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Involvement of MAPKs and NF-╨Ю╤ФB in tumor necrosis factor ╨Ю┬▒induced vascular cell adhesion molecule 1 expression in human rheumatoid arthritis synovial fibroblasts.

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ARTHRITIS & RHEUMATISM
Vol. 62, No. 1, January 2010, pp 105–116
DOI 10.1002/art.25060
© 2010, American College of Rheumatology
Involvement of MAPKs and NF-␬B
in Tumor Necrosis Factor ␣–Induced
Vascular Cell Adhesion Molecule 1 Expression
in Human Rheumatoid Arthritis Synovial Fibroblasts
Shue-Fen Luo,1 Rou-Yi Fang,1 Hsi-Lung Hsieh,2 Pei-Ling Chi,1 Chih-Chung Lin,1
Li-Der Hsiao,1 Chi-Chuan Wu,1 Jong-Shyan Wang,1 and Chuen-Mao Yang1
Objective. To investigate the roles of MAPKs and
NF-␬B in tumor necrosis factor ␣ (TNF␣)–induced
expression of vascular cell adhesion molecule 1
(VCAM-1) in human rheumatoid arthritis synovial fibroblasts (RASFs).
Methods. Human RASFs were isolated from synovial tissue obtained from patients with RA who underwent knee or hip surgery. The involvement of MAPKs
and NF-␬B in TNF␣-induced VCAM-1 expression was
investigated using pharmacologic inhibitors and transfection with short hairpin RNA (shRNA) and measured
using Western blot, reverse transcriptase–polymerase
chain reaction, and gene promoter assay. NF-␬B translocation was determined by Western blot and immunofluorescence staining. The functional activity of
VCAM-1 was evaluated by lymphocyte adhesion assay.
Results. TNF␣-induced VCAM-1 expression,
phosphorylation of p42/p44 MAPK, p38 MAPK, and
JNK, and translocation of NF-␬B were attenuated by
the inhibitors of MEK-1/2 (U0126), p38 (SB202190),
JNK (SP600125), and NF-␬B (helenalin) or by transfection with their respective shRNA. TNF␣-stimulated
translocation of NF-␬B into the nucleus and NF-␬B
promoter activity were blocked by Bay11-7082, but not
by U0126, SB202190, or SP600125. VCAM-1 promoter
activity was enhanced by TNF␣ in RASFs transfected
with VCAM-1-Luc, and this promoter activity was inhibited by Bay11-7082, U0126, SB202190, and
SP600125. Moreover, up-regulation of VCAM-1 increased the adhesion of lymphocytes to the RASF monolayer, and this adhesion was attenuated by pretreatment
with helenalin, U0126, SP600125, or SB202190 prior to
exposure to TNF␣ or by anti–VCAM-1 antibody before
the addition of lymphocytes.
Conclusion. In RASFs, TNF␣-induced VCAM-1
expression is mediated through activation of the p42/
p44 MAPK, p38 MAPK, JNK, and NF-␬B pathways.
These results provide new insights into the mechanisms
underlying cytokine-initiated joint inflammation in RA
and may inspire new targeted therapeutic approaches.
Drs. Luo, Lin, and Yang’s work was supported by the Chang
Gung Medical Research Foundation (grant CMRPG350642 to Dr.
Luo, grant CMRPG350652 to Dr. Lin, and grants CMRPD150253 and
CMRPD180061 to Dr. Yang). Drs. Luo and Yang’s work was also
supported by the National Science Council, Taiwan (grants NSC962314-B-182-012-MY3 and NSC95-2320-B-182-047-MY3, respectively).
1
Shue-Fen Luo, MD, Rou-Yi Fang, MS, Pei-Ling Chi, MS,
Chih-Chung Lin, MD, PhD, Li-Der Hsiao, MS, Chi-Chuan Wu, MD,
Jong-Shyan Wang, PhD, Chuen-Mao Yang, PhD: Chang Gung University and Chang Gung Memorial Hospital, Kwei-San, Tao-Yuan,
Taiwan; 2Hsi-Lung Hsieh, PhD: Chang Gung Institute of Technology,
Kwei-San, Tao-Yuan, Taiwan.
Address correspondence and reprint requests to Chuen-Mao
Yang, PhD, Department of Pharmacology, Chang Gung University,
259 Wen-Hwa First Road, Kwei-San, Tao-Yuan 33302, Taiwan. Email: chuenmao@mail.cgu.edu.tw.
Submitted for publication July 22, 2009; accepted in revised
form September 25, 2009.
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by thickening of the synovial lining layer and infiltration of the synovial membrane by inflammatory cells. The cellular infiltration is
due to up-regulation of adhesion molecules including
intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) (1–4). The
induction of cell adhesion molecules mediates the tight
adhesiveness of polymorphonuclear cells (PMNs) and
thus facilitates PMN migration across the vascular endothelial barrier (5–7). RA synovial fibroblasts (RASFs)
are versatile cells with the potential to activate an array
of genes that are able to initiate and propagate inflammation in RA-affected joints. Up-regulation of
105
106
VCAM-1 has been shown in the synovial lining of RA
patients by immunohistochemical staining (2) and in
cultured human RASFs by Western blotting (8). However, the molecular mechanisms by which cytokines
induce VCAM-1 expression in RASFs remain unclear.
