Báo cáo Y học: CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK), and proliferation through a p44/42 MAPK/extracellular-signal regulated kinase (ERK) doc

CTGF/Hcs24 induces chondrocyte differentiation through a p38
mitogen-activated protein kinase (p38MAPK), and proliferation
through a p44/42 MAPK/extracellular-signal regulated kinase (ERK)
Gen Yosimichi
1,2
, Tohru Nakanishi
1
, Takashi Nishida
1,3
, Takako Hattori
1
, Teruko Takano-Yamamoto
2
and
Masaharu Takigawa
1,3
1
Department of Biochemistry and Molecular Dentistry, and
2
Department of Orthodontics, Okayama University Graduate School of
Medicine and Dentistry, Okayama, Japan;
3
Biodental Research Center, Okayama University Dental School, Japan
Connective tissue growth factor/hypertrophic chondrocyte
specific gene product 24 (CTGF/Hcs24) promotes pro-
liferation and differentiation of chondrocytes in culture. We
investigated the roles of two major types of mitogen acti-
vated protein kinase (MAPK) in the promotion of prolif-
eration and differentiation by CTGF/Hcs24. Here we report
the effects of the MAPKK/MEK 1/2 inhibitor, PD098059,
and p38 MAPK inhibitor, SB203580, in a human chondro-

sarcoma-derived chondrocytic cell line (HCS-2/8) and
rabbit growth cartilage (RGC) cells treated with CTGF/
Hcs24. In the proliferation phase, CTGF/Hcs24 induced a
< fivefold increase in the phosphorylation of p44/42
MAPK/ERK and a < twofold increase in that of p38
MAPK in an in vivo kinase assay. These inhibitors
of MAPKK and MAPK suppressed phosphorylation of
ets-like gene-1 (Elk-1) and nuclear activating transcription
factor-2 (Atf-2) induced by CTGF/Hcs24 in a dose-dependent
manner, respectively. Western blot analysis showed that
phosphorylation of ERK was induced from 30 to 60 min
and phosphorylation of p38 MAPK from 10 to 15 min after
the addition of CTGF/Hcs24 in confluence HCS-2/8 cells.
PD098059 suppressed the DNA synthesis of HCS-2/8 cells
and RGC cells, while SB203580 did not. On the other hand,
the p38 MAPK inhibitor, SB203580, completely inhibited
the CTGF/Hcs24-induced synthesis of proteoglycans in
HCS-2/8 cells and RGC cells but the MEK1/2 inhibitor,
PD098059, did not. These results suggest that ERK mediates
the CTGF/Hcs24-induced proliferation of chondrocytes, and
that p38 MAPK mediates the CTGF/Hcs24-induced
differentiation of chondrocytes.
Keywords: connective tissue growth factor; hypertrophic
chondrocyte specific gene product (CTGF/Hcs24); MAPK;
chondrocyte; MAPK inhibitor; signal transduction.
Connective tissue growth factor/hypertrophic chondrocyte
specific gene product 24 (CTGF/Hcs24) is a cysteine-rich,
heparin-binding protein. Its gene and two other genes, cef10/
cyr61 and nov, belong to the CCN gene family [1 –5].
Recently, new members of this family, wisp2/rcop-1 [6],

wisp3/elm1 [7,8], and ctgf-L [9], were isolated, but the
functions of these genes are unknown. We cloned a mRNA
preferentially expressed in chondrocytes from a human
chondrosarcoma-derived chondrocytic cell line, HCS-2/8
[10,11] by differential display-PCR [12]. The gene product
was identical to CTGF. CTGF/Hcs24 is strongly expressed
on hypertrophic chondrocytes in growth plate of cartilage
[12], and promotes proliferation and differentiation of chon-
drocytes in culture [13]. In addition, CTGF/Hcs24 promotes
both proliferation and differentiation of osteoblasts [14].
Although a CTGF/Hcs24-specific receptor [15] is yet to be
cloned, a CTGF/Hcs24-receptor complex with an apparent
molecular mass of 280 kDa was tyrosine-phophorylated
in HCS-2/8 cells (data not shown). CTGF/Hcs24 has also
multiple effects on fibroblasts [16], endothelial cells [17,18],
and tumor cells [19]. CTGF/Hcs24 is highly expressed in the
process of wound healing, and mediates fibrotic disorder
[4,17,18]. CTGF/Hcs24 and Cyr61 are potential angio-
genetic factors [3 –5,18], and directly bind to integrin a
V
b
3
and a
IIb
b
3
on fibloblasts [20,21]. CTGF/Hcs24 and Cyr61
induce adhesion of human fibloblasts mediated by integrin
a
6

