Smad signal pathway in BMP-2-induced osteogenesis－
a mini review
CHIU-JOU WU 1
1
2
HSEIN-KUN LU 1,2
School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan, ROC.
Clinical Periodontics, Dental Department of Taipei Medical University Hospital, Taipei, Taiwan, ROC.
Bone morphogenetic protein-2 (BMP-2) is a multifunctional growth factor. It strongly induces bone
formation and belongs to the transforming growth factor-β (TGF-β) superfamily. Recently, recombinant
human BMP-2 (rhBMP-2) and rhBMP-7 (also called osteogenic protein-1; OP-1) has become available for
clinical applications. This article focuses on the influence of BMP-2 on intracellular Smad signal
transduction pathways of osteoblasts in osteogenesis. After BMP-2 binds to the BMP receptor (BMPR)
on cell membranes, the BMPR activates receptor-specific Smads (R-Smads) which then associate with
the common-mediator Smad (Co-Smad). This complex is translocated into the nucleus and interacts with
several transcription factors such as Runx2/Cbfα1, Osx, Dlx5, and Msx2. These molecules mediate the
transcription of related genes to induce osteogenesis. （J Dent Sci, 3(1)：13-21 , 2008）
Key words: BMP-2, signal transduction pathway, Smad, transcription factor.
Bone morphogenetic proteins (BMPs) are
secretary growth factors which can induce ectopic
bone formation and chondrogenesis1-3. Recently, it was
also found that BMPs play important roles as
multifunctional regulators in morphogenesis4-9. BMPs
can induce endochondral bone formation, that is,
stimulate mesenchymal stem cell differentiation into
chondrocytes, and after cartilage formation, it is
replaced by bone tissue10-12. BMPs can also directly
induce intramembranous bone formation as well.
BMPs are local factors regulating osteoblast
differentiation13,14. Several experiments with BMP
knockout mice have elucidated the roles of BMPs in
bone formation and development15,16.
Alkaline phosphatase (ALP) and osteocalcin are
widely accepted bone markers17. Treatment with
BMP-2 can stimulate the expression of ALP by
activating the BMP receptor (BMPR)18, receptorspecific Smads (R-Smads)19, and the expressions of
Dlx5 and Runx2/Cbfα117. In addition, research
has found that osteoblast markers of pluripotent
mesenchymal stem cell cultures are upregulated by
BMP-2. This indicates that BMPs may mediate the
specific differentiation pathways of uncommitted
cells20,21.
By transgenic and knockout approaches and
animal models of naturally occurring mutations in
BMP and related genes, it was shown that BMPs
play critical roles in mesoderm formation, heart
development, cartilage development11, and postnatal
bone formation. Clinical studies have shown that
recombinant human BMP-2 (rhBMP-2) and rhBMP-7
can be applied to many diseases22-31 such as bone
defects32, non-union fractures, spinal fusions26,32-35,
osteoporosis36, and osteogenesis imperfecta37. They
are also a great help in dental procedures38, including
bone augmentation39, root canal surgery, repairing
severe bone loss with periodontal disease, stimulation
of osseointegration40-43, oral surgery, craniofacial
reconstruction35, autogenous bone grafts in sinus
augmentations, and extraction socket-related alveolar
ridge augmentation44.
Received: January 4, 2008
Accepted: February 28, 2008
Reprint requests to: Dr. Hsein-Kun Lu, College of Oral Medicine, Taipei
Medical University, No. 250, Wu-Xing Street, Taipei,
Taiwan 11042, ROC.
Smad pathways of BMP-2 in osteogenesis
J Dent Sci 2008‧Vol 3‧No 1
Osteoblasts and other mesenchymal cell lineages
such as chondrocytes, myoblasts, and bone marrow
13
C.J. Wu and H.K. Lu.
stromal cells, including adipocytes, originate from a
common progenitor. During their development, BMPs
are the strongest inducers and stimulators of cell
differentiation. BMPs not only stimulate osteoprogenitors to differentiate into mature osteoblasts, but
also induce non-osteogenic cells to differentiate into
osteoblast lineage cells21,45,46.
