Báo cáo khoa học: Cartducin stimulates mesenchymal chondroprogenitor cell proliferation through both extracellular signal-regulated kinase and phosphatidylinositol 3-kinase⁄Akt pathways pot

Cartducin stimulates mesenchymal chondroprogenitor cell
proliferation through both extracellular signal-regulated
kinase and phosphatidylinositol 3-kinase⁄ Akt pathways
Hironori Akiyama
1,2
, Souhei Furukawa
2
, Satoshi Wakisaka
1
and Takashi Maeda
1
1 Department of Anatomy and Cell Biology, Graduate School of Dentistry, Osaka University, Japan
2 Department of Radiology, Graduate School of Dentistry, Osaka University, Japan
Cartilage formation is driven by mesenchymal chon-
droprogenitor cells that proliferate and differentiate
into chondrocytes. The molecular mechanisms by
which growth factors regulate the fate of these cells
are not well defined. We recently discovered a secre-
tory protein, cartducin, which is an  27 kDa polypep-
tide containing an N-terminal signal peptide, a short
variable domain, a collagen-like domain, and a C-ter-
minal C1q-like globular domain. Its mRNA is
predominantly and highly expressed in developing
cartilages [1]. Interestingly, cartducin has structural
homology with a 30 kDa serum protein Acrp30 ⁄ adipo-
nectin, which is exclusively and highly expressed in
differentiated adipocytes [2], and has been shown to be
dysregulated in various forms of obesity in mice and
humans [3,4]. Because cartducin is a paralog of
Acrp30 ⁄ adiponectin, cartducin is a novel member of a
newly designated C1q family of proteins [5,6]. The

Keywords
cartducin; chondroprogenitor cells; MAPK;
PI3K ⁄ Akt; proliferation
Correspondence
T. Maeda, Department of Anatomy and Cell
Biology, Graduate School of Dentistry,
Osaka University, 1–8 Yamadaoka, Suita,
Osaka 565–0871, Japan
Fax: +81 6 6879 2875
Tel: +81 6 6879 2874
E-mail:
(Received 20 November 2005, revised 20
February 2006, accepted 20 March 2006)
doi:10.1111/j.1742-4658.2006.05240.x
Cartducin, a paralog of Acrp30 ⁄ adiponectin, is a secretory protein pro-
duced by both chondrogenic precursors and proliferating chondrocytes,
and belongs to a novel C1q family of proteins. We have recently shown
that cartducin promotes the growth of both mesenchymal chondroprogeni-
tor cells and chondrosarcoma-derived chondrocytic cells in vitro. However,
the cartducin-signaling pathways responsible for the regulation of cell pro-
liferation have not been documented. In this study, we examined whether
cartducin exists in serum and further investigated the intracellular signaling
pathways stimulated by cartducin in mesenchymal chondroprogenitor cells.
Western blot analysis showed that, unlike Acrp30⁄ adiponectin, cartducin
was undetectable in mouse serum. Next, mesenchymal chondroprogenitor
N1511 cells were stimulated with cartducin, and three major groups of
mitogen-activated protein kinase (MAPK) pathways and the phosphatidyl-
inositol 3-kinase (PI3K) ⁄ Akt signaling pathway were examined. Cartducin
activated extracellular signal-regulated kinase 1 ⁄ 2 (ERK1 ⁄ 2) and Akt, but
not c-jun N-terminal kinase (JNK) nor p38 MAPK. The MEK1 ⁄ 2 inhib-