b
1
and cell surface heparan sulfate proteoglycans, and
activate intracellular signaling molecules including focal
adhesion kinase (FAK), paxillin, and Rac, and sustained
phosphorylation of ERK [22]. Recently, the transcriptional
mechanism of CTGF/Hcs24 was revealed [23], and it was
also reported that intracellular CTGF/Hcs24 may act as an
antimitotic agent [24]. But the mechanisms of the multiple
functions of CTGF/Hcs24 described above are not well
understood.
MAPK pathways are essential mitogenic pathways in
many cell lines and responsible for various growth factors.
MAPK is activated by a wide variety of growth factors such
Correspondence to M. Takigawa, Department of Biochemistry and
Molecular Dentistry, Graduate school of Medicine and Dentistry,
Okayama University Dental School, 2-5-1 Shikata-cho Okayama
700-8525, Japan. Fax: 1 81 86 2356649, Tel.: 1 81 86 235 6645,
E-mail:
(Received 21 May 2001, revised 1 August 2001, accepted 21 September
2001)
Abbreviations: CTGF/Hcs24, connective tissue growth factor/
hypertrohic chondrocyte specific gene product 24; Elk-1, ets-like
gene-1; Atf-2, nuclear activating transcription factor-2; DMEM,
Dulbecco’s modified Eagle’s medium; aMEM, alpha minimal essential
medium; MAPK, mitogen-activated protein kinase; ERK, extracellular-
signal regulated kinase; HCS-2/8, human chondrosarcoma-derived
chondrocytic cell line clone 2/8; RGC, rabbit growth cartilage cells;
GDF-5, growth differentiation factor-5; PTH, parathyroid hormone;
EGF, epidermal growth factor.

Eur. J. Biochem. 268, 6058–6065 (2001) q FEBS 2001
as epidermal growth factor (EGF) [25], nerve growth factor
(NGF) [26], fibroblast growth factor (FGF) [27], and trans-
forming growth factor (TGF)-b [28,29], and directs phos-
phorylation of transcription factors, such as ets-like gene-1
(Elk-1) [30], nuclear activating transcription factor-2 (Atf-2)
[31], and c-Jun [32], and other kinases. MAPK cascades are
composed of many kinds of kinases and are intricately
regulated. However, downstream of these pathways can
simply be classified into three major groups mediated by the
following kinases; p44/42 MAPK/ERK, p38 MAPK, and
c-jun N-terminal kinase (JNK). It is reported that Elk-1 and
Atf-2 are phosphorylated by ERK and p38 MAPK, respec-
tively [30,33,34].
In this study, we investigated the signal transducible
pathways of CTGF/Hcs24 responsible for its multiple
roles in the proliferation and differentiation of chondrocytes,
and analyzed the relationship between the MAPK pathways
and the proliferation and differentiation of CTGF/Hcs24,
using two MAPK inhibitors, the MEK1/2-specific inhibitor,
PD098059 [35,36], and the p38 MAPK inhibitor, SB203580
[37,38]
MATERIALS AND METHODS
Cell culture and materials
A human chodrosarcoma-derived chondrocytic cell line,
HCS-2/8, was inoculated at a density of 2 Â10
4
per cm
2
in

96-well plates, 24-well plates, and six-well plates
(Sumitomo Bakelite Co. Ltd, Tokyo, Japan) and cultured
in Dulbecco’s modified Eagle’s medium (DMEM; Nissui
Pharmaceutical Co. Ltd, Tokyo, Japan) containing 10% fetal
bovine serum (Upsdate Biotechnology, Inc., Lake Placid,
NY, USA), and 50 ng
:
mL
21
of human recombinant CTGF/
Hcs24 (rCTGF/Hcs24). For the inhibitor assay, 50 m
M of
MEK1/2 inhibitor (PD098059, Calbiochem, San Diego,
CA, USA), or 10 m
M of p38 MAPK inhibitor (SB203580,
Calbiochem) was added to the culture after 24 h of serum
depletion, and cells were harvested after 12 h of incubation.
Rabbit growth cartirage (RGCs) cells were isolated from
growth cartilage of ribs of young rabbits as described
previously [13,39,40]. The isolated cells were inoculated at
a density of 1 Â10
4
per cm
2
in 96-well plates, 24-well plates,
and six-well plates (Sumitomo Bakelite Co. Ltd), and
cultured in aMEM (alpha minimal essential medium, ICN
Biomedicals, Inc., Costa Mesa, CA, USA) containing 10%
fetal bovine serum.
In vivo