The signal transduction pathways of BMPs
include the main Smad pathways19 and non-Smad
pathways. Many studies have shown that R-Smads
including Smad 147, 5, and 8, which are downstream
molecules of BMP receptors, play central roles in
signal transduction pathways of BMPs. Smads
mediate several different subsequent biological effects.
As to signal transduction pathways of BMP-2 in
osteogenesis, BMP receptors ligand-dependently
phosphorylate only R-Smad 1 and 534,48-54.
BMP-2 activates BMP receptors
BMPs exert different biological effects on 2 types
of transmembrane receptors, types I (BMPR-I) and II
BMP receptors (BMPR-II), both of which possess
intrinsic serine/threonine kinase activity18,52,55. Now 3
type I receptors are known: the type IA BMP receptor
(BMPR-IA, also called ALK3), type IB BMP receptor
(BMPR-IB, also called ALK6), and type IA activin
receptor (ActR-IA, also called ALK-2), which binds
to BMP ligands28,52,53,56-61. There are also 3 type II
receptors known: the type II BMP receptor (BMPR-II),
type II activin receptor (ActR-II), and type IIB activin
receptor (ActR-IIB)28,52,53,59,60,62,63.
After BMP dimeric ligands bind to receptors, 2
pairs of BMPR-I and BMPR-II form a heterotetrameric-activated receptor complex64. Smad proteins19
are one of the BMPR-I substrates, and they play
critical roles in relaying BMP signals from receptors
to target genes in the nucleus. That is to say, after
dimeric ligands bind to heterotetrameric BMP
receptors, the intrinsic serine/threonine kinase activity
is activated, and then R-Smads are phosphorylated.
Similarly, through BMPR-IA and BMPR-IB, BMP-2
can phosphorylate the intracellular transducers,
Smad 1 and 5, which results in inhibition of the
differentiation of myoblasts and the induction of
osteoblast differentiation45,50,65.
BMP-2-induced R-Smad activation
BMP dimers initiate signaling by binding to both
14
types I and II serine/threonine kinase receptors and the
phosphorylation of type I receptors upon ligand
binding66. Activation of BMP receptors initiates
phosphorylation of the downstream effector proteins,
known as receptor-regulated Smads, Smad 1 and 5,
leading to signal transduction. After activated
R-Smads are released from their receptors, they
combine with Co-Smad (also called commonmediator Smad or common-partner Smad), that is,
Smad 4, to form hetero-oligomeric complexes. These
complexes are then translocated to the nucleus to
interact with other transcription factors in order to
mediate target gene transcription67.
The BMP signaling cascade is closely regulated
by anti-Smads (also called I-Smads or inhibitory
Smads), i.e., Smad 6 and 7, and the intracellular
signaling inhibitor, Smad ubiquitination regulatory
factor-1 (Smurf1). Smad 6 and 7 can inhibit R-Smad
phosphorylation. In addition, Smad 6 can inhibit the
association of Smad 1 and 434. Therefore anti-Smads
can block the intracellular signaling cascade, and
negatively regulate BMP-2 signal transduction to
assure that BMP-2 expression maintains its normal
function18. Smurf1 can interact with Smad 1 and 5
specific to the BMP pathway to trigger their
ubiquitination and proteasomal degradation68. Thus,
Smurf1 can inhibit BMP signal transduction by
increasing the degradation of Smad 1 and 5 and then
decreasing their intracellular levels19,28,32,34,53,59,69-76. On
the other hand, several extracellular antagonists such
as protein noggin77 and chordin78 can directly bind to
BMP-2 and -4 with high affinity in order to interfere
with the combination of BMPs and BMP receptors.
These inhibitors carefully regulate the signal
transduction of BMP-2 to avoid carcinogenesis due to
overexpression72,79-82.