itor, U0126, blocked cartducin-stimulated ERK1 ⁄ 2 phosphorylation and
suppressed the DNA synthesis induced by cartducin in N1511 cells. The
PI3K inhibitor, LY294002, blocked cartducin-stimulated Akt phosphoryla-
tion and a decrease in cartducin-induced DNA synthesis in N1511 cells
was also observed. These data suggest that cartducin is a peripheral skeletal
growth factor, and that the proliferation of mesenchymal chondroprogeni-
tor cells stimulated by cartducin is associated with activations of the
ERK1 ⁄ 2 and PI3K ⁄ Akt signaling pathways.
Abbreviations
BrdU, bromodeoxyuridine; ERK, extracellular signal-regulated kinase; JNK, c-jun N-terminal kinase; MAPK, mitogen-activated protein kinase;
MEK, MAPK ⁄ ERK kinase; a-MEM, alpha-minimal essential medium; PI3K, phosphatidylinositol 3-kinase.
FEBS Journal 273 (2006) 2257–2263 ª 2006 The Authors Journal compilation ª 2006 FEBS 2257
members of this family contain a C-terminal C1q-like
globular domain and are involved in processes as
diverse as host defense, inflammation, apoptosis, auto-
immunity, cell differentiation, organogenesis, hiberna-
tion and insulin-resistant obesity.
More recently, we found that cartducin is strongly
expressed in both the chondrogenic cell lineage in
the sclerotome and proliferating chondrocytes in the
growth plate of cartilage, and it promotes the prolifer-
ation of mesenchymal chondroprogenitor cells and
chondrocytes in vitro [7]. Although cartducin is a bio-
logically active molecule involved in both embryonic
cartilage development and postnatal longitudinal bone
growth, the signaling pathways activated upon cartdu-
cin stimulation have not been documented. Because a
high mitogenic activity of mesenchymal chondropro-
genitor cells is initially required to produce enough
cells for the process of chondrogenesis [8], elucidation

of the signaling pathways responsible for cartducin-
induced DNA synthesis by mesenchymal chondropro-
genitor cells is necessary to more fully understand the
molecular mechanisms of cartilage formation.
Mitogen-activated protein kinase (MAPK) pathways
are essential mitogenic pathways in many cell lines and
are responsible for various growth factors. Down-
stream of these pathways can simply be classified into
three major groups mediated by the following kinases:
extracellular signal-regulated kinase (ERK), c-Jun
N-terminal kinase (JNK), and p38 MAPK [9,10].
Another signaling pathway associated with the control
of mitogenesis is the phosphatidylinositol 3-kinase
(PI3K) ⁄ Akt pathway. It is also an important mediator
of cell growth and survival in response to growth
factors and other signals. PI3K activates Akt serine ⁄
threonine kinase by generating specific inositol phos-
pholipids, which recruit Akt to the cell membrane and
enable its activation [11]. In this study, we examined the
possibility that cartducin is a serum protein and further
investigated the possible signaling pathways activated
by cartducin in the regulation of DNA synthesis using
the mouse mesenchymal chondroprogenitor cell line,
N1511 [12]. The results show that cartducin, a local reg-
ulatory factor, exerts its effects on the proliferation of
mesenchymal chondroprogenitor cells by activating
both the ERK1 ⁄ 2 and PI3K ⁄ Akt pathways.
Results
Cartducin is a local regulatory factor but not a
systemic hormone

Cartducin is a novel growth factor which belongs to
a newly designated family of proteins, the C1q ⁄ TNF
superfamily, all of which contain a C-terminal C1q-like
globular domain and most of which contain a colla-
genous region. Based on the structural similarities of
cartducin to the Acrp30 ⁄ adiponectin, which has been
shown to play important roles in the regulation of glu-
cose and lipid metabolism as a hormone, we investi-
gated the possibility that cartducin is also a serum
protein. A protein of identical molecular mass to cart-
ducin can be detected in the total tissue protein extrac-
ted from mouse growth plate cartilage. In contrast,
unlike Acrp30 ⁄ adiponectin, we failed to detect cartdu-
cin in mouse serum using western blot analysis
(Fig. 1). These results suggest that cartducin exerts its
effects in vivo in an autocrine ⁄ paracrine manner.
Cartducin activates the ERK1 ⁄ 2 pathway in
mesenchymal chondroprogenitor N1511 cells
Cartducin is known to play important roles in regula-
ting both chondrogenesis and cartilage development by
its direct stimulatory action on the proliferation of
chondrogenic precursors and chondrocytes. MAPK
pathways are essential mitogenic pathways in many
cells and are responsible for various growth factors.
Three major groups of MAPKs, such as ERK, JNK,
and p38 MAPK, have been identified. Therefore, we
analyzed the effects of cartducin on the phosphoryla-
tion of these three groups of MAPKs in N1511
cells. Western blot analysis showed that increased
ERK1 ⁄ 2 phosphorylation in N1511 cells treated with

