TGF - Eli and Edythe Broad Center of Regeneration Medicine and

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TGF-β Family Signaling in
Skeletal Development,
Maintenance, and Disease
Tamara Alliston
Department of Orthopaedic Surgery
University of California
San Francisco, California 94143
Ester Piek
Department of Applied Biology
Faculty of Science
Radboud University Nijmegen
6525 ED Nijmegen, The Netherlands
Rik Derynck
Department of Cell and Tissue Biology
University of California
San Francisco, California 94143
T
HE TRANSFORMING GROWTH FACTOR-β (TGF-β) FAMILY is critically involved
in the development and maintenance of skeletal tissues. In searches for factors with potent cartilage and bone inductive activities, TGF-β was isolated
as cartilage-inducing factor (Seyedin et al. 1987), whereas bone morphogenetic proteins (BMPs) were isolated as factors able to induce cartilage and
bone formation (Urist 1965; Luyten et al. 1989). The TGF-β family shows
extensive redundancy of ligands, receptors, agonists, and antagonists, yet the
diverse temporal and spatial patterns of expression of each pathway component allow these signaling factors to direct skeletal development and
homeostasis by regulating patterning, cell-fate determination, cell differentiaton, and bone remodeling. The critical roles of each factor are evident in
mice with mutations in components of these pathways, whereas in vitro
The TGF-β Family ©2008 Cold Spring Harbor Laboratory Press 978-087969752-5
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T. Alliston, E. Piek, and R. Derynck
studies have elucidated the mechanisms for the action of the TGF-β family
in mesenchymal and skeletal cells. Because the TGF-β family has a central
role in the regulation of mesenchymal differentiation into skeletal cells, its
role in osteoblast and chondrocyte development is detailed in Chapter 21.
Here we discuss, at the tissue level, the impact of TGF-β family ligands and
signaling pathways in skeletal patterning, skeletal development, and skeletal
maintenance and metabolism, as well as the effects of their deregulation in
many skeletal diseases.
IMPLICATION AND LOCALIZATION OF TGF-β FAMILY SIGNALING
COMPONENTS IN THE SKELETON
Many members of the TGF-β family, their receptors, and signaling effectors, the Smads, are widely expressed in mesenchymal tissues throughout
development and, in particular, at sites of skeletal patterning and bone
and cartilage formation. Furthermore, TGF-β family members and their
signaling mediators are expressed in differentiating chondrocytes and
osteoblasts that deposit cartilage and bone matrix, respectively, and in
osteoclasts, whose function is to resorb calcified hypertrophic cartilage or
bone matrix. Among the TGF-β family members, the expression and roles
of several BMPs, growth and differentiation factors (GDFs), and TGF-βs
have been well studied.
Localization and Function of BMPs and GDFs, Their
Antagonists and Receptors
Since their discovery as inducers of ectopic cartilage and bone formation,
BMPs have been studied as key regulators of early embryogenesis, pattern
formation, and organogenesis. BMP signaling in skeletal structures is tightly
regulated by the localized expression and activities of BMP proteins, their
receptors, and extracellular antagonists. BMP-1 to BMP-7 have different but
overlapping expression patterns in the developing skeleton (Solloway et al.
1998). Like most TGF-β family receptors, BMP type II receptor (BMPRII)
and the type I receptor BMPRIA/ALK-3 are expressed nearly ubiquitously
in development (Kawabata et al. 1995); however, the expression of the
BMPRIB/ALK-6 type I receptor is restricted to precartilaginous condensations, developing cartilage and bone (Ishidou et al. 1995). The expression
of BMPRIA, BMPRIB, and BMPRII is induced in bone fracture repair
(Onishi et al. 1998). In addition, statins, estrogen, and Wnt3a induce BMP
expression as part of their osteoblast differentiation promoting activities
(Rickard et al. 1998; Mundy et al. 1999; Rawadi et al. 2003). BMP-2 and
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TGF-β Family in Skeletal Development and Disease
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BMP-4 also induce their own expression through the osteogenic transcription factor Runx2 (Helvering et al. 2000; Ghosh-Choudhury et al. 2001).
BMP activity is tightly controlled by secreted agonists and antagonists
including noggin, sclerostin (SOST), twisted gastrulation (Tsg), chordin,
gremlin, follistatin, Dan, Dante, and PRDC (protein related to Dan and
Dante), as discussed in Chapter 8. Each of these is expressed in skeletal
cells (for review, see Canalis et al. 2003), allowing them to dampen or facilitate BMP binding to their receptors. Remarkably, BMPs induce the
expression of their own antagonists, including the secreted noggin, gremlin, and sclerostin and the inhibitory Smad6 and Smad7 (Gazzerro et al.
1998; Takase et al. 1998; Ishisaki et al. 1999; Pereira et al. 2000; van
Bezooijen et al. 2005). The expression of BMP ligands, their receptors, and
extracellular agonists and antagonists is often regulated through positive
or negative feedback loops that respond to other signaling pathways,
including fibroblast growth factor (FGF) and sonic hedgehog (Shh), in
limb morphogenesis, endochondral bone formation, or pathological conditions. Following binding and activation of BMP receptor complexes, the
BMPs activate Smad-mediated and non-Smad signaling pathways, which
are critical for BMP action in skeletogenesis. BMP-activated signaling
through Smad1, Smad5, and Smad8 is antagonized by Tob, c-Ski, menin,
and Smad6 and Smad7 (Berk et al. 1997; Hayashi et al. 1997; Imamura
et al. 1997; Nakao et al. 1997; Yoshida et al. 2000; Sowa et al. 2004), whose
expression patterns are again regulated during skeletal development or in
skeletal cell differentiation. Together, this complex network regulates the
temporal and spatial expression and amplitude of the BMP activity that
controls skeletal patterning, as well as the organization of growth plates
and postnatal maintenance of bone mass.
GDF-5, -6, and -7, also referred to as cartilage-derived morphogenetic
proteins (CDMP-1, -2, and -3) (Chang et al. 1994; Storm et al. 1994), were
first identified in a screen for BMP-related genes possibly corresponding
to the brachypodism mouse locus (Storm et al. 1994). Null mutations in
Gdf5 cause the brachypodism phenotype, characterized by reduced length
of long bones in the limbs and altered patterning of segments in the digits, indicating crucial roles for GDF-5 in the patterning of the appendicular skeleton, chondrogenesis, and longitudinal bone growth (Storm et al.
1994; Storm and Kingsley 1996, 1999). This is consistent with the striking
localization of GDF-5 expression to sites of joint formation (Merino et al.
1999b). GDFs can induce osteogenic differentiation in vitro (Erlacher
et al. 1998b; Yeh et al. 2005) and endochondral bone formation when
implanted intramuscularly (Hotten et al. 1996) and are involved in bone
formation in vivo (Mikic et al. 2002).
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Expression of TGF-βs and Their Receptors
The three TGF-βs—TGF-β1, TGF-β2, and TGF-β3—signal through
receptor complexes of TGF-β type II receptor (TβRII) and TβRI, whereas
the type III receptor, betaglycan, facilitates the presentation of TGF-β
ligands to the heteromeric receptor complex. Although the three ligands
function similarly at the cellular level, mice with targeted inactivation for
each of the corresponding genes exhibit unique phenotypes, thus
reflecting their different spatiotemporal distributions and roles. For
example, rescue of the severe inflammatory phenotype in TGF-β1-null
mice revealed defects in bone quality, detailed in Chapter 21 (Shull et al.
1992; Kulkarni et al. 1993; Geiser et al. 1998). Deletions of TGF-β2 or
TGF-β3 cause perinatal lethality resulting from defective palatogenesis
and other developmental malformations, in part due to impaired
epithelial–mesenchymal transdifferentiation and cell proliferation
(Kaartinen et al. 1995; Proetzel et al. 1995; Sanford et al. 1997). TGF-β2null mice also possess defects in axial, appendicular, and craniofacial
skeleton patterning (Sanford et al. 1997).
The unique and overlapping expression patterns of the TGF-β
ligands throughout skeletal development are also regulated by feedback
loops involving TGF-β family members and other signaling pathways.
Some discrepancies between the mRNA and protein expression patterns
may reflect the regulation of TGF-β storage in cartilage or bone matrix
(Alliston and Derynck 2000). The interactions of TGF-β with extracellular matrix proteins allow for high levels of TGF-β storage, especially
TGF-β1, in bone (Pelton et al. 1991). All three TGF-β isoforms are
expressed in mesenchyme, and their expression increases during mesenchymal condensation. TGF-β3 is expressed at high levels in early development of cartilage rudiments for ribs and vertebrae, whereas TGF-β1
and TGF-β2 expression is very low (Pelton et al. 1990, 1991). Later in
development, TGF-β3 levels are reduced and TGF-β1 and TGF-β2
levels are increased (Pelton et al. 1990). Often, TGF-β3 levels are highest in the soft tissues associated with skeletogenesis, whereas TGF-β2
is up-regulated at sites of new mineralization.
In cartilage, TGF-β3 is expressed at higher levels than the other
TGF-βs in the perichondrium (Pelton et al. 1990, 1991). In the growth
plate, TGF-β1 and TGF-β3 are primarily expressed in the proliferative
and hypertrophic zones, whereas TGF-β2 is expressed in all zones of the
growth plate, with its highest levels in the hypertrophic and mineralizing
zones (Sandberg et al. 1988; Millan et al. 1991; Thorp et al. 1992; Horner
et al. 1998). The periosteum of long bones expresses high levels of TGF-β1,
whereas the periosteum of intramembranous bone expresses high levels
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of TGF-β2 (Pelton et al. 1989). TGF-β1 is the predominant isoform
expressed by osteoclasts. All three isoforms are expressed by osteoblasts.
Vitamin D3, parathyroid hormone (PTH), and estrogen induce the
expression of TGF-β by osteoblasts in culture (Oursler et al. 1993; Streuli
et al. 1993; Wu et al. 1999), although the effects in cultured osteoblasts
may not be indicative of in vivo mechanisms.
TβRI and TβRII are also expressed throughout the developing skeleton (Horner et al. 1998), but their expression is reduced or lost in
hypertrophic chondrocytes and mineralized osteophyte tissue, respectively (Horner et al. 1998). The presumed reduction of TGF-β responsiveness in osteophytes is consistent with an osteoarthritic phenotype in
mice that express a dominant-negative TβRII, or mice null for the
expression of Smad3. Expression of the TGF-β-responsive Smads is also
regulated in skeletal tissues (Sakou et al. 1999). Smad2 is preferentially
expressed in proliferating chondrocytes, whereas Smad3 is expressed at
higher levels in mature chondrocytes and Smad4 is expressed throughout the growth plate. The inhibitory Smad6 and Smad7 are expressed by
the most mature chondrocytes.