MAP kinase luciferase assay
ERK phosphorylation was quantified using an MAPK
in vivo kinase assay kit and p38K in vivo kinase assay kit
(Clontech) according to the manufacturer’s protocol. This
system is designed to detect endogeneous ERK and p38
MAPK activity in vivo. A pTet-Elk or pTet-Atf vector
expresses a fusion protein with the functional domain of Elk
or Atf and Tet repressor (TetR) domain. The reporter vector,
pTRE-Luc, contains a tet-responsive element (TRE)
upstream of the luciferase gene. Phosphorylation of Elk by
ERK, or Atf by p38 MAPK causes homodimeriization of
these proteins that induces DNA binding through the TRE
element, and results in the activation of the reporter gene.
For this experiment, 3 Â10
5
HCS-2/8 cells in a 35-mm
tissue culture dish were transiently cotransfected with 1 mg
of pTRE-luc, 1 mg of pTet-Elk or pTet-Atf by FuGENE
TM
6
(Roche, Indianapolis, IN, USA). An internal control plasmid,
0.5 mg of pRL-TK, was also cotransfected for monitoring
transfection efficiency. At 24 h post-transfection, the cells
were incubated with various concentrations of PD098059 or
SB203580 for 1 h, and cells were cultured with or without
50 ng
:
mL
21
of rCTGF/Hcs24 for 24 h. The cell lysates

were prepared and assayed for luciferase activity using the
Dual-Luciferase
TM
Reporter Assay System (Promega)
according to the manufacturer’s instructions. Light emission
was measured for 12–24 s with a luminometer (TD-20/20:
Tuner Design, Sunnyvale, CA, USA).
Western blotting
Total cellular protein was prepared by lysing cells in lysis
buffer [20 m
M Tris/HCl (pH 8.0), 150 mM NaCl, 1 mM
EDTA, 1% Triton X-100, 1 mM Na (VO
4
), 5% glycerol,
40 m
M ammonium molybdate, and 1 mM phenylmethane-
ulfonyl flyoride]. Then, 4 mg of the protein was separated by
4–20% gradient SDS/PAGE and electrophoretically trans-
ferred to poly(vinylidene difluoride) filters (Bio-Rad). The
filters were blocked with 5% nonfat drymilk in Tris-buffered
saline (pH 7.5) containing 0.1% Tween 20 for 30 min at
room temparature and then incubated with anti-ERK Ig,
anti-(active-ERK) Ig, anti-(p38 MAP) Ig or anti-(active-p38
MAPK) Ig (New England BioLaboratories, Bevely, MA,
USA, Calbiochemistry, San Diego, CA, USA and Santa
Cruz Biochemistry, Santa Cruz, CA, respectively) for 24 h
at 4 8C. The filters were then incubated with the secondary
antibodies [horseradish peroxidase-conjugated anti-(rabbit
IgG) Ig (DAKO, Trappes, France), and alkaline phospha-
tase-conjugated anti-(rabbit IgM) Ig (Cappel, Durham, NC,

USA)], and the signal was detected by enhanced
chemiluminescence (Amersham Pharmacia), or colored
with nitro blue tetrazolium and 5-bromo-4-chloroindolyl
phosphate (BCIP).
DNA synthesis
For measurement of DNA synthesis, HCS-2/8 cells were
inoculated at a density of 2 Â 10
4
per well in 96-well
multiplates with 100 mL of DMEM containing 10% fetal
bovine serum. When they reached subconfluence, the
medium was changed to fetal-bovine-serum-free DMEM
and preincubated for 24 h. In the case of RGC cells, the cells
were inoculated at a density of 1 Â 10
4
per well in 100 mLof
aMEM containing 10% fetal bovine serum. When they
reached confluence, the medium was changed to fetal-
bovine-serum-free aMEM, and preincubated for 48 h. MAPK
inhibitors (50 m
M of PD098059, 10 mM of SB203580) were
added to the culture 1 h before the addition of 50 ng
:
mol
21
of
rCTGF/Hcs24. After 22 h, [
3
H]thymidine (9925 Tbq
:

nmol
21
;
Amersham Pharmacia Biotech), dissolved in DMEM was
added to the cultures at a final concentration of
740 KBq
:
mL
21
, and cells were incubated for another 4 h.
After labeling, the cell layers were washed three times with
NaCl/P
i
and treated successively with 5% trichloroacetic
acid and ethanolethyl ether (3 : 1, v/v). Radioactivity in the
residual materials was measured using a Micro b-PLUS
(Pharmacia Biotech), as described previously [13,15].
q FEBS 2001 MAPK mediates CTGF actions on chondrocytes (Eur. J. Biochem. 268) 6059
Proteoglycan synthesis
Proteoglycan synthesis was assayed as described previously
[10,15] with slight modification. HCS-2/8 cells were grown
to confluence in 24-well microplates in DMEM containing
10% fetal bovine serum. They were then preincubated in
DMEM without fetal bovine serum for 24 h and incubated
in the same medium with rCTGF/Hcs24 (50 ng
:
mL
21
) for
5 h. Then, [