Elevated levels of proinflammatory cytokines including tumor necrosis factor ␣ (TNF␣) in the synovial
LUO ET AL
fluid have been detected in RA patients (9–11). TNF␣
acts as a potent stimulus of inflammatory responses
through up-regulation of many genes, including those
for cytokines, chemokines, proteinases, cyclooxygenase,
and adhesion molecules (12–15). VCAM-1 may be the
predominant adhesion molecule responsible for migration and adherence of lymphocytes to inflamed syno-
Figure 1. Tumor necrosis factor ␣ (TNF␣) induces expression of vascular cell adhesion molecule 1 (VCAM-1) in rheumatoid arthritis synovial
fibroblasts (RASFs). A, Primary cultured RASFs were identified by immunofluorescence (IF) staining using an antibody specific for the fibroblast
protein marker vimentin. The nucleus was stained with 4⬘,6-diamidino-2-phenylindole (DAPI). (Original magnification ⫻ 1,000). B, To assess
VCAM-1 protein expression, RASFs were treated with TNF␣ for the indicated periods of time. C, To determine TNF␣-induced VCAM-1 mRNA
expression, RASFs were incubated with TNF␣ for the indicated periods of time. VCAM-1 mRNA expression was analyzed by reverse
transcriptase–polymerase chain reaction (RT-PCR). D, TNF␣ induction of VCAM-1 expression in primary cultured osteoarthritis synovial
fibroblasts (OASFs) was assessed as a control. OASFs were treated with TNF␣ for the indicated periods of time. VCAM-1 protein and mRNA
expression were analyzed by Western blot and RT-PCR. E and F, Shown are the effects of actinomycin D (E) and cycloheximide (F) on VCAM-1
expression induced by TNF␣. Cells were pretreated with actinomycin D or cycloheximide and then incubated with TNF␣ for 8 hours. The cell lysates
were prepared and blotted using antibodies to VCAM-1 or GAPDH (as a control). Data are summarized from the time course study and expressed
as the mean and SEM from 4 independent experiments. In B and C, ⴱ ⫽ P ⬍ 0.05; # ⫽ P ⬍ 0.01, versus cells incubated with vehicle alone. In E
and F, ⴱ ⫽ P ⬍ 0.05; # ⫽ P ⬍ 0.01, versus cells incubated with TNF␣ alone.
TNF␣-INDUCED VCAM-1 EXPRESSION IN SYNOVIAL FIBROBLASTS
vium in RA, and levels of soluble VCAM-1 have been
shown to correlate with clinical severity and progression
of RA (16,17). Therefore, cytokine-triggered upregulation of VCAM-1 expression on RASFs may be the
key factor responsible for the targeted transmigration of
lymphocytes into the interstitial space under conditions
of inflammation (5,7).
Although the roles of cytokines and adhesion
molecules in PMN adhesion to endothelial cells have
been well described, little is known about the mechanism(s) underlying the interaction between lymphocytes
and human RASFs. The induction of VCAM-1 by
cytokines such as TNF␣ and interleukin-1␤ has been
demonstrated in various cell types (12–20). TNF␣ has
also been reported to activate all of the 3 MAPKs
(21,22), including p42/p44 MAPK (23), p38 MAPK (24),
and JNK (25). The relationship between the activation
of these pathways and expression of adhesion molecules,
however, has been controversial. For example, TNF␣induced VCAM-1 and ICAM-1 expression in mouse
Sertoli cells does not require the activation of p38
MAPK, whereas activation of JNK is essential for these
responses (26). In endothelial cells, JNK and protein
kinase C activation is required for TNF␣-mediated
ICAM-1 expression. In contrast, cardiac cells require
p38 MAPK activation for VCAM-1 and ICAM-1 expression (27). Moreover, p38 MAPK and JNK oppositely
regulate TNF␣-induced VCAM-1 expression in chondrosarcoma cells (18).
These discrepancies imply that there are divergent pathways leading to expression of adhesion molecules induced by TNF␣, depending on the nature of the
stimulus, cell types, and target genes. Moreover, the
expression of VCAM-1 and other genes appears to be
highly regulated by MAPKs and NF-␬B in various cell
types (12,28–30). Both ICAM-1 and VCAM-1 promoters contain NF-␬B binding sites, which are regulated by
TNF␣ through MAPKs in several cell types (29,30).
Therefore, it is worthwhile to examine whether the
activation of these MAPK and NF-␬B pathways is
involved in the TNF␣-induced VCAM-1 expression in
RASFs.
In addressing these questions, we performed
experiments to investigate the roles of MAPKs and
NF-␬B in TNF␣-induced VCAM-1 expression in
RASFs. We found that activation of p42/p44 MAPK,
p38 MAPK, JNK, and NF-␬B is required for maximal
induction of VCAM-1 gene expression by RASFs. The
increased VCAM-1 expression correlates with enhanced
adhesion of lymphocytes to TNF␣-stimulated RASFs.
107
MATERIALS AND METHODS
Materials. Dulbecco’s modified Eagle’s medium
(DMEM)–Ham’s F-12, fetal bovine serum (FBS), and TRIzol
were purchased from Invitrogen (Carlsbad, CA). Hybond C
membrane, the enhanced chemiluminescence (ECL) Western
blotting detection system, and Hyperfilms were from Amersham Biosciences (Little Chalfont, UK). Polyclonal antibodies
against VCAM-1, I␬B␣, and NF-␬B (p65 subunit) were from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GAPDH
antibody was from Biogenesis (Bournemouth, UK). PhosphoPlus p42/p44 MAPK, p38 MAPK, and JNK antibody kits
were from Cell Signaling Technology (Beverly, MA). U0126,
SB202190, SP600125, Bay11-7082, and helenalin were from
Biomol (Plymouth Meeting, PA). TNF␣ was from PeproTech
(Rocky Hill, NJ). The BCA Protein Assay Reagent kit was
from Pierce (Rockford, IL). Enzymes and other chemicals
were from Sigma (St. Louis, MO).