10 lgÆmL
)1
of cartducin was detected after 5 min, with
maximal increase occurring after 15 min of treatment
and decreasing after 1 h (Fig. 2A). A dose-dependent
increase in ERK1 ⁄ 2 phosphorylation in N1511 cells
after 30 min of treatment was detected at 2, 5, and
10 lgÆmL
)1
of cartducin (Fig. 2B). In contrast, cartdu-
cin had no effect on the activities of JNK1 ⁄ 2 and p38
37
25
Cartducin
kDa
Cartilage
Serum
Fig. 1. Cartducin is not a serum protein. One microliter of mouse
serum was boiled for 5 min in 2 · sample buffer and analyzed by
SDS ⁄ PAGE and western blot using anti-mouse IgG. Antibody was
visualized with an anti-goat IgG coupled to alkaline phosphatase.
A protein of identical molecular mass to cartducin can be detected
in the total tissue protein extracted from mouse growth plate carti-
lage. However, cartducin could not be detected in serum from
mice.
Cartducin activates MAPK and PI3K ⁄ Akt H. Akiyama et al.
2258 FEBS Journal 273 (2006) 2257–2263 ª 2006 The Authors Journal compilation ª 2006 FEBS
MAPK, and none of their phosphorylated forms was
detected (Fig. 3). Both ERK1 and ERK2 have been
shown to be activated by their upstream activators,

MEK1 and MEK2. We next examined the action of
the specific MEK1 ⁄ 2 inhibitor, U0126, on ERK1 ⁄ 2
pathway activation. Phosphorylation of ERK1 ⁄ 2
induced by cartducin was completely inhibited by pre-
treatment with U0126 (10 lm) (Fig. 4).
ERK1 ⁄ 2 pathway activation is involved in
cartducin-induced proliferation of mesenchymal
chondroprogenitor N1511 cells
Because western blot analysis confirmed cartducin-
induced ERK1 ⁄ 2 pathway activation in N1511 cells,
we next determined whether cartducin-induced mesen-
chymal chondroprogenitor cell proliferation is medi-
ated through activation of the ERK1 ⁄ 2 pathway.
N1511 cells were pretreated with the MEK1 ⁄ 2 inhib-
itor U0126 (10 lm), JNK inhibitor SP600125 (20 lm),
or p38 MAPK inhibitor SB203580 (10 lm) for 1 h
before and for the duration of the stimulation, and
DNA synthesis analysis by estimating the incorpor-
ation of bromodeoxyuridine (BrdU) was performed
(Fig. 5). Cartducin induced about a twofold increase
in DNA synthesis in N1511 cells compared with the
buffer-treated control culture. Treatment with U0126,
SP600125, or SB203580 suppressed the basal level of
DNA synthesis in N1511 cells compared with the
dimethylsulfoxide-treated control culture, respectively.
Fig. 3. Effects of cartducin on phosphorylation of JNK and p38
MAPK in N1511 cells. Mesenchymal chondroprogenitor N1511 cells
stimulated with cartducin (10 lgÆmL
)1
) for 5, 15, 30, and 60 min

were analyzed by western blot for phosphorylated JNK1 ⁄ 2 (p-
JNK1 ⁄ 2), total JNK1 ⁄ 2, phosphorylated p38 MAPK (p-p38 MAPK),
and total p38 MAPK. Cartducin had no effect on the phosphoryla-
tion of JNK1 ⁄ 2 and p38 MAPK, and none of their phosphorylated
forms were detected.
Cartducin: – –+
++––
+
U0126:
ERK1/2
p-ERK1/2
Fig. 4. Effects of MAPK inhibitor on phosphorylation of ERK in
N1511 cells. Mesenchymal chondroprogenitor N1511 cells were
pretreated with MEK1 ⁄ 2 inhibitor, U0126 (10 l
M), for 1 h, stimula-
ted with cartducin (10 lgÆmL
)1
) for 15 min, and then analyzed by
western blot for phosphorylated ERK1 ⁄ 2 (p-ERK1 ⁄ 2). Treatment
with U0126 inhibited cartducin-induced ERK1 ⁄ 2 phosphorylation.
A
B
Fig. 2. Effects of cartducin on phosphorylation of ERK in mesen-
chymal chondroprogenitor N1511 cells. (A) Subconfluent cultures of
N1511 cells stimulated with cartducin (10 lgÆmL
)1
) for 5, 15, 30,
and 60 min were analyzed by western blot for phosphorylated
ERK1 ⁄ 2 (p-ERK1 ⁄ 2) and total ERK1 ⁄ 2. Cartducin increased the
p-ERK1 ⁄ 2 level after 5 min incubation. Peak activation of ERK1 ⁄ 2