35
S]sulfate (37 MBq
:
mL
21
) dissolved in NaCl/
P
i
was added to the cultures to a final concentration of
370 kBq
:
mL
21
, and incubation was continued for another
17 h. After labeling, the cultures were digested with
1mg
:
mL
21
actinase E (Kaken Pharmaceuticals, Tokyo,
Japan), and the radioactivity of the material precipitated
with cetylpyridium chloride was measured in a scintillation
counter.
Statistical analysis
Unless otherwise specified, all experiments were repeated at
least twice, with similar results. One-way analysis of variance
(
ANOVA) was used for statistical evaluation. Statistical
analysis was performed by the Dunnett test if necessary.
Data are expressed as the mean ^ SD and P , 0.05 was

considered significant.
RESULTS
In vivo
MAP kinase and p38 MAPK luciferase assay
HCS-2/8 cells were transiently cotransfected with an Elk or
Atf expression vector, pTet-Elk or pTet-Atf, together with a
pTRE-luc plasmid that contains a luciferase reporter gene.
Transfected cells were subsequently treated with various
concentrations of rCTGF/Hcs24. The luciferase activity in
this experiment reflects endogeneous ERK and p38 MAPK
phosphorylation activity. The luciferase activity increased
on incubation with rCTGF/Hcs24 in a dose-dependent
manner (Fig. 1). A high dose of CTGF/Hcs24 (50 ng
:
mL
21
)
caused a fivefold increase in the MAPK luciferase activity
(Fig. 1A), but the same concentration of rCTGF/Hcs24
caused only a twofold increase in the p38 MAPK luciferase
activity (Fig. 1B). To check the phosphorylation of Elk by
ERK and Atf by p38 MAPK through the activation of
MEK1/2 and p38 MAPK, we used PD098059, a MEK1/
2-specific inhibitor and SB203580, a p38 MAPK inhibitor in
the same assay (Fig. 2). Phosphorylation of Elk (Fig. 2A) or
Atf (Fig. 2B) was suppressed by each inhibitor dose-
dependently.
Effects of rCTGF/Hcs24 on phosphorylation of ERK and
p38 MAP kinase in HCS-2/8 cells
We analyzed the effects of rCTGF/Hcs24 on the phosphory-

lation of two major types of MAP kinases, ERK and p38
MAP kinase, by Western blotting in HCS-2/8 cells (Fig. 3).
We determined that CTGF/Hcs24 stimulated the phosphory-
lation of the kinases with different time kinetics. Interest-
ingly, CTGF/Hcs24 induced a slow phosphorylation of ERK
from 10 min after the treatment with a maximal effect
observed at 30 min (Fig. 3A). This was different from other
growth factors that promote proliferation. On the other hand,
rCTGF/Hcs24 induced a rapid phosphorylation of p38
MAPK from 10 to 30 min after the addition (Fig. 3B). The
MEK1/2-specific inhibitor, PD098059, and p38 MAPK-
specific inhibitor, SB203580, inhibited the phosphorylation
of each MAP kinase induced by rCTGF/Hcs24 (Fig. 4A,B).
But neither inhibitor had an effect on the different
phosphorylation of MAP kinase.
Effects of MAP kinase inhibitors on DNA synthesis in
HCS-2/8 and RGC cells
To investigate the signal pathways for proliferation of
chondrocytes induced by CTGF/Hcs24, we performed DNA
Fig. 1. In vivo MAPK and p38 MAPK luciferase activity induced
by CTGF/Hcs24 in the sparse phase of HCS-2/8 cells. Elk-1 (A) and
Atf-2 (B) phosphorylation induced by CTGF/Hcs24 through endo-
geneous ERK and p38 MAPK, respectively. HCS-2/8 cells were
cotransfected with the fusion transactivater plasmid, pTet-Elk (A) and
pTet-Atf (B) and the reporter plasmid, pTRE-luc, as described in
Materials and methods. At 24 h after transfection, the cells were serum-
deprived for 24 h, and incubated with the indicated concentrations of
CTGF/Hcs24 for 24 h. Extracts prepared thereafter were assayed for
luciferase activity using the Dual-Luciferase
TM