Cell cultures. This study was approved by the Institutional Review Board, Chang Gung Memorial Hospital. Human
RASFs were isolated from synovial tissue obtained from
patients with RA who underwent knee or hip surgery. Synovial
strips were cut into small pieces and placed in 10-cm dishes.
Osteoarthritis synovial fibroblasts (OASFs) were isolated from
synovial tissue obtained from patients with OA who underwent
knee or hip joint surgery. These cells were grown in DMEM–
Ham’s F-12 containing 10% (volume/volume) FBS and antibiotics (100 units/ml penicillin G, 100 ␮g/ml streptomycin, and
250 ng/ml Fungizone) at 37°C in a humidified 5% CO2
atmosphere. When the cultures reached confluence, cells were
treated with 0.05% (weight/volume) trypsin/0.53 mM EDTA
for 5 minutes at 37°C. The cell suspensions were diluted with
DMEM–Ham’s F-12 containing 10% FBS to a concentration
of 2 ⫻ 105 cells/ml. More than 95% of the cells were
fibroblasts, as characterized by immunofluorescence staining
using an antibody specific for the fibroblast protein marker
vimentin (Figure 1A). Culture medium was changed after 24
hours and then every 3 days. Experiments were performed
using cells from passages 3–8.
Preparation of cell extracts and Western blot analysis.
RASFs were plated onto 12-well culture plates and made
quiescent at confluence by incubation in serum-free DMEM–
Ham’s F-12 for 24 hours. Growth-arrested cells were incubated
with different concentrations of TNF␣ at 37°C for the indicated periods of time. The cell lysates were collected and
subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% (w/v) running gel, transferred to nitrocellulose membranes, and blotted using antibodies against phospho-p42/p44 MAPK, p38 MAPK, JNK, and
VCAM-1 as described previously (31).
Plasmids and transfection. The plasmids encoding
dominant-negative mutants of NF-␬B–inducing kinase (NIK)
(KKAA), IKK␣ (KM), and IKK␤ (KM) were kindly provided
by Dr. M. Karin (Department of Pharmacology, University of
California, San Diego). The plasmids encoding short hairpin
RNA (shRNA) of ERK-2, p38, and JNK-2 were kindly provided by Dr. C. P. Tseng (Department of Medical Biotechnology and Laboratory Science, Chang Gung University). All
plasmids were prepared using plasmid DNA preparation kits
(Qiagen, Chatsworth, CA).
RASFs were plated at 3 ⫻ 105 cells/ml in 12-well
108
culture plates for 24 hours. Cells were transfected with 1
␮g/well of dominant-negative mutants or shRNA using DNA
Lipofectamine Plus (Invitrogen) and incubated at 37°C for 3
hours. One milliliter of DMEM–Ham’s F-12 containing 10%
FBS was added and incubated for an additional 19 hours. The
cells were washed twice with phosphate buffered saline and
maintained in DMEM–Ham’s F-12 containing 1% FBS for 24
hours before treatment with TNF␣. The transfection efficiency
(⬃60%) was determined by transfection with an enhanced
green fluorescent protein plasmid.
Total RNA extraction and reverse transcriptase–
polymerase chain reaction (RT-PCR) analysis. Total RNA was
isolated from RASFs treated with TNF␣ for the indicated
periods of time in 10-cm culture dishes with TRIzol, according
to the protocol of the manufacturer (Invitrogen). Complementary DNA obtained from 0.5 ␮g total RNA was used as a
template for PCR amplification, as previously described (31).
NF-␬B translocation. RASFs were seeded in a 10-cm
dish. When they reached 90% confluence, the cells were
starved in serum-free DMEM–Ham’s F-12 for 24 hours. The
cells were incubated with TNF␣ for the indicated time intervals, harvested, sonicated for 10 seconds at output 4 with a
sonicator (Misonix, Farmingdale, NY), and centrifuged at
8,000 revolutions per minute for 5 minutes at 4°C. The
supernatant was collected as the cytosolic fraction and the
pellet as the nuclear fraction. Proteins were subjected to
SDS-PAGE and transferred to a nitrocellulose membrane.
The degradation of I␬B␣ and translocation of NF-␬B were
identified and quantified by Western blot analysis using antibodies against I␬B␣ or NF-␬B (p65), respectively. The immunoreactive bands detected with ECL reagents were developed
using Hyperfilm ECL.
Immunofluorescence staining. RASFs were plated on
6-well culture plates with coverslips. Cells were further cultured in serum-free DMEM–Ham’s F-12 for 24 hours and
treated with 15 ng/ml TNF␣ for 30 minutes. Cells were fixed,
permeabilized, and stained using an anti–p65 NF-␬B polyclonal antibody as described previously (31). The nucleus was
stained with 4⬘,6-diamidino-2-phenylindole.
Measurement of NF-␬B and VCAM-1 promoter luciferase activities. For construction of the ␬B-Luc and VCAM1-Luc plasmids, human VCAM-1 promoter (⫺1,716 to ⫺119
bp) (kindly provided by Dr. W. C. Aird, Beth Israel Deaconess
Medical Center, Boston, MA), was cloned into pGL3-basic
vector (Promega, Madison, WI). VCAM-1-Luc activity was
determined as previously described (12), using a luciferase
assay system (Promega) according to the manufacturer’s instructions. Firefly luciferase activity was normalized to
␤-galactosidase activity.
Lymphocyte adhesion assay. Lymphocyte–RASF adhesion was measured with a parallel plate chamber according
to methods previously described (31). Nonadherent lymphocytes were washed away from the slide. Following flow perfusion, the number of lymphocytes adhering to the RASF
monolayer was analyzed with an inverted light microscope
(Nikon, Tokyo, Japan) connected to an image analysis system
(Micro-optic Industrial, Richmond Hill, Ontario, Canada).