occurred at 15 min. (B) Cells were stimulated with increasing doses
of cartducin (2–10 lgÆmL
)1
) for 30 min. Cartducin induced a dose-
dependent activation of ERK1 ⁄ 2.
Fig. 5. Effects of MAPK inhibitors on DNA synthesis in mesenchy-
mal chondroprogenitor N1511 cells. Subconfluent cultures of
N1511 cells were serum starved, preincubated with MEK1 ⁄ 2 inhib-
itor U0126 (10 l
M), JNK inhibitor SP600125 (20 lM), or p38 MAPK
inhibitor SB203580 (10 l
M) for 1 h before cartducin treatment
(8 lgÆmL
)1
), and bromodeoxyuridine (BrdU) incorporation was
measured. Data are represented as the mean ± SD of triplicate
determinations. P < 0.05 versus cartducin-untreated each control.
Similar results were obtained from two independent experiments.
H. Akiyama et al. Cartducin activates MAPK and PI3K ⁄ Akt
FEBS Journal 273 (2006) 2257–2263 ª 2006 The Authors Journal compilation ª 2006 FEBS 2259
Inhibition of the ERK1 ⁄ 2 pathway by pretreatment
with U0126 led to a block of cartducin-induced N1511
cell proliferation. By contrast, inhibition of JNK or
p38 MAPK pathways by SP600125 or SB203580 did
not affect cartducin-induced N1511 cell proliferation.
Cartducin activates the PI3K ⁄ Akt pathway
in N1511 cells
The PI3K ⁄ Akt pathway is also an important mediator
of cell growth and survival in response to growth
factors and other signals. We therefore analyzed the

effects of cartducin on activation of the PI3K ⁄ Akt
pathway in mesenchymal chondroprogenitor N1511
cells. Western blot analysis showed that increased
Akt phosphorylation in N1511 cells treated with 10
lgÆmL
)1
of cartducin was detected after 5 min, with
maximal increase occurring after 30 min of treatment
and decreasing after 1 h (Fig. 6A). A dose-dependent
increase in Akt phosphorylation in N1511 cells after
30 min of treatment was detected at 2, 5, and
10 lgÆmL
)1
of cartducin (Fig. 6B). Next, the action of
the specific PI3K inhibitor, LY294002, on Akt phos-
phorylation was examined. Phosphorylation of Akt
induced by cartducin was inhibited completely by pre-
treatment with LY294002 (25 lm) (Fig. 6C).
PI3K ⁄ Akt pathway activation is involved in
cartducin-induced proliferation of mesenchymal
chondroprogenitor N1511 cells
Because western blot analysis confirmed cartducin-
induced PI3K ⁄ Akt pathway activation in N1511 cells,
we further determined whether cartducin-induced
mesenchymal chondroprogenitor cell proliferation is
mediated through activation of the PI3K ⁄ Akt path-
way. N1511 cells were pretreated with the PI3K inhib-
itor, LY294002 (1–25 lm), for 1 h before and for the
duration of the stimulation, and DNA synthesis analy-
sis was performed (Fig. 7). Similar to treatments with

