0163-769X/00/$03.00/0
Endocrine Reviews 21(4): 393– 411
Copyright © 2000 by The Endocrine Society
Printed in U.S.A.
Regulation of Osteoblast Differentiation Mediated by
Bone Morphogenetic Proteins, Hedgehogs, and Cbfa1
AKIRA YAMAGUCHI, TOSHIHISA KOMORI,
AND
TATSUO SUDA
Department of Oral Pathology (A.Y.), Nagasaki University School of Dentistry, 1–7-1 Sakamoto,
Nagasaki 852, Department of Molecular Medicine (T.K.), School of Medicine, Osaka University, 2–2
Yamada-oka, Suita, Osaka 565 and “Form and Function”, PRESTO, Japan Science and Technology
Corporation, and Department of Biochemistry (T.S.), School of Dentistry, Showa University, 1–5-8
Hatanodai, Shinagawa-ku, Tokyo 142, Japan
ABSTRACT
Osteoblasts arise from common progenitors with chondrocytes,
muscle and adipocytes, and various hormones and local factors regulate their differentiation. We review here regulation of osteoblast
differentiation mediated by the local factors such as bone morphogenetic proteins (BMPs) and hedgehogs and the transcription factor,
core-binding factor ␣-1 (Cbfa1). BMPs are the most potent regulators
of osteoblast differentiation among the local factors. Sonic and Indian
hedgehogs are involved in osteoblast differentiation by interacting
with BMPs. Cbfa1, a member of the runt domain gene family, plays
a major role in the processes of a determination of osteoblast cell
lineage and maturation of osteoblasts. Cbfa1 is an essential transcription factor for osteoblast differentiation and bone formation,
because Cbfa1-deficient mice completely lacked bone formation due to
maturation arrest of osteoblasts. Although the regulatory mechanism
of Cbfa1 expression has not been fully clarified, BMPs are an important local factor that up-regulates Cbfa1 expression. Thus, the intimate interaction between local factors such as BMPs and hedgehogs
and the transcription factor, Cbfa1, is important to osteoblast differentiation and bone formation. (Endocrine Reviews 21: 393– 411, 2000)
I. Introduction
II. Origin of Osteoblasts
III. Regulation of Osteoblast Differentiation by Bone Morphogenetic Proteins (BMPs) and Hedgehogs
A. BMPs
B. Sonic and Indian hedgehogs
IV. Transcription Factors That Regulate Osteoblast Differentiation and Bone Formation
A. Transcription factors involved in osteoblast differentiation
B. Cbfa1 is an important transcription factor regulating
osteoblast differentiation
C. Absence of ossification in Cbfa1-deficient mice
D. Role of Cbfa family transcription factors in osteoblast
differentiation
E. Cbfa1 is involved in chondrocyte maturation
F. Cbfa1 is involved in osteoclastogenesis
G. Heterozygous mutations of Cbfa1 locus cause cleidocranial dysplasia
V. Summary
phenotypes depending on their maturation during differentiation. Osteoblasts express various phenotypic markers such
as high alkaline phosphatase (ALP) activity and synthesize
collagenous and noncollagenous bone matrix proteins including osteocalcin (4). The most important function of osteoblasts is to form mineralized bones. Osteoblasts express
receptors for various hormones including PTH (8, 9), 1␣,25dihydroxyvitamin D3 [1␣,25(OH)2D3] (10), estrogen (11, 12),
and glucocorticoids (13, 14), which are involved in the regulation of osteoblast differentiation. Osteoblast differentiation is also regulated by various local factors in a paracrine
and/or an autocrine fashion (4). To investigate the roles of
these hormones and local factors in osteoblast differentiation,
various osteoblastic cell lines have been successfully established (15–17). It is also possible to use multipotent mesenchymal progenitors to examine the differentiation process of
osteoblasts in vitro (18, 19). Experiments using these in vitro
assay systems have yielded a great deal of information concerning local factors that regulate osteoblast differentiation.
Indeed, several research groups, including ourselves, using
various culture systems have demonstrated that bone morphogenetic proteins (BMPs) are potent local factors that regulate osteoblast differentiation (18 –22).
The hedgehog signaling pathway mediates inductive
events during development in invertebrates and vertebrates
(23). In higher vertebrates, the Hedgehog gene family consists
of at least three members, Sonic, Indian, and Desert hedgehog
(Shh, Ihh, and Dhh, respectively) (23, 24). Among these, Shh
and Ihh are involved in the skeletal formation during development (25–27) and in skeletal repair (27–29). In Drosophila,
hedgehog signaling induces expression of decapentaplegic
(dpp), which is a homolog of vertebrate BMP, in adjacent
cells, in which dpp acts as a secondary signaling molecule to
I. Introduction
T
HE skeletal tissue is composed of various types of mesenchymal cells such as osteoblasts, chondrocytes, myoblasts and bone marrow stromal cells including adipocytes.
These cell lineages are believed to originate from common
mesenchymal progenitors (1–7) called pluripotent mesenchymal stem cells (5–7). These progenitors acquire specific
Address reprint requests to: Akira Yamaguchi, D.D.S., Ph.D., Department of Oral Pathology, Nagasaki University School of Denistry,
1-7-1 Sakamoto, Nagasaki 852, Japan. E-mail: [email protected]
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control the fate of these cells (23). Similar interactions between hedgehogs and BMPs were also demonstrated in several organs during vertebrate development (30). Thus, the
hedgehog-BMP interaction is highly conserved in the patterning process of various organs including skeletons in
higher vertebrates.
Transcription factors that determine the differentiation
pathways of specific cell types have been identified in several
cell lineages. In the case of skeletal muscles, the musclespecific transcription factors of the MyoD family, which belong to the basic helix-loop-helix (HLH) family, are necessary
for determining the pathway of differentiation into the muscle lineage and are required for the differentiation of committed myoblasts to fully differentiated myotubes (31). In
addition, peroxisome proliferator-activated receptor ␥2
(PPAR␥2) has been reported to play an important role in
determining the differentiation pathway of adipocyte lineage
cells (32). The specific transcription factors that determine
osteoblast differentiation remained unclear. Recently, several research groups independently reported that Cbfa1
[Core binding factor ␣1, also called Pebp2␣A (Polyomavirus
enhancer binding protein 2 ␣A), AML3 (acute myelocytic
leukemia 3) and OSF2 (osteoblast specific factor 2)], which
belongs to the runt-domain gene family, is an important
transcription factor for osteoblast differentiation and bone
formation (33–36). Since it has been reported that BMP upregulates expression of Cbfa1 during osteoblast differentiation (33), Cbfa1 seems to be a downstream factor controlled
by BMP. In contrast to MyoD and PPAR␥2, Cbfa1 is necessary but not sufficient to support differentiation to the mature osteoblast phenotype (37).
Although osteoblast differentiation is regulated by many
factors, the three molecules described above, BMP, hedgehogs, and Cbfa1, play important roles in the differentiation
process with intimate interaction between them. In this article we review recent advances regarding the regulation of
osteoblast differentiation mediated by BMP, hedgehogs, and
Cbfa1.
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chondral ossification, mesenchymal cells first condense to
form a cartilage model, and then bone formation occurs
replacing this cartilage. This type of ossification forms most
of the bones including the axial and appendicular skeletons.
Several in vivo and in vitro experiments have demonstrated
the presence of osteoprogenitors in both bones and extraskeletal tissues in the postnatal state. In bone tissues, osteoprogenitors are present in bone marrow and the periosteum.
Friedenstein and colleagues (39 – 42) have proved that osteoprogenitors are present in bone marrow. They showed
that bone marrow cells harvested from confluent in vitro
cultures of marrow cells retained the ability to form osteogenic tissues when cultured in vivo within diffusion chambers. Then they demonstrated by various in vivo and in vitro
experiments that the single cell-derived fibroblastic colonies,
termed CFU-F (colony forming units-fibroblastic) (41), retained osteogenic potential (42). Other groups also demonstrated that bone marrow cells, including that harvested from
human marrow, contained mesenchymal progenitors, which
differentiated into osteogenic, chondrogenic, and adipogenic
lineage cells (4 –7, 43). Further characterization of human
mesenchymal stem cells is important to develop new therapeutic drugs for bone diseases such as osteoporosis. The
osteogenic potential of the periosteum was also shown by
several experiments. In vivo experiments using [3H]-thymidine as a tracer demonstrated that the cells located in the
outer layer of the periosteum differentiated into mature osteoblasts and osteocytes (44). Periosteum or periosteumderived cells generate bone nodules in in vitro cultures (45).
These osteoprogenitors in the periosteum contribute to formation of bone callus during fracture repair.
Transplantation of BMPs into muscle or subcutaneous
sites induces ectopic bone formation (46, 47), indicating that
osteoprogenitors, which respond to BMPs, are also present
at extraskeletal sites. These osteoprogenitors may have BMP
receptors, but other characteristics of these cells have not
been analyzed in detail. Further characterization of these
cells is important to develop effective cell therapy for bone
repair by transplantation of such extraskeletal osteoprogenitors after appropriate in vitro culture (48).
II. Origin of Osteoblasts
Several lines of evidence from classical embryology have
established that two different embryonic lineages, neural
crest and mesoderm, form the early skeleton (4, 38). The
branchial arch derivatives of the craniofacial skeleton originate from neural crest, whereas the axial skeleton, ribs, appendicular skeletons, and the skull base arise from mesoderm. Among the skeletal tissues formed by the mesoderm,
the axial skeleton originates from the sclerotome, and the
appendicular skeleton arises from the lateral plate mesoderm.