Statistical analysis. Concentration–effect curves were
made and 50% maximum response concentration values were
estimated using the GraphPad Prism Program (GraphPad
Software, San Diego, CA). Data were expressed as the mean ⫾
LUO ET AL
SEM and analyzed using one-way analysis of variance with
Bonferroni adjustment for multiple comparisons. P values less
than 0.05 were considered significant.
RESULTS
TNF␣ induces de novo VCAM-1 protein and gene
expression. To determine the effect of TNF␣ on the
expression of VCAM-1 protein and messenger RNA
(mRNA), RASFs were incubated with various concentrations of TNF␣ for various periods of time. As shown
in Figure 1B, TNF␣ induced VCAM-1 protein expression in a time-and concentration-dependent manner, as
revealed by Western blot analysis. A significant increase
in this response was observed within 4 hours. A maximal
response was achieved within 16 hours and was sustained over 24 hours. The amount of VCAM-1 expression was increased with increasing concentrations of
TNF␣ (Figure 1B). The blots were stripped and reprobed using an anti-GAPDH antibody as an internal
control. To further examine whether the effect of TNF␣
on VCAM-1 expression occurs at the level of transcription, the level of VCAM-1 mRNA was determined by
RT-PCR. As shown in Figure 1C, TNF␣ induced VCAM-1
mRNA accumulation in a time-dependent manner. A
maximal response was obtained within 4 hours and was
sustained over 6 hours during the period of observation. In
addition, we prepared OASFs as the control cell population to determine the effect of TNF␣ on this cell type. As
shown in Figure 1D, TNF␣ also induced VCAM-1 protein
and mRNA expression in OASFs.
Next, to determine whether the effect of TNF␣
on VCAM-1 expression was dependent on de novo
protein synthesis, we used the transcription inhibitor
actinomycin D and the translation inhibitor cycloheximide. As shown in Figures 1E and F, pretreatment with
either actinomycin D or cycloheximide attenuated
TNF␣-induced VCAM-1 expression in RASFs in a
concentration-dependent manner, suggesting that
VCAM-1 expression induced by TNF␣ is mediated
through transcription and translation.
Involvement of p42/p44 MAPK in TNF␣-induced
VCAM-1 expression. Proinflammatory cytokines have
been shown to induce the outgrowth of synovial cells by
up-regulation of synoviolin via the ERK-1/2–Ets-1 pathway
(32). We first investigated whether TNF␣-induced expression of VCAM-1 in RASFs is mediated via p42/p44
MAPK. As shown in Figure 2A, pretreatment with the
MEK-1/2 inhibitor U0126 (33) for 1 hour prior to exposure
to TNF␣ for 8 hours attenuated TNF␣-induced VCAM-1
expression in a concentration-dependent manner.
TNF␣-INDUCED VCAM-1 EXPRESSION IN SYNOVIAL FIBROBLASTS
109
sion, activation of these kinases was assayed using an
antibody specific for the phosphorylated, active forms of
Figure 2. Involvement of p42/p44 MAPK in TNF␣-induced VCAM-1
expression in RASFs. A, Cells were pretreated with U0126 for 1 hour
and then incubated with TNF␣ for 8 hours. B, TNF␣ stimulated
p42/p44 MAPK phosphorylation was assessed. Cells were incubated
without (control) or with U0126 for 1 hour and then stimulated with 15
ng/ml TNF␣ for the indicated periods of time. C, To verify the
involvement of p42/p44 MAPK in TNF␣-induced VCAM-1 expression
in RASFs, the cells were transfected with an empty plasmid (vector) or
a selective short hairpin RNA (shRNA) of p42 MAPK (shRNA of
ERK-2, or sh-ERK2) and then incubated with 15 ng/ml TNF␣ for 8
hours. The cell lysates were prepared and blotted using anti–VCAM-1
(A and C), anti–phospho–p42/p44 MAPK (B), or anti–ERK-2 or
anti-GAPDH (C). Data are expressed as the mean and SEM from 3
independent experiments. ⴱ ⫽ P ⬍ 0.05; # ⫽ P ⬍ 0.01, versus cells
incubated with TNF␣ alone. See Figure 1 for other definitions.
To determine whether p42/p44 MAPK phosphorylation is necessary for TNF␣-induced VCAM-1 expres-
Figure 3. TNF␣-induced VCAM-1 expression is mediated through
p38 MAPK in RASFs. A, Cells were pretreated with SB202190 for 1
hour and then incubated with TNF␣ for 8 hours. B, TNF␣ stimulated
p38 MAPK phosphorylation was assessed. Cells were incubated without (control) or with SB202190 for 1 hour and then stimulated with 15
ng/ml TNF␣ for the indicated periods of time. C, To verify the
involvement of p38 MAPK in TNF␣-induced VCAM-1 expression in
RASFs, the cells were transfected with an empty plasmid (vector) or a
selective short hairpin RNA (shRNA) of p38 MAPK (shRNA of p38,
or sh-p38) and then incubated with 15 ng/ml TNF␣ for 8 hours. The
cell lysates were prepared and blotted using anti–VCAM-1 (A and C),
anti–phospho–p38 MAPK (B), or anti-p38 or anti-GAPDH (C). Data
are expressed as the mean and SEM from 3 independent experiments.
ⴱ ⫽ P ⬍ 0.05; # ⫽ P ⬍ 0.01, versus cells incubated with TNF␣ alone.
See Figure 1 for other definitions.