Reporter Assay System.
In these system, firefly luciferase is used as an expression reporter of
TRE (pTRE-Luc), and TK drived renilla luciferase is usd as a
transfection control (pRL-TK). These plasmids were cotransfected to
HCS-2/8 cells. F/R ratio indicates the ratio of luciferase activity of
firefly (F) and renilla (R). Points and bars are the mean ^ SD for
duplicate cultures. *, P , 0.05; **, P , 0.01 (significantly different
from the control culture).
6060 G. Yosimichi et al. (Eur. J. Biochem. 268) q FEBS 2001
synthesis analysis by estimating incorporation of [
3
H]thymi-
dine in HCS-2/8 (Fig. 5A) and RGC cells (Fig. 5B). rCTGF/
Hcs24 at a concentration of 50 ng
:
mL
21
induced about a
1.5-fold increase in DNA synthesis both in HCS-2/8 and in
RGC cells (Fig. 5A,B, bar 2) compared to the control
culture (Fig. 5A,B, bar 1). In HCS-2/8 cells, treatment with
50 m
M of the MEK1/2 inhibitor, PD098059, suppressed the
degree of DNA synthesis in the control culture by about half,
either with (Fig. 5A, bar 3) or without rCTGF/Hcs24
(Fig. 5A, bar 5). On the other hand, 10 m
M of the p38
MAPK inhibitor, SB203580 suppressed DNA synthesis in
HCS-2/8 cells induced by CTGF/Hcs24, but had no effect
in the control culture (Fig. 5A, bars 1 and 6) without rCTGF/

Hcs24 (Fig. 5A, bar 6). In RGC cells (Fig. 5B), rCTGF/
Hcs24 induced about a 1.5-fold increase in the synthesis of
DNA similar (Fig. 5B, bar 2) to the control (Fig. 5B, bar 1).
PD098059 inhibited the effect of CTGF/Hcs24 (Fig. 5B, bar
3), but had no effect on the basal level of DNA synthesis in
RGC cells (Fig. 5B, bar 5). SB203580 treatment did not
suppress the synthesis of DNA in RGC cells with or without
rCTGF/Hcs24 (Fig. 5B, bar 6). SB203580 did not decrease
the synthesis further in PD098059-treated cells (Fig. 5B,
bar7).
Effects of MAP kinase inhibitors on proteoglycan
synthesis of HCS-2/8 and RGC cells
Previously we reported that CTGF/Hcs24 dose-dependently
enhanced proteoglycan synthesis in HCS-2/8 cells and RGC
cells [13]. To investigate the intracellular pathways for the
stimulatory action of CTGF/Hcs24 on chondrocyte differ-
entiation, we examined proteoglycan synthesis in HCS-2/8
cells (Fig. 6A) and RGC cells (Fig. 6B) in the presence of
MAP kinase inhibitors. Addition of 50 ng
:
mL
21
of rCTGF/
Hcs24 promoted the synthesis of proteoglycans by about
twofold in both HCS-2/8 cells (Fig. 6A, bar 2) and RGC
cells (Fig. 6B, bar 2) relative to control cultures (Fig. 6A,B,
bar 1). Treatment with 50 m
M of the MEK1/2 inhibitor,
PD098059 did not inhibit the stimulatory effect of rCTGF/
Hcs24 (Fig. 6A,B, bar 3), but 10 m

M of the p38 MAPK
inhibitor, SB203580 suppressed the stimulatory effects of
rCTGF/Hcs24 in HCS-2/8 cells (A bar 4) and RGC cells (B
bar 4). The inhibitors had no effect on the basal level of
Fig. 3. Effects of CTGF/Hcs24 on phosphorylation of ERK (A) and
p38 MAPK (B) in confluent HCS-2/8 cells. Western blot analysis
revealed that stimulation with CTGF/Hcs24 increased the phosphoryl-
ation of ERK from 10 min to 30 min (A). Stimulation with CTGF/
Hcs24 also increased the phosphorylation of p38 MAPK peaked from
10 to 30 min (B).
Fig. 2. Effects of MAP kinase inhibitors (PD098059, SB203580) on
phosphorylation of Elk-1 and Atf-2 induced by CTGF/Hcs24. HCS-
2/8 cells were cultured under the same conditions as for Fig. 1. CTGF/
Hcs24 (50 ng
:
mL
21
) was added after 1 h pretreatment with various
concentrations of MAP kinase inhibitors (PD098059, A; SB203580, B).
CTGF/Hcs24-induced phosphorylation of Elk-1 (A) and Atf-2 (B)
through endogeneous ERK was suppressed by PD098059 (A) and
SB203580 (B), respectively, dose-dependently. F/R ratio indicates the
ratio of luciferase activity of firefly (F) and renilla (R). Points and bars
are the mean ^ SD for duplicate cultures. *, P , 0.05; **, P , 0.01
(significantly different from the control culture).
Fig. 4. Effects of MAPK inhibitors (50 mM PD098059 and 10 mM
SB203580) on phosphorylation of ERK (A) and p38 MAPK (B)
determined by Western blotting. (A) Phosphorylation of ERK was
enhanced by CTGF/Hcs24 at 30 min. Treatment with 50 m
M PD098059