As discussed in Chapter 7, TGF-β is secreted as an inactive complex
of the mature TGF-β dimer noncovalently interacting with the amino-terminal part of the TGF-β propeptide (latency-associated peptide or LAP),
which in turn can bind to one of the latent TGF-β-binding proteins
(LTBPs). Among the four isoforms of LTBPs, all but LTBP-2 bind to the
TGF-β propeptide. These LTBPs facilitate the deposition and storage of
TGF-β in the bone matrix. Although all four LTBPs are broadly expressed
and regulated, LTBP-3 expression is high in bone, and LTBP-2 is abundantly expressed in chondrogenic condensations (Shipley et al. 2000). The
expression of LTBP-1 is regulated by PTH (Kwok et al. 2005). The release
of active TGF-β from these complexes is also regulated, as discussed in
Chapter 7. In addition to proteases such as plasmin, the acidic microenvronment created by osteoclasts during bone resorption effectively releases
active TGF-β (Oreffo et al. 1989; Oursler 1994; Dallas et al. 2002). Therefore, several levels of regulation determine the availability of active TGF-β
in the skeleton, including the expression of TGF-β ligands, receptors, and
LTBPs; deposition and activation of TGF-β; and presentation of ligands
to TGF-β receptor complexes.
Localization of Activins, Inhibins, Follistatin, and Activin Receptors
The activins are involved in skeletal development and metabolism. Activins
and activin receptors are expressed in prechondrogenic limb condensations
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and developing limbs during chondrogenesis (Roberts et al. 1991; Feijen
et al. 1994; Merino et al. 1999a). The activin βA subunit (encoded by the
Inhba gene) and ActRII (type II activin receptor) are expressed in proliferating but not hypertrophic chondrocytes in the growth plate. ActRII is
highly expressed in newborn rat skeletal tissues and fracture calluses
in vivo, particularly in osteoblasts at sites of active proliferation and early
intramembranous and endochondral bone formation (Shuto et al. 1997).
Activin receptors are also expressed during ectopic bone formation induced
by implantation of the demineralized bone matrix (Funaba et al. 1996).
Inhibin, a heterodimer consisting of an inhibin α with a βA or βB
subunit (see Chapters 4 and 27), may compete with activin for binding
to ActRII and with BMP for binding to ActRII or BMPRII (Lewis et al.
2000), indicating that, also in the context of skeletal development and
homeostasis, inhibin may act as a physiological antagonist of activins and
BMPs. Activin function is regulated by binding to the extracellular
antagonist follistatin, which also binds some BMPs including BMP-2,
BMP-4, and BMP-7, thus preventing ligand binding to their receptors
(Nakamura et al. 1990; Iemura et al. 1998; Abe et al. 2004; Thompson
et al. 2005). Follistatin is expressed in developing limb digits, and its
expression is regulated by activin, particularly in zones of digital ray formation at sites that mark the positions of developing tendons (Merino
et al. 1999a). Follistatin is expressed by osteoblasts in culture and at sites
of endochondral bone formation in vivo and its expression can be
induced by BMP-2 (Hashimoto et al. 1992; Funaba et al. 1996; Kearns
and Demay 2000; Abe et al. 2004).
ROLES OF TGF-β FAMILY IN SKELETAL PATTERNING
TGF-β family signaling has key roles in the patterning of the cranial,
axial, and appendicular skeletons, with different TGF-β family members
shown to be functionally important in different skeletal elements. The
cross-talk of TGF-β family signals with other signaling pathways, including those activated by Shh, Wnt, PTH-related protein (PTHrP), and
FGFs, results in synergies and antagonisms that restrict or define the sizes
and shapes of the bones and cartilage structures as well as the joints.
Determination of Left–Right Patterning and
Development of Axial Skeleton
Before limb development, members of the TGF-β family act to specify
embryonic germ layers, establish body axes, and initiate left–right
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patterning (Chang et al. 2002). Defects in these fundamental aspects of
embryogenesis can result in skeletal manifestations. The activin-related
nodal is required for mesoderm induction and left–right patterning
(Schier and Shen 2000). Because the transcription factor Zic3 is required
for nodal expression, Zic3–/– mice exhibit axial skeleton malformations
because of insufficient nodal expression and signaling in early embryogenesis (Purandare et al. 2002). Nodal signals through a Smad2-dependent
pathway. Although Smad2–/– and Nodal –/– mice are embryonic-lethal, the
double heterozygous Smad2+/–;Nodal+/– mice are viable, but they die perinatally with defective left–right patterning and craniofacial malformations (Nomura and Li 1998).
Normal development of the axial skeleton requires specification of
the dorsal midline by regulated BMP signaling. Uninhibited BMP signaling in the dorsal mesoderm during somitogenesis causes abnormal
development of the vertebrae and ribs of the axial skeleton (Hogan 1996;
Dale and Jones 1999; Schier 2001). For example, targeted deletion of the
secreted BMP antagonist noggin results in severe rib and vertebral malformations and other skeletal abnormalities (Wijgerde et al. 2005). The
axial skeletal defects of Noggin–/– mice can be rescued by heterozygous
deletion of Bmp4 (Wijgerde et al. 2005). Axial skeletal defects are also
observed in Bmp5 –/–;Bmp7 –/– mice (Dudley et al. 1995; Luo et al. 1995;
Solloway and Robertson 1999).
Regulation of Limb Development by BMP Signaling
BMP signaling is required to establish the limb buds, which give rise to
the appendicular skeleton (Wan and Cao 2005). Two signaling centers—
the apical ectodermal ridge (AER) and the zone of polarizing activity
(ZPA)—communicate to establish a gradient of FGF, Shh, BMP, GDF, and
Wnt signaling that collectively defines the sizes, shapes, axes, and locations of the joints within the appendicular skeleton. Active BMP signaling is also required to establish the AER, as shown by loss of AER
formation and abnormal limb development in mice with a conditional
BMPRIA deletion in the limb ectoderm (Ahn et al. 2001).
Once the AER is established, BMP activity must be tightly regulated. The AER expresses BMP-2 and BMP-4, which direct the dorsal–ventral patterning of the limb mesoderm, establish boundaries to
limit the size of the AER, and eventually contribute to AER regression
(Pizette and Niswander 1999; Ahn et al. 2001; C.K. Wang et al. 2004). The
AER also secretes FGFs, whereas the ZPA produces Shh (Fig. 1). These
FGFs and Shh reinforce the continued expression of each other through
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Figure 1. BMP signaling in the developing limb bud. BMP is required to establish
the apical ectodermal ridge (AER). Later, BMP-2 and BMP-4, produced by the AER,
limit the growth of the AER and zone of polarizing activity (ZPA) by opposing FGF
and sonic hedgehog (Shh) signaling. Gremlin and noggin, whose expression is
induced by FGF, antagonize BMP signaling. Abnormal regulation of BMP activity in
the developing limb results in severe malformation of the appendicular skeleton.
a positive feedback loop that supports the growth and maintenance of
the AER and ZPA. BMPs antagonize this positive feedback loop and
consequently limit the expansion of the AER and ZPA (Niswander and
Martin 1993; Laufer et al. 1994). Insufficient and excessive levels of
BMP activity in the developing limb bud both result in deregulated
appendicular skeletogenesis. Insufficient BMP signaling leads to abnormal expansion of the AER and ZPA, which in turn results in syndactyly,
polydactyly, lack of interdigital apoptosis, and severe limb malformation (C.K. Wang et al. 2004). In contrast, excessive BMP signaling, due
to loss of gremlin, is the cause of the naturally occurring limb deformity (ld) mouse mutation (Zuniga et al. 2004). Gremlin, a secreted
BMP antagonist, is expressed in the developing limb and represses BMP
signaling to allow maturation of ZPA and AER signaling centers. In
gremlin-null mice, the ZPA and AER disintegrate prematurely, resulting in impaired limb outgrowth and digit and bone agenesis (Khokha
et al. 2003).
The same signals that define the ZPA and AER also define the cell
populations in the developing limb that undergo apoptosis. BMP-2,
BMP-4, and BMP-7 are expressed in the interdigital mesenchyme, where
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they promote apoptosis through Smad and p38 mitogen-activated protein (MAP) kinase-dependent pathways (Francis et al. 1994; Lyons
et al. 1995; Yokouchi et al. 1996; Zou and Niswander 1996; Laufer
et al. 1997; Macias et al. 1997). FGF signaling counteracts the apoptotic BMP signals and thereby inhibits apoptosis (Buckland et al. 1998).
Cross-talk with the Wnt pathway has been implicated in the induction
of limb mesenchyme apoptosis, although the precise mechanisms
remain unclear (Soshnikova et al. 2003). The syndactyly observed in
the absence of BMP signaling resembles that observed in transgenic
mice with Noggin expression from the Msx2 promoter (they also have
reduced BMP signaling in the limb mesenchyme) and results from
inhibition of mesenchyme apoptosis (C.K. Wang et al. 2004).
In addition to the role of gremlin in limb outgrowth, other secreted
BMP antagonists have been implicated in limb development, including
noggin, chordin, Tsg, follistatin, and Dan (see Chapter 8). Each of these
is expressed in unique temporal-spatial patterns in the developing limb
and has specific binding affinities for select TGF-β family ligands.
Noggin binds BMP-2, BMP-4, GDF-5, GDF-6, and GDF-7 with picomolar affinity (Re’em-Kalma et al. 1995; Zimmerman et al. 1996; Chang
et al. 1999) and is expressed in the limb mesenchyme and in cartilage
condensations where it regulates joint specification (Brunet et al. 1998).
As part of the cross-talk between FGF and BMP signaling, noggin
expression is induced by FGFs (Warren et al. 2003). A signaling feedback loop between BMP and noggin controls the pattern of BMPRIB
expression, which is thought to control digit size and shape (Merino et
al. 1998). As mentioned, Noggin–/– mice have severe malformations of
the axial and appendicular skeleton (Brunet et al. 1998; McMahon et al.
1998). The observation that these axial malformations, but not their
appendicular defects, are rescued by reduction of BMP levels, suggests
that noggin may antagonize GDF signaling in the appendicular skeleton
(Wijgerde et al. 2005).
Skeletal Malformations in Mice Lacking BMP Ligands or Antagonists
GDF and BMP interactions in skeletal patterning are also apparent in
Gdf5–/– and Bmp5–/– mice (Kingsley 1994; Storm et al. 1994). Inactivation
of Bmp5, which is mutated in the “short ear” mutant mouse, causes defects
in appendicular and axial skeletal morphogenesis (Kingsley 1994).
Although the GDF-5 and BMP-5 expression patterns do not overlap,
Gdf5 –/–;Bmp5–/– mice exhibit sternum malformations that are more severe
than those in either single mutant line alone (Storm and Kingsley 1996).