110
Figure 4. TNF␣ induces VCAM-1 protein expression via JNK phosphorylation in RASFs. A, Cells were preincubated with SP600125 for
1 hour and then incubated with TNF␣ for 8 hours. B, TNF␣ stimulated
JNK phosphorylation was assessed. Cells were incubated without (control) or with SP600125 for 1 hour and then stimulated with 15 ng/ml
TNF␣ for the indicated periods of time. C, To verify the involvement of
JNK in TNF␣-induced VCAM-1 expression in RASFs, the cells were
transfected with an empty plasmid (vector) or a selective short hairpin
RNA (shRNA) of JNK-2 (sh-JNK2) for 4 hours and then incubated with
15 ng/ml TNF␣ for 8 hours. The cell lysates were prepared and blotted
using anti–VCAM-1 (A and C), anti–phospho–JNK-1/2 (B), or anti–
JNK-2 or anti-GAPDH (C). Data are expressed as the mean and SEM
from 3 independent experiments. ⴱ ⫽ P ⬍ 0.05; # ⫽ P ⬍ 0.01, versus cells
incubated with TNF␣ alone. See Figure 1 for other definitions.
p42/p44 MAPK, and the level of activity was determined
by Western blotting. As shown in Figure 2B, TNF␣
stimulated p42/p44 MAPK phosphorylation in a time-
LUO ET AL
dependent manner. Maximal response was obtained
within 30 minutes. To further examine the involvement
of MEK-1/2 in p42/p44 MAPK phosphorylation, RASFs
were pretreated with U0126 and then stimulated with
TNF␣. Pretreatment with 1 ␮M U0126 for 1 hour almost
completely inhibited TNF␣-stimulated p42/p44 MAPK
phosphorylation during the period of observation (Figure 2B). To determine whether activation of p42/p44
MAPK is required for TNF␣-induced VCAM-1 expression, RASFs were transfected with an shRNA of p42
MAPK (shRNA of ERK-2) and then treated with
TNF␣. As shown in Figure 2C, transfection with shRNA
of ERK-2 significantly down-regulated ERK-2 protein
expression and reduced TNF␣-induced VCAM-1 expression. These results suggest that there is a link
between activation of MEK-1/2–p42/p44 MAPK and
induction of VCAM-1 expression by TNF␣ in RASFs.
TNF␣-induced VCAM-1 expression is mediated
through p38 MAPK. To further determine whether p38
MAPK is also involved in TNF␣-induced VCAM-1
expression in RASFs, we used SB202190, a p38 inhibitor
(34). As shown in Figure 3A, pretreatment with
SB202190 attenuated TNF␣-induced VCAM-1 expression in a concentration-dependent manner. Next, to
determine whether p38 MAPK phosphorylation is involved in the responses, activation of these kinases was
assayed using an anti–phospho–p38 MAPK antibody,
and the level of activity was determined by Western
blotting. As shown in Figure 3B, TNF␣ stimulated
phosphorylation of p38 MAPK in a time-dependent
manner; maximal response was observed within 15 minutes and was sustained over 30 minutes. Moreover,
pretreatment with 10 ␮M SB202190 for 1 hour almost
completely inhibited TNF␣-stimulated p38 phosphorylation during the period of observation. In addition, to
examine the involvement of p38 MAPK phosphorylation
in the responses, RASFs were transfected with an
shRNA of p38 MAPK (shRNA of p38) and then treated
with TNF␣. Transfection with shRNA of p38 significantly reduced the p38 MAPK protein level and blocked
VCAM-1 induction by TNF␣ (Figure 3C). These results
suggest that p38 MAPK is involved in TNF␣-induced
VCAM-1 expression in RASFs.
TNF␣ induces VCAM-1 expression via the JNK
pathway. In addition to p42/p44 and p38 MAPK, we also
determined whether JNK is essential for TNF␣-induced
VCAM-1 expression in RASFs; for this purpose, we
used SP600125, a JNK inhibitor (35). As shown in Figure
4A, pretreatment of RASFs with SP600125 attenuated
VCAM-1 expression in a concentration-dependent man-
TNF␣-INDUCED VCAM-1 EXPRESSION IN SYNOVIAL FIBROBLASTS
111
Figure 5. NF-␬B is required for TNF␣-induced VCAM-1 expression in RASFs. A, Cells were preincubated with helenalin and then incubated with
TNF␣ for 8 hours. B, TNF␣ stimulates phosphorylation and degradation of I␬B␣ and translocation of NF-␬B (p65 subunit). Cells were treated with
TNF␣ for the indicated periods of time. The cytosolic and nuclear fractions were prepared and analyzed. C, To determine the effect of inhibitors
on p65 translocation, cells were pretreated with helenalin (HLN), U0126 (U0), SB202190 (SB), or SP600125 (SP) and then incubated with TNF␣
for 30 minutes. The translocation of p65 was determined by immunofluorescence staining, and individual cells were imaged (n ⫽ 3 experiments)
(original magnification ⫻ 1,000). D, For NF-␬B activity, cells were transfected with p␬B-Luc plasmid and then incubated with TNF␣ for 4 hours in
the presence of Bay11-7082 (Bay; 1 ␮M), U0126 (1 ␮M), SB202190 (10 ␮M), or SP600125 (1 ␮M). Luciferase activity was analyzed and normalized.
E and F, Involvement of NF-␬B in the response was confirmed by transfection with short hairpin RNA (shRNA) of p65 (sh-p65) (E) or
dominant-negative mutants of NF-␬B–inducing kinase (NIK) (⌬NIK), IKK␣ (⌬IKK␣), and IKK␤ (⌬IKK␤) (F) followed by incubation with TNF␣
for 8 hours. The p65 and VCAM-1 protein levels were determined by Western blot. Data are expressed as the mean and SEM (n ⫽ 3 experiments).