decreased the amount of phosphorylated ERK but treatment with
10 m
M SB203580 had no effect. (B) Phosphorylated p38 MAPK was
increased at 15 min and 30 min, and the increase was suppressed by
SB203580. PD098059 treatment had no effect on the amount of
phosphorylated p38 MAPK.
q FEBS 2001 MAPK mediates CTGF actions on chondrocytes (Eur. J. Biochem. 268) 6061
proteoglycan synthesis in HCS-2/8 cells (Fig. 6A, bars 5
and 6) and RGC cells (Fig. 6B, bars 5 and 6).
DISCUSSION
In this study, we first investigated the phosphorylation of
two major types of MAP kinase induced by CTGF/Hcs24.
The MAPK cascade is known to be activated by receptor
tyrosine kinase activity induced by growth factors, such as
EGF [35], FGF [27], and parathyroid hormone (PTH) [41]
and serine threonine kinase activity by TGF-b [28,29] and
growth differentiation factor-5 (GDF-5) [42] in chondro-
cytes. We previously reported that a 280-kDa CTGF/
Hcs24-receptor complex was present in HCS-2/8 cells [14],
and a human osteosarcoma cell line, Saos-2 [15], and this
receptor complex was thyrosine-phosphorylated by CTGF/
Hcs24 (data not shown). It was also reported that Nov, a
member of the CCN family, induced thyrosine-phosphoryl-
ation of a 221-kDa protein in fibloblasts [43]. These
Fig. 5. Effects of MAP kinase inhibitors on DNA synthesis in
HCS-2/8 (A) and RGC cells (B). For the evaluation of DNA synthesis,
HCS-2/8 cells were inoculated at a density of 2 Â 10
4
per well into
96-well multiplates and cultured in 100 mL of DMEM containing 10%

fetal bovine serum, while RGC cells were inoculated at a density of
1 Â 10
4
per well into 96-well multiplates and cultured in 100 mLof
aMEM containing 10% fetal bovine serum. When they reached
subconfluence, HCS-2/8 cells were incubated in 100 mL of serum-free
DMEM for 24 h, and RGC cells were incubated in 100 mL of serum-
free aMEM for 24 h, and then 50 ng
:
mL
21
of CTGF/Hcs24 was added
to the cultures. Next, 50 m
M PD098059 and 10 mM SB203580 were
added 1 h before the addition of rCTGF/Hcs24. Dimethylsulfoxide was
added to the control culture. DNA synthesis was measured 22 h later as
described in Materials and methods. (A) control (bar 1), 50 ng
:
mL
21
of
CTGF/Hcs24 (bar 2), 50 m
M PD098059 with 50 ng
:
mL
21
of rCTGF/
Hcs24 (bar 3), 10 m
M SB203580 with 50 ng
:

mL
21
of CTGF/Hcs24 (bar
4), 50 m
M PD098059 (bar 5), 10 mM SB203580 (bar 6), both of the
inhibitors with 50 ng
:
mL
21
of rCTGF/Hcs24 (bar 7). Columns and bars
are mean ^ SD for triplicate cultures. **, P , 0.01, significantly
different from the control cultures. *, P , 0.05, significantly different
from the CTGF/Hcs24 containing cultures.
Fig. 6. Effects of MAP kinase inhibitors on proteoglycan synthesis
in HCS-2/8 (A) and RGC cells (B). For estimation of proteoglycan
synthesis, confluent cultures of HCS-2/8 cells (A) and RGC cells
(B) were preincubated in serum-free DMEM for 24 h and then
incubated in the same medium with or without 50 ng
:
mL
21
of rCTGF/
Hcs24. Next, 50 m
M PD098059 and 10 mM SB203580 was added
1 h before the addition of rCTGF/Hcs24. Dimethylsulfoxide was added
to the control culture. and then [
35
S]sulfate (37 Mbq
:
mL