Roles of osteogenic transcription factors and
their interactions in the BMP-2 signal
transduction pathway
Runt-related transcription factor 2 (Runx2)/core
binding factor alpha 1 (Cbfα1)
Runx2 is also called core-binding factor α1
(Cbfα1), polyomavirus enhancer binding protein 2αA
(PEBP2αA), and acute myeloid leukemia 3 (AML3).
It is the transcription factor of the runt domain gene
family83. Runx2/Cbfα1 can regulate the gene expressions of several types of osteoblasts and plays
J Dent Sci 2008‧Vol 3‧No 1
Smad pathway of BMP-2 in osteogenesis
important roles in skeletal development at 2 stages:
commitment of skeletal lineage cells and maturation
of osteoblasts in postnatal development. In addition,
Runx2/Cbfα1 may regulate the bone resorption ability
of osteoclasts by affecting mRNA levels of RANKL.
Therefore, Runx2/Cbfα1 is the critical transcription
factor regulating osteoblast differentiation and bone
formation84,85.
The maturation of osteoblast ceases and bone
formation is absent in Runx2/Cbfα1-knockout mice86.
Runx2/Cbfα1 plays an important role in the expression of osteoblast marker genes87,88. The human
RUNX2/CBFα1 gene has been mapped to chromosome 6p21, and this gene mutation causes
cleidocranial dysplasia (CCD), an autosomaldominant disease89. In mice, heterozygous loss of
functional alleles causes the same disease phenotypes
of open fontanelles and hypoplastic clavicles90.
BMPs are important local factors regulating
osteoblast differentiation and the transcription factor,
Runx2/Cbfα191, which plays a critical role in determining the osteoblast cell lineage and osteoblast
maturation. It is an essential transcription factor in
osteoblast differentiation and bone formation. Although the regulatory mechanism of Runx2/Cbfα1
expression is not clearly known, BMPs are important
factors in its upregulation92. As a result, interactions
between BMPs and the Runx2/Cbfα1 transcription
factor are very important to osteoblast differentiation
and bone formation46,74,84,88,93.
BMP-2 can upregulate Runx2/Cbfα1 mRNA
expressions of an immortalized human bone marrow
stromal cell line [hMC(2–6)]94, C2C12 cells95,96, and
2T3 cells97. Nishimura et al.96 reported that BMP-2 can
induce Cbfα1 mRNA in C2C12 myoblasts, and this
induction was abolished by the overexpression of
dominant-negative Smad 1, 4, and 5. In addition,
Hanai et al.98 showed that Smad 1 or 5 and Cbfα1
formed complexes, indicating close interactions
among these molecules during osteoblast differentiation. The above results suggest that
Runx2/Cbfα1 is the target of BMP signaling in the
nucleus during osteoblast differentiation. Also,
Komori et al.86 found that calvaria-derived cells
isolated from Cbfα1-deficient embryos increased
production of osteocalcin in response to BMP-2,
although it was less than that produced by wild-type
embryos. This indicates that besides Runx2/Cbfα1,
other transcription factors play roles in the production
of BMP-2-induced osteocalcin at least in vitro. Lee et
J Dent Sci 2008‧Vol 3‧No 1
al.95 demonstrated that BMP-2 and TGF-β transiently
upregulated the expression of Runx2/Cbfα1 mRNA in
C2C12 cells, but only BMP-2 induced osteoblast
differentiation-related mRNAs.
Osterix (Osx)
Nakashima et al.99 identified osterix (Osx), a
newly found zinc finger-containing transcription
factor. It is specifically expressed only in all types of
developing bone tissues and is related to osteoblast
differentiation and bone formation. Osx-knockout
mice completely lose the ability to form bone, but
they can still normally express Runx2/Cbfα1. Thus, it
is known that although Osx is not the transcription
factor required for Runx2/Cbfα1 expression, it may
occur downstream of Runx2/Cbfα1 in the osteoblast
differentiation pathway to regulate osteoblast formation. Preosteoblast differentiation requires the
presence of Osx. Osx may be the negative regulator of
the transcription factor, Sox9, and chondrocytes. It
may prevent Osteo-Chondro progenitor cells from
differentiating into chondrocytes100. Runx2/Cbfα1
functions from the commitment step to the point
at which Osteo-Chondro progenitor cells appear,
whereas Osx acts mainly during the terminal differentiation of osteoblasts, and distinguishes osteogenic from chondrogenic pathways101.