During skeletogenesis, bone is formed in two different
manners, intramembranous ossification and endochondral
ossification, regardless of the embryonic lineage. In the case
of intramembranous ossification, osteogenesis occurs directly in the condensed mesenchymal cells. Ossification generated in this fashion is responsible for forming the flat bones
of the skull, part of the clavicle, and the additional bone on
the periosteal surface of long bones. In the process of endo-
III. Regulation of Osteoblast Differentiation by BMPs
and Hedgehogs
Osteoblast differentiation and bone formation are regulated by many local factors. Among these, BMPs are one of
the most potent factors. Recent studies revealed that specific
signaling systems through receptors and the specific inhibitors such as noggin and chordin regulate BMP function at
the extracellular level. In addition, several lines of evidence
indicate that hedgehogs modulate BMP function during pattern formation including skeletal formation in vertebrates.
To investigate the roles of local factors involved in osteoblast differentiation and bone formation, it is important to
establish in vitro culture systems that reflect the different
stages of maturation during osteogenesis. Although many
cell lines are available for such investigations, we have used
several cell lines that are useful for investigating osteoblast
differentiation in vitro (Table 1). Using these cell lines, we can
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REGULATION OF OSTEOBLAST DIFFERENTIATION
395
TABLE 1. Characteristics of useful cultured cells for analyzing the differentiation pathway of osteoblasts in vitro
Name of cells
Origin
Characteristics
C3H10T1/2 clone 8
Early mouse embryo
ROB-C26
Newborn rat calvaria
MC3T3-E1
Calvaria of a late stage mouse embryo
ST2
Bone marrow of adult mouse
C2C12
A subclone isolated from parental C2 myoblasts,
which were established from the regenerating
thigh muscle of an adult mouse
Calvaria from late embryos or newborn rats
Calvaria-derived primary
osteoblastic cells
explore the roles of BMPs and hedgehogs that regulate osteoblast differentiation and bone formation. In this section,
we describe details of the roles of two important local factors,
BMPs and hedgehogs, in osteoblast differentiation.
A. BMPs
Urist (46) reported that implantation of decalcified bone
matrix into muscular tissues induced new ectopic bone formation associated with endochondral bone formation. He
named the factor contained in the decalcified bone matrix
BMP (49). A number of investigators attempted to isolate
BMP from the decalcified bone matrix (50 –52), but it was
very difficult to obtain BMP as a single protein until the late
1980s. In 1988, Wozney et al. (47) first cloned four cDNAs for
human BMPs [BMP-1, BMP-2A (BMP-2), BMP-2B (BMP-4)
and BMP-3]. At present, at least 15 BMPs have been cloned,
and with the exception of BMP-1, these belong to the transforming growth factor-␤ (TGF-␤) superfamily (53). Recombinant proteins of several BMPs with the ability to induce
ectopic bone formation in vivo have been successfully generated (20, 47, 54 –56). Several research groups, including
ourselves, have examined the effects of recombinant human
BMPs (rhBMPs) on the differentiation of skeletal mesenchymal cells using various types of cells in vitro. Recombinant
human BMPs are expected to be potent local factors that
promote bone formation in bone defects, fracture repair, and
periodontal diseases.
Since various members of the BMP family are expressed
during skeletogenesis, localization of such BMPs provides
important information to understand the role of each BMP in
skeletal development. For example, BMP-5 mRNA is localized to mesenchymal condensations before cartilage development (57), whereas mRNAs for BMP-2, BMP-4, and BMP-7
(OP-1:osteogenic protein 1) are present in the mesenchyme
surrounding cartilaginous anlage (53, 58). The expression of
mRNAs for these BMPs continue to be present at perichondrium and periosteum at later stages (53, 57, 58). Growth/
differentiation factor5 (GDF5), a member of the BMP family, is
weakly expressed at perichondrium, whereas its expression
is strong at the interface between cartilage anlages where
joints will later form (59). This expression pattern correlates
closely with joint patterning defects in GDF5 mutant (brachypodism) mice (59, 60). Skeletal analysis in null mutation mice
A pluripotent cell line, which can differentiate into
osteoblasts, chondrocytes, myotubes, and adipocytes
An osteoblast precursor cell line, which is capable of
differentiating into myotubes and adipocytes
A monopotential cell line, which can only differentiate
into osteoblast lineage cells
A bone marrow stromal cell line that can differentiate
into osteoblasts
A myogenic cell line with characteristics of satellite
cells. This cell line differentiates into osteoblasts on
treatment with BMPs
Primary culture of osteoblastic cells. These cells are a
mixed population of several cell types, but retain
various osteoblastic phenotypes including bone-nodule
formation
by targeted disruption of BMP genes will enable us to understand the role of each BMP in skeletogenesis. Although
studies using such mutant mice revealed important functions
of BMPs in mesodermal induction (61– 63) and organogenesis (63– 66), they failed to provide much information on the
role of each BMP in skeletogenesis. BMP-2 and BMP-4 knockout mice die during early gastrulation due to failure of mesoderm induction (61, 67, 68). BMP-7 null mutation mice die
shortly after birth due to severe renal failure and eye defects,
and they exhibited mild skeletal changes such as polydactyly
and occasional abnormalities of ribs (65). Normal skeletons
formed in these mice might be rescued by the redundant
function of other BMPs, which were expressed cooperatively
with BMP-7. The generation of conditional knockout mice for
each BMP will provide more important information concerning the roles of each BMP in skeletogenesis.
1. BMP receptors and signal transduction systems. Similarly to
TGF-␤, BMPs bind to two types of serine-threonine receptors,
termed BMP type I and type II receptors (69, 70). Both types
of receptors are necessary to transduce BMP signals. BMP
type I receptors (BMPR-I) also bind BMPs directly in the
absence of BMP type II receptor (BMPR-II), whereas the
TGF-␤ type I receptor does not bind ligands in the absence
of the TGF-␤-type II receptor (71–73). Two kinds of BMPR-Is,
BMPR-IA and BMPR-IB, have been cloned in mammals (74 –
76). During embryogenesis, BMPR-IA is more widely expressed than BMPR-IB in various tissues (77, 78). BMPR-IA
is also expressed in various types of cultured cells including
MC3T3-E1 cells, C2C12 cells (79), C3H10T1/2 cells, and primary osteoblasts isolated from newborn rat calvariae (78),
but BMPR-IB is highly expressed in a limited number of
osteoblastic cells such as ROB-C26 (55) and primary osteoblasts isolated from calvariae. More importantly, immunohistochemical and in situ hybridization analyses demonstrated that osteoblasts express BMPs and their receptors in
the process of bone formation during skeletal development
and fracture repair (53, 57–59, 77, 78, 80 – 85), suggesting that
BMPs are involved in the differentiation process of osteoblasts from osteoprogenitors to mature osteoblasts.
Genetically engineered mutant forms of BMPRs are useful
for exploring the functions of these proteins. A soluble form
of BMPR-IA lacking the transmembrane and cytoplasmic
domains can bind ligands and antagonize the action of BMP
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YAMAGUCHI, KOMORI, AND SUDA
(85). Truncated or kinase-inactivated forms of BMPR-Is are
also capable of blocking the activity of BMPs (76, 79, 86). In
contrast, constitutively active forms of BMPRs can induce the
action of BMP in the absence of ligand (87). These mutant
BMPRs have been successfully used to investigate the signal
transduction pathways during osteoblast differentiation.
Signal-transducing molecules of the TGF-␤ superfamily,
termed Smads, have been identified (69, 70, 88) (Figs. 1 and
2). At present, eight mammalian Smad proteins, Smad1
through Smad8, have been isolated (69, 70, 89). These are
classified into three subgroups according to their structures
and functions (69, 70). The first subgroup is pathwayrestricted Smad (R-Smad). Smads belonging to this subgroup
are ligand specific and activated by the binding of ligands to
type I receptors. Among these, Smad1, Smad5, and Smad8
are involved in BMP signaling (89 –95), and Smad2 and
Smad3 mediate TGF-␤/activin signaling (96, 97). The second
subgroup of Smads is the common mediator Smads (CSmads). Smad4 (also termed DPC-4) belongs to this subgroup (98, 99). R-Smads are phosphorylated by the serine/
threonine kinase receptors that interact with C-Smads,
forming a heterodigomeric complex. This complex is translocated into the nucleus and regulates the transcription of
target genes through direct binding to DNA as well as association with other DNA-binding proteins (70). The third
subgroup of Smads is the inhibitory Smads (I-Smads). Smad6
and Smad7 comprise this subgroup (100 –103). These Smads
inhibit ligand activity by stably binding to type I receptors.
Smad6 binds to the TGF-␤ type I receptor, activin type IB
receptor, and BMPR-IB (101), while Smad7 binds to TGF-␤
type I receptor (100, 102). I-Smads compete with R-Smads for
binding to type I receptors. Smad6 also competes for binding
of activated Smad1 with Smad4 (103).