MAPK inhibitors, treatment with LY294002 also sup-
pressed the basal level of DNA synthesis in N1511
cells. Inhibition of the PI3K ⁄ Akt pathway by pretreat-
ment with LY294002 significantly reduced DNA syn-
thesis in N1511 cells in a dose-dependent manner after
cartducin stimulation, suggesting involvement of the
PI3K ⁄ Akt pathway in the ability of cartducin to sti-
mulate proliferation of these cells.
Discussion
Skeletal development requires the condensation of
multipotential mesenchymal cells to differentiate
A
B
C
Fig. 6. Cartducin-induced Akt phosphorylation is dependent on PI3K
activity. (A) Time course of cartducin-induced phosphorylation of Akt
in N1511 mesenchymal chondroprogenitor cells. The cells stimula-
ted with cartducin (10 lgÆmL
)1
) for 5, 15, 30, and 60 min were ana-
lyzed by western blot for phosphorylated Akt (p-Akt) and total Akt.
Cartducin increased the p-Akt level after 5 min incubation. Peak acti-
vation of Akt occurred at 30 min. (B) Dose–response of cartducin-
induced phosphorylation of Akt in N1511 cells. The cells were
stimulated with increasing doses of cartducin (2–10 lgÆmL
)1
) for
30 min. Cartducin induced a dose-dependent activation of Akt. (C)
Effects of PI3K inhibitor on phosphorylation of Akt in N1511 cells.
Cells were pretreated with the PI3K inhibitor, LY294002 (25 l

M), for
1 h, stimulated with cartducin (10 lgÆmL
)1
) for 30 min, and then
analyzed by western blot for phosphorylated Akt (p-Akt). Treatment
with LY294002 inhibited cartducin-induced Akt phosphorylation.
Fig. 7. Effects of PI3K inhibitors on DNA synthesis in mesenchymal
chondroprogenitor N1511 cells. Subconfluent cultures of N1511
cells were serum starved, preincubated with PI3K inhibitor,
LY294002 (1–25 l
M), for 1 h before cartducin treatment
(8 lgÆmL
)1
), and BrdU incorporation was measured. Treatment of
N1511 cells with LY294002 inhibited the cartducin-stimulated DNA
synthesis in a concentration-dependent manner. Data are represen-
ted as the mean ± SD of triplicate determinations. P < 0.05 versus
cartducin-untreated control. Similar results were obtained from two
independent experiments.
Cartducin activates MAPK and PI3K ⁄ Akt H. Akiyama et al.
2260 FEBS Journal 273 (2006) 2257–2263 ª 2006 The Authors Journal compilation ª 2006 FEBS
toward the various cell types. One such process is
cartilage formation from undifferentiated mesenchy-
mal cells. Discoveries of diffusible factors that act
on the chondrogenic cell lineage and ⁄ or chondrocytes
have dramatically improved our understanding of
the molecular mechanisms of skeletal development.
Cartducin is a recently described cartilage-derived
secretory protein [1], and it has been indicated to
play important roles in regulating both embryonic

cartilage development and postnatal longitudinal
bone growth by directly promoting the proliferation
of mesenchymal chondroprogenitor cells and chond-
rocytes [7]. Because cartducin shares a similar modu-
lar organization to the serum protein Acrp30 ⁄
adiponectin, which has been shown to play import-
ant roles as a hormone in the regulation of glucose
and lipid metabolism, we considered the possibility
that cartducin might be not only a local regulatory
factor, but also a systemic hormone. However, we
could not detect cartducin in 1 lL (Fig. 1) or more
(data not shown) of mouse serum using western blot
analysis. These data suggested that cartducin is not
a serum protein, and thus cartducin may exert its
stimulatory action on the proliferation of chondro-
genic precursors and chondrocytes in vivo in an
autocrine ⁄ paracrine fashion only.
The expression of cartducin starts in the sclero-
tome, which contains a chondrogenic cell lineage
during mouse embryogenesis. Recombinant cartducin
induced DNA synthesis in mesenchymal chondropro-
genitor N1511 cells in a dose-dependent manner [7].
In contrast to Acrp30 ⁄ adiponectin [13], a cartducin-
specific receptor has not yet been identified and
cloned, and little is known about its signaling path-
ways. To gain insight into the mechanisms by which
cartducin promotes proliferation of mesenchymal
chondroprogenitor cells, we evaluated the signaling
events. MAPKs are well known to play an essential
role in controlling cell proliferation and differentia-

