Role of CCN2 in Amino Acid Metabolism of Chondrocytes Yurika Murase,1,2 Takako Hattori,1 Eriko Aoyama,3 Takashi Nishida,1 Aya Maeda-Uematsu,1 Harumi Kawaki,1 Karen M. Lyons,4 Akira Sasaki,2 Masaharu Takigawa,1,3* and Satoshi Kubota1,3* 1Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan 2Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan 3Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama, Japan 4Department of Orthopaedic Surgery, UCLA School of Medicine, Los Angeles, California ABSTRACT CCN2/connective tissue growth factor (CTGF) is a multi-functional molecule that promotes harmonized development and regeneration of cartilage through its matricellular interaction with a variety of extracellular biomolecules. Thus, deficiency in CCN2 supply profoundly affects a variety of cellular activities including basic metabolism. A previous study showed that the expression of a number of ribosomal protein genes was markedly enhanced in Ccn2-null chondrocytes. Therefore, in this study, we analyzed the impact of CCN2 on amino acid and protein metabolism in chondrocytes. Comparative metabolome analysis of the amino acids in Ccn2-null and wild-type mouse chondrocytes revealed stable decreases in the cellular levels of all of the essential amino acids. Unexpectedly, uptake of such amino acids was rather enhanced in Ccn2-null chondrocytes, and the addition of exogenous CCN2 to human chondrocytic cells resulted in decreased amino acid uptake. However, as expected, amino acid consumption by protein synthesis was also accelerated in Ccn2-null chondrocytes. Furthermore, we newly found that expression of two genes encoding two glycolytic enzymes, as well as the previously reported Eno1 gene, was repressed in those cells. Considering the impaired glycolysis and retained mitochondrial membrane potential in Ccn2-null chondrocytes, these findings suggest that Ccn2 deficiency induces amino acid shortage in chondrocytes by accelerated amino acid consumption through protein synthesis and acquisition of aerobic energy. Interestingly, CCN2 was found to capture such free amino acids in vitro. Under physiological conditions, CCN2 may be regulating the levels of free amino acids in the extracellular matrix of cartilage. J. Cell. Biochem. 117: 927–937, 2016. © 2015 Wiley Periodicals, Inc. KEY WORDS: CCN2; CTGF; CARTILAGE; CHONDROCYTES; METABOLISM The CCN family is a group of matricellular proteins with aconserved molecular structure and unique functionality that integrates extracellular signaling networks. This family name was given in 1993, by assembling the initials of the original names of its three classical members: cysteine-rich protein 61 (CYR61), connective tissue growth factor (CTGF), and nephroblastoma-overexpres.

1. Role of CCN2 in Amino Acid Metabolism of Chondrocytes
Yurika Murase,1,2 Takako Hattori,1 Eriko Aoyama,3 Takashi
Nishida,1
Aya Maeda-Uematsu,1 Harumi Kawaki,1 Karen M. Lyons,4
Akira Sasaki,2
Masaharu Takigawa,1,3* and Satoshi Kubota1,3*
1Department of Biochemistry and Molecular Dentistry,
Okayama University Graduate School of Medicine, Dentistry
and Pharmaceutical Sciences, Okayama, Japan
2Department of Oral and Maxillofacial Surgery, Okayama
University Graduate School of Medicine, Dentistry and
Pharmaceutical Sciences, Okayama, Japan
3Advanced Research Center for Oral and Craniofacial Sciences,
Okayama University Dental School, Okayama, Japan
4Department of Orthopaedic Surgery, UCLA School of
Medicine, Los Angeles, California
ABSTRACT
CCN2/connective tissue growth factor (CTGF) is a multi-
functional molecule that promotes harmonized development and
regeneration of
cartilage through its matricellular interaction with a variety of
extracellular biomolecules. Thus, deficiency in CCN2 supply
profoundly affects
a variety of cellular activities including basic metabolism. A
previous study showed that the expression of a number of
ribosomal protein genes
was markedly enhanced in Ccn2-null chondrocytes. Therefore,

2. in this study, we analyzed the impact of CCN2 on amino acid
and protein
metabolism in chondrocytes. Comparative metabolome analysis
of the amino acids in Ccn2-null and wild-type mouse
chondrocytes revealed
stable decreases in the cellular levels of all of the essential
amino acids. Unexpectedly, uptake of such amino acids was
rather enhanced in
Ccn2-null chondrocytes, and the addition of exogenous CCN2 to
human chondrocytic cells resulted in decreased amino acid
uptake. However,
as expected, amino acid consumption by protein synthesis was
also accelerated in Ccn2-null chondrocytes. Furthermore, we
newly found that
expression of two genes encoding two glycolytic enzymes, as
well as the previously reported Eno1 gene, was repressed in
those cells.
Considering the impaired glycolysis and retained mitochondrial
membrane potential in Ccn2-null chondrocytes, these findings
suggest that
Ccn2 deficiency induces amino acid shortage in chondrocytes
by accelerated amino acid consumption through protein
synthesis and
acquisition of aerobic energy. Interestingly, CCN2 was found to
capture such free amino acids in vitro. Under physiological
conditions, CCN2
may be regulating the levels of free amino acids in the
extracellular matrix of cartilage. J. Cell. Biochem. 117: 927–
937, 2016.
© 2015 Wiley Periodicals, Inc.
KEY WORDS: CCN2; CTGF; CARTILAGE;
CHONDROCYTES; METABOLISM
The CCN family is a group of matricellular proteins with
aconserved molecular structure and unique functionality that

