
with Freund’s adjuvant (Sigma). New Zealand white rab-
bits were obtained from Orient Bio (Gyeonggi-do, South
Korea) and were maintained in the Animal Care Facility
of Sungkyunkwan University according to the Korean
Experimental Animal Care Guidelines.

Cell proliferation assay
MH7A cells were seeded in 96-well plates overnight at a
density of (1–5) · 10
3
cells per well in 100 lL of RPMI1640
containing 10% fetal bovine serum. Cells that had been
pretreated with appropriate reagents or transfected with
siRNA were cultured in the presence or absence of various
concentrations of GM-CSF. After 24 h, 3-(4,5-dim-
ethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
dye solution (20 lL per well, Promega, Madison, WI,
USA) was added to each well, and incubated for another
4 h at 37 °C. The reaction was stopped by the addition of
stop solution (150 lL per well), and the absorbance of each
sample was subsequently measured by a spectrophotometer
(Molecular Device, Union City, CA, USA) at 570 nm.
Western blot analysis
Each experiment was conducted as described previously [7].
In brief, cell lysates were normalized with Bradford reagent
(Bio-Rad, Hercules, CA, USA), and 40–70 lg of lysate
was subjected to 8–12% SDS-PAGE and transferred to
poly(vinylidene difluoride) membranes (Millipore, Eschborn,
Germany). The membranes were blocked and probed with
appropriate antibody as described previously [7], and were
then analyzed by an enhanced chemiluminescence western
blotting system (Millipore ⁄ Amersham Biosciences, Freiburg,
Germany).
Quantitative RT-PCR
Quantitative RT-PCR was conducted as described previously
[7,32]. In brief, total RNAs were extracted using Tri-zol

reagent (Invitrogen) and were then normalized. RT-PCR was
conducted using the pre-Mix kit (Intron Biotech, Seoul,
Korea) and the following primer pairs: MLN51: sense,
5¢-AAGACACCGAGGACGAGGAATC-3¢; anti-sense,
5¢-CCTTCCATAGCTTTCGCTGACG-3¢; FLIP: sense,
5¢-GAATGTGGAATTCAAGGCTCA-3¢; anti-sense, 5¢-AT
ACAGGTACCCACACCCACA-3¢; Bcl-2: sense, 5¢-TTC
CTCTGGGAAGGATGGCG-3¢; anti-sense, 5¢-CGTCCC
TGAAGAAGCTCCTCC-3¢; IAP: sense, 5¢-TGTTGTGGC
CTGATGCTGGA-3¢; anti-sense, 5¢-CAGGCAAAGCAAG
CCACTCTG-3¢; XIAP: sense, 5¢-TGGTGACCAAGTGC
AGTGCT-3¢; anti-sense, 5¢-AGGGTCTTCACTGGGCTT
J E. Ha et al. FLIP and MAPK in MLN51-mediated FLS hyperproliferation
FEBS Journal 275 (2008) 3546–3555 ª 2008 The Authors Journal compilation ª 2008 FEBS 3553
CC-3¢; NF-kB(p50): sense, 5¢-AGTTTCGGCGGTGGT
AGTGG-3¢; anti-sense, 5¢-GCCAGCAGCATCTTCACG
TC-3¢; NF-kB(p65): sense, 5¢-GACAATCGTGCCCCCAA
CAC-3¢; anti-sense, 5¢-TGGGTCCGCTGAAAGGACT-3¢;
human b-actin: sense, 5¢-TGACGGGGTCACCCACACT
GTGCCCATCTA-3¢; anti-sense, 5¢-AGTCATAGTCCGC
CTAGAAGCATTTFCGGT-3¢.
siRNA transfection
Human MLN51 and c-FLIP siRNAs were designed and
synthesized by Invitrogen (StealthÔ) with sequences of
5¢-GGGCCCUAAGCAUUUGGAUGAUGAU-3¢ and 5 ¢-CC
CUGGGCUAUGAAGUCCAGAAAUU-3¢, respectively.
Cell transfection with siRNA was conducted using Lipo-
fectamine 2000 (Invitrogen) according to the protocol of
the manufacturer. After 5 h of incubation, the media were
completely replaced and incubated further.

Apoptotic analysis by flow cytometry
MH7A cells were cultured in six-well plates at 5 · 10
5
cells
per well. After 24 h, cells were transfected with MLN51 or
FLIP siRNA, and cultured in the presence or absence of
100 ngÆmL
)1
of GM-CSF at 37 °C for an additional 24 h.
The cells were harvested and incubated for 15 min with
fluorescein isothiocyanate-conjugated Annexin V antibody
(Becton Dickinson) at room temperature in the dark. The
cells were then analyzed using a FACS Calibur system
(Becton Dickinson) with cell quest software.
Acknowledgement
This work was supported by Bio New Drug grants
(A060115 and A040010) from the Korean Ministry of
Health and Welfare.
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