tion [9,10]. Indeed, the ERK1 ⁄ 2 pathway is involved
in controlling mesenchymal chondroprogenitor cell
proliferation [14]. In this study, we found that cart-
ducin induces activation of ERK1 ⁄ 2 in mesenchymal
chondroprogenitor N1511 cells but has no effect on
activation of JNK and p38 MAPK. Next, we investi-
gated the effects of MAPK inhibitors on the prolif-
eration of cartducin-stimulated N1511 cells by
analyzing DNA synthesis. Inhibition of the ERK1 ⁄ 2
signaling pathway by the specific MEK1 ⁄ 2 inhib-
itor, U0126, but not the specific JNK inhibitor,
SP600125, nor the specific p38 MAPK inhibitor,
SB203580, blocked cartducin-stimulated proliferation
of N1511 cells. The data suggest that the ERK1 ⁄ 2
pathway has an important role in the proliferation
of mesenchymal chondroprogenitor cells stimulated
by cartducin. JNK or p38 MAPK pathways do not
seem to be involved in the cartducin-stimulated pro-
liferation of these cells.
Another signaling pathway associated with the con-
trol of cell proliferation is the PI3K ⁄ Akt pathway. It is
also an important mediator of cell growth and survival
in response to growth factors and other signals. PI3K
activates Akt serine ⁄ threonine kinase by generating
specific inositol phospholipids, which recruit Akt to
the cell membrane and enable its activation [11]. Akt
mediates cell survival and growth signals by phos-
phorylating and inactivating proapoptic proteins [15].
Moreover, growth retardation has been reported in
knockout mice for Akt1 gene, which demonstrates that

Akt is important for normal growth [16]. In the pre-
sent study, we also found that the PI3K ⁄ Akt pathway
was activated by cartducin stimulation, and inhibition
of this signaling pathway by a specific PI3K inhibitor,
LY294002, blocked the cartducin-stimulated pro-
liferation of N1511 cells. The data suggest that the
PI3K ⁄ Akt pathway is also involved in cartducin-stimu-
lated proliferation of mesenchymal chondroprogenitor
cells.
Our results indicated that cartducin initiates
MAPK and PI3K signaling cascades in mesenchymal
chondroprogenitor cells, leading to the phosphoryla-
tion of ERK1 ⁄ 2 and Akt, respectively, and both
pathways are required for the cells to complete pro-
gression through the mitotic cycle. This finding is
consistent with the well-established roles of these
signaling pathways in the propagation of mitogenic
signals [9,15]. Although Akt has been thought to
play a major role, mainly in cell survival, recent data
suggest that PI3K activation is required for the pro-
gression of mitosis, promoting the entry of quiescent
cells into the S phase, and the downstream phos-
phorylation of Akt is in part responsible for the
propagation of this signal [17]. In particular, the
mitogenic response to platelet-derived growth factor
involves the sequential activation of the MAPK and
PI3K pathways, and these different phases of signa-
ling are a general requirement in mitogenic signa-
ling [18]. The inhibition of the mitogenic response to
cartducin by the specific PI3K inhibitor, LY294002,

is consistent with this role for PI3K in mitogenesis.
In conclusion, we have demonstrated that cartdu-
cin, a novel C1q family member, is a peripheral skel-
etal growth factor, and mitogenic response to
cartducin by mesenchymal chondroprogenitor cells
requires the activation of both the ERK1 ⁄ 2 and
PI3K ⁄ Akt pathways.
H. Akiyama et al. Cartducin activates MAPK and PI3K ⁄ Akt
FEBS Journal 273 (2006) 2257–2263 ª 2006 The Authors Journal compilation ª 2006 FEBS 2261
Experimental procedures
Reagents
Mouse recombinant cartducin was prepared as described
[7]. Anti-mouse cartducin (also called CORS26) serum was
purchased from R&D Systems (Minneapolis, MN). Anti-
(ERK1 ⁄ 2), anti-(JNK1 ⁄ 2), and anti-(p38 MAPK) sera were
purchased from Sigma-Aldrich (St. Louis, MO). Anti-
(p-ERK1 ⁄ 2), anti-(p-JNK1 ⁄ 2), anti-(p-p38 MAPK), anti-
Akt, and anti-(p-Akt) sera were purchased from Cell
Signaling Technology, Inc. (Beverly, MA). MEK1 ⁄ 2 inhi-
bitor U0126, JNK inhibitor SP600125, p38 MAPK inhi-
bitor SB203580, and PI3K inhibitor LY294002 were
purchased from Calbiochem (San Diego, CA).
Cell lines and cell culture
The mouse mesenchymal chondroprogenitor cell line
N1511 [12] was cultured in alpha-minimal essential med-
ium (a-MEM; Sigma-Aldrich) supplemented with 10%
fetal bovine serum (FBS; PAA Laboratories, Linz, Aus-
tria), 2 mml-glutamine, and penicillin ⁄ streptomycin at
37 °C in a humidified atmosphere containing 5% CO
2

