Signaling responses of osteoblast cells to hydroxyapatite: the activation of ERK and SOX9.

Abstract

The intracellular signaling cascade triggered as a result of the surface chemistry of the bone mineral hydroxyapatite (HA) remains largely unknown. In this study, we found that the ERK signaling molecule is activated in response to HA. Moreover, we have performed the first systemic analysis of expression profiles using microarray technology. Eleven genes, including those involved in calcium regulation and bone matrix formation, showed a greater than 2.0-fold change in expression level in response to HA. Among those genes upregulated by HA was the gene encoding SOX9, whose expression we confirmed by real-time PCR analysis with a 5.7-fold increase in expression. Taken together, our results suggest that the activation of ERK and SOX9 by HA could have important implications for understanding the mechanisms by which cells respond on a molecular level to HA.

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ORIGINAL ARTICLE
J Bone Miner Metab (2008) 26:138–142
DOI 10.1007/s00774-007-0804-6
© Springer 2008
J.-H. Song · H.-E. Kim
School of Materials Science and Engineering, Seoul National
University, Seoul, Korea
J.-H. Kim · S. Park · W. Kang · J.-H. Jang (*)
Department of Biochemistry and BK21 Center for Advanced
Medical Education, Inha University School of Medicine, Incheon
400-712, Korea
Tel. +82-32-890-0933; Fax +82-32-882-1877
e-mail: juhjang@inha.ac.kr
H.-W. Kim
Department of Biomaterial Science, Dankook University School of
Dentistry, Cheonan, Korea
Ju-Ha Song · Ji-Hyun Kim · Soonok Park · Wonmo Kang
Hae-Won Kim · Hyoun-Ee Kim · Jun-Hyeog Jang
Signaling responses of osteoblast cells to hydroxyapatite: the activation of
ERK and SOX9
Abstract The intracellular signaling cascade triggered as a
result of the surface chemistry of the bone mineral hydroxy-
apatite (HA) remains largely unknown. In this study, we
found that the ERK signaling molecule is activated in
response to HA. Moreover, we have performed the fi rst
systemic analysis of expression profi les using microarray
technology. Eleven genes, including those involved in
calcium regulation and bone matrix formation, showed a
greater than 2.0-fold change in expression level in response
to HA. Among those genes upregulated by HA was the
gene encoding SOX9, whose expression we confi rmed by
real-time PCR analysis with a 5.7-fold increase in expres-
sion. Taken together, our results suggest that the activation
of ERK and SOX9 by HA could have important implica-
tions for understanding the mechanisms by which cells
respond on a molecular level to HA.
Key words cellular signaling · ERK · hydroxyapatite ·
microarray · osteoblast
Introduction
Bone is a mineralized tissue that confers multiple mechani-
cal and metabolic functions to the body [1]. The majority of
bone consists of extracellular matrix proteins and the
mineral hydroxyapatite (HA). Although HA has been well
characterized chemically and mechanically, little is known
about how cells respond on a molecular level to HA. Inter-
actions of bone cells with HA surfaces are mediated by
adhesion receptors belonging to the integrin superfamily
that recognize binding domains within proteins of the
extracellular matrix (ECM). Integrin-mediated adhesion to
extracellular proteins activates multiple cytoskeletal-
associated and intracellular signaling proteins, such as focal
adhesion kinase (FAK). FAK associates with Shc protein
activating Ras, which leads to the stimulation of the extra-
cellular regulated kinases (ERK) signaling cascade [2]. The
activation of the ERK cascade leads to the expression of
several genes expressed by osteoblasts such as osteocalcin
and type I collagen [3,4]. Thus, ERK plays an important
role in cytoskeletal-associated and intracellular signaling
[5–7]. In a previous study, we reported that activation of
ERK is involved in the fi bronectin-mediated cell adhesion
signaling pathway of osteoblasts [8]. In this study, we found
that the ERK is activated in response to HA.
Although ERK is one of the important signaling mole-
cules involved in osteoblastic responses, the intracellular
signaling cascade that is triggered as a result of the surface
chemistry of HA remains largely unknown. Therefore, in
this study, we chose to investigate the cell’s global pattern
of gene expression involved in osteoblastic responses to
HA using cDNA microarray technology. Moreover, this
approach could lead to the identifi cation of so far unidenti-
fi ed osteoblast-specifi c genes and novel key regulators of
this process. Here we have explored the genome-wide gene
expression profi le of osteoblast cells in response to HA for
the fi rst time.
Methods
HA preparation
Hydroxyapatite [Ca10(PO4)6(OH)2; Alfa Aesar, Ward Hill,
MA, USA] powders were pressed in a metallic mold with a
diameter of 2.5 cm and cold-isostatically pressed at 150 MPa.
The disks of HA green bodies were sintered at 1250°C for
Received: April 30, 2007 / Accepted: August 10, 2007