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This synergy of GDF-5 and BMP-5 between two independent cell populations illustrates a principle of TGF-β family signaling in skeletal development. Despite extensive redundancy, the specific expression patterns
and unique functions of each family member are required for the intricate patterning, development, and maintenance of the skeleton (Storm
and Kingsley 1996).
The same principle helps to explain the phenotypes of Bmp5–/–,
Bmp6 –/–, and Bmp7 –/– mice. Bmp6 –/– mice exhibit delayed chondrocyte
hypertrophy and sternum malformations (Solloway et al. 1998). BMP-6
is coexpressed with BMP-2 and BMP-7 in long bones, which largely
compensates for loss of BMP-6. In sternocostal joints, however, BMP-6
is expressed without BMP-2 and BMP-7, explaining the sternal malformations observed in Bmp6–/– mice (Solloway et al. 1998). Deletion of
BMP-7, which is expressed in bone (Lyons et al. 1995), results in lethal
defects in kidney development as well as polydactyly and abnormal rib
development (Dudley et al. 1995; Luo et al. 1995). As in Bmp6–/– mice,
defects in Bmp7–/– mice occur at sites that exclusively express BMP-7,
suggesting that enough redundancy exists at other locations to compensate for loss of BMP-7 (Dudley and Robertson 1997). In addition,
Bmp5–/–;Bmp7–/– mice have skeletal defects that are much more severe
than in either single knockout, illustrating the functional redundancy of
BMP-5 and BMP-7 (Dudley et al. 1995; Luo et al. 1995; Solloway and
Robertson 1999). Similar compensation among BMPs is observed when
crossing Bmp7–/– mice with Bmp4+/– mice (Katagiri et al. 1998).
Deletion of follistatin, chordin, and Tsg in mice also results in skeletal malformations. Follistatin, which antagonizes activin and BMP signaling, is expressed in proliferating chondrocytes and osteoblasts, and its
expression is regulated by BMP (Funaba et al. 1996). Exogenous
application of follistatin to developing limb buds blocks digit formation,
whereas Fst–/– mice have various skeletal and nonskeletal abnormalities
(Hashimoto et al. 1992; Matzuk et al. 1995b). Tsg is expressed by
osteoblasts and can form a complex with chordin, short gastrulation
(Sog), and BMP, which then acts as a more efficient BMP antagonist than
any component alone (Canalis et al. 2003). However, Tsg can also promote BMP signaling by facilitating the degradation of chordin by tolloidlike proteases (Blader et al. 1997; Piccolo et al. 1997). The phenotype of
Tsg–/– mice reflects both roles of Tsg. Its role as a BMP antagonist may
contribute to the osteoporotic phenotype in the appendicular skeleton,
whereas its BMP promoting function may be important for embryonic
head development (Zakin and De Robertis 2004). Chordin binds BMPs
but not other TGF-β family members. Chordin–/– mice exhibit cranio-
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facial malformations similar to those seen in DiGeorge syndrome (see
further below). Chordin is expressed by chondrocytes and acts as a particularly important regulator of chondrocyte maturation. The malformations of endochondral bone in chordin-null mice result from its action
on chondrocytes rather than on osteoblasts (Zhang et al. 2002; Bachiller
et al. 2003).
TGF-β and Activin Signaling in Skeletal Patterning
Many components of the TGF-β pathway have been evaluated in mice by
targeted deletion, including the three TGF-β ligands, TβRI and TβRII,
Smad2, Smad3, Smad4, LTBP-3, and LTBP-4. Because of their critical
roles in early development, deletions of the genes encoding TβRI, TβRII,
or Smad4 cause early embryonic lethality, precluding analysis of these
proteins in skeletogenesis. Among the mice with targeted gene inactivation who survive through skeletogenesis, inactivation of each individual
gene for an effector of the TGF-β–Smad pathway (except LTBP-4) results
in a bone phenotype. Additional mouse models that delete or overexpress
components of the TGF-β pathways in a tissue-specific manner have been
particularly informative of the role of TGF-β family signaling molecules
in skeletogenesis.
For example, tissue-specific deletion of TβRII under the control of
the Col2a1 (α1(II) procollagen gene) promoter showed that TGF-β
responsiveness of Col2a1-expressing cells is required for normal patterning of the axial skeleton (Baffi et al. 2004). Mutant mice exhibit several
defects in the vertebrae and intervertebral discs. This is most likely due
to loss of TβRII expression in the sclerotome, which expresses Col2a1 and
gives rise to the axial skeleton. Interestingly, chondrocyte differentiation
is normal in these mice, which highlights the importance of the perichondrium in mediating the effects of TGF-β on chondrocytes (Baffi
et al. 2004). TGF-β2 participates in patterning of the digits. TGF-β2 is
expressed in condensing mesenchyme that gives rise to the digits (Pelton
et al. 1989). At that stage, TGF-β prevents apoptosis of interdigital cells.
Increased TGF-β expression can promote the formation of extra digits
based on its chondrogenic activity (Ganan et al. 1996).
TGF-β family signaling also instructs the formation of the bones and
cartilages of the jaw, head, and neck from cranial neural crest cells (Wan
and Cao 2005). Deletion of ALK-2, a type I receptor for BMPs, in the
neural crest results in craniofacial defects, cleft palate, and hypomorphic
mandible formation (Dudas et al. 2004b). Studies in mice and in
humans have revealed a particularly critical role for TGF-β signaling in
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palate formation. Human mutations in TGFBR1 and TGFBR2 genes
(encoding TβRI and TβRII, respectively) are associated with craniosynostoses and cleft palate, as well as several soft-tissue anomalies (Loeys
et al. 2005). Members of the TGF-β family signaling network that are
required for palatogenesis include TGF-β3, TβRI/ALK-5, TβRII, and
Smad2, as revealed from mouse models. For example, cleft palates in
Tgfb3–/– mice are rescued by activation of the ALK-5 receptor or by
targeted overexpression of Smad2 in the medial edge epithelium of the
palate (Dudas et al. 2004; Cui et al. 2005). In addition to cleft palate, targeted deletion of TβRII also results in calvarial agenesis and other skull
defects. These defects were shown to result from impaired cell proliferation and defective signaling interactions with other cranial neural-crestderived tissues, such as the dura mater (Ito et al. 2003). Homozygous or
heterozygous deletion of the MH1 domain of Smad2 results in early
embryonic lethality (Nomura and Li 1998). Smad2+/– mice die later in
development than their homozygote-null littermates and exhibit
mandibular agenesis or hypoplasia. LTBP-3-null mice also have craniofacial malformations, as well as progressive osteosclerosis and osteoarthritis
(Dabovic et al. 2002). The high bone mass phenotype of LTBP-3-null
mice is consistent with a reduction in TGF-β signaling due to insufficient storage or stabilization of TGF-β in the bone matrix (Dabovic
et al. 2005).
Finally, deletion of activin or activin receptor IIB (ActRIIB) in mice
also results in defects in skeletal patterning. Activin βA-deficient mice
exhibit cleft palates (Matzuk et al. 1995c), whereas ActRIIB-deficient
mice exhibit a disruption of axial patterning as specified by the Hox
code, resulting in homeotic transformations of vertebrae, as well as
other major nonskeletal abnormalities (Oh and Li 1997). Exogenous
application of activins in the interdigital space during development
induces formation of extra digits, through functional interactions with
other members of the TGF-β family, Wnts and FGFs (Merino et al.
1999a).
ROLES OF TGF-β FAMILY IN BONE REMODELING
Signaling by TGF-β family proteins regulates the differentiation and
function of the bone-matrix-depositing osteoblasts (discussed in
Chapter 21) and of the bone-matrix-resorbing osteoclasts, as well as the
cross-talk between both cell types, which controls bone remodeling and
homeostasis. Osteoclasts are derived from the monocyte/macrophage
lineage and, when fully differentiated, are large multinucleated cells
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Figure 2. Communication between osteoblasts and osteoclasts during osteoclast
differentiation. Osteoblasts produce M-CSF, RANKL, and OPG, each of which regulates
specific aspects of osteoclast proliferation, differentiation, and survival. TGF-β family
members can direct osteoclastogenesis directly through receptors on osteoclasts or
indirectly through receptors on osteoblasts. Signaling by TGF-β family members in
osteoblasts regulates the expression of M-CSF, RANKL, and OPG, as well as other factors.
(Takahashi et al. 2002). They secrete proteases and create an acidic
microenvironment that mediates resorption of the organic and mineral
components of the bone matrix. Several critical factors are required for
the differentiation of the progenitor cells into this highly specialized cell
type (Fig. 2). Among these, macrophage colony-stimulating factor (M-CSF)
promotes the proliferation of monocyte/macrophage precursors and
induces osteoclast differentiation. In addition, the transmembrane and
soluble forms of RANK ligand (RANKL), expressed by osteoblasts,
bind to the RANK receptor on osteoclasts to activate signaling through
the NF-κB pathway. RANK signaling is required for osteoclast differentiation, function, and survival. Osteoblasts also secrete a soluble
inhibitor of osteoclast differentiation, osteoprotegerin (OPG), which
acts as a “decoy” receptor for RANKL. OPG inhibits activation of the
RANK receptor. A balance of these osteoclast promoting and inhibitory
signals allows calibration and coordination of bone depostion and bone
resorption (Takahashi et al. 2002).
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Direct Actions of TGF-β on Osteoclast Function and Differentiation
Manipulation of TGF-β family signaling by deletion or overexpression of
their signaling mediators often results in changes in bone mass resulting
from altered communication between the osteoblasts and the osteoclasts.
Because osteoclasts depend on osteoblast signals for their differentiation,
function, and survival, the effects of TGF-β family members on osteoblasts
often elicit effects on both bone deposition and bone resorption that result
in changes in bone mass.
TGF-β itself is a major regulator of osteoclast function that acts on
osteoclasts directly and indirectly through osteoblasts. Although the
importance of TGF-β signaling is clear, the literature on this topic seems
confusing. This complexity reflects the cross-talk between osteoblasts and
osteoclasts, the dose-dependent activities of TGF-β on both the osteoblasts
and osteoclasts, and the unique actions of TGF-β at each stage of the
osteoclast life cycle.
Because osteoclasts express TGF-β1 and TGF-β receptors, they can
respond directly to TGF-β signaling (Kaneda et al. 2000). During bone
resorption, osteoclasts release active TGF-β from the bone matrix
through the combined activities of the matrix metalloproteinases
MMP-2 and MMP-9, which proteolytically release active TGF-β from
the latent complex, and the acidic microenvironment that directly
activates the TGF-β ligand (Oreffo et al. 1989; Oursler 1994; Dallas et al.
2002). The release of active TGF-β by osteoclasts is a key factor in the
progression of osteolytic tumor metastases, as described later in this
chapter.