2. Roles of BMPs in differentiation of osteoblast lineage cells.
a. BMP role in differentiation of multipotential mesenchymal
cells into osteochondrogenic lineage cells. It is important to understand the regulatory mechanism underlying the differentiation process of osteochondrogenic cells from mesenchymal stem cells. Multipotent mesenchymal cell lines are
very useful to gain insight into such mechanisms. C3H10T1 /2
clone 8 (C3H10T1/2) cells, a fibroblastic cell line isolated from
an early mouse embryo, is such a cell line. Untreated control
C3H10T1/2 cells expressed no or extremely low levels of phenotypic characteristics related to osteoblasts, chondrocytes,
myoblasts, and adipocytes (18). Treatment with 5-azacytidine
FIG. 1. The mammalian Smad family is comprised of three classes of
protein, R-Smad (pathway-restricted Smad), C-Smad (common mediator Smad), and I-Smad (inhibitory Smad). [Adapted from M. Kawabata et al.: Cytokine Growth Factor Rev 9:49 – 61, 1998 (70).]
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FIG. 2. Possible mechanism of BMP signaling through BMP receptors and Smads. BMPs bind to BMP receptors (BMPR-I and BMPR-II)
in the target cells, and Smad1 and Smad5 transduce BMP signals
interacting with Smad4. Smad6 inhibits BMP activity by binding to
BMPR-I. Smads1/4/5 act positively, and Smad6 acts negatively in
BMP signaling. [Adapted from M. Kawabata et al.: Cytokine Growth
Factor Rev 9:49 – 61, 1998 (70).]
induced C3H10T1/2 cells to differentiate into chondrocytes,
myotubes, and adipocytes, indicating the multipotential of this
cell line (1). We and others investigated whether BMPs could
induce C3H10T1/2 cells to differentiate into osteoblast lineage
cells (18, 104, 105). Control C3H10T1/2 cells exhibited an extremely low level of ALP activity as well as mRNAs for BMP-2
and BMP-4, and neither production of PTH-dependent cAMP
and osteocalcin nor expression of mRNAs for BMP-5, BMP-6,
and BMP-7 was observed (18, 106). BMP-2 and BMP-7 enhanced or induced osteoblast-related markers in C3H10T1/2
cells (18, 33, 104, 105). These results indicated that BMP-2 and
BMP-7 induce C3H10T1/2 cells to differentiate into osteogenic
lineage cells. In addition, these BMPs induced C3H10T1/2 cells
to differentiate into not only osteoblasts but also chondrocytes
(104, 105). Ahrens et al. (107) also demonstrated that transfection
of cDNAs encoding human BMP-2 and BMP-4 into C3H10T1/2
cells induced the cells to differentiate into both osteoblasts and
chondrocytes. We confirmed that C3H10T1/2 cells formed
mineralized bone as well as cartilage in diffusion chambers,
when the cells placed in a BMP-2-coated diffusion chamber
were transplanted into the peritoneal cavity of athymic mice (A.
Yamaguchi, unpublished data). In this case, histological examination revealed that bone and cartilage were formed separately
in diffusion chambers, suggesting that C3H10T1/2 cells differentiated into osteoblasts and chondrocytes independently.
BMPs (BMP-2, BMP-4, and BMP-7) also induced C3H10T1/2
cells to differentiate into adipocytes (104, 105) as described later.
These results suggested that BMPs induce the synthesis of transcription factors involved in regulation of differentiation pathways into osteoblasts, chondrocytes, and adipocytes, respectively, in C3H10T1/2 cells. In the differentiation of osteoblasts,
it is notable that BMP-7 induced the expression of Cbfa1 mRNA
before induction of osteocalcin mRNA in C3H10T1/2 cells (33).
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REGULATION OF OSTEOBLAST DIFFERENTIATION
BMPs may induce some transcription factor(s) involved in determination of chondrocyte differentiation in C3H10T1/2 cells.
b. BMP and differentiation of osteoblast precursor cells. ROBC26 is a committed osteoprogenitor cell line, retaining the
differentiation potential to form myotubes and adipocytes
(3). The developmental potential of this cell line is similar to
that of RCJ 3.1, which is one of the osteoblastic cell lines
isolated from fetal rat calvariae by Aubin et al. (108) and
characterized by Grigoriadis et al. (2). RCJ 3.1 cells are capable
of differentiating into chondrocytes in addition to osteoblasts, adipocytes, and myotubes, while ROB-C26 cells lack
the potential to differentiate into chondrocytes. Kellermann
and colleagues (109, 110) established a mesodermal tripotential progenitor cell line (C1) from mouse teratocarcinoma,
which differentiated into three types of cells including osteoblasts, chondroblasts, and adipocytes. These cell lines are
also useful for studying the regulatory mechanism of osteoblast differentiation from mesenchymal progenitors. Among
these, ROB-C26 cells have been frequently used to investigate the effects of BMPs on osteoblast differentiation. BMP-2
stimulated ALP activity and PTH-dependent cAMP production and induced osteocalcin synthesis in ROB-C26 cells (19).
Gitelman et al. (22) reported that overexpression of BMP-6
accelerated osteoblast differentiation in ROB-C26 cells, and
this effect was antagonized by the addition of a neutralizing
antibody against BMP-6. Nishitoh et al. (55) demonstrated
that GDF-5 stimulated ALP activity in ROB-C26, which was
mediated by BMPR-IB and BMPR-II. BMP-7 also bound predominantly to BMPR-IB in ROB-C26 cells, and Smad5 was a
key component in the intercellular signaling of BMP-7 (111).
These results indicated that BMPs are important regulators
of osteoblast differentiation from multipotent mesenchymal
cells.
There are several osteoblast precursor cell lines, the differentiation potential of which are restricted to the osteoblast
lineage. Among these, MC3T3-E1, which is a clonal osteoblastic cell line isolated from calvariae of a late stage mouse
embryo (17), is most frequently used to study osteoblast
differentiation. This cell line expresses various osteoblast
functions including formation of mineralized bone nodules
in long-term culture. In MC3T3-E1 cells, BMP-2 and BMP-7
increased ALP activity, PTH responsiveness, and osteocalcin
production (112, 113), suggesting that BMPs promote differentiation of osteoblast precursors to more mature osteoblasts. BMP-7 stimulated osteoblast differentiation in
ROS17/2.8 cells, a typical osteoblastic cell line isolated from
rat osteosarcoma, by increasing synthesis of collagen and
osteocalcin, ALP activity, and PTH responsiveness (114).
BMP-12, alternatively called GDF7, increased ALP activity
within 24 h of treatment in ROS17/2.8 cells (115), whereas it
failed to increase ALP activity in this cell line after treatment
for 6 days (116). The effects of BMP-12 on osteoblast differentiation should be more extensively studied using other
osteoblastic cell lines.
Osteoblastic cells isolated from the calvariae of newborn
rats (117) or the bone marrow of adult rats (primary osteoblasts) (118) provide a suitable model in which to explore the
bone formation process in vitro, because these cells generate
numerous mineralized bone nodules when cultured in the
397
presence of ␤-glycerophosphate and ascorbic acid. Since only
a limited number of clonal cell lines retain the capacity to
form mineralized bones in vitro (17), primary osteoblasts are
important tools for analyzing the differentiation process of
osteoblasts from osteoprogenitors to bone-forming osteoblasts. To explore the roles of BMPs in formation of bone
nodules, we investigated the distributions of BMPs and their
receptors in osteoblastic cells isolated from newborn rat calvariae (119). In situ hybridization studies detected strong
signals for BMP-2 and BMP-4 mRNAs in bone nodule-forming cells, but not in the cells located in internodular regions.
In addition, immunohistochemical analysis using an antibody reactive with both BMP-2 and BMP-4 demonstrated
that positive cells first appeared in unmineralized nodules
and were then localized preferentially in mineralized nodules at a later stage in culture. BMP receptors such as BMPRIA, BMPR-IB, and BMPR-II were preferentially expressed at
the sites of nodule formation in calvarial culture (119). Harris
et al. (120) demonstrated by Northern blotting analysis that
not only BMP-2 and BMP-4 but also BMP-6 mRNAs were
expressed during bone nodule formation by osteoblasts isolated from fetal rat calvariae. The maximal levels of expression of each BMP mRNA coincided with the formation of
mineralized bone nodules. These results suggested that several BMPs are involved in the mechanism of bone nodule
formation by osteoblasts in vitro. Hughes et al. (121) compared the effects of BMP-2, BMP-4, and BMP-6 on the formation of bone nodules by rat calvaria-derived osteoblastic
cells. BMP-2 was less potent than BMP-4 and BMP-6 in this
assay system. Boden et al. (122) reported that glucocorticoidinduced formation of bone nodules in fetal rat calvarial osteoblasts was mediated by BMP-6. Glucocorticoids preferentially increased expression of BMP-6 mRNA, and the
antisense oligonucleotide corresponding to BMP-6 strongly
inhibited formation of bone nodules. BMP-7 also increased
formation of bone nodules by rat calvarial osteoblasts (123,
124). The effects of BMPs on osteoblast differentiation were
also investigated using human osteoblastic cells. Lecanda et
al. (125) reported that BMP-2 had profound effects on proliferation, expression of most of the bone matrix proteins, and
the mineralization of human osteoblastic cells. We also demonstrated that BMP-2 stimulated ALP activity and PTHdependent cAMP production in primary osteoblastic cells
isolated from human bones (126). Taken together, these observations indicated that various BMPs play important roles
in the process of osteoblast differentiation in a paracrine
and/or autocrine fashion.