# ⫽ P ⬍ 0.01 versus cells incubated with TNF␣ alone. See Figure 1 for other definitions.
ner. To determine whether JNK phosphorylation is
involved in VCAM-1 induction by TNF␣, activation of
these kinases was assayed using an antibody specific for
the phosphorylated, active forms of JNK-1/2, and the
level of activity was determined by Western blotting. As
shown in Figure 4B, TNF␣ stimulated a time-dependent
phosphorylation of JNK-1/2, with maximal response
within 15 minutes that was sustained over 30 minutes.
Moreover, pretreatment with 1 ␮M SP600125 resulted in
a significant attenuation of the TNF␣-stimulated JNK1/2 phosphorylation during the period of observation
(Figure 4B). To determine whether JNK-1/2 is truly
essential for TNF␣-induced VCAM-1 expression,
RASFs were transfected with an shRNA of JNK-2 and
then treated with 15 ng/ml TNF␣. As shown in Figure
4C, transfection with shRNA of JNK-2 significantly
112
down-regulated expression of JNK-2 protein and inhibited VCAM-1 expression induced by TNF␣. The data
indicated that JNK plays a crucial role in the responses.
NF-␬B is required for TNF␣-induced VCAM-1
expression. Inflammatory responses following stimulation with cytokines such as TNF␣ are highly dependent
on activation of the transcription factor NF-␬B. Moreover, TNF␣ has been shown to be involved in VCAM-1
gene expression through the NF-␬B cascade in human
tracheal smooth muscle cells (12). Therefore, the involvement of NF-␬B in VCAM-1 induction by TNF␣ in
RASFs was confirmed using the NF-␬B pharmacologic
inhibitor helenalin, a specific sesquiterpene lactone
compound that is known to inhibit NF-␬B (36). As
shown in Figure 5A, pretreatment with helenalin for 1
hour prior to exposure to TNF␣ caused a concentrationdependent attenuation of VCAM-1 expression in
RASFs.
It has been shown that cell activation by cytokines
leads to the degradation of I␬B␣, accompanied by
NF-␬B translocation to the nucleus. To determine
whether TNF␣ stimulates phosphorylation and degradation of I␬B␣ and translocation of NF-␬B, the cells were
stimulated with 15 ng/ml TNF␣ for the indicated periods
of time. Cytosolic and nuclear fractions were prepared
to determine the phosphorylation and degradation of
I␬B␣ and translocation of NF-␬B by Western blot
analysis using anti–phospho-I␬B␣, anti-I␬B␣, and anti–
NF-␬B (anti-p65), respectively. As shown in Figure 5B,
TNF␣ rapidly stimulated phosphorylation and degradation of I␬B␣ within 5 minutes; maximal degradation was
reached within 15 minutes and was sustained over 60
minutes during the period of observation. In contrast,
TNF␣ stimulated translocation of NF-␬B (p65 subunit)
into the nucleus within 5 minutes, and this was sustained
over 60 minutes.
Since p42/p44 MAPK, p38 MAPK, JNK, and
NF-␬B were involved in TNF␣-induced VCAM-1 expression in RASFs, it was important to determine
whether these MAPKs were associated with NF-␬B
activation. To test this possibility, activation (translocation) of NF-␬B was assessed following TNF␣ stimulation
in the presence of inhibitors for MEK-1/2, p38, JNK, and
NF-␬B. TNF␣-stimulated translocation of NF-␬B (p65
subunit) was significantly inhibited by pretreatment with
helenalin, but not by pretreatment with U0126,
SB202190, or SP600125 (Figure 5C). Similarly, immunofluorescence staining also showed that TNF␣-induced
p65 translocation into the nucleus was blocked by pretreatment with helenalin, but not by pretreatment with
the pharmacologic inhibitors of these MAPKs (Figure
LUO ET AL
5C). To further determine whether NF-␬B transcriptional activity is stimulated by TNF␣, RASFs were
transfected with a promoter reporter construct containing the NF-␬B binding site (p␬B-Luc). The TNF␣stimulated increase of NF-␬B transcriptional activity was
attenuated by pretreatment with Bay11-7082, but not by
pretreatment with the 3 MAPK inhibitors (Figure 5D),
suggesting that NF-␬B activation by TNF␣ is independent of MAPKs.
To further verify that activation of NF-␬B is
required for TNF␣-induced expression of VCAM-1,
RASFs were transfected with shRNA of p65 and
dominant-negative mutants of NF-␬B upstream molecules, including NIK, IKK␣, and IKK␤, and then incubated with 15 ng/ml TNF␣. As shown in Figure 5E,
transfection with shRNA of p65 significantly downregulated the p65 protein and inhibited VCAM-1 expression induced by TNF␣. Moreover, transfection with
NIK, IKK␣, or IKK␤ all significantly attenuated TNF␣induced VCAM-1 expression (Figure 5F). These results
demonstrated that TNF␣-stimulated NF-␬B activation
via a traditional NIK/IKK␣/IKK␤ pathway may be essential for VCAM-1 up-regulation and independent of
activation of MAPKs in RASFs.
MAPKs and NF-␬B are vital to TNF␣-induced
VCAM-1 promoter activity, mRNA up-regulation, and
lymphocyte adhesion. We further examined whether
these MAPKs and NF-␬B are involved in VCAM-1
expression occurring at the transcriptional level in these
cells. First, the up-regulation of VCAM-1 gene transcription was confirmed by gene promoter luciferase
activity assay. As shown in Figure 6A, TNF␣ stimulated
a significant increase in VCAM-1 promoter activity
within 6 hours; this reached a maximum within 16 hours
and then declined by 24 hours. Moreover, TNF␣stimulated VCAM-1 promoter activity was significantly
inhibited by pretreatment with the inhibitors of NF-␬B
(Bay11-7082), MEK-1/2 (U0126), p38 (SB202190), or
JNK (SP600125) (Figure 6B). TNF␣-induced VCAM-1
mRNA expression was also attenuated by pretreatment
with helenalin, U0126, SB202190, and SP600125, as
determined by RT-PCR (Figure 6C).