The direct effects of TGF-β on osteoclast differentiation and function
depend on the stage of osteoclast differentiation. TGF-β can inhibit the
recruitment of osteoclast precursors to bone in fetal bone cultures
(Pfeilschifter et al. 1988). However, following infiltration of osteoclast precursors into fetal bones, TGF-β enhances bone resorption by stimulating
the proliferation and differentiation of osteoclast precursors (Tashjian et al.
1985; Shinar and Rodan 1990; Dieudonné et al. 1991). Although the mechanism is as yet uncharacterized, the stimulatory effect of TGF-β on the
differentiation of monocyte/macrophage precursors into osteoclasts is so
potent that many studies include TGF-β in the culture medium to potentiate osteoclast differentiation. One possible underlying mechanism is the
induction by TGF-β of SOCS, a negative regulator of the proinflammatory
Jak-Stat pathway (Fox et al. 2003). SOCS overexpression drives the differentiation of the monocyte/macrophage precursors into the osteoclast
rather than the macrophage lineage. Other investigators have suggested that
the stimulatory effects on early osteoclast differentiation require activation
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of the MAP kinase pathway by TGF-β (Karsdal et al. 2003). TGF-β also
induces the expression of RANK, the receptor for RANKL, which promotes
osteoclast differentiation (Yan et al. 2001). In contrast, TGF-β inhibits later
stages of osteoclast differentiation and function. Accordingly, long-term
treatment with TGF-β results in reduced expression of RANK and other
osteoclast markers and impaired bone resorption (Karsdal et al. 2003).
Regulation of the Cross-talk between Osteoblasts and
Osteoclasts by TGF-β
Other effects of TGF-β on osteoclasts derive from its actions on
osteoblasts. TGF-β regulates osteoblast expression of M-CSF, RANKL,
and OPG, which have key roles in osteoclast differentiation. At low
doses, TGF-β treatment generally results in increased M-CSF expression
and prostaglandin production and an increase in the relative expression
of RANKL to OPG, resulting in promotion of osteoclastogenesis (Karst
et al. 2004). In contrast, high TGF-β levels repress M-CSF and RANKL
expression while increasing OPG expression (Murakami et al. 1998;
Thirunavukkarasu et al. 2001). Because high levels of TGF-β do not
inhibit osteoclastogenesis in pure osteoclast cultures, the inhibitory effects
of TGF-β at high doses are mediated by osteoblasts and thus may serve
as a negative feedback loop to limit bone resorption. The high levels of
TGF-β released during bone resorption switch osteoblasts to an osteoclast-inhibitory pattern of gene expression.
The cross-talk between osteoblasts and osteoclasts is well illustrated
by the phenotypes of mice with increased or decreased autocrine TGF-β
signaling in differentiating osteoblasts. Increased TGF-β expression in
osteoblasts leads to increased bone deposition by osteoblasts, yet progressive bone loss. Bone loss is due to an increase in bone resorption
that outweighs bone deposition (Erlebacher and Derynck 1996). Conversely, decreased TGF-β responsiveness of the osteoblasts due to transgenic expression of a dominant-negative TβRII mutant in osteoblasts
causes a progressive increase in bone mass and trabecular bone volume
(Filvaroff et al. 1999). This increase primarily results from decreased
bone resorption. Therefore, autocrine TGF-β signaling by osteoblasts
directs not only bone matrix deposition, but also bone resorption by
osteoclasts. Consistent with these findings, mice that lack the TGF-βinducible transcription factor TIEG1 have increased numbers of
osteoblasts with reduced expression of differentiation markers. TIEG1null mice exhibit decreased osteoclastogenesis, in part, because of
reduced RANKL and increased OPG expression (Subramaniam et al.
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2005). These findings further illustrate the cross-talk between osteoblasts
and osteoclasts and the role of TGF-β in this communication.
Estrogen is well known to inhibit osteoclast activity and bone
resorption. Several reports suggest that TGF-β is downstream from
estrogen signaling in bone resorption. First, estrogen induces the expression of TGF-β in osteoblast cultures (Oursler et al. 1991; Robinson et al.
1996; Yang et al. 1996). Antibodies against TGF-β repress the osteoclastinhibitory effect of estrogen in osteoblast cocultures (Hughes et al. 1996).
Second, TGF-β has been implicated in the proapoptotic effects of estrogen on osteoclasts (Hughes et al. 1996). Finally, injection of TGF-β into
the marrow cavity decreases the extent of ovariectomy-induced bone loss
(Beaudreuil et al. 1995). Additional work is needed to better understand
the role of TGF-β and other intermediates in the bone-protective effects
of estrogen.
Roles of BMPs in Bone Remodeling
Osteoclasts can also respond directly to BMP signals, consistent with their
expression of BMPRIA (but not BMPRIB), BMPRII, Smad1, Smad5, and
Smad4. Thus, BMP treatment of highly purified primary osteoclast cultures results in phosphorylation of the BMP-specific R-Smads in osteoclast precursors (Okamoto et al. 2006) and increased resorptive activity
(Kaneda et al. 2000). In addition, BMP limits the progression of bone
resorption by inhibiting expression of collagenase-3, which digests collagens I and II (Varghese and Canalis 1997; Zhao et al. 1999). Although
osteoclasts can respond directly to BMPs, most known actions of BMPs
on osteoclasts are indirectly mediated by the osteoblasts. For example,
BMP-induced osteoblast maturation results in increased expression of the
osteoclast differentiation factors M-CSF (Ghosh-Choudhury et al. 2006)
and RANKL (Okamoto et al. 2006). This indirect effect of BMPs on
osteoclasts is evident in mice, in which BMPRIA is conditionally deleted
in osteoblasts (Mishina et al. 2004). These mice exhibit a low bone mass
due to insufficient osteoblast differentiaton and function, which is rescued within 6 months because of insufficient bone resorption. The
osteoblast-specific Bmpr1a deletion results in reduced osteoclast number
and function due to insufficient expression by osteoblasts of RANKL,
which is required for osteoclast differentiaton, function, and survival. A
similar reduction in bone mass is observed when noggin is overexpressed
in osteoblasts (Devlin et al. 2003). The osteoblast number is normal in
noggin-overexpressing mice, but osteoblast function is reduced, resulting
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in reduced osteoclast number and activity. However, a direct effect of
noggin on osteoclast BMP signaling cannot be excluded (Wu et al. 2003).
Therefore, BMPs have an indirect permissive role in osteoclast
differentiation and function by promoting osteoblast differentiation and
function. In addition to this permissive role, BMP inhibits osteoclast differentiation and function by inducing the expression of OPG in
osteoblasts (Wan et al. 2001). Thus, BMP-activated Smad1 displaces the
transcriptional repressor, Hoxc8, to activate transcription from the OPG
promoter (Wan et al. 2001). The inhibitory Smad6, whose expression is
also induced by BMPs, binds Hoxc8 to prevent Hoxc8 displacement from
DNA by Smad1 (Bai et al. 2000).
Effects of Activins on Bone Remodeling
Activin A (inhibin-βA dimer) is secreted by bone marrow cells and, in
addition to regulating erythroid differentiation (Meunier et al. 1988;
Shiozaki et al. 1992; Yamashita et al. 1992), is thought to regulate
osteoclast differentiation of bone marrow macrophages. Activin A can
promote osteoclastogenesis in bone marrow cultures as well as in calvarial organ cultures in the absence of exogenous RANKL and M-CSF (Sakai
et al. 1993; Gaddy-Kurten et al. 2002). Conversely, inhibin blocks osteoclast differentiation in bone marrow cultures, but this effect cannot be
overcome by excess activin or BMP-2, possibly because inhibin acts on
osteoclastogenesis by alternative pathways (Gaddy-Kurten et al. 2002).
Follistatin, which binds and thereby inactivates activin, as well as an antiactivin A antibody, inhibits osteoclastogenesis in cocultures of bone marrow cells and primary osteoblasts, suggesting that activin A produced by
osteoblasts regulates osteoclastogenesis (Murase et al. 2001).
Activin also synergizes with RANKL and M-CSF to stimulate osteoclastogenesis in bone marrow macrophages by increasing osteoclast lineage commitment (Murase et al. 2001; Koseki et al. 2002; Sugatani et al.
2003). However, the terminal differentiation, survival, and activation of
osteoclasts are not affected by activin (Koseki et al. 2002; Sugatani et al.
2003). The mechanism by which activin synergizes with RANKL is not
fully understood, but enhanced nuclear levels of phosphorylated NF-κB,
elevated expression of RANK or JunB (a component of the AP-1 transcription complex), and increased phosphorylation of p38 MAP kinase,
Erk MAP kinase, and Smad2 may all contribute to potentiation of osteoclastogenesis (Murase et al. 2001; Koseki et al. 2002; Sugatani et al. 2003).
Although JNK MAP kinase is essential for osteoclastogenesis (Wagner
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and Karsenty 2001), it does not appear to be activated by the combination of RANKL, M-CSF, and activin in bone marrow macrophages
(Koseki et al. 2002; Sugatani et al. 2003).
ROLES OF TGF-β FAMILY IN TOOTH MORPHOGENESIS
The development of the tooth represents a striking example of how mesenchymal and epithelial cells communicate to define each other’s differentiation. The outer layer of the fully developed tooth is enamel,
deposited by ameloblasts that are of epithelial origin. Inside the enamel
is dentin, which is deposited by the mesenchymally derived odontoblasts.
The center of the tooth contains the pulp, which is analogous to the bone
marrow cavity and consists of mesenchymal and endothelial cells and
contains the nerve endings. TGF-β family signaling regulates tooth
patterning and development.
Among the BMPs, BMP-2, BMP-4, and BMP-7 are expressed in specific patterns in the enamel knot and the dental mesenchyme. As in limb
bud patterning, BMPs interact with Shh and FGFs to pattern teeth
(Hogan 1996). BMP induces the expression of the transcription factor
Islet1 at the sites of incisor formation. Islet1 then induces BMP expression, thus mediating a positive feedback loop that promotes incisor
development while repressing molar development. Islet1 represses the
expression of Barx1, the transcription factor that promotes molar development. Accordingly, antagonism of BMP activity by noggin promotes
Barx1 expression and molar formation (Tucker et al. 1998; Mitsiadis
et al. 2003). In addition, conditional deletion of Bmpr1a in surface
epithelium arrests tooth morphogenesis (Andl et al. 2004).
GDF-5, GDF-6, and GDF-7 are expressed during odontogenesis
in vivo and can be detected in dental follicle tissue at the root-forming
stage (Morotome et al. 1998; Sena et al. 2003), suggesting that GDFs may
have a role in the formation of the dental attachment, including the periodontal ligament. The human bone disorder angel-shaped phalangoepiphyseal dysplasia (ASPED) is caused by heterozygous loss-of-function
mutations in GDF5 and, among other skeletal defects, is characterized by
dental abnormalities including abnormally positioned teeth, premature
loss of teeth, and multiple caries (Holder-Espinasse et al. 2004), indicating that GDF-5 has an important role in positioning, development,
and/or maintenance of teeth.