3. integrates extracellular signaling networks. This family name
was
given in 1993, by assembling the initials of the original names
of its
three classical members: cysteine-rich protein 61 (CYR61),
connective
tissue growth factor (CTGF), and nephroblastoma-
overexpressed (NOV)
gene product [Bork, 1993]. After the establishment of this
family, three
additional members were discovered by independent research
groups
and became widely known as Wnt-inducible secretory proteins
(WISP)-
1, -2, and -3 [Leask and Abraham, 2006; Jun and Lau, 2011;
Kubota
and Takigawa, 2012]. For these six family members, new names
CCN1-
6 were assigned in order, based on the unified nomenclature
[Brigstock
et al., 2003]. All of the members consist of four conserved
modules:
insulin-like growth factor binding protein (IGFBP) module, von
Willebrand factor type C repeat (VWC) module,
thrombospondin
type 1 repeat (TSP1) module, and carboxyl-terminal (CT)
module. Only
this last one is absent in CCN5. Every module is highly
interactive with
a variety of molecular counterparts. Generally, by interacting
with such
cofactors as cell-surface receptors and extracellular signaling
molecules, the CCN family proteins play a role in the regulation
of

4. The authors have no conflicts of interest to declare.
Grant sponsor: Japan Society for the Promotion of Science;
Grant numbers: 25462886, 24390415, 15H0514.
*Correspondence to: Dr. Satoshi Kubota and Dr. Masaharu
Takigawa, Department of Biochemistry and Molecular
Dentistry, Okayama University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences, and Advanced
Research Center for Oral and Craniofacial Sciences, Okayama
University Dental School, 2-5-1 Shikata-cho, Okayama
700-8525, Japan.
E-mail: [email protected] (S.K.), [email protected] (M.T.)
Manuscript Received: 28 May 2015; Manuscript Accepted: 10
September 2015
Accepted manuscript online in Wiley Online Library
(wileyonlinelibrary.com): 14 September 2015
DOI 10.1002/jcb.25377 � © 2015 Wiley Periodicals, Inc. 927
ARTICLE
Journal of Cellular Biochemistry 117:927–937 (2016)
various cellular activities, such as cell-cycle control, adhesion,
migration, and extracellular matrix (ECM) remodeling [Perbal
and
Takigawa, 2005; Kubota and Takigawa, 2007, 2015].
Among the six family members, CCN2 has been the best
investigated upto today, and a number of reports revealed the
multiple functionality of CCN2 in a variety of tissues and
organs,
under either physiological or pathological biological conditions.
During normal animal development, CCN2 plays a critical role
in the
proper development of the skeleton, central nervous system,
pancreas, hair, and teeth [Charrier and Brigstock, 2013; Kubota

5. and Takigawa, 2015]. On the other hand, during abnormal tissue
remodeling, CCN2 not only mediates the development of
fibrotic
disorders [Leask and Abraham, 2006] but also modulates the
development of tumors in a variety of tissues and organs [Jun
and
Lau, 2011; Kubota and Takigawa, 2013]. In most cases, with the
exception being ovarian, oral, and lung cancers, CCN2 is
observed to
promote the development of tumors including breast cancer,
prostate cancer, glioma, pancreatic cancer, colon cancer, thyroid
carcinoma, chondrosarcoma, gallbladder carcinoma, melanoma,
and leukemia. These effects of CCN2 can be principally
ascribed to
the angiogenic property of the molecule [Babic et al., 1999;
Shimo
et al., 1999, 2001; Kubota and Takigawa, 2007].
In relation to skeletal development, the role of CCN2 in
endochondral ossification, which determines the size of long
bones,
is of particular note. CCN2, predominantly produced by pre-
hypertrophic chondrocytes, promotes all of the processes
involved
by encouraging the participation of a variety of the cells that
operate in endochondral ossification [Babic et al., 1999; Shimo
et al.,
1999; Nakanishi et al., 2000; Nishida et al., 2000; Safadi et al.,
2003;
Smerdel-Ramoya et al., 2008; Kawata et al., 2012; Takigawa,
2013;
Kubota and Takigawa, 2015]. In fact, CCN2-deficient cartilage
exhibits abnormal organization of growth plate chondrocytes
with
delayed ossification, which results in remarkable skeletal
defects