TNF␣ has been recognized as a potent proinflammatory mediator that increases the expression of
adhesion molecules in RASFs (8) and the adhesiveness
between lymphocytes and resident cells in several tissues
(12,37). To test the functional activity of VCAM-1
expressed on RASFs, we assessed the ability of purified
lymphocytes to adhere to TNF␣-stimulated RASFs. As
shown in Figure 6D, the amount of lymphocyte adhesion to the RASF monolayer was significantly increased
TNF␣-INDUCED VCAM-1 EXPRESSION IN SYNOVIAL FIBROBLASTS
113
Figure 6. Involvement of MAPKs and NF-␬B in VCAM-1 promoter activity, mRNA expression, and lymphocyte adhesion stimulated by TNF␣. A,
For VCAM-1 promoter activity, RASFs were transiently transfected with a VCAM-1-Luc reporter gene and then treated with 15 ng/ml TNF␣ for
the indicated periods of time. B, Cells were pretreated with 1 ␮M U0126 (U0), 10 ␮M SB202190 (SB), 1 ␮M SP600125 (SP), or 1 ␮M Bay11-7082
(Bay) and then stimulated with TNF␣ for 6 hours. Promoter luciferase activity was analyzed and normalized to ␤-galactosidase activity. B ⫽ basal.
C, For assessment of VCAM-1 mRNA expression, cells were pretreated with helenalin (HLN), U0126, SB202190, or SP600125 and then incubated
with TNF␣ for 4 hours. The RNA samples were analyzed by RT-PCR. D, For adhesion activity, cells were pretreated with helenalin, U0126,
SB202190, or SP600125 for 1 hour and then incubated with TNF␣ for 8 hours prior to the addition of lymphocytes. Functional expression of
VCAM-1 was determined by lymphocyte adhesion assay in the absence or presence of anti–VCAM-1 antibody (VCAM-1-Ab; 1:100 dilution). Data
are expressed as the mean and SEM of 3 separate experiments. In A, # ⫽ P ⬍ 0.01 versus cells incubated with vehicle alone. In B and D, ⴱ ⫽ P
⬍ 0.05; # ⫽ P ⬍ 0.01, versus cells incubated with TNF␣ alone. See Figure 1 for other definitions.
(⬃12.5-fold) by stimulation with 15 ng/ml TNF␣ for 8
hours (P ⬍ 0.01 compared with the basal level; n ⫽ 3
experiments). The enhanced adhesion was attenuated by
pretreatment with helenalin (10 ␮M), U0126 (1 ␮M),
SP600125 (10 ␮M), or SB202190 (30 ␮M) prior to
exposure to TNF␣. To further determine the surface
molecules responsible for lymphocyte adhesion to RASF
monolayers, lymphocyte adhesion was assessed in the
presence of antibody to VCAM-1. Lymphocyte adhesion
to TNF␣-stimulated RASF monolayers was significantly
inhibited by preincubation with the antibody to the
adhesion molecule (Figure 6D). Taken together, these
results demonstrated that in RASFs, up-regulation of
VCAM-1 expression by TNF␣ through activation of
p42/p44 MAPK, p38 MAPK, JNK, and NF-␬B occurred
mainly at the transcriptional level and then enhanced
lymphocyte adhesion.
DISCUSSION
Up-regulation of adhesion molecules on the surface of the synovial lining may play a key role in
recruitment and infiltration of lymphocytes at sites of
inflammation in RA (2,8). TNF␣ has been confirmed to
induce the expression of VCAM-1 in a variety of fibroblasts, including synovial fibroblasts (38–41), but the
intracellular signaling mechanisms remain unclear.
Moreover, TNF␣ has also been shown to activate all 3
MAPK pathways in several cell types (17,22). We undertook to investigate the still-unknown mechanisms
underlying TNF␣-induced VCAM-1 expression in
RASFs. In this study, TNF␣-induced VCAM-1 expression was attenuated by the inhibitors of MEK-1/2, p38
MAPK, JNK, and NF-␬B or by transfection with respective shRNA and dominant-negative mutants. Furthermore, activation of NF-␬B was inhibited by helenalin,
but not by U0126, SB202190, or SP600125. In this study,
pretreatment with these inhibitors and transfection of
shRNA had no cytotoxicity on cultured RASFs as assessed
by XTT assay (data not shown). These results suggest that
activation of p42/p44, p38 MAPK, JNK, and NF-␬B by
TNF␣ is essential for VCAM-1 up-regulation and thus
enhances the adhesion of lymphocytes to RASFs.
114
First, our results demonstrated that these 3
MAPKs (p42/p44 MAPK, p38 MAPK, and JNK) are
necessary for TNF␣-induced VCAM-1 expression in
RASFs. An inhibitor of MEK-1/2, U0126, attenuated
TNF ␣ -induced VCAM-1 expression and p42/p44
MAPK phosphorylation in these cells (Figures 2A and
B). The finding that transfection with shRNA of ERK-2
reduced TNF␣-induced VCAM-1 expression (Figure
2C) suggests that the MEK-1/2–p42/p44 MAPK cascade
is required for TNF␣-induced VCAM-1 expression.
These results were consistent with those regarding
TNF␣-induced VCAM-1 expression in astrocytes (28)
and human tracheal smooth muscle cells (12).