TGF-β-activated Smad2 and Smad3 are expressed in temporally and
spatially regulated patterns in the developing tooth (Xu et al. 2003) in
both the enamel epithelium and dental mesenchyme. In mandible
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explant cultures, Smad2 antisense oligonucleotides cause hyperproliferation of ameloblasts and advanced tooth development. In contrast, Smad7
antisense oligonucleotides inhibit tooth development and apoptosis of
the enamel depositing epithelium (Ito et al. 2001).
An essential role for activin signaling in tooth patterning and development has been revealed in mice with targeted gene inactivation (Matzuk
et al. 1995a,b,c; Ferguson et al. 1998). Mice defective in activin A expression by deletion of the Inhba gene show impaired development of incisors
and mandibular molars but normal development of maxillary molars. A
similar phenotype is also observed with low penetrance in mice that lack
ActRII (Ferguson et al. 2001) or when soluble extracellular ActRII
domains are administered during tooth development of maxillary and
mandibular explant cultures (Ferguson et al. 2001). In addition, Smad2+/–,
but not Smad3–/–, mice exhibit alterations in tooth development (Ferguson et al. 2001), further suggesting the importance of activin signaling in
tooth development. Activin expression in the mesenchyme, which is maintained by FGF-8 secreted from the oral epithelium (Ferguson et al. 1998),
is required before tooth bud formation and enables incisor and mandibular molar tooth germs to progress beyond the bud stage.
The expression of several genes known to be essential for tooth
development, including Barx1, Msx1, Pax9, and Dlx2, is unaffected in
activin βA-deficient tooth germs (Ferguson et al. 1998). In contrast, the
expression of the homeobox gene Irx1 and follistatin is lost in dental
epithelium of activin βA-deficient mice, including in the maxillary molar
epithelium. Because maxillary molars of activin βA-deficient mice
develop normally, Irx1 and follistatin are not required for development
of these teeth (Ferguson et al. 1998, 2001). Follistatin is involved in early
patterning of tooth development and is expressed in the dental epithelium of all teeth adjacent to and in a complementary pattern to mesenchyme-expressed activin (Matzuk et al. 1995b; Ferguson et al. 1998).
Follistatin is also a critical regulator of tooth crown morphogenesis,
enamel knot formation, and patterning of tooth cusps (Wang et al.
2004b). Follistatin integrates the antagonistic signaling between activin
and BMP-4 in the differentiation of dental epithelium into ameloblasts.
Activin induces the expression of follistatin on the lingual surface of
rodent incisors, where it inhibits ameloblast differentiation induced by
BMP-4 that is secreted by odontoblasts. Its expression is down-regulated
during BMP-4-induced ameloblast differentiation in the labial epithelium
of the developing tooth. Follistatin thereby regulates asymmetric distribution of enamel, which is only deposited on the labial side of the rodent
incisor by differentiated ameloblasts (Wang et al. 2004a).
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ROLES OF TGF-β FAMILY IN HUMAN SKELETAL
PATTERNING DEFECTS
Mutations in GDF5 and BMPR1B Cause
Chondrodysplasias and Brachydactyly
Consistent with the roles of TGF-β family signaling in mammalian patterning, discussed above, various skeletal patterning defects in humans
have been associated with mutations in genes encoding TGF-β family ligands or their signaling mediators (see Table 1). Homozygous mutations
in GDF5, also known as CDMP1, provide the basis for the acromesomelic
chondrodysplasia Hunter-Thompson type (CHTT), Grebe type (CGT),
and DuPan syndromes in humans (Thomas et al. 1996, 1997; Faiyaz-UlHaque et al. 2002a,b), whereas heterozygous mutations in GDF5 can
cause brachydactyly types A2 (BDA2) and C (BDC), symphalangism
(SYM1), and ASPED (Polinkovsky et al. 1997; Holder-Espinasse et al.
2004; Seemann et al. 2005; Kjaer et al. 2006). Homozygous loss of
BMPR1B is implicated in a novel subtype of acromesomelic chondrodysplasia that is accompanied by genital defects (Demirhan et al.
2005), whereas heterozygous missense mutations in BMPR1B cause
BDA2 (Lehmann et al. 2003). Chondrodysplasias are generally characterized by appendicular bone dysmorphogenesis, showing a proximal–distal
gradient in severity, whereas brachydactylies involve shortening and
sometimes the loss of some phalanges. ASPED highly resembles BDC and
is further characterized by angel-shaped phalanges, hip dysplasia, and
dental abnormalities (Holder-Espinasse et al. 2004).
The broad phenotypic variations within and between the various
chondrodysplasias and brachydactylies are partly due to the unique but
overlapping expression patterns and the differential effects of each
mutation in GDF5 and BMPR1B. Missense mutations in GDF5 or
BMPR1B can cause more severe phenotypes than loss-of-function mutations, due to dominant-negative effects by the mutant proteins. For
example, CGT is caused by a mutation in the cystine knot of mature
GDF-5 (CDMPC400Y) that results in impaired secretion and bioavailability
not only of GDF-5, but also of other TGF-β family members with which
it can heterodimerize (Thomas et al. 1997). In BDA2, missense mutations
of BMPR1B cause dominant-negative inhibition of receptor kinase
activation, receptor endocytosis, or transphosphorylation by BMPRII
(Lehmann et al. 2003, 2006). In contrast, other TGF-β family ligands or
receptors may partly compensate for the homozygous loss-of-function
mutations in GDF5 or BMPR1B, as observed in CHTT (Thomas et al.
1996), thus resulting in a less severe phenotype.
heterozygous Cys400Tyr, Arg438Cys,
or Cys498Ser mutation in cystine
knot of GDF-5
heterozygous Ile200Lys mutation in
glycine-serine (GS) domain of
BMPRIB
heterozygous Arg486Trp or Arg486Gln
mutation in NANDOR domain of
BMPRIB
heterozygous Leu441Pro mutation in
receptor interaction interface of
GDF-5
homozygous Leu441Pro mutation
in receptor interaction interface
of GDF-5
heterozygous Arg438Leu mutation
in receptor interaction interface
of GDF-5
Trp217Gly mutation in noggin
Brachydactyly type C
(BDC)
Dawson et al. (2006);
Seemann et al. (2005)
Gong et al. (1999);
Marcelino et al. (2001)
abolished secretion and dimerization
of functional noggin dimers
Faiyaz-Ul-Haque et al.
(2002a)
increased affinity for BMPRIA,
functionally resembling BMP-2
mutant GDF-5 has lost affinity for
BMPRIA and BMPRIB
Kjaer et al. (2006);
Seemann et al. (2005)
Lehmann et al. (2003,
2006)
(Continued)
SYM1, multiple
synostosis syndrome
(SYNS1)
SYNS1
DuPan syndrome
dominant-negative inhibition of
BMPRIB endocytosis or
transphosphorylation by BMPRII
mutant GDF-5 has lost affinity for
BMPRIA and BMPRIB
Everman et al. (2002);
Polinkovsky et al.
(1997)
Lehmann et al. (2003)
Thomas et al. (1997)
2:00 PM
BDA2 or BDC/proximal
symphalangism
(SYM1)-like
BDA2
impaired secretion of (mutant) GDF-5
and of TGF-β family heterodimerization partners
mutations affect normal folding of
GDF-5 and result in early
degradation
dominant-negative inhibition of
BMPRIB kinase activity
References
8/13/07
Brachydactyly type A2
(BDA2)
heterozygous Cys400Tyr mutation
in cystine knot of GDF-5
Chondrodysplasia Grebe
type (CGT)
orders, for which the molecular mechanism causing the disorder has been elucidated
Disorder
Mutation
Functional consequence
Table 1. Overview of mutations in genes encoding TGF-β family ligands or their signaling mediators, identified in various skeletal dis-
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687
single-nucleotide polymorphism
rs15705 in highly conserved motif
of 3′UTR of BMP2 mRNA
heterozygous Arg206His mutation
in GS domain of BMP type I
receptor ALK-2
Osteoporosis
Fibrodysplasia ossificans
progressiva (FOP)
C-509→T polymorphism in promoter
region of TGFB1 gene
increased decay of BMP2 mRNA due
to altered affinity of specific proteins
for SNP sequence
unstable GS domain likely resulting in
increased ALK-2 activation
CC genotype correlates with increased
TGF-β1 serum levels in Japanese
population; TT genotype correlates
with increased TGF-β1 serum levels
in European population
TT genotype correlates with increased
TGF-β1 serum levels
reduced secretion and dimerization
of functional noggin dimers
impaired Smad signaling due to
inability of mutant Runx2 to
interact with receptor-activated
Smads
Functional consequence
Grainger et al. (1999);
Langdahl et al. (2003);
Yamada (2001)
Fritz et al. (2006);
Styrkarsdottir et al.
(2003)
Shore et al. (2006)
Hinke et al. (2001);
Langdahl et al. (2003);
Yamada (2000, 2001)
Gong et al. (1999);
Marcelino et al. (2001)
Otto et al. (2002);
Zhang et al. (2000)
References
11:44 AM
Osteoporosis
Gly189Cys or Pro223Leu
mutation in noggin
heterozygous CCDαA376
mutation, resulting in truncated
Runx2 lacking part of
carboxy-terminal activation
domain
Leu10Pro polymorphism due to
T→C transition in TGF-β1
signal peptide
Mutation
8/15/07
Osteoarthritis (OA),
osteoporosis
Cleidocranial dysplasia
(CCD)
SYM1
Disorder
688
Table 1. (Continued)
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Leu10-12 duplication in exon 1,
encoding signal peptide of TGF-β1
Tyr81His missense mutation in
exon 1 of TGFB1, coding for aminoterminal sequence of LAP
loss-of-function, nonsense, splicesite mutations in SOST
CED
CED
homozygous 52-kb deletion
downstream from SOST
total LEMD3 deletion or heterozygous
loss-of-function mutations in
LEMD3
Osteopoikilosis
Hellemans et al. (2004);
Menten et al. (2007)
Balemans et al. (2001);
Brunkow et al. (2001);
van Bezooijen et al.
(2007)
Balemans et al. (2002);
van Bezooijen et al.