Several experiments concerning the regulation of BMP
activity have been reported. IL-1␤ synergistically increased
BMP-2-induced ALP activity in MC3T3-E1 cells, but tumor
necrosis factor-␣ (TNF␣) inhibited BMP-2-induced ALP activity in this cell line (113). Insulin-like growth factor I (IGF-I)
synergistically enhanced BMP-7-induced osteoblast differentiation in primary culture of fetal rat calvaria (124). These
results suggest that the action of BMP is modulated by various local factors. Rickard et al. (127) investigated the effects
of estrogen on BMP production using two estrogen-responsive human immortalized osteoblastic cell lines (hFOB/ER3
and hFOB/ER9). Interestingly, estrogen (17␤-estradiol: 10⫺10
to 10⫺7 m) increased the expression level of BMP-6 mRNA
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and production of BMP-6 protein, while levels of mRNAs
encoding TGF-␤1, TGF-␤2, and BMPs-1 through -5 and -7
were unchanged (127). They suggested that some of the
skeletal effects of estrogen on bone might be mediated by
increased production of BMP-6 by osteoblasts. However,
further experiments are needed to confirm such a role for
estrogen, because estrogen suppresses the rate of bone remodeling in vivo. Recently, Mundy et al. (128) searched 30,000
small molecule compounds that activated the promoter of
BMP-2 and found that the statins, lovastatin and simvastatin,
drugs used for lowering serum cholesterol, had such activity.
In addition, they demonstrated by an organ culture system
and in vivo subcutaneous injection that statins stimulated
new bone formation associated with an increased expression
level of BMP-2 mRNA. This suggests a therapeutic application of statins for osteoporosis.
Thus, various BMPs promote osteoprogenitors to differentiate into more mature osteoblasts. However, it has not
been established which BMP is the most potent in osteoblast
differentiation, because these studies were conducted using
different cell types and different culture conditions. More
extensive in vitro studies using a standardized culture system
are necessary to evaluate the potential of each BMP. It is also
important to investigate further the regulation of BMP activity by local and systemic factors.
c. BMP and differentiation of bone marrow stromal cells. The
osteogenic potential of bone marrow stromal cells has been
demonstrated by studies using in vivo and in vitro culture
systems. Bone marrow-derived clonal cell lines and freshly
isolated bone marrow stromal cells have often been used in
such studies. Various bone marrow-derived cell lines show
the characteristics of preadipocytes. More importantly, several cell lines retain the capacity to support hematopoiesis
including osteoclastogenesis (129).
Thies et al. (130) reported that BMP-2 induced the mouse
bone marrow-derived cell line W-20 –17 to exhibit osteoblast
phenotypic markers using an in vitro culture system. We
investigated the effects of BMPs on osteoblast differentiation
using two mouse bone marrow stromal cell lines (131), ST2
(132) and MC3T3-G2-PA6 (PA6)(133), because the two cell
lines had preadipocytic properties and retained the capacity
to support hematopoiesis including osteoclastogenesis (129).
Neither ST2 nor PA6 cells exhibited features typical of osteoblast phenotype under control culture conditions. BMP-2,
BMP-4, and BMP-6 induced ST2 cells to express osteoblast
phenotypic markers such as elevated levels of ALP activity,
PTH-dependent production of cAMP, and the synthesis of
osteocalcin (131). Ascorbic acid also induced osteoblast differentiation in ST2 cells via the action of BMP (134). In contrast, the stimulatory effects of the BMPs on ALP activity and
PTH-dependent production of cAMP were weaker in PA6
cells than in ST2 cells, and BMPs failed to induce the synthesis of osteocalcin in PA6 cells (131). These results indicated
that the effects of BMPs on osteoblast differentiation of bone
marrow stromal cells differ between different cell lines. It
will be interesting to explore differences in BMP receptorsignaling systems in these cell lines. Rickard et al. (127) reported that BMP-2 induced osteoblast differentiation in primary cultures of rat bone marrow cells. In this case, BMP-2
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exerted synergistic effects on bone nodule formation with
dexamethasone (127).
Adipocytes are an important component of bone marrow
stromal cells derived from common progenitors with osteoblasts. As described above, BMP-2 and BMP-4 promoted
C3H10T1/2 cells to differentiate into not only osteochondrogenic cells but also adipocytes (104, 105). Chen et al. (135)
investigated roles of BMPRs in the process of BMP-induced
differentiation of osteoblasts and adipocytes using 2T3 cells,
which were derived from the calvariae of transgenic mice
expressing T antigen driven by the BMP-2 promoter. BMP-2
induced this cell line to differentiate into mature osteoblasts
or adipocytes. Overexpression of a kinase domain-truncated
BMPR-IB in 2T3 cells completely inhibited osteoblast differentiation in this cell line, and the decreased level of ALP
activity in the 2T3 cells with the truncated BMPR-IB was
rescued by transfection with wild-type BMPR-IB. In addition, overexpression of constitutively active BMPR-IB induced formation of bone in 2T3 cells in the absence of BMP-2.
In contrast, overexpression of a kinase domain-truncated
BMPR-IA blocked adipocyte differentiation, whereas transfection of constitutively active BMPR-IA induced adipocyte
differentiation, increasing expression levels of adipocyte differentiation-related genes such as adipsin and PPAR␥2 in
2T3 cells. These results suggested that BMPR-IA and
BMPR-IB have different functions in the differentiation of
osteoblasts and adipocytes in 2T3 cells: BMPR-IB is the major
receptor involved in osteoblast differentiation and BMPR-IA
is the major receptor for adipocyte differentiation. As described below, however, an important role of BMPR-IA has
been demonstrated in the process of BMP-2-induced osteoblast differentiation in C2C12 myoblasts (79). In contrast to
the stimulatory effects of BMP-2 on adipocyte differentiation,
Gimble et al. (136) reported that BMP-2 and BMP-4 inhibited
adipocyte differentiation of murine bone marrow stromal
cells. Inhibitory effects of BMP-2 on adipocyte differentiation
were also demonstrated in the immortalized human bone
marrow stromal cell line [hMS (2– 6)] (137). Since reciprocal
regulation of osteogenesis and adipogenesis in the bone marrow microenvironment has been suggested (136, 137), further investigation of the regulatory mechanism involved in
lineage determination of osteoblasts and adipocytes is
important to understand the pathogenesis of osteopenic diseases such as osteoporosis. Lecka-Czernik et al. (138) reported interesting findings in this regard. They demonstrated that overexpression of PPAR␥2 in mouse bone
marrow cells stimulated adipocyte differentiation and inhibited osteoblast differentiation by suppressing expression
of Cbfa1 mRNA. This suggests that PPAR␥2 negatively regulates osteoblast differentiation of bone marrow stromal cells
by suppressing Cbfa1 expression.
Thus, BMPs play important roles in the process of cell
lineage determination of osteoblasts and adipocytes from
bone marrow stromal cells. In this process, BMPs promote
osteoblastic differentiation, but they exert diverse effects on
adipocyte differentiation depending on cell type. The diverse
actions of BMPs on adipocyte differentiation might be caused
by different usage of the BMP receptor-signaling systems
and the transcription factors relating to cell lineage determination such as Cbfa1 and PPAR␥2.
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REGULATION OF OSTEOBLAST DIFFERENTIATION
d. Role of BMPs in osteogenic transdifferentiation of myogenic
cells. Classical transplantation experiments of BMPs into
muscular sites demonstrated that BMPs induced ectopic cartilage and bone formation (46, 47). In addition, tissue culture
experiments showed that muscle cultured on decalcified
bone generated chondrogenic cells (139). These findings suggest that muscles contain osteochondrogenic progenitor
cells, and that BMPs divert the differentiation pathway of
myogenic cells into osteochondrogenic lineage cells.
We first investigated the effects of BMP-2 on myogenic
differentiation in ROB-C26, which is an osteoblast precursor
cell line with the capacity to differentiate into myogenic cells
(3). BMP-2 inhibited myogenic differentiation with concomitant stimulation of osteoblast differentiation in this cell line
(19).
To further investigate the regulatory mechanism of myogenic differentiation by BMP-2, Katagiri et al. (21) used
C2C12 myoblasts, which originated from muscular tissue
satellite cells. Both BMP-2 and TGF-␤1 inhibited myotube
formation completely in C2C12 cells, but only BMP-2 induced them to differentiate into osteoblast lineage cells (21).
BMP-2 exerted effects similar to those observed in C2C12
cells in the primary muscle cells isolated from newborn mice
(21). In the process of myogenic inhibition in C2C12 cells,
both BMP-2 and TGF-␤1 strongly down-regulated the levels
of expression of mRNAs encoding MyoD and myogenin (21,
140), which are critical transcription factors regulating myogenic differentiation. Chalaux et al. (141) demonstrated the
involvement of JunB in the early steps of inhibition of myogenic differentiation by BMP-2 and TGF-␤1. Thus, BMP-2
and TGF-␤1 have similar inhibitory effects on myotube formation, but only BMP-2 induced osteoblast differentiation,
indicating different functional effects on osteoblast differentiation between these two molecules.
To understand the molecular mechanism involved in osteogenic transdifferentiation in C2C12 cells induced by
BMP-2, the roles of BMPRs and Smads were investigated.