.
To investigate the effects of cartducin on the MAPK or
PI3K ⁄ Akt signaling pathways, cells were seeded at a den-
sity of 1 · 10
4
cells per well in 24-well plates and grown
for 48 h. The cells were then washed and cultured for
24 h in the medium without serum. Subsequently,
10 lgÆmL
)1
of recombinant cartducin was added to the
medium for 5, 15, 30, and 60 min. In another set of
experiments, cells were treated with an increasing doses
of recombinant cartducin (2–10 lgÆmL
)1
) for 30 min. For
experiments with protein kinase inhibitors, cells were pre-
treated with specific inhibitors for 1 h prior to cartducin
treatment.
Western blot analysis
Total cellular protein was prepared by lysing cells in Cel-
Lytic lysis buffer (Sigma-Aldrich) containing protease
inhibitor (Sigma-Aldrich) and phosphatase inhibitor
(Sigma-Aldrich) cocktails. Total tissue protein was extrac-
ted from rib growth plate cartilage from 10-day-old mice.
The proteins (20 lg) were separated by SDS ⁄ PAGE (10%
polyacrylamide gel) and transferred to a poly(vinylidene
difluoride) membrane (Bio-Rad, Hercules, CA) as described
previously [19]. Membranes were blocked for 30 min
at room temperature and then incubated with anti

p-(ERK1 ⁄ 2), anti-(p-JNK1 ⁄ 2), anti-(p-p38 MAPK), or
anti-(p-Akt) sera for 18 h at 4 °C. In other cases, the
membranes were incubated with first antibodies for 1 h at
room temperature. The detection of bound antibodies
was performed by the WesternBreeze Chromogenic Detect-
ion System (Invitrogen, Carlsbad, CA) using alkaline
phosphatase-conjugated donkey anti-goat IgG, or anti-
rabbit IgG serum (Promega, Madison WI).
Measurement of DNA synthesis
To determine the growth-stimulatory effect of cartducin on
mesenchymal chondroprogenitor cells, a BrdU assay was
performed as described previously [7]. In brief, N1511 cells
were seeded at a density of 1 · 10
4
cells per well in 96-well
plates and grown for 24 h. The cells were then washed and
the medium was replaced with a-MEM containing 0.3%
FBS for 24 h. Subsequently, 8 lgÆmL
)1
of recombinant
cartducin was added to the medium, incubated for 24 h,
and labeled with BrdU for the last 3 h of incubation. To
determine the effects of inhibitors on the cartducin-induced
proliferation of N1511 cells, cells were treated with different
inhibitors for 1 h before and for the duration of the stimu-
lation with cartducin. In the control experiments, 50 mm
NaH
2
PO
4

(pH 8.0) containing 1 mm EDTA and ⁄ or dimeth-
ylsulfoxide was added to the culture.
Statistical analysis
An unpaired Student’s t-test was used for statistical analysis
of the experiments. Error bars represent SD, and P < 0.05
was taken as the level of significance.
Acknowledgements
This work was supported by Grants-in-aid for Scienti-
fic Research (No. 16791142) and the 21st Century
Center of Excellence Program from the Ministry of
Education, Culture, Sports, Science and Technology of
Japan.
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