(2007)
Janssens et al. (2003)
Janssens et al. (2003)
Janssens et al. (2003);
Kinoshita et al. (2000);
Saito et al. (2001)
1:48 PM
van Buchem disease
impaired secretion and intracellular
retention of TGF-β1, resulting in
increased TGF-β signaling
impaired secretion of TGF-β1 and
enhanced intracellular TGF-β
signaling
loss of function of SOST protein,
resulting in unopposed Wnt
signaling downstream from BMP
signaling during bone formation
down-regulation of SOST gene
expression, resulting in unopposed
Wnt signaling downstream from
BMP signaling during bone formation
nonsense-mediated decay of LEMD3
or truncated LEMD3 lacking carboxyterminal domain, resulting in impaired
antagonism of Smad activity
increased secretion of bioactive TGF-β1
and enhanced TGF-β signaling
8/15/07
Sclerosteosis
TGFB1 missense mutations in exon 4,
coding for region in LAP that is
responsible for homodimerization
Camurati–Engelmann
disease (CED)
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Mutations in the receptor interaction interface of mature GDF-5 cause
skeletal malformations by affecting the affinity or specificity of GDF-5 for
its receptors. For example, heterozygous or homozygous point mutations
in GDF5 involving the exchange of Leu441 for proline (L441P) result in
BDA2 or the more severe DuPan syndrome, respectively (Faiyaz-Ul-Haque
et al. 2002a; Seemann et al. 2005; Kjaer et al. 2006). The L441P mutant
has lost affinity for BMPRIA and BMPRIB and is functionally strongly
impaired (Seemann et al. 2005; Kjaer et al. 2006). Two different GDF5
point mutations, replacing Arg438 by leucine (R438L) (Seemann et al.
2005; Dawson et al. 2006) or Glu491 by lycine (E491K) (Wang et al. 2006),
were identified in families with proximal symphalangism (SYM1) or multiple synostosis syndrome (SYNS1), limb disorders that were only known
to be caused by mutations in the NOG gene (see below). The R438L
mutant has normal affinity for BMPRIB, but it has increased affinity for
BMPRIA and functionally resembles BMP-2, resulting in loss of receptor
specificity and the SYM1 and SYNS1 phenotypes (Seemann et al. 2005).
NOG Mutations Cause Symphalangism and Synostoses Syndromes
Heterozygous mutations in the NOG gene, encoding the secreted BMP
antagonist noggin, form the basis for various patterning disorders affecting joint development, including SYM1, SYNS1, tarsal/carpal coalition
syndrome (TCC), and autosomal dominant stapes ankylosis. SYM1,
SYNS1, and TCC are generally characterized by the absence of joint cartilage and fusion of carpal and tarsal bones and phalanges (Gong et al.
1999; Dixon et al. 2001). Patients with congenital stapes ankylosis suffer
mainly from conductive hearing loss and share several skeletal features
with SYM1 and SYNS1 patients but do not display symphalangism
(Brown et al. 2002). As these disorders start in childhood and continue
through adult life, noggin seems to be essential to preserve joints during
adult life, and loss of BMP antagonism results in joint fusion.
The NOG mutations identified in SYM1, SYNS1, and TCC are predominantly single missense substitutions of evolutionary conserved
amino acids (Gong et al. 1999; Debeer et al. 2004). Heterozygous NOG
mutations identified in congenital stapes ankylosis are predicted to disrupt the cysteine-rich carboxy-terminal domain of noggin (Brown et al.
2002). Deletions of the carboxy-terminal domain of noggin have also been
reported in a family with SYM1 and in a family with SYNS1 (Takahashi
et al. 2001). The different phenotypes associated with similar or identical
NOG mutations (Dixon et al. 2001; Takahashi et al. 2001; Brown et al.
2002) might be explained by allelic differences in noggin modifier genes.
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Functional analysis of the SYM1 and SYNS1 noggin mutants revealed
that secretion and dimerization of the SYNS1 mutant is abrogated,
whereas the two SYM1 mutations reduce secretion of the noggin dimer
(Marcelino et al. 2001), possibly explaining the more severe phenotype
of SYNS1. The mutants did not interfere with secretion of the wild-type
noggin dimer. Moreover, the noggin mutants are able to interact with
GDF-5 and antagonize BMP signaling in Xenopus embryos (Marcelino
et al. 2001), suggesting that reduced secretion rather than functional
impairment of noggin contributes to development of SYM1 and SYNS1.
Noggin is broadly expressed during development, and it is interesting that heterozygous mutations of NOG mainly affect joint morphogenesis without affecting other tissues. This may result from the
functional redundancy by other antagonists or differences in dosage
requirements for BMPs in various tissues.
Functional Haploinsufficiency of Runx2 Causes Cleidocranial Dysplasia
Smad proteins physically interact with the osteoblast transcription factor
Runx2, and their cooperation is important for osteoblast differentiation
(see Chapter 21). Thus, the balance of cooperation with interacting
Smads defines the Runx2 function. Heterozygous loss-of-function mutations in RUNX2 have been identified in cleidocranial dysplasia (CCD)
(Otto et al. 2002), a bone disorder involving defective endochondral and
intramembranous bone formation that results in multiple skeletal
abnormalities such as persistently open or delayed fontanelle closure,
additional cranial plates, rudimentary clavicles, and short stature, all
indicative of defective patterning. Runx2–/– mice demonstrated that
Runx2 is required for maturation of osteoblasts and bone formation,
whereas the Runx2+/– phenotype or radiation-induced mutation of
Runx2 results in symptoms that closely resemble those observed in CCD
patients (Mundlos et al. 1997; Otto et al. 1997).
The RUNX2 nonsense mutation CCDαA376, identified in three
unrelated CCD patients (Otto et al. 2002), encodes a truncated Runx2
protein that lacks a large part of its carboxy-terminal activation domain.
This truncated protein exerts a dominant-negative effect on wild-type
Runx2, likely through its Runt domain, which retains the ability to bind
DNA and interact with CBFβ (also known as PEBP2β). The CCDαA376
Runx2 protein fails to interact with BMP or TGF-β receptor-activated
Smads and displays severely impaired transactivation activity. Moreover,
whereas overexpression of Runx2 in BMP-2–treated C2C12 myoblasts
activates alkaline phosphatase expression, overexpression of CCDαA376
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Runx2 does not (Zhang et al. 2000). Thus, the phenotypic manifestations
of CCD might be explained in part by impaired Smad signaling due to
functional haploinsufficiency of the osteoblast-specific transcriptional
coregulator Runx2. Furthermore, mice that overexpress TGF-β2 in bone
also exhibit phenotypic features of CCD, consistent with the ability of
TGF-β to repress Runx2 function (Erlebacher and Derynck 1996; Alliston et al. 2001).
THE TGF-β FAMILY IN RHEUMATOID
ARTHRITIS AND OSTEOARTHRITIS
Rheumatoid arthritis and osteoarthritis are joint diseases characterized
by destruction of articular cartilage and bone, fibrosis, and loss of joint
function. Osteoarthritis is additionally characterized by sclerosis of subchondral bone and formation of osteophytes, that is, cartilage outgrowths
at the joint margins undergoing endochondral ossification. Rheumatoid
arthritis is a chronic autoimmune disease that primarily results in inflammation of the synovial membrane of peripheral joints. Synovial cells proliferate and develop into an invasive lesion that infiltrates cartilage and
bone. Already at early stages of rheumatoid arthritis, chondrocytes synthesize reduced levels of proteoglycans that causes loss of cartilage
matrix. Biomechanical, metabolic, and genetic factors, rather than
inflammation, are thought to contribute to osteoarthritis, primarily
affecting articular cartilage. However, elevated expression of the proinflammatory cytokine interleukin 1 (IL-1) is often detected in the synovium of joints of osteoarthritis. The pathology of osteoarthritis reflects
attempted repair of the damaged extracellular matrix by chondrocytes,
overruled by matrix-degrading enzymes produced by chondrocytes.
Several TGF-βs, BMPs, and GDFs are expressed at elevated levels in
synovial tissue of patients with rheumatoid arthritis and osteoarthritis,
although their roles in the progression of these diseases are poorly understood (Fava et al. 1989; Lafyatis et al. 1989; Lories et al. 2003; Nakase
et al. 2003). TGF-β is thought to protect against the development of
rheumatoid arthritis through its potent antiinflammatory activity,
thereby inhibiting macrophage and T-cell proliferation (Lafyatis et al.
1989; Lotz et al. 1990). Overexpression of dominant-negative TβRII or
TGF-β1 in murine cells indicated that functional TGF-β signaling in lymphoid cells protects against rheumatoid arthritis (Chernajovsky et al.
1997; Schramm et al. 2004). Furthermore, TGF-β counteracts the
destructive activity of IL-1 on articular cartilage through down-regulation of IL-1 receptor expression (Redini et al. 1993; Takahashi et al. 2005).
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Endogenous TGF-β signaling is also involved in the development of
osteoarthritis (Serra et al. 1997; Yang et al. 2001; Li et al. 2006; Tchetina et
al. 2006). A missense mutation in the linker region of Smad3 has been
detected in a patient with osteoarthritis and was shown to confer elevated
serum levels of the matrix-degrading metalloproteinases MMP-2 and MMP9 (Yao et al. 2003). An additional link is provided by the small extracellular
matrix protein asporin, a leucine-rich proteoglycan that is abundantly
expressed in articular cartilage of patients with osteoarthritis and binds
TGF-β, thus inhibiting TGF-β’s chondrogenic activity. A human Asp14 allele
is overrepresented in individuals with osteoarthritis and confers a greater
inhibition of TGF-β signaling than wild-type asporin (Kizawa et al. 2005).
Blocking endogenous TGF-β activity in murine models of osteoarthritis by
injection of soluble TβRII ectodomain or expression of the LAP of TGF-β1
or Smad7, or deletion of histone deacetylase 4, revealed a protective role for
TGF-β in articular cartilage damage and adverse effects of TGF-β signaling
on osteophyte formation (Scharstuhl et al. 2002, 2003; Vega et al. 2004), suggesting a dual role for TGF-β in the pathogenesis of osteoarthritis.
TGF-β protects against cartilage destruction by inducing differentiation
in synoviocytes, resulting in increased cartilage matrix protein expression
(Lafyatis et al. 1989). Moreover, prolonged exposure to TGF-β sensitizes
normal articular chondrocytes to TGF-β-induced proteoglycan synthesis
(van Beuningen et al. 1994; Gelse et al. 2001), consistent with the stronger
induction by TGF-β of proteoglycan synthesis by osteoarthritic cartilage
than by healthy cartilage (Lafeber et al. 1993). Excessive TGF-β signaling can
cause joint space narrowing due to cartilage overgrowth. Whereas BMP-2
and BMP-9 induce proteoglycan synthesis in normal articular cartilage, they
do not restore cartilage loss in a mouse model of arthritis, due to their
inability to counteract IL-1 activity (Glansbeek et al. 1997). GDFs are able
to reconstitute proteoglycan content in matrix-depleted cartilage explants
and stimulate articular chondrocytes of osteoarthritis to synthesize proteoglycans (Erlacher et al. 1998a; Bobacz et al. 2002).