Although BMP-2 treatment stimulates Runx2/
Cbfα195,102 and Osx99 mRNA levels, pretreatment with
cycloheximide, a de novo protein synthesis inhibitor,
blocks BMP-2-induced Runx2/Cbfα1103 and Osx100
mRNA expressions. This suggests that the osteogenic
master genes are not the direct targets of the BMP
signaling cascade, and their expressions require the
intermediation of newly synthesized proteins.
Distal-less homeobox 5 (Dlx5)
Dlx5 is an essential regulator of BMP-2-induced
osteoblast differentiation104. It is a bone-inducing
homeodomain transcription factor that is expressed in
the latter stages of osteoblast differentiation105. Forced
expression of Dlx5 in cell culture causes expression of
osteocalcin and full matrix mineralization106,107.
Normally, Dlx5 is detected in discrete neuronal tissues
and developing skeletal elements such as cartilage,
bone, and teeth90,108. In addition, Dlx5-deficient mice
show severe craniofacial abnormalities such as
delayed cranial ossification and abnormal osteogenesis109,110. This indicates that Dlx5 plays important
roles in the development of mineralized tissues.
15
C.J. Wu and H.K. Lu.
When Dlx5 and BMP-2 or BMP-4 are coexpressed in vivo111, Dlx5 may become the target of the
BMP signaling cascade. BMP-2 treatment, active
forms of BMPR-IA or IB, or the overexpression of
Smad 1 or 5 can stimulate Dlx5 transcription112.
However, in contrast to Runx2/Cbfα1 or Osx
expression induced by BMP-2, cycloheximide
pretreatment does not affect BMP-2-induced Dlx5
transcription95. This indicates that Dlx5 may be the
upstream regulator of Runx2/Cbfα1 and Osx in
BMP-2 signal transduction. This is also supported by
the fact that inhibition of Dlx5 expression with
antisense techniques completely blocks Runx2/Cbfα1
and Osx expressions113. Furthermore, Dlx5 overexpression induces Runx2/Cbfα1 expression even
without BMP-2 treatment. The above results clearly
show that Dlx5 is a necessary regulator of osteogenic
master gene expression and osteoblast differentiation.
The interaction between Dlx5 and the msh homeobox
homolog 2 (Msx2)
Dlx5 and Msx2 proteins antagonize each other
during osteoblast differentiation. Dlx5 functions in
later stages at the same time as expression105. It was
also found that Dlx5 activates the promoter of bone
marker genes17,114,115 and stimulates osteoblast differentiation while Msx2 seems to play a contrary role.
Msx2 stimulates cell differentiation and inhibits
osteogenic differentiation116,117. Its expression precedes
that of osteocalcin and inhibits osteoblast terminal
differentiation118.
There are many models to explain the antagonistic actions between Dlx5 and Msx2. Because
these 2 proteins obscure DNA-binding homeodomains,
they may interact to become a functionally inactive
complex119,120. Runx2/Cbfα1 and Msx2 interact to
become a complex, and Msx2-bound Runx2/Cbfα1 is
transcriptionally inactive. However, adding Dlx5 to
Runx2/Cbfα1-Msx2 causes the release of Runx2/
Cbfα1, because Dlx5 sequesters the Msx2 protein and
then restores the transcription activity of Runx2/
Cbfα1121. Moreover, homeodomains of Dlx5 and Msx2
proteins may compete for binding to common response elements in bone-specific marker genes such
as osteocalcin105,114,119,122 and ALP17 in the Runx2-P1
promoter111. Therefore, Dlx5 and Msx2 may regulate
each other at the transcription level, that is, Msx2
inhibits Dlx5 expression and vice versa.