Wild-type C2C12 cells expressed BMPR-IA and BMPR-II
mRNAs, but not BMPR-IB mRNA (79). A subclonal cell line
of C2C12 stably expressing a kinase domain-truncated
BMPR-IA generated numerous myotubes but failed to differentiate into ALP-positive cells after treatment with BMP-2
(79). When wild-type BMPR-IA was transiently transfected
into the BMPR-IA mutant cells, BMP-2 inhibited myogenic
differentiation and induced ALP-positive cells (79). BMP-2
did not induce ALP-positive cells in BMPR-IA mutant cells
transfected with wild-type BMPR-IB (79). These results suggest that BMP-2 signals inhibiting myogenesis and inducing
osteoblast differentiation are transduced via BMPR-IA, at
least in C2C12 cells. Interestingly, Akiyama et al. (87) demonstrated that C2C12 cells stably transfected with constitutively active BMPR-IB exhibited osteoblast phenotypic markers, but did not express myogenic phenotypic markers. These
results suggest that a common signal transducer(s) including
Smads is involved in the signal transduction pathway via
BMPR-IA and BMPR-IB during differentiation of C2C12
cells. C2C12 cells constitutively expressed Smad1, Smad2,
Smad4, and Smad5 mRNAs (94). Yamamoto et al. (94) demonstrated that Smad1 and Smad5, which belong to the RSmad family and mediate BMP signaling, are involved in the
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process of myogenic inhibition and induction of osteoblast
differentiation in C2C12 cells. Nishimura et al. (95) demonstrated that BMP-2 caused serine phosphorylation of Smad1
and Smad5, unlike TGF-␤. They also showed that the activation of Smad5 and subsequent formation of the complex of
Smad5 and Smad4, which is alternatively called DPC4 and
belongs to the C-Smad family, were key steps in the process
of BMP-2-induced osteoblast differentiation in C2C12 cells
(95). Overexpression of I-Smads (Smad6 and Smad7) repressed ALP activity induced by BMP-6 in C2C12 cells (142),
whereas BMP-2 or BMP-7 markedly induced mRNA encoding Smad6 in C2C12 cells (143). These results suggest that
Smad6 is involved in a feedback loop to regulate the signaling activity of BMPs.
Using primary cells isolated from human muscle, we reported that BMP-2 inhibited myotube formation and stimulated ALP activity, but failed to induce osteocalcin production (126). In addition, transplantation of these myogenic
cells with BMP-2 using diffusion chambers into athymic mice
induced ALP-positive cells in the chambers but did not induce formation of bone or cartilage. These results suggested
that the capacity of human muscular cells to differentiate into
the osteoblast lineage is more restricted than that in rodents.
Taken together, the findings obtained from in vitro experiments show that BMPs are important local factors regulating
the differentiation pathway of mesenchymal cell lineages
into osteoblasts, chondrocytes, adipocytes, and muscles. Furthermore, BMPs promote osteoblastic and chondrocytic differentiation, inhibit myogenic differentiation, and exert diverse actions on adipogenic differentiation.
3. Extracellular regulation of BMP activity. Recent molecular
embryological findings have shown that BMPs play crucial
roles in the induction and patterning of ventral mesoderm at
an early stage of development (63). During gastrulation, the
Spemann organizer provides essential patterning information to the adjacent mesoderm and the overlying ectoderm.
In 1996, noggin (144) and chordin (145), which are the Spemann organizer signals, were demonstrated to bind BMP-4
with high affinities at an extracellular region and to antagonize the action of BMP (Fig. 3A). Subsequently, two other
molecules, gremlin (146) and follistatin (147), were found to
antagonize the action of BMP at the extracellular level. Indeed, noggin, chordin, and gremlin inhibited BMP-induced
ALP activity in W-20 –17 bone marrow stromal cells and
C3H10T1/2 cells (144 –146). During mouse embryogenesis,
noggin is expressed not only in the node, notochord, and
dorsal somite, but also in the condensing cartilage and immature chondrocytes (148, 149). Experiments in noggin null
mutant mice indicated that this molecule plays important
roles in normal patterning of the neural tube, somites, and
cartilage including joint formation (148, 149). These results
indicated that BMP activity is also regulated by BMP antagonists such as noggin, chordin, follistatin, and gremlin at the
extracellular level.
Xolloid, which is a secreted metalloprotease in Xenopus
and a tolloid-related protein in Drosophila, was found to cleave
chordin and the chordin/BMP-4 complex (150) (Fig. 3B).
Xolloid digestion released biologically active BMPs from an
inactive chordin/BMP complex (150). Interestingly, BMP-1,
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YAMAGUCHI, KOMORI, AND SUDA
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FIG. 3. Mechanisms of extracellular regulation of BMP activity. A, An inhibitory mechanism of chordin by competing with BMP ligands for receptor binding at an extracellular
level. B, Xolloid, a human BMP-1 homolog in
Xenopus, cleaves chordin, and reactivates
BMP.
which is a metalloprotease isolated from demineralized bone
extracts and purified together with BMP-2 and BMP-3 (47),
is a human homolog of Xolloid and Tolloid (150, 151). These
results suggest important roles of BMP-1 in the regulation of
BMP activity in mammalian bones. Further investigations
are necessary to identify other proteases, which can cleave
the noggin/BMP complex, because noggin is expressed at an
early stage of skeletogenesis (149) and Xolloid cannot cleave
the noggin/BMP complex (150).
Recently, Engstrand et al. (152) reported that BMP-3 antagonized BMP-2-induced osteoblastic differentiation in
W-20 –17 cells. They also demonstrated increased bone formation and bone density in BMP-3-deficient mice compared
with wild-type controls. These observations suggest that
BMP-3 is an inhibitory regulator of bone formation. Further
studies of the regulatory mechanism of action of BMP by
antagonistic molecules at the extracellular level will provide
deeper insight into the mechanism of osteoblast differentiation and bone formation by BMPs.
B. Sonic and Indian hedgehogs
1. Involvement of Sonic and Indian hedgehogs in skeletogenesis.
The gene hedgehog is a segment polarity gene regulating
embryonic segmentation and patterning in Drosophila and is
highly conserved in vertebrates (23). In higher vertebrates,
the Hedgehog gene family consists of at least three members,
Shh, Ihh, and Dhh (23). Shh has multiple functions during
formation of various organs and tissues including formation
of skeletal tissues in vertebrae and limbs (25). The phenotypes observed in Shh knockout mice indicated that Shh
plays a critical role in patterning of embryonic tissues, including the brain, the spinal cord, the eyes, and the skeleton
(25). They completely lacked vertebrae and partly lacked
autopods (25). These results suggested that Shh mutations
cause some malformations in humans. Indeed, the similarity
of forebrain development between Shh mutant mice and
cases of human holoprosencephary with SHH mutation is
reported (24, 153–155). In addition, mutation of human
PATCHED, which encodes a transmembrane protein that
negatively regulates Shh signaling in target cells, causes the
human autosomal disease termed nevoid basal cell carcinoma syndrome (156). Developmental skeletal abnormalities
and a high risk of various forms of cancers, mainly basal cell
carcinoma, characterize this syndrome. Mutations in the
human SHH gene and genes that encode components of its
downstream intracellular signaling pathway also cause
three distinct congenital disorders, Greig syndrome, Pallister-Hall syndrome, and isolated postaxial polydactyly
(157). Thus, SHH signaling is involved in the pathogenesis
of several diseases including those of skeletal tissues in
humans.
Bitgood and McMahon (30) first reported that Ihh is expressed in cartilage during skeletogenesis in mouse embryos.
Vortkamp et al. (26) demonstrated that Ihh regulated chondrocyte differentiation through regulation of PTHrP in
chicken embryos. They also showed that the hedgehogresponsive genes Patched and Gli (transcription factor) were
highly expressed in the perichondrium, where formation of
bone collar occurred directly from perichondrial cells (26, 27).
These results suggested that the target cells for Ihh are located
in the perichondrium, and that Ihh induces adjacent perichondrial cells to differentiate into bone-forming osteoblasts.
Since Shh and Ihh have similar functions in chondrocyte
differentiation (26, 158), it is likely that these hedgehog proteins are involved in osteoblast differentiation as well as
chondrocyte differentiation in vertebrates. Indeed, this was
supported by the recent report that Ihh null mutant mice
exhibited failure of osteoblast development in endochondral
bones as well as markedly reduced chondrocyte proliferation
and maturation (159).
The hedgehog family retains structural and functional
similarities between Drosophila and vertebrates. In Drosophila, a major role of hedgehog signaling is the activation of
additional signals including dpp, which is a homolog of vertebrate BMP, and wingless. Laufer et al. (160) reported that
Shh is capable of regulating the expression of BMP-2 in
chicken limb buds, because BMP-2 mRNA was expressed
adjacent to Shh-expressing cells and the ectopic transplantation of Shh-expressing cells induced BMP-2 expression in
the cells around the transplanted cells. By in situ hybridization using serial sections, Bitgood and McMahon (30)
showed an intimate correlation between the expression of
mouse Shh/Ihh genes and BMPs in various tissues. These
findings prompted us to investigate whether Shh and Ihh are
involved in osteoblast differentiation by a mechanism involving BMPs.
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REGULATION OF OSTEOBLAST DIFFERENTIATION
2. Regulation of osteoblast differentiation by hedgehogs. Several
lines of evidence obtained from in vitro experiments indicate
that hedgehogs regulate osteoblast differentiation. We first
examined the effects of hedgehogs on osteoblast differentiation using the conditioned media collected from Shh- or
Ihh-overexpressing chicken embryonic fibroblasts (106, 161).