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2 h. The specimens were formed into disks 19–20 mm in
diameter and 1–1.2 mm in height. For cell culture, all the
specimens were immersed in 100% ethanol for 24 h and
then dried.
Cell culture
MC3T3-E1 preosteoblastic cells were cultured in α-
minimum essential medium (Invitrogen) containing 10%
heat-inactivated fetal calf serum (Invitrogen), 100 units/ml
penicillin G sodium, 100 µg/ml streptomycin sulfate, and
0.25 µg/ml amphotericin B (Invitrogen) in a 5% CO2 atmo-
sphere at 37°C.
Western blot analysis
Activation of ERK was determined by using antiphospho-
tyrosine antibody (Santa Cruz). Proteins binding to the
primary antibodies were detected with a horseradish
peroxidase-conjugated secondary antibody (Santa Cruz);
positive bands were detected using chemiluminescence
detection according to protocols supplied by the manufac-
turer (Pierce).
Microarray analysis
MC3T3-E1 cells were seeded on HA in six-well plates at a
concentration of 2 × 104 cells/well and were harvested after
24 h. RNA was extracted from the cells by using an RNeasy
Mini kit (Qiagen, Chatsworth, CA, USA), and 100 µg total
RNA was used for each sample. We used a cDNA microar-
ray Mac Array-II 10 K chip (Macrogen, Seoul, Korea), as
described on the website maintained by the company (http://
www.macrogen.com), and monitored the expression of
10 108 genes with good reproducibility (70% of the gene
tested had a gene expression variation lower than 10%)
(data not shown). Genepix software was used to analyze the
data. The gene expression of cells on untreated six-well
plates was used as the reference. After removing local back-
ground, genes with expression levels of more than twofold
were the basis of the analysis because expression less than
twofold was not reliable at that detection level.
Real-time PCR assay
First-strand cDNA was synthesized from total RNA (2 µg)
using the SuperScript fi rst-strand synthesis system for
reverse transcriptase-polymerase chain reaction (RT-PCR)
(Invitrogen) according to the manufacturer’s instructions.
Volume of the reaction mixture was made up to 50 µl. Real-
time quantitative PCR was performed using SYBR
GreenER qPCR SuperMix reagents (Invitrogen) and a Bio-
Rad iCycler. Relative transcript quantities were calculated
using the ∆∆Ct method with β-actin as the endogenous ref-
erence gene amplifi ed from the samples. Primer sequences
were as follows: SOX9 forward: 5′-AGGAAGCTGGCA
GACCAGTA,
AAC; β-actin forward: 5′-GGAAATCGTGCGTGACAT
TA, reverse: 5′-GGAGCAATGATCTTGATCTTC.
reverse: 5′-CCCTCTCGCTTCAGATC
Results
Activation of the ERK signaling pathway by HA
Immunoreactivity of phosphorylated ERK is shown in Fig.
1. The levels of phosphorylated ERK were found to increase
after seeding on HA, which was sustained up to 1 h. It is
known that ERK has been shown to be involved in the
proliferative response of osteoblasts. Thus, our Western
blotting results showed that the ERK signaling molecule is
involved in the HA-induced signaling pathway.
Signifi cant differentially expressed genes in response
to HA
To further investigate the signaling events elicited by
HA, we performed systemic analysis of expression profi les
using microarray technology, providing primary informa-
tion on transcriptional changes in response to the HA
surface.
Of the 10 108 genes examined, 11 were upregulated and
6 were downregulated at least 2-fold by HA (Fig. 2). Those
Fig. 1. Extracellular regulated kinase (ERK) activation by hydroxy-
apatite (HA) shown by Western blot analysis for phosphorylated ERK
in MC3T3-E1 osteoblastic cells seeded on an HA disk for 0, 15, 30, 45,
or 60 min

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microarray data, which revealed a 4.4-fold increase in
SOX9 expression. Haploinsuffi ciency of SOX9 has been
shown to lead to defective cartilage primordia and prema-
ture mineralization in cranial bones. A possible direct role
of SOX9 on both bone and cartilage formation has been
suggested by other investigators [9]. Therefore, it is not
surprising that the microarray experiment reveals that
SOX9 is upregulated by HA. However, the molecular
mechanism of SOX9 regulating signaling pathways in bone
needs further study.
Fig. 2. Scatter plot of the 10 108 genes from MC3T3-E1 osteoblastic
cells relative to those seeded on HA (MC3T3-HA). cDNA from
MC3T3-E1 scatter data were normalized by modifi ed intensity depen-
dence normalization (print-tip normalization)
Table 1. The differentially expressed genes in MC3T3 osteoblastic cells seeded on hydroxyapa-
tite (HA)
Gene SymbolFold change
Ca2+ metabolism
Pancreatic stone protein
Guanylate cyclase activator 1B
Extracellular matrix proteins
Alpha-1,4-N-acetylglucosaminyltransferase
Fibulin
Hyaluroran and proteoglycan linkprotein 2
Signaling/transcription
SOX9
4.5 LIM domains 3
NGFI-A binding protein (EGR1-binding protein)
Ras-related GTP-binding protein
Others
Mucin 5
Phosphoserine phosphatase
Decreased
Testis-specifi c leucine-rich repeat protein
Opioid growth factor receptor
Angiotensin II receptor
Hedgehog interacting protein
Calpain 9
3-Phosphoinositide-dependent protein kinase-1
REG1A
GUGA1B
4.003
3.230
A4GNT
FBLN1
HAPLN2
2.953
2.673
2.343
SOX9
FHL3
NAB1
RRAGA
4.418
3.912
3.224
2.927
MUC5B
PSPH
5.278
3.905
TSLRP
OGFR
AGTR2
−7.101
−3.973
−3.855
−3.592
−3.073
−2.579
CAPN9
PI3KS
NGF, nerve growth factor; EGR, epidermal growth receptor; GTP, guanosine triphosphate
genes with an established function and at least a 2-fold
change over the control are listed in Table 1. Among those
genes upregulated by HA, a particularly interesting gene
was SOX9, whose expression we confi rmed by real-time
PCR analysis. SOX9 is a transcription factor that is essen-
tial for chondrocyte differentiation and cartilage formation.
Real-time PCR analysis verifi ed that SOX9 is indeed the
highly upregulated gene in response to HA, with an
approximate 5.7-fold increase in expression relative to
control cells (Fig. 3). This result is consistent with our
Fig. 3. Quantitative real-time reverse transcriptase-polymerase chain
reaction (qRT-PCR) analysis of SOX9 gene expression. Primers and
PCR conditions used are described under Materials and methods. Data
(mean ± SE) are from at least three separate sets of cells and were
normalized relative to β-actin and calculated by the 2∆∆Ct method