Endogenous TGF-β and BMP contribute to pathogenesis of
osteoarthritis by inducing osteophyte formation and synovial thickening,
which can be reduced by overexpression of Smad6 and Smad7 as seen in
experimental models of osteoarthritis (Scharstuhl et al. 2003; Blaney
Davidson et al. 2006). Synovial macrophages contribute significantly to
TGF-β-induced osteophyte formation and produce factors, possibly
BMP-2 and BMP-4, that, in concert with TGF-β, can induce cartilage formation by mesenchymal cells in vitro (van Lent et al. 2004).
Adverse effects of TGF-β may result partly from synergy with IL-1
(Takahashi et al. 2005) and induction of expression of aggrecanase-1 and
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urokinase-type plasminogen activator (uPA) in synovial fibroblasts
(Hamilton et al. 1991; Yamanishi et al. 2002), possibly contributing to
matrix degradation as seen upon increased TGF-β1 expression in antigen-induced arthritic rabbit knees (Mi et al. 2003).
Osteoarthritis seems to protect against osteoporotic fractures
(Dequeker et al. 2003), and an inverse correlation has been found
between the occurrence of a Leu10Pro polymorphism in the TGF-β1 signal peptide sequence, caused by a T→C base substitution, and the frequency of spinal osteoarthritis and osteoporosis in the Japanese
population. The CC genotype correlates with a higher risk for
osteoarthritis than the TC or TT genotype, and the C allele is associated
with elevated TGF-β serum levels. On the other hand, the T allele is a
risk factor for genetic susceptibility to postmenopausal osteoporosis in
Japanese women (Yamada et al. 1998; Yamada 2000). Recently, it was
shown that the T allele is also associated with increased inflammatory
activity, poor long-term disease outcome, and mortality in rheumatoid
arthritis of Northern European patients (Mattey et al. 2005).
TGFB1 POLYMORPHISMS AND OSTEOPOROSIS
Osteoporosis commonly affects postmenopausal women and the
elderly and is characterized by increased fracture risk resulting from
reduced bone quality, including low bone mass and deteriorated
micro-architecture of the bone. Multiple factors contribute to the etiology of osteoporosis, and the genetic susceptibility of low bone density may result from combined effects of polymorphisms in a number
of genes involved in bone homeostasis. Although many polymorphisms in the TGFB1 gene exist, only a few are associated with bone
mineral density, osteoporosis, and/or fracture risk. The pathophysiological mechanisms by which these TGFB1 polymorphisms affect bone
density and susceptibility to bone fracture are not known. The availability of TGF-β1 is likely altered as a result of altered splicing or stability of the TGF-β1 mRNA, intracellular trafficking and/or efficiency
of export of the TGF-β1 preproprotein, or transcriptional activation
of the TGFB1 promoter (Hinke et al. 2001; Yamada 2001; Dick et al.
2003; Langdahl et al. 2003). Recently, TGF-β was shown to regulate
material properties of bone matrix, but the role of these properties in
osteoporosis requires further investigation (Balooch et al. 2005). Polymorphisms in the BMP2 and BMP4 genes have also been linked to low
bone mass and osteoporosis (Styrkarsdottir et al. 2003; Ramesh Babu
et al. 2005; Fritz et al. 2006).
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ECTOPIC BONE FORMATION
Fibrodysplasia ossificans progressiva (FOP), also known as myositis
ossificans progressiva, is a rare genetic connective tissue disorder characterized by progressive postnatal endochondral ossification of tendons, ligaments, fascia, and striated muscle. In affected individuals, osteogenic lesions
often arise spontaneously, but they can be induced by surgery, trauma, or
intramuscular injections. Interestingly, normal skeletogenesis is not affected.
Although the pathophysiology of initiation and development of early
FOP lesions is largely unknown, the current hypothesis is that BMP-4
overexpressing lymphocytes infiltrate connective tissue after local soft-tissue injury. The elevated local BMP-4 levels may cause recruitment of
mesenchymal stem cells, which in turn produce autocrine BMP-4 and
develop into fibroproliferative and, ultimately, osseous lesions (Shafritz
et al. 1996; Kan et al. 2004). In support of this hypothesis, BMP signaling is up-regulated in several ways in lymphoblastoid cell lines (LCLs)
and fibroblast-like lesional cells from FOP patients (Shafritz et al. 1996;
Gannon et al. 1997). Specifically, these cells display increased BMP-4
transcription (Olmsted et al. 2003), increased levels of hyperphosphorylated BMPRIA at the cell surface (de la Pena et al. 2005), increased BMPinducible MAP kinase activation (Fiori et al. 2006), and decreased
expression of BMP antagonists (Ahn et al. 2003). Targeted overexpression
of BMP-4 in similar cell types of mice also results in an FOP-like phenotype (Kan et al. 2004). Although these studies suggest that increased
BMP signaling contributes to the development of FOP, linkage analysis
and mutational screening indicate that the BMP4 or BMPR1A genes are
not the genetic cause of FOP. In fact, the genetic defect involves a heterozygous Arg206His mutation in the glycine-serine domain of the BMP
type I receptor ALK-2, causing instability of the receptor and possibly
resulting in increased activation of ALK-2 (Shore et al. 2006). More
research is needed to understand the molecular mechanisms of FOP.
SCLEROSING BONE DISORDERS
Activating Mutations in TGFB1 Cause Camurati–Engelmann Disease
TGFB1 mutations resulting in a mutant signal peptide or the propeptide
LAP form the pathological basis for Camurati–Engelmann disease (CED),
also referred to as progressive diaphyseal dysplasia. CED is a craniotubular dysplasia, characterized by hyperostosis and sclerosis of the diaphyses
of the long bones and base of the skull. Progressive muscle fibrosis is also
frequently observed. Most TGFB1 mutations associated with CED are
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located in exon 4 (Janssens et al. 2000, 2006; Kinoshita et al. 2000, 2004),
which encodes the carboxy-terminal sequence of LAP and is important
for its interaction with mature TGF-β1 (see Chapter 7). These mutations
result in a significant increase in bioactive TGF-β1 secreted from the cell
and correlate with enhanced TGF-β signaling (Saito et al. 2001; Janssens
et al. 2003). Other CED-associated mutations in the signal peptide of the
TGF-β1 precursor or in the amino-terminal segment of the LAP interfere with intracellular trafficking or secretion. These mutations enhance
intracellular activation of the TGF-β pathway (Janssens et al. 2003). Thus,
elevated TGF-β1 signaling that favors bone formation over bone resorption appears to underlie the pathophysiological mechanism of CED.
As TGF-β1 is made by many cell types, it is intriguing that CED patients
predominantly show hyperostosis and muscle fibrosis without other tissues
being affected. Bone abnormalities may prevail in CED patients because
osteoblasts, in contrast to many other cell types, also secrete small latent
TGF-β complexes lacking LTBPs (Bonewald et al. 1991; Dallas et al. 1994).
LTBPs facilitate folding and secretion of the TGF-β1 complex and can modulate TGF-β bioavailability (see Chapter 7). Interestingly, LTBP-3-null mice
show osteosclerotic bone malformations that affect the long bones and skull
(Dabovic et al. 2002). Locus heterogeneity has been described to underlie
CED (Hecht et al. 2001; Nishimura et al. 2002), and the identification of
another modifying gene or locus linked to CED may provide clues as to why
no other tissues are affected by the TGF-β1 mutations.
Loss of Sclerostin, a BMP Antagonist, Leads to
Sclerosteosis and van Buchem Disease
Among the BMP antagonists is sclerostin, a member of the DAN family of secreted cystine-knot-containing glycoproteins that is encoded by
the SOST gene. Mutations in SOST are the genetic basis for sclerosteosis and van Buchem disease, two progressive craniotubular hyperostotic
disorders. Loss-of-function mutations in SOST prevail in sclerosteosis,
whereas in van Buchem disease, a homozygous 52-kbp deletion downstream from SOST has been identified, probably harboring regulatory
elements that control SOST expression (Balemans et al. 2001, 2002;
Brunkow et al. 2001). Loss of SOST expression results in increased
osteoblast activity, causing thicker than normal trabeculae and cortical
bone, increased bone mineral density, and increased bone strength. This
leads to massive bone overgrowth throughout postnatal life and entrapment of cranial nerves and mandibular overgrowth. Tall stature and syndactyly discriminates sclerosteosis from the milder van Buchem disease.
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Although sclerostin can bind to various BMPs and interferes with
their binding to BMP receptors (Winkler et al. 2003), sclerostin inhibits
BMP-stimulated bone formation indirectly through binding to the LRP5
and LRP6 Wnt coreceptors, thus antagonizing downstream Wnt signaling (Li et al. 2005; Semenov et al. 2005; Ellies et al. 2006; van Bezooijen
et al. 2007). Excessive postnatal bone deposition in sclerosteosis patients
likely results from unopposed Wnt signaling due to loss of sclerostin
expression in osteocytes. Underscoring the role of sclerostin in bone
homeostasis, mice overexpressing SOST under control of the osteocalcin
promoter display an osteopenic phenotype with reduced bone formation
and fragile bones, resembling osteoporosis (Winkler et al. 2003).
Loss of LEMD3, a Smad Antagonist, Results in Osteopoikilosis
Osteopoikilosis is a skeletal dysplasia characterized by symmetric but
unequal distribution of hyperostotic areas in different parts of the skeleton. Buschke-Ollendorff syndrome (BOS) and melorheostosis are allelic
variants of osteopoikilosis with additional abnormalities, including sclerotic skin lesions. Linkage analysis revealed that these disorders are caused
by total deletion of LEMD3 (LEM domain-containing 3, also called MAN1)
or heterozygous loss-of-function mutations in LEMD3 (Hellemans et al.
2004; Menten et al. 2007). LEMD3 is an integral protein of the inner
nuclear membrane and antagonizes Smad activity through interaction of
its RNA recognition motif (RRM) region with the MH2 domain of BMPor TGF-β-activated Smads. This prevents R-Smad–Smad4 interaction and
nuclear translocation, resulting in impaired TGF-β- and BMP-induced
gene expression (Osada et al. 2003; Hellemans et al. 2004; Lin et al. 2005).
The LEMD3 loss-of-function mutations identified so far result in
either decay of mRNA from the mutant allele or expression of truncated
LEMD3 protein lacking the Smad-interacting segment (Hellemans et al.
2004, 2006). Expression of loss-of-function mutants fails to reduce
TGF-β-induced luciferase reporter activation; moreover, skin fibroblasts
from an affected person showed enhanced expression of a TGF-β-responsive gene (Hellemans et al. 2004). These findings suggest that haploinsufficiency of LEMD3 confers increased BMP and/or TGF-β signaling,
which in turn leads to hyperostotic bone lesions in osteopoikilosis, BOS,
and melorheostosis. As LEMD3 appears to be ubiquitously expressed (Lin
et al. 2000), it is unclear at present why a specific bone phenotype is
observed in osteopoikilosis.