21
) dissolved
in NaCl/P
i
was added to the cultures (370 kBq
:
mL
21
final
concentration), and incubation was continued for another 17 h.
Proteoglycan synthesis was measured as described in Materials
and methods. Control (bar 1), 50 ng
:
mL
21
of rCTGF/Hcs24 (bar 2),
50 m
M PD098059 with 50 ng
:
mL
21
of rCTGF/Hcs24 (bar 3), 10 mM
SB203580 with 50 ng
:
mL
21
of rCTGF/Hcs24 (bar 4), 50 mM
PD098059 (bar 5), 10 mM SB203580 (bar 6), both of the inhibitors
with 50 ng
:

mL
21
of rCTGF/Hcs24 (bar 7). Columns and bars are
mean ^ SD for duplicate cultures. **, P , 0.01, significantly different
from the control cultures. *, P , 0.05, significantly different from the
CTGF/Hcs24-added cultures.
6062 G. Yosimichi et al. (Eur. J. Biochem. 268) q FEBS 2001
observations indicate a possible relation between CTGF/
Hcs24-signal transduction and MAPK cascades. The present
study demonstrated that CTGF/Hcs24 potentiated phosphory-
lation of Elk-1, downsream of ERK [30], and Atf-2,
downstream of p38 MAPK [31] in a dose-dependent manner
in HCS-2/8 cells (Fig. 1). Both ERK1 and ERK2 have been
shown to be activated by their upstream activaters, MEK1 and
MEK2. A recently developed MEK1/2 inhibitor, PD098059,
has been reported to bind to MEK1/2 and inhibit MEK1/2
phosphorylation activated by either c-Raf or MEKK [35,36].
PD098059 efficiently and specifically suppresses the
activation of ERK in response to various growth factors
[25,26,28,44]. The inhibitory effect of PD098059 on MEK2
is less potent than that on MEK1 (IC
50
values of PD098059
for MEK1 and MEK2 are 4 and 50 m
M, respectively) [35].
On the other hand, p38 MAPK is known to be a stress signal
transducer, and phosphorylation of p38 MAPK in chondro-
cytes by EGF [25], PTH [41], and GDF-5 [42] regulates
differentiation of chondrocytes. p38 MAPK is phosphory-
lated by MKK3, and MKK6 [45], and Atf-2 is one of the

transcription factors downstream of p38 MAPK [31]. A
specific p38 MAPK inhibitor, SB203580 [38], suppresses two
isoforms of p38 MAPK, p38a,andb2, but not p38g [37,46].
In this study, the MEK1/2 inhibitor, PD098059, dose-
dependently suppressed phosphorylation of ERK, and Elk-1
downstream of ERK in the growth phase of HCS-2/8 cells
treated with CTGF/Hcs24 (Fig. 2). Similarly, the p38
MAPK inhibitor, SB203580, suppressed phosphorylation of
p38 MAPK, and Atf-2 downstream of p38 MAPK (Fig. 2).
Western blot analysis revealed that CTGF/Hcs24 increased
phosphorylation of ERK and p38 MAPK in confluent HCS-
2/8 cells (Fig. 3). PD098059 suppressed the phosphoryl-
ation of ERK, but had no effect on the phosphorylation of
p38 MAPK. In the same way, SB203580 suppressed
phosphorylation of p38 MAPK but not ERK (Fig. 4). These
results indicate that both MAPK pathways contributed
selectively to the effects of CTGF/Hcs24.
Next, we showed the effects of MAP kinase inhibitors in
both HCS-2/8 cells and RGC cells induced by CTGF/Hcs24.
Previously, we reported that CTGF/Hcs24 induced DNA
synthesis, and proteoglycan synthesis in HCS-2/8 cells and
RGC cells [13]. We investigated the effects of MAP kinase
inhibitors on pathways of proliferation by analyzing DNA
synthesis, and pathways of differentiation by analyzing
proteoglycan synthesis, in HCS-2/8 and RGC cells. In the
case of HCS-2/8 cells, the MEK1/2 inhibitor, PD098059,
suppressed the synthesis of DNA under all conditions, and
the p38 MAPK inhibitor, SB203580, suppressed the syn-
thesis induced by CTGF/Hcs24 to the control level (Fig. 5A,
bar 4), but had no effect on the basal level (Fig. 5A, bar 6).