Addition of each conditioned medium increased ALP activity in C3H10T1/2 and MC3T3-E1 cells and increased the level
of osteocalcin mRNA expression in MC3T3-E1 cells. Chicken
embryonic fibroblasts used for the transfection of Shh or Ihh
constitutively expressed substantial levels of mRNAs for
BMP-2 and BMP-4. In addition, each conditioned medium
induced no apparent increases in BMP-2, BMP-4, or BMP-6
mRNAs in C3H10T1/2 and MC3T3-E1 cells, but the increase
in ALP activity induced by the conditioned media was abolished by addition of soluble BMPR-IA (Ref. 161 and T. Yuasa,
and A. Yamaguchi, unpublished data), which antagonized
the action of BMP on osteoblast differentiation in vitro (85).
These results suggest that the stimulatory effects induced by
addition of the conditioned media might be synergistically
induced with Shh or Ihh and the BMPs produced by chicken
fibroblasts themselves. Indeed, recombinant Shh (rShh) synergistically stimulated the BMP-2-induced ALP activity and
the expression level of osteocalcin mRNA in C3H10T1/2 cells
(T. Yuasa and A. Yamaguchi, unpublished data). Since the
cooperative action of Shh and BMP-7 was reported in the
induction of forebrain ventral midline cells by prechordal
mesoderm (162), a cooperative effect of Shh and BMPs might
be important in osteoblast differentiation as well. Murtaugh
et al. (163) reported that chondrogenesis of somitic tissues is
regulated by intimate interaction between Shh and BMPs.
The intimate link between Ihh and the BMP/noggin signaling pathway during chondrocyte differentiation is also suggested by other investigators (164 –166). Therefore, it is likely
that Shh and BMPs act cooperatively during differentiation
of osteochondrogenic cells, but further studies are necessary
to determine the precise interaction between Shh and BMPs
in this process.
3. Role of hedgehogs in bone formation. To investigate whether
Shh and Ihh induce ectopic bone formation, we transplanted
Shh- or Ihh-overexpressing chicken fibroblasts cultured on
type I collagen gel into intraperitoneal sites in athymic mice
(161). Endochondral bone formation was induced at the site
of transplantation (106, 161). Since the transplanted chicken
embryonic fibroblasts expressed low levels of mRNAs encoding BMP-2 and BMP-4, it should be elucidated whether
such endochondral bone formation is due to the direct effect
of hedgehog proteins alone or the synergistic effects of Shh/
Ihh and BMPs. Further studies using recombinant proteins
of hedgehogs are currently underway in our laboratory.
Important roles of Ihh during bone repair have been suggested by in vivo experiments. Vortkamp et al. (27) investigated the expression patterns of Ihh and BMPs during fracture repair. The fracture site expressed neither type X collagen,
which is a marker of hypertrophic chondrocytes, nor Ihh at
an early stage (within 3 days after fracture), but both mRNAs
were strongly expressed in cartilaginous callus by 7 days
after fracture. Ferguson et al. (28) also reported a similar
expression pattern of Ihh during fracture repair. When the
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cartilage was completely replaced by bone at 3 weeks after
fracture, expression of both mRNAs encoding type X collagen
and Ihh disappeared (27). Interestingly, BMP-2 and BMP-4
were expressed in a number of chondrocytes of the healing
callus overlapping the Ihh-expressing cells, suggesting some
interaction between Ihh and BMPs during fracture repair
(27). Although these observations suggest that Ihh is involved in fracture repair, further investigations are needed
to explore a more precise role for Ihh because Ito et al. (29)
reported that up-regulation of Ihh mRNA occurred within
hours after fracture of mouse ribs.
Thus, hedgehogs, by interacting with BMPs, may play an
important role in bone formation, especially at early stages
of skeletogenesis and fracture repair.
IV. Transcription Factors That Regulate Osteoblast
Differentiation and Bone Formation
A. Transcription factors involved in osteoblast
differentiation
Many transcription factors are involved in the regulatory
mechanism of differentiation of different cell types. Among
them, cell lineage-specific transcription factors play crucial
roles in determining the fate of each cell type. Such transcription factors have been identified in several cell lineages
including myoblasts and adipocytes, e.g., the MyoD family in
myoblasts (31) and PPAR␥-2 in adipocytes (32) (Fig. 4).
Proliferation and differentiation of osteoblasts are regulated by many transcription factors including members of the
families of HLH proteins, leucine zipper proteins, zinc finger
proteins, and runt-domain proteins, and the protooncogenes
such as c-myc, c-jun, and c-fos (4). In the case of osteoblasts,
the specific transcription factors that regulate their differentiation have not been identified. To identify these transcription factors, many investigators have examined the regulatory mechanism of osteocalcin gene expression, since this gene
is expressed only in osteoblasts with the exception of
megakaryocytes (10, 167). Two laboratories independently
identified three osteocalcin genes in mice (168, 169). Two of
these, osteocalcin gene 1 (OG1) (169) [alternatively called
mOC-A (168)] and osteocalcin gene 2 (OG2) (169) [alternatively
called mOC-B (168)], are uniquely expressed in bone. The
FIG. 4. Osteoblasts, chondrocytes, myotubes, and adipocytes arise
from a common pluripotent mesenchymal cell. Each differentiation
pathway is regulated by the cell lineage-specific transcription factors.
The transcription factor(s) that determines chondroblast differentiation has not been clarified.
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YAMAGUCHI, KOMORI, AND SUDA
other gene, osteocalcin-related gene (ORG) (169) [alternatively
called mOC-X (168)], is not expressed in bone but only in the
kidney (169). Extensive analyses of the osteocalcin gene promoter have been conducted to investigate the mechanism
controlling osteoblast-specific gene expression, and several
functional domains have been identified. The osteocalcin box
I (OC Box I) conserved the homeodomain-containing protein
binding motif, and MSX1 and MXS2 bind to this particular
sequence (170, 171). Tamura and Noda (172) identified an E
box to which HLH protein could bind in the osteocalcin
promoter, but the factor interacting with osteocalcin E box
has not been identified (172, 173). The osteocalcin box II (OC
Box II) contains runt-domain protein recognition sites, which
were extensively analyzed by two research groups. Stein and
colleagues (174, 175) identified three runt-domain protein
recognition sites, one site in OC Box II and other two sites in
the distal promoter in the rat osteocalcin gene. Karsenty and
colleagues (176, 177) also identified two distinct DNA sequences, designated osteoblast-specific element 1(OSE1) and
osteoblast-specific element 2 (OSE2), in the promoter of the
mouse osteocalcin gene (OG2). OSE2 also showed conserved
consensus binding sequences for runt-domain protein (176).
The two groups demonstrated that nuclear extracts of osteoblastic cells, designated nuclear matrix protein 2 (NMP2)
(174, 175) or osteoblast specific factor 2 (OSF2) (177), bound
to the runt-domain protein recognition site, and its binding
was blocked by antibodies against AML1-B (Cbfa2)(175, 176,
178) and Cbfa1 (36). These results indicate that a transcription
factor immunologically related to Cbfa family proteins is
involved in osteoblast-specific transcription of osteocalcin.
There are three Cbfa transcription factors, Cbfa1, Cbfa2 (also
called Pepb2␣B and AML1), and Cbfa3 (also called Pepb2␣C
and AML2), in the mouse and in humans. Among these,
several lines of evidence demonstrated that Cbfa1 plays a
critical role in osteoblast differentiation and bone formation
as described below.
Recently, Schinke and Karsenty (179) purified osteoblast
specific factor 1 (OSF1) as a 40-kDa protein, which specifically bound to distinct DNA sequences designated OSE1 in
the OG2 promoter. They also suggested that OSF1 regulated
Cbfa1 transcription by binding to the OSE1 sequence in
Cbfa1 itself (179).
B. Cbfa1 is an important transcription factor regulating
osteoblast differentiation
Two laboratories independently demonstrated that the
osteoblast-specific DNA binding activity, designated OSF2
and NMP-2, was identical to Cbfa1 (33, 36). Thereafter, several laboratories including these two showed that Cbfa1 regulated the expression of various genes expressed in osteoblasts (33, 36, 180 –182). Overexpression of Cbfa1 in
nonosteogenic cells such as C3H10T1/2 cells and skin fibroblasts induced them to express osteoblast-related genes (33,
182). Cbfa1 was highly expressed in osteoblast lineage cells
(33, 34). Antisense oligonucleotides for Cbfa1 down-regulated expression of osteoblast-related mRNAs in ROS17.2/8
osteoblastic cells (33). Using rat primary osteoblasts, Banerjee
et al. (36) also demonstrated that antisense oligonucleotides
for Cbfa1 inhibited osteoblast differentiation including for-
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mation of bone nodules in vitro. These results indicated that
Cbfa1 plays a crucial role in osteoblast differentiation.
Ducy et al. (33) demonstrated that BMP-7 induced expression of Cbfa1 mRNA before induction of osteocalcin mRNA.
BMP-2 also increased the level of Cbfa1 mRNA expression in
an immortalized human bone marrow stromal cell line
[hMC(2– 6)] (137), C2C12 cells (37, 183), and 2T3 cells (135).