In conclusion, during the differentiation process
from bone marrow mesenchymal stem cells to
16
osteocytes, Runx2/Cbfα1 and osterix are 2 important
transcription factors. Both in vivo and in vitro studies
have shown that Runx2/Cbfα1 is indispensable for
osteogenesis of bone marrow mesenchymal stem cells,
and Runx2/Cbfα1 genes are the key genes in bone
formation. Dlx5 and Msx2 proteins antagonize
each other during osteoblast differentiation. Dlx5 is
expressed in later stages to stimulate osteoblast
differentiation, while Msx2 stimulates cell proliferation and inhibits osteoblast terminal differentiation. Among the downstream master osteogenic
transcription factors, Runx2/Cbfα1 may be the earliest
master molecules which regulate osteoblast differentiation, while Dlx5 may be the earliest target of
BMP-2-activated R-Smads, and it independently
regulates Runx2/Cbfα1 and Osx.
Transcription of downstream target genes
The above-described master osteogenic transcription factors, the Runx2/Cbfα1, Osx, Dlx5, and
Msx2 molecules, independently or cooperatively
stimulate osteoblast target genes, type I collagen and
fibronectin, in the early stages, and ALP17 and
osteocalcin105,114,119,122 in later stages of differentiation.
BMP-2 treatment can stimulate ALP expression by
activating BMP receptors, R-Smads, Dlx5, and
Runx2/Cbfα1 expressions. Numerous studies have
shown that BMP-2, -3, -4, and -7 can mediate the
differentiation of mature osteoblasts and upregulate
the expression of ALP activity on a short-term basis
and osteocalcin expression on a long-term basis20.
CONCLUSIONS
We schematically summarize the BMP-2-induced
signal transduction pathway of Smads in Figure 1.
Smad-dependent molecular cascades can be summarized as follows.
1. During embryonic bone development, BMP-2
dimers bind to BMP receptors, and then 2 pairs of
BMPR-I and BMPR-II form a heterotetramericactivated receptor complex, activating intrinsic
serine/threonine kinase activity.
2. BMP-2 activates BMPR-I and phosphorylates its
downstream molecules, R-Smads.
3. BMP-2-activated R-Smads are associated with
Co-Smad to form a complex. They then enter
the nucleus and interact with several transcription
factors.
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Smad pathway of BMP-2 in osteogenesis
BMP-2
Antagonist：
Chordin
Noggin
DNA family
Cell membrane
BMPR-II
BMPR-I
R-Smad:
Smad1
Smad5
R-Smad:
Smad1
Smad5
Anti-Smad/I-smad：
Smad6
Smad7
Co-Smad:
Smad4
Smurf1
Nucleus
Transcription
factors
Msx2
Dlx5
Runx2/Cbfα1
Stem cell
Osx
Preosteoblast
Commitment
Target genes:
type I collagen,
fibronectin
Osteoblast
Osteoblast
differentiation
Target genes:
ALP, osteocalcin
Figure 1. Smad pathways of bone morphogenetic protein (BMP)-2 in osteogenesis. “ ” indicates
phosphorylation; BMPR, bone morphogenetic protein receptor; Smurf1, Smad ubiquitination regulatory factor-1;
Msx, msh homeobox homolog 2; Dlx5, Distal-less homeobox 5; Runx2, Runt-related transcription factor 2; Cbfα1,
core binding factor alpha 1; Osx, Osterix; ALP, alkaline phosphatase.
J Dent Sci 2008‧Vol 3‧No 1
17
C.J. Wu and H.K. Lu.
4. Dlx5 may be the earliest target of BMP-2-activated
R-Smads and independently regulates Osx and
Runx2/Cbfα1, the earliest master molecules to
regulate osteoblast differentiation. Dlx5 expression
stimulates osteoblast differentiation while Msx2
stimulates cell proliferation.
5. These molecules regulate target genes, and then
affect the translation of related downstream proteins
to induce osteogenesis.
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