6. [Ivkovic et al., 2003; Kawaki et al., 2008; Takigawa, 2013]. The
cartilage-regenerating activity of CCN2 and its derivatives is
also of
note [Nishida et al., 2004; Abd El Kader et al., 2014].
In a recent study, an interesting functional aspect of CCN2 was
uncovered by metabolomic and transcriptomic investigation of
Ccn2-null chondrocytes. According to the results of that study,
Ccn2-null chondrocytes revealed a stable reduction in their
cellular ATP level [Maeda-Uematsu et al., 2014]. The results of
both microarray and quantitative RNA analyses showed down-
regulation of a gene encoding one of the glycolytic enzymes,
Eno1,
in response to Ccn2 deletion. Therefore, it has been suggested
that
CCN2 plays an important role in endochondral ossification by
enhancing ATP production, where it is required, by enhancing
the
gene expression of Eno1. Microarray analysis also picked up a
number of genes that are rather up-regulated by Ccn2 deletion.
Of
note, among these genes were a number of ribosomal protein
genes
highly suspected of affecting the protein synthesis and amino
acid
metabolism in Ccn2-null chondrocytes. However, no further
investigation to clarify the role of CCN2 therein was conducted
at that time.
Based on this previous finding, here we investigated the
functional impact of CCN2 on amino acid metabolism, mainly
utilizing Ccn2-null chondrocytes. Taken together with
subsequent
analysis in vitro, a novel role of CCN2 in amino acid
metabolism in

7. cartilage was indicated.
MATERIALS AND METHODS
CELL CULTURE
Primary costal and epiphyseal chondrocytes were isolated from
rib
and epiphyseal cartilage of Ccn2-null mice and wild-type
littermates
at E17.5, 18.5, or E19.5, following an established protocol as
previously described [Kawaki et al., 2008; Hattori et al., 2010].
Briefly, after careful elimination of soft tissues by digestion
with
0.25% trypsin for 5 min at 37°C, the cartilage was digested with
1.5 mg/ml collagenase A (Roche, Basel, Switzerland) for 2 or 3
h at
37°C to liberate the chondrocytes. Isolation of the cells was
performed according to the Guidelines for Animal Research of
Okayama University and was approved by the animal
committee.
These cells were then inoculated at a density of 1.5 � 105
cells/dish
into 3.5 cm dishes or 3 � 104 cells/well into 24-well multiwell
plates
containing Dulbecco0s modified Eagle0s medium (DMEM)
supple-
mented with 10% fetal bovine serum (FBS) and then incubated
at
37°C under 5% CO2 in air. Cells of the human chondrosarcoma-
derived cell line HCS-2/8 [Takigawa et al., 1989] were
inoculated at a
density of 5 � 105 cells/dish into 3.5 cm dishes containing
DMEM
supplemented with 10% FBS, followed by incubation at 37°C
under
5% CO2 in air.

8. MICROARRAY ANALYSIS
Comparative transcriptomic analysis was performed by using a
mouse Panorama Micro Array (Sigma–Aldrich, St. Louis, MO)
following the manufacturer0s instruction. Total RNA was
extracted
from eight individual mouse embryos from four different litters,
as
previously described [Maeda-Uematsu et al., 2014], and the
RNA
mixture was subjected to labeling. Signals were quantified and
analyzed by use of a GenePix 4000B (Molecular Devices,
Sunnyvale, CA).
METABOLOME ANALYSIS
Extraction of total metabolites from murine chondrocytes was
performed by the method recommended by Human Metabolome
Technologies (Tsuruoka, Japan), as described previously
[Maeda-
Uematsu et al., 2014]. Briefly, chondrocytes isolated from
mouse
cartilage were inoculated into a 6-well multiwell plate at a
density
of 3.6 � 105 cells/well and cultured to confluence. Then, the
cells
were washed with 5% mannitol solution (Wako, Osaka, Japan),
after which methanol containing 10 mM Internal Standard
Solution
(Human Metabolome Technologies) was added; and
then the cells were collected. Prior to the analysis, these

9. samples
were mixed with chloroform and water and centrifuged at
5,000�g
for 5 min at 4°C. After the removal of proteins by filtration (5
kDa,
Millipore, Billerica, MA), the samples were subjected to
cationic
and anionic metabolite analysis with a capillary electrophoresis
time-of-flight mass spectrometer (CE-TOFMS; Agilent CE-
TOFMS
system, Agilent Technologies Japan, Ltd., Tokyo, Japan).
Cationic
metabolites were analyzed by using a fused silica capillary (i.d.
50 mm � 80 cm), with Cation Buffer