Nishimura et al. (183) reported that BMP-2 induced Cbfa1
mRNA in C2C12 myoblasts, and this induction was abolished by overexpression of dominant-negative Smad1,
Smad4, and Smad5. In addition, Hanai et al. (184) demonstrated that Smad 1 or Smad 5 and Cbfa1 formed complexes,
indicating an intimate interaction between these molecules
during osteoblast differentiation. These results suggest that
Cbfa1 is a nuclear target of BMP signaling in osteoblast
differentiation. On the other hand, we found that calvariaderived cells isolated from Cbfa1-deficient embryos increased production of osteocalcin in response to BMP-2, although it was less than that produced by wild-type embryos
(34). This suggests that transcription factors other than Cbfa1
also play some roles in BMP-2-induced osteocalcin synthesis,
at least in vitro. Lee et al. (37) demonstrated that both BMP-2
and TGF-␤ transiently up-regulated expression of Cbfa1
mRNA in C2C12 cells, but only BMP-2 induced expression
of osteoblast differentiation-related mRNAs. Recently, Wang
et al. (185) isolated several subclones from the MC3T3-E1
osteoblastic cell line. Characterization of each subclone indicated that the presence of Cbfa1 in a subclone was not
sufficient for osteoblast differentiation (185). Taken together,
these observations indicated that Cbfa1 plays a crucial role
in the differentiation process of osteoblasts, but it is not a
sufficient transcription factor for osteoblast differentiation.
Isolation of cell lines from Cbfa1-deficient mice may provide
useful tools for investigating transcription factors, other than
Cbfa1, involved in osteoblast differentiation. Such studies are
important to understand the regulatory mechanism of osteoblast differentiation, and they are currently underway in
our laboratories.
C. Absence of ossification in Cbfa1-deficient mice
To investigate the precise function of Cbfa1, we disrupted
exon 1 of the Cbfa1 gene, which contained the first 41 amino
acids of the runt-domain (34). We extensively examined skeletal changes in Cbfa1-deficient mice on embryonic day 18.5
(E18.5) because Cbfa1-deficient mice died soon after birth due
to respiratory insufficiency. In Cbfa1-deficient embryos at
E18.5, only parts of the tibia, radius, and vertebrae were
weakly calcified, and no calcification occurred in the skull,
mandible, humerus, or femur, while wild-type embryos at
E18.5 exhibited extensive calcification of all the skeletons on
soft x-ray examination (Fig. 5). Histological examination revealed that Cbfa1-deficient embryos completely lacked ossification. Interestingly, ALP-positive cells surrounded calcified cartilage such as the tibia and radius in Cbfa1-deficient
embryos, whereas no ALP-positive cells appeared around
uncalcified cartilage such as the humerus and femur. These
findings suggest that calcified cartilage contains some factor(s) inducing early differentiation of osteoblast lineage cells
even in Cbfa1-deficient embryos. In E18.5 Cbfa1-deficient em-
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REGULATION OF OSTEOBLAST DIFFERENTIATION
FIG. 5. Soft x-ray of E18.5 wild-type (⫹/⫹) and Cbfa1-deficient (⫺/⫺)
embryos. Wild-type embryo exhibits well calcified skeletons, but mutant embryos had barely calcified skeletons.
bryos, only a thin layer of the fibrous connective tissue was
observed between the brain and subcutaneous connective
tissue. ALP-positive cells were detected in the fibrous connective tissues, but no calcified bone was observed. Similar
skeletal changes in Cbfa1-deficient mice were reported by
Otto et al. (35). These morphological changes were confirmed
extensively at ultrastructural and histochemical levels by
Hoshi et al. (186). These results demonstrated that Cbfa1 is an
important transcription factor for bone formation.
Many mRNAs related to bone matrix proteins such as
osteocalcin, osteopontin, and ␣1(I) collagen have Cbfa1 binding
sites in their promoter regions (33). As expected from these
promoter sequences, Cbfa1 mutant mice expressed extremely
low levels of osteopontin and ␣1(I) collagen, and no osteocalcin
in their skeletons (34). These indicated that maturational
arrest of osteoblasts caused the lack of bone formation in
Cbfa1-deficient mice.
Since Cbfa1-deficient mice die soon after birth, it is difficult
to explore the exact role of Cbfa1 in growing mice. To investigate the function of Cbfa1 in growing mice, Ducy et al.
(187) generated transgenic mice overexpressing the Cbfa1
DNA-binding domain (⌬Cbfa1) driven by the OG2 promoter.
⌬Cbfa1 was expressed in differentiated osteoblasts only postnatally and acted in a dominant-negative fashion due to a
higher affinity for DNA than Cbfa1 itself. The skeleton of
⌬Cbfa1-transgenic mice was normal at birth, but they suffered from osteopenia due to a decrease in bone formation
rate 3 weeks after birth. These results indicate that Cbfa1
plays a crucial role in not only osteoblast differentiation but
also osteoblast function.
D. Role of Cbfa family transcription factors in osteoblast
differentiation
Several isoforms of Cbfa1 produced by the differential
promoter usage have been identified. One isoform originally
cloned from ras-transformed NIH3T3 cells was named
Pebp2␣A by Ogawa et al. (188, 189) (tentatively referred to as
type I isoform, which begins with the N-terminal amino acid
sequence MRIPVD). Subsequently, two other isoforms of
403
Cbfa1 have been identified from osteoblasts and lymphoblasts (33, 190). In these two isoforms, two methionine residues were found in the novel N-terminal region: one was a
shorter isoform translated from the second methionine residue (tentatively referred to as type II isoform, which begins
with the N-terminal amino acid sequence MASNSL), and the
other was a longer isoform translated from the first methionine residue (tentatively referred to as type III isoform,
which begins with the N-terminal amino acid sequence
MLHSPH). Type II isoform was originally reported as til-I by
Stewart et al. (190), and type III was first identified as Cbfa1/
Osf2 by Ducy et al. (33). The expression pattern of these Cbfa1
isoforms in various cell types has not been fully investigated.
We demonstrated by RT-PCR analysis that these three isoforms were expressed in adult mouse bones (182). Xiao et al.
(191) extensively analyzed genomic structure and isoform
expression of mouse, rat, and human Cbfa1. They demonstrated that type II isoform was expressed in osteoblasts of
all species, and type III isoform was recognized in osteoblasts
of the mouse and rat but not in human osteoblasts. These
expression patterns suggest that type II isoform, rather than
type III isoform, plays an important role in osteoblast differentiation. The expression of Cbfa1 mRNA in nonosteogenic cells is still controversial. We demonstrated that
C3H10T1/2 cells expressed undetectable levels of mRNAs
for three isoforms of Cbfa1 in control culture (182). Ducy et
al. (33) also reported that C3H10T1/2 cells expressed an
undetectable level of Type III isoform of Cbfa1 in control
culture, but another group showed that C3HT101/2 cells as
well as NIH3T3 fibroblasts constitutively exhibited a substantial level of mRNA for Type I isoform (180). The discrepant results of Cbaf1 expression among laboratories using
the same cells might arise from the different culture conditions employed.
As described above, Ducy et al. (33) demonstrated that
transfection of type III isoform of Cbfa1 into nonosteogenic
cells induced gene expression related to osteoblast differentiation, but functional differences between the three isoforms
of Cbfa1 have not been clarified. Harada et al. (182) investigated the functional differences in these isoforms of Cbfa1 by
transfection of the respective isoforms into C3H10T1/2 cells
and transcription assay using Cbfa1 target gene promoter
driven-luciferase reporter genes. Both transient and stable
transfection with type I and type II Cbfa1 isoforms, but not
with type III isoform, induced ALP activity in C3H10T1/2
cells. All of the Cbfa1 isoforms induced or up-regulated expression of osteocalcin, osteopontin, and type I collagen mRNAs
in stable transformants, although the cells transfected with
type II isoform exhibited the highest level of osteocalcin
mRNA expression. Luciferase reporter gene assay using
6XOSE2-SV40 promoter (six tandem binding elements for
Cbfa1 ligated in front of the SV40 promoter sequence) and
mouse osteocalcin promoter revealed differences in the transcriptional induction of target genes by each Cbfa1 isoform.
These findings were supported in a recent similar study by
Xiao et al. (191). Although all three Cbfa1 isoforms might be
involved in stimulation of osteoblast differentiation, the expression pattern of Cbfa1 isoforms and the transfection experiments of these isoforms suggest that the type III isoform
has much less activity than the type II isoform. The lower
404
YAMAGUCHI, KOMORI, AND SUDA
translational efficiency of type III isoform compared with
type II isoform (192) supports this notion.
It has been shown that Cbfa1 and Ets1, which is a nuclear
phosphoprotein of the Ets transcription factor family modulating cell proliferation, differentiation, and oncogenic
transformation (193), synergistically enhanced promoter activity of osteopontin in skeletal tissue (194). The molecular
mechanism of DNA binding of Cbfa2 and Ets-1 has been well
investigated as a model system of combinatorial control that
utilizes multiple transcription factors (195, 196). Both Cbfa2
and Ets-1 contain a negatively regulatory domain for DNA
binding in their sequences, and interaction between each
negative regulatory domain is necessary and sufficient for
cooperative DNA binding (195). Further investigation is necessary to gain deep insights into the regulatory mechanism
of osteoblast differentiation by Cbfa1.