As HCS-2/8 cells were tumor cells, their basal levels of
DNA synthesis were high, and proliferation was not under
normal control. Therefore we analyzed normal chondrocytic
response in RGC cells, PD098059 suppressed DNA syn-
thesis induced by CTGF/Hcs24 to the control level (Fig. 5B,
bar 3) and had no effect on the basal level. SB203580 did not
suppress DNA synthesis induced by CTGF/Hcs24, and had
no effect in the absence of CTGF/Hcs24 (Fig. 5B, bars 3 and
5). These findings suggest that the ERK pathway has an
important role in chondrocyte proliferation induced by
CTGF/Hcs24.
Next, we analyzed proteoglycan synthesis to investigate
the effects of MAP kinase inhibitors on the differentiation of
chondrocytes induced by CTGF/Hcs24. We previously
reported that CTGF/Hcs24 induced synthesis of proteo-
glycans in HCS-2/8 cells and RGC cells [13]. In the present
study, as shown in Fig. 6, PD098059 had no effect on
proteoglycan synthesis in the presence or absence of CTGF/
Hcs24 (Fig. 6A,B, bars 3 and 5). On the other hand,
SB203580 inhibited the increase in proteoglycan synthesis
evoked by CTGF/Hcs24 in HCS-2/8 and RGC cells
(Fig. 6A,B bar 4). These findings suggest that the p38
MAPK pathway is the most important signalling pathway in
the differentiation of chondrocytes. The ERK pathway does
not seem to be involved in the differentiation of
chondrocytes especially when induced by CTGF/Hcs24.
Concerning crosstalk between ERK and p38 MAPK, it is
reported that GDF-5 phosporylated p38 MAPK and ERK,
and p38 MAPK inhibitor inhibited, but MEK1/2 inhibitor
enhanced, the chondrogenic response induced by GDF-5 in

ATDC5 cells [42]. EGF inhibited the chondrogenic differen-
tiation of mesenchymal cells, and activated ERK but
inhibited p38 MAPK [25]. Related to the effect of CTGF/
Hcs24 on chondrocytes, luciferase assay revealed phosphory-
lation of Elk-1 was inhibited by the MEK inhibitor but
slightly enhanced by treatment with the p38 MAPK inhibitor
(data not shown). and phosphorylation of Atf-2 was inhibited
by the p38 MAPK inhibitor and also inhibited slightly by the
MEK inhibitor (data not shown). These results suggest
that ERK activates both Elk-1 and Atf-2 (Fig. 7), and has a
dominant effect in the growth phase of cells. Furthermore,
DNA synthesis indicated that both the ERK and p38 MAPK
pathways are involved in the proliferation of chondrocytes
induced by CTGF/Hcs24 in tumor cells. Also Western
blotting shows that SB203580 treatment increased phos-
phorylation of ERK (Fig. 4, at 0 min), but the effect was not
related to synthesis of DNA or proteoglycan (Figs 5 and 6,
bars 5 and 6). These results suggest that inhibition of one
pathway induces activation of another.
In summary, the present results emphasized the functional
importance of two MAP kinase cascades, the ERK and p38
MAPK pathways, in the promotion of the chondrogenesis
Fig. 7. Possible scheme of MAPK signaling cascades in CTGF/
Hcs24-induced chondrogenesis. E, extracellular; M, cell membrane;
C, cytoplasm; N, nucleus.
q FEBS 2001 MAPK mediates CTGF actions on chondrocytes (Eur. J. Biochem. 268) 6063
mediated by CTGF/Hcs24 in HCS-2/8 and RGC cells. The
ERK pathway plays an important role in the proliferation
of chondrocytes, while the p38 MAPK pathway plays a role
in the differentiation of chondrocytes induced by CTGF/

Hcs24. Our findings suggest that two MAP kinase cascades
mediate various important roles of CTGF/Hcs24 in the
proliferation and differentiation of chondrocytes during
endochondoral ossification.
ACKNOWLEDGEMENTS
This work was supported in part by Grants-in-Aid for Scientific
Research from the Ministry of Education, Science,Sports and Culture of
Japan (to T.N., T.N., and M.T.), and grants from the Foundation for
Growth Science in Japan (to M.T.), the Sumitomo Foundation (to M.T.)
and the Research for the Future Programme of The Japan Society for the
Promotion of Science (JSPS) (Project: Biological Tissue Engineering,
JSPS-RFTF98100201). We thank Drs Satoshi Kubota, Takanori Eguchi,
and Seiji Kondo for useful discussions, Drs Kumiko Nawachi and
Norifumi Moritani for technical assistance, Dr Daisuke Ekuni for
performing the Dunnett test, and Miss Yuki Nonami for secretarial
assistance.
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