Next, we demonstrated that p38 MAPK is involved in TNF␣-induced VCAM-1 expression, using a
p38 MAPK inhibitor, SB202190, and performing transfection with shRNA of p38 (Figures 3A–C). These
results indicated that p38 MAPK participates in TNF␣induced VCAM-1 expression, consistent with the expression of VCAM-1 and ICAM-1 in cardiac cells (27),
chondrosarcoma cells (18), human tracheal smooth muscle cells (12), and vascular smooth muscle cells (42). In
addition, TNF␣-stimulated JNK-1/2 activation has also
been demonstrated in several cell types (12,43). Here we
also investigated the involvement of JNK in TNF␣induced VCAM-1 expression, using a JNK inhibitor,
SP600125, and performing transfection with shRNA of
JNK-2. Pretreatment with SP600125 and transfection
with shRNA of JNK-2 attenuated TNF␣-induced
VCAM-1 expression and JNK phosphorylation (Figures
4A–C), consistent with reports that activation of JNK is
essential for the up-regulation of ICAM-1 and VCAM-1
in various cell types (12,26,42,44). Taken together, these
findings indicated that TNF␣-induced VCAM-1 expression in RASFs is mediated through p42/p44 MAPK, p38
MAPK, and JNK pathways.
It has been well established that inflammatory
responses following exposure to cytokines are highly
dependent on activation of NF-␬B, which plays an
important role in expression of several inflammatory
genes (8,30,45,46). Our previous study demonstrated
that activation of NF-␬B is essential for expression of
VCAM-1 induced by TNF␣ in human tracheal smooth
muscle cells (12). In the present study, VCAM-1 expression induced by TNF␣ was abolished by pretreatment
with an NF-␬B inhibitor, helenalin (Figure 5A) and by
transfection with shRNA of p65 (Figure 5E), indicating
that NF-␬B plays an important role in TNF␣-induced
VCAM-1 expression. Moreover, we found that the increase in NF-␬B (p65) translocation correlated with the
rapid and transient degradation of I␬B␣ in the cytosol of
LUO ET AL
RASFs treated with TNF␣ (Figure 5B). The rapid
translocation of NF-␬B (p65) following TNF␣ exposure
was inhibited by helenalin (Figure 5C). These results are
consistent with findings in our previous study (12),
indicating that TNF␣-induced VCAM-1 expression is
mediated through NF-␬B activation in human tracheal
smooth muscle cells.
Interestingly, activation of p42/p44 MAPK, p38
MAPK, and JNK as well as NF-␬B appears to be
involved in TNF␣-induced VCAM-1 expression in various cell types. However, it remains unclear how the
activation of p42/p44 MAPK, p38 MAPK, and JNK is
associated with VCAM-1 gene expression in RASFs. It
has been established that MEKK-1 induces activation of
both IKK␣ and IKK␤, leading to NF-␬B activation
(29,45). Thus, p42/p44 MAPK activation may be required for NF-␬B activation following exposure to
TNF␣. In the present study, TNF␣-stimulated NF-␬B
translocation and transcriptional activity were not inhibited by pretreatment with the 3 MAPK inhibitors U0126
(inhibitor of MEK-1/2), SB202190 (inhibitor of p38
MAPK), and SP600125 (inhibitor of JNK) (Figures 5C
and D), indicating that both the MAPK cascade and
NF-␬B independently regulated VCAM-1 expression in
RASFs. Furthermore, we demonstrated that NF-␬B
activation may be mediated through a traditional pathway from NIK/IKK␣/IKK␤ linking to NF-␬B in RASFs
(Figure 5F). These results suggest that the activation of
NF-␬B and the activation of p42/p44 MAPK, p38
MAPK, and JNK are mediated through distinct pathways (12,47), or that these MAPK pathways may converge at a step downstream of NF-␬B transactivation
(47,48).
Moreover, our results showed that pretreatment
with Bay11-7082 (another NF-␬B inhibitor), U0126,
SB202190, and SP600125 attenuated VCAM-1 promoter
luciferase gene activity stimulated by TNF␣ (Figure 6B),
suggesting that these 3 MAPKs and NF-␬B pathways
independently regulated VCAM-1 expression at the
transcriptional level. However, it remains to be investigated how the VCAM-1 gene is regulated by TNF␣
through p42/p44 MAPK, p38 MAPK, and JNK.
To our knowledge, this study is the first to define
the roles of p42/p44 MAPK, p38 MAPK, JNK, and
NF-␬B in TNF␣-induced VCAM-1 expression in human
RASFs. We conclude that activation of MAPKs and
NF-␬B pathways by TNF␣ is required for VCAM-1
expression in RASFs, which correlates with adhesion of
lymphocytes to RASFs. Since cell adhesion is important
in initiation and propagation of joint inflammation in
RA, the mechanisms by which TNF␣ induces VCAM-1
TNF␣-INDUCED VCAM-1 EXPRESSION IN SYNOVIAL FIBROBLASTS
expression and lymphocyte adhesion in RASFs provide
an important link regarding the pathogenesis and risk of
RA and may inspire development of new targeted
therapeutic approaches.
ACKNOWLEDGMENTS
The authors are grateful to Drs. M. Karin (Department
of Pharmacology, University of California, San Diego), W. C.
Aird (Department of Molecular Medicine, Beth Israel Deaconess Medical Center, Boston, MA), and C. P. Tseng (Department of Medical Biotechnology and Laboratory Science,
Chang Gung University) for providing dominant-negative mutants (NIK [KKAA], IKK␣ [KM], and IKK␤ [KM]), VCAM1-Luc constructs, and shRNA (of ERK-2, p38, and JNK-2),
respectively.
AUTHOR CONTRIBUTIONS
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. Yang 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. Luo, Hsieh, Lin, Yang.
Acquisition of data. Fang, Chi, Hsiao, Wu, Wang.
Analysis and interpretation of data. Luo, Hsieh, Lin, Yang.
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