E. Cbfa1 is involved in chondrocyte maturation
Cbfa1 was apparently expressed in hypertrophic chondrocytes (34, 197). In Cbfa1-deficient mice, calcification of cartilage occurred in the distal limbs (tibia, fibula, radius, and
ulna), and almost all other cartilage remained uncalcified
(34). These observations suggested that Cbfa1 played some
roles in chondrocyte differentiation. We investigated expression patterns of cartilage-related mRNAs in Cbfa1-deficient
mice by in situ hybridization. In the distal limbs showing
calcification, hypertrophic chondrocytes expressed Ihh, type
X collagen, and BMP-6, but did not express osteopontin or
collagenase 3 (197). In the humerus and femur in Cbfa1-deficient mice, however, chondrocytes expressed no detectable
levels of mRNAs encoding PTH/PTHrP receptor, Ihh, type X
collagen, or BMP-6, indicating that chondrocyte differentiation was blocked before prehypertrophic chondrocytes in
these skeletal structures (197). Similar findings concerning
maturational arrest of chondrocytes were reported in other
Cbfa1-deficient mice (198) generated by Otto et al. (35). These
observations suggest that Cbfa1 plays an important role in
chondrocyte maturation.
F. Cbfa1 is involved in osteoclastogenesis
In 1981, Rodan and Martin (199) proposed an important
hypothesis concerning the possible involvement of osteoblast lineage cells in the hormonal control of bone resorption. They suggested the potential direct activation of osteoclasts by the products of osteoblast lineage cells in
response to bone-resorbing hormones. A series of experiments have confirmed this hypothesis (200 –202), but the
precise molecular mechanism involved in the interaction
between osteoblast lineage cells and osteoclasts has not
been clarified.
Recently, two molecules produced by osteoblast lineage
cells, which play important roles in osteoclastogenesis, were
identified. One is osteoprotegerin (OPG) (203), which is identical to osteoclastogenesis-inhibitory factor (OCIF) (204, 205).
OPG is a secretary protein belonging to the TNF receptor
family (203–205). This protein inhibited not only formation
of osteoclast-like cells (OCLs) in culture but also bone resorption both in vitro and in vivo (203–205). In addition, OPG
Vol. 21, No. 4
knockout mice exhibited severe osteopenia due to accelerated bone resorption (206, 207). The other molecule is
RANKL (receptor activator of NF-kB ligand) (208), which is
identical to OPG ligand (OPGL) (209), TRANCE (TNFrelated activation-induced cytokine) (210), and osteoclast differentiation factor (ODF)(211). RANKL belongs to the TNF
ligand family and binds to OPG. A soluble form of RANKL
(soluble RANKL) together with macrophage colony-stimulating factor induced formation of OCLs from spleen cells in
the absence of osteoblast lineage cells in vitro (209, 211).
Recently, Kong et al. (212) reported that OPGL- deficient mice
exhibited severe osteopetrosis and completely lacked osteoclasts as a result of an inability of osteoblasts to support
osteoclastogenesis. The formation of OCLs induced by soluble RANKL was completely abolished by the addition of
OPG (209, 211), indicating a specific interaction between
RANKL and OPG in osteoclastogenesis.
We reported that osteoclastogenesis was markedly retarded in Cbfa1-deficient mice (34). These results suggested
that the maturational arrest of osteoblasts caused by disruption of the Cbfa1 gene might be related to the insufficient
osteoclastogenesis in Cbfa1-deficient mice. These observations also allowed us to speculate on the role of Cbfa1 in the
regulation of RANKL and OPG, because both are synthesized by osteoblast lineage cells.
We investigated the mechanism involved in retarded
osteoclastogenesis in Cbfa1-deficient mice (213). Cocultures of calvarial cells isolated from embryos with three
different Cbfa1 genotypes (Cbfa1⫹/⫹, Cbfa1⫹/⫺, and
Cbfa1⫺/⫺) and normal spleen cells generated TRAP-positive OCLs in response to 1␣,25(OH)2D3 and dexamethasone, but the number and bone-resorbing activity of OCLs
formed in coculture with Cbfa1⫺/⫺ calvarial cells were
significantly decreased in comparison with those formed
in cocultures with Cbfa1⫹/⫹ or Cbfa1⫹/⫺ calvarial cells. The
expression of RANKL mRNA was increased by treatment
with 1␣,25(OH)2D3 and dexamethasone in calvarial cells
from Cbfa1⫹/⫹ and Cbfa1⫹/⫺ mouse embryos, but not in
those from Cbfa1⫺/⫺ embryos. In contrast, the expression
of OPG mRNA was inhibited by 1␣,25(OH)2D3 and dexamethasone to a similar extent in all three types of calvarial
cells. RANKL and OPG mRNAs were highly expressed in
the tibia and femur of Cbfa1⫹/⫹ and Cbfa1⫹/⫺ embryos. In
the tibia and femur of Cbfa1⫺/⫺ embryos, however,
RANKL mRNA was undetectable, and the expression of
OPG mRNA was also decreased compared with those in
Cbfa1⫹/⫹ and Cbfa1⫹/⫺ embryos. Thus, it is likely that
Cbfa1 is involved, at least in part, in osteoclastogenesis by
regulating the expression of RANKL. This was supported
by recent reports by O’Brien et al. (214) and Kitazawa et al.
(215). They identified potential Cbfa1 binding sites in the
promoter region of murine RANKL, suggesting that Cbfa1
may directly regulate RANKL expression. More extensive
studies on the regulation of RANKL by Cbfa1 will provide
insight into the molecular mechanism involved in the classical hypothesis proposed by Rodan and Martin (199) concerning the interaction between osteoblasts and osteoclasts during bone remodeling.
August, 2000
REGULATION OF OSTEOBLAST DIFFERENTIATION
405
G. Heterozygous mutations of Cbfa1 locus cause
cleidocranial dysplasia
Cleidocranial dysplasia (CCD) is an autosomal-dominant
disease showing hypoplastic clavicles, open fontanelles, supernumerary teeth, short stature, and other skeletal changes
(216, 217). Mice heterozygous for mutation in the Cbfa1 locus
(Cbfa1⫹/⫺) (34, 35) exhibited similar skeletal changes to CCD
(218, 219). They exhibited hypoplastic frontal, parietal, interparietal, temporal, and supraoccipital bones with open
fontanelles and sutures. They also showed hypoplastic clavicles and nasal bones. Development of the primordia of tooth
structures, however, was slightly delayed but structurally
normal in heterozygous Cbfa1 mice (35). In homozygously
mutated mice, developmental arrest was observed at the cap
stage in molar tooth germs, and neither the differentiation of
mesenchymal cells in dental papilla to preodontoblasts nor
the differentiation of epithelial cells to preameloblasts was
observed (Ref. 220 and S. Miyake and T. Komori, unpublished data). The lack of supernumerary teeth in heterozygous Cbfa1 mutant mice can be explained by the fact that mice
have only one set of teeth, and deciduous teeth are not
affected in humans (218). Although clefts involving hard and
soft palates have been often described in CCD patients, we
could not find apparent cleft palates in heterozygous Cbfa1
mutant mice (A. Yamaguchi and T. Komori, unpublished
results). One family with CCD showed a microdeletion in
chromosome 6p21 (218). CBFA1 is mapped to chromosome
6p12-p21 (221, 222). Mutations of CBFA1 have been identified
in patients with CCD (221, 223).
V. Summary
Osteoblasts originate from common progenitors, which
are capable of differentiating into other mesenchymal cell
lineages such as chondrocytes, myoblasts, and bone marrow
stromal cells including adipocytes. During the differentiation
process from mesenchymal progenitors, various hormones
and cytokines regulate osteoblast differentiation. Among
these, BMPs are the most potent inducers and stimulators of
osteoblast differentiation: BMPs not only stimulate osteoprogenitors to differentiate into mature osteoblasts but also
induce nonosteogenic cells to differentiate into osteoblast
lineage cells. Sonic and Indian hedgehogs also play important roles in regulation of osteoblast differentiation by interacting with BMPs. Cbfa1, a transcription factor belonging
to the runt-domain gene family, is essential but not sufficient
for osteoblast differentiation and bone formation. Cbfa1deficient mice completely lacked bone formation due to maturational arrest of osteoblasts. Overexpression of Cbfa1 induces nonosteogenic cells to express osteoblast-related genes
in vitro. BMPs are important local factors that up-regulate
Cbfa1 expression. Cbfa1 plays important roles in skeletal
development at two stages, for commitment to skeletal lineage cells and for maturation of osteoblasts in postnatal
development. The phenotype of the heterozygous Cbfa1 mutation is similar to that of CCD. Patients suffering from this
disease exhibit CBFA1 mutations. Thus, the intimate interaction between the local factors including hedgehogs and
BMPs and the transcription factor Cbfa1 play crucial roles in
FIG. 6. A hypothetical molecular pathway involved in osteoblast differentiation. Shh and Ihh may act cooperatively with BMPs. Noggin
and chordin inhibit BMP action by competing for binding to BMPRs
at an extracellular region. BMP signals may up-regulate expression
of Cbfa1 through Smads. Cbfa1 effectively transcribes various osteoblast-related genes to induce osteoblast differentiation. Some transcription factor(s) other than Cbfa1 may be involved in BMP-induced
osteoblast differentiation. Solid arrows indicate direct interactions
and dashed arrows indicate hypothetical interactions.
the process of osteoblast differentiation and bone formation.
A hypothetical molecular pathway involved in osteoblast
differentiation is summarized in Fig. 6.
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