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Discussion
In the present study, we have explored intracellular signal-
ing molecules to investigate how osteoblast cells respond to
HA. Our study was the fi rst study to performed systemic
analysis of expression profi les, revealing that 11 genes
including those involved in calcium regulation and bone
matrix formation were at least 2-fold upregulated while 6
genes were 2-fold downregulated in response to HA.
Although the effects of HA on bone formation have been
investigated in detail, few target genes have been identifi ed
so far. Here we report several genes whose expression was
signifi cantly modulated by the HA. Among those genes
upregulated by HA was SOX9, whose mRNA expression
we confi rmed by real-time PCR analysis with a 5.7-fold
increase in expression. Interestingly, we found that the
ERK signaling molecule is activated in response to HA.
ERK has been shown to be involved in the regulation of
SOX9 expression for chondrocyte differentiation [10,11].
Taken together, these results strongly suggest that the
increase of SOX9 expression was mediated by the ERK
pathway. Despite the increase in SOX9, we did not observe
an increase in chondrocyte-specifi c marker Col2a1 mRNA
levels (data not shown).
Calcium-related proteins (pancreatic stone protein and
guanylate cyclase activator), extracellular matrix proteins
(alpha-1,4-N-acetylglucosaminyltransferase, fi bulin, hyal-
uroran, and proteoglycan link protein 2), and genes involved
in cell signaling (SOX9, FHL3, NGFI-A binding protein,
and Ras-related GTP-binding protein) were identifi ed as
HA-inducible genes. The HA effect on cellular Ca2+ regula-
tion has not been previously well studied. The microarray
results reveal that HA may modify several other signaling
pathways related with Ca2+. For example, REG1A, a pan-
creatic glycoprotein, was increased by HA. This pancreatic
stone protein has been reported to be involved in pancreatic
stone formation [12]. Guanylate cyclase activator proteins
(GCAP1 and GCAP2), which are expressed by the guanyl-
ate cyclase activator 1B (GUCA1B), are calcium-binding
proteins [13]. Taken together, these observations suggest
that the signals from HA may modulates several genes
involved in Ca2+ regulation.
ECM proteins also play important roles in bone forma-
tion [14]. Our microarray experiments suggest that several
genes related with ECM were upregulated by HA. HAPLN2,
one of the vertebrate hyaluronan and proteoglycan binding
link protein gene family, was upregulated by HA, refl ecting
the fundamental importance of the hyaluronan-dependent
extracellular matrix to tissue architecture and function in
osteoblast cells [15]. Fibulin-1 (FBLN1) is an extracellular
matrix protein often associated with fi bronectin in vivo,
modulating the adhesion, spreading, and motility-promot-
ing activities of fi bronectin [16].
Overall, the microarray data verify that HA-inducible
genes are involved in bone formation. To the best of our
knowledge, we have performed the fi rst systemic analysis
of expression profi les, revealing a list of 17 known genes
that are regulated by HA with at least twofold expression
changes in osteoblastic cells. These genes are involved in
many signal transduction pathways related with calcium
regulation and bone matrix formation, suggesting that HA-
inducible genes may play an important role in the osteocon-
ductivity of HA.
In conclusion, the present study has demonstrated that
the activation of ERK and SOX9 by HA could have impor-
tant implications for understanding the mechanisms by
which cells respond on a molecular level to HA.
Acknowledgments This work was supported by the Korea Research
Foundation Grant funded by the Korean Government (MOEHRD)
(KRF-2005-042-D00155), and the Korea Science and Engineering
Foundation (KOSEF) grant funded by the Korea government (MOST)
(R01-2007-000-20183-0).
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