Other genetic factors may contribute to the additional abnormalities
in BOS and melorheostosis, as no somatic mutations in the second
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LEMD3 allele occur in skin and bone lesions of these patients (Hellemans
et al. 2004, 2006; Mumm et al. 2007). Increased TGF-β signaling has been
implicated in the development of skin fibrosis and may contribute to the
skin lesions associated with BOS and melorheostosis.
CANCER METASTASIS TO BONE
Osteolytic Breast Cancer Bone Metastases
Bone provides an attractive microenvironment for the homing and
metastasis of particular cancer types. Breast tumor cells preferentially and
frequently metastasize to bone where they give rise to osteolytic lesions,
causing hypercalcemia, bone pain and fracture, and nerve compression
syndromes (Pluijm et al. 2000).
An important mediator of breast-tumor-induced bone destruction is
PTHrP (parathyroid-hormone-related protein), which stimulates RANKL
expression and inhibits OPG expression by osteoblasts, thereby activating osteoclasts (Guise and Chirgwin 2003). Autocrine PTHrP secretion
by the breast tumor cells causes bone destruction that results in release
of TGF-β from the bone matrix. In turn, TGF-β promotes progression of
osteolytic bone metastases by inducing PTHrP expression by breast
tumor cells in a Smad- and p38 MAP kinase-dependent manner (Yin
et al. 1999; Kakonen et al. 2002), thus establishing a positive feedback
loop for mutual control of PTHrP and TGF-β expression in tumorinduced bone destruction (Guise and Chirgwin 2003).
In addition to stimulating osteoclast activity, breast tumor cells may
aggravate osteolytic bone lesions by inhibiting osteoblast differentiation
and function. TGF-β produced by the breast cancer cells mediates this
inhibition and may furthermore reduce adhesion and induce apoptosis
of osteoblasts (Mastro et al. 2004; Mercer et al. 2004). Breast tumor cells
metastasized to bone show a prominent increase in expression of the
angiogenic connective tissue growth factor (CTGF) and IL-11, an activator of osteoclast differentiation (Kang et al. 2003). TGF-β induces the
expression of IL-11 and CTGF (Chirgwin and Guise 2000; Kang et al.
2003, 2005). Smad4 is essential for induction of IL-11 and contributes to
the formation of osteolytic bone metastases (Kang et al. 2005; Deckers et
al. 2006). Combined overexpression of IL-11 and CTGF together with
osteopontin endows breast cancer cells with potent metastatic activity,
suggesting mechanisms by which TGF-β may promote breast-cancerinduced bone destruction.
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Osteoblastic Prostate Cancer Bone Metastasis
In contrast to breast cancer cells that generally cause osteolytic bone
metastases, dissemination of prostate cancer cells to bone typically results
in osteoblastic metastases, resulting in bone pain and increased risk of
bone fracture due to the poor quality of newly formed bone. The molecular mechanisms that contribute to osteoblastic metastasis are poorly
understood, but a role for TGF-β and BMPs is suspected.
Elevated TGF-β expression by prostate cancer cells correlates with
poor prognosis (Ivanovic et al. 1995). Prostate tumor cells with increased
TGF-β1 expression have lost sensitivity to the growth-inhibitory effects of
TGF-β and instead show enhanced tumorigenicity and metastasis (Steiner
and Barrack 1992), to which various mechanisms may contribute. Thus,
bone-derived TGF-β1 is chemotactic and stimulates invasion of prostate
cancer cells in vitro (Festuccia et al. 1999a,b, 2000). TGF-β also induces
the expression of proteases by these cells, including prostate-specific antigen (PSA) and uPA, which may not only facilitate tumor invasiveness, but
also activate latent TGF-β (Killian et al. 1993; Festuccia et al. 1999a, 2000).
TGF-β induces the expression of the α2β1 and α3β1 integrins by prostate
cancer cells, thus resulting in increased adhesion to type I collagen
(Kostenuik et al. 1997; Festuccia et al. 1999a), which may enable disseminating prostate cancer cells to adhere to bone matrix.
Increased expression of BMP-6 or BMP-7 (Hamdy et al. 1997;
Masuda et al. 2003) and reduced expression of BMP-2, Smad4, Smad8,
or BMP receptors have been detected in bone metastases of prostate cancer and correlated with poor prognosis (Ide et al. 1997; Kim et al. 2000;
Horvath et al. 2004). Proliferation and tumorigenicity of prostate tumor
cell lines can be inhibited by increasing BMP signaling (Brubaker et al.
2004; Miyazaki et al. 2004), and reduced Smad and BMP receptor
expression during malignant progression might allow prostate tumor
cells to escape the growth-inhibitory effects of BMPs. BMPs expressed by
prostate cancer cells can induce osteoblasts to form new bone, resulting
in osteoblastic lesions (Dai et al. 2005).
FRACTURE HEALING
Fracture healing largely recapitulates embryonic endochondral bone
formation and involves repair of broken bone to original quality and
function. Upon injury, chemotactic factors including TGF-β and BMPs
are released from the bone matrix and platelets. In addition, inflamma-
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tory cells, such as monocytes, macrophages, and T cells, that are recruited
into the fracture site (Andrew et al. 1994) secrete cytokines and growth
factors, including TGF-β1 and BMP-2 (Champagne et al. 2002), that are
chemotactic and osteoinductive to mesenchymal precursor cells, preosteoblasts, and chondroblasts (Cunningham et al. 1992; Lind et al.
1996). These cells consequently proliferate and undergo chondrogenic
and/or osteogenic differentiation to form a callus that fills the fracture
gap. Although the osteoinductive capacity of TGF-β is overall weak in
comparison to BMPs, TGF-β1 synergizes with BMPs and insulin-like
growth factor-1 in fracture healing (Schmidmaier et al. 2003; Centrella
et al. 1994), likely by enhancing recruitment and proliferation of progenitor cells to the fracture site. The distinct spatial and temporal expression patterns of various TGF-βs, BMPs, and GDFs during fracture healing
(Cho et al. 2002) suggest that they may have unique roles during bone
healing. In addition, the expression of distinct BMP type I and type II
receptors is up-regulated during fracture healing, and the spatial distribution of these receptors resembles that of BMP-2, BMP-4, and BMP-7
(Ishidou et al. 1995; Onishi et al. 1998).
Although their effects on bone are complex, ectopic application of
TGF-β, BMP-2, BMP-4, or BMP-7 in animal models of fracture healing as
well as in patients revealed a potent role for these growth factors in regeneration of bone at the fracture site, repair of critical-size defects, improved
healing of non-union fractures, and accelerating fracture healing, resulting
in increased bone or callus formation and mechanical stability (Joyce et al.
1990; Yasko et al. 1992; Lind et al. 1993; Cook et al. 1994; Nielsen et al.
1994; Bostrom et al. 1996; Friedlaender et al. 2001; Govender et al. 2002;
Einhorn et al. 2003; Edwards et al. 2004). Calibration of growth factor dose
and timing will improve therapeutic outcomes by targeting specific cell
populations at the appropriate stage of differentiation.
THERAPEUTIC POTENTIAL
The role of TGF-β family members in skeletal disorders involves, on the
one hand, increased signaling as seen in conditions such as FOP and sclerosing bone disorders, osteosarcomas with expression of BMPs and/or
BMPRII, pathological conditions of ectopic ossification affecting the
spinal ligament, and traumatic heterotopic ossification as is frequently
seen in patients that have had head and neck traumas or total hip
replacement. On the other hand, reduced signaling by TGF-β family
members can also contribute to development of skeletal disorders, as is
the case in slowly healing or non-union fractures.
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For those disorders that result from excessive BMP signaling, BMP
antagonists such as noggin might restore the balance in BMP activity.
Thus, intramuscular gene transfer of Noggin prevents onset and progression of the spondyloarthropathy ankylosing enthesitis (Lories et al. 2005),
a chronic inflammatory joint disorder characterized by joint ankylosis due
to new bone formation in which BMP-2, BMP-6, and BMP-7 are thought
to have a role. Promise holds for deletion mutants of stable osteoinhibitory
BMP folding variants, which were even more effective than noggin in
inhibiting BMP activity by competing with natural BMPs for binding to
the BMP receptors (Weber et al. 2003).
Drugs that selectively enhance the expression of TGF-β or BMPs may
provide therapeutic approaches for diseases such as arthritis and osteoporosis. Diacerein and avocado/soya unsaponifiables have been shown to
be beneficial both in animal models of osteoarthritis and in patients with
osteoarthritis (Maheu et al. 1998; Dougados 2001) and are thought to act
by inducing expression of TGF-β1 and TGF-β2 (Boumediene et al. 1999;
Felisaz et al. 1999). Isoflavones extracted from Sophorae fructus act like
estrogen by binding to estrogen receptors and may be useful in the treatment of estrogen-deficient osteoporosis patients, because these isoflavones were shown to induce expression of TGF-β (Joo et al. 2004).
Combined therapies might be necessary for diseases such as rheumatoid arthritis and osteoarthritis whereby TGF-β has a protective role as
well as a pathological role. Thus, application of TGF-β to reduce inflammation and promote cartilage repair, combined with a BMP antagonist to
reduce osteophyte formation, might control the disease. BMP-2, BMP-7,
and GDF-5 have been shown to be effective in animal models of
(osteo)chondral defects in cartilage repair or intervertebral disc cartilage
repair (Mont et al. 2004; Chujo et al. 2006; Masuda et al. 2006). Studies
involving in vivo gene therapy of arthritis look promising (Evans et al.
2006), but clinical application will depend on the development of safe and
effective vectors. Cell-mediated gene transfer provides an alternative, safer
method for gene therapy. Injection of ex-vivo-transduced mesenchymal
cells with adenoviral BMP-2 enables repair of articular cartilage defects in
rodents, although osteophyte formation was observed (Gelse et al. 2003).
BMPs are attractive molecules for orthopedic applications and are
shown to be effective and safe in the healing of critical-size defects and
delayed or non-union fractures (Friedlaender et al. 2001; Govender et al.
2002) (see Chapter 33). BMP-2 and BMP-7 have been approved by the
U.S. Food and Drug Administration for treatment of open tibial fractures
and spinal fusion. BMP-9 and BMP mixtures extracted from bovine bone
are under investigation in clinical trials.
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For regeneration of cartilage and bone, optimal effects are achieved
when BMPs are applied in combination with a carrier that prolongs the
residence time and function of BMPs at target sites and allows for a significant reduction in the quantity of BMP required compared to bolus
injections (Seeherman and Wozney 2005). In humans, BMPs loaded onto
collagen type I carriers are as effective as autogenous bone grafting
(Friedlaender et al. 2001; Johnsson et al. 2002) and optimization of
dosage, mixture of BMPs added, time course, release dynamics, and composition of matrix carrier may ultimately result in therapeutic application of BMPs that are superior to bone grafts.
When designing experiments to study the role of a particular TGF-β
family member in a certain disease process, it should be realized that
these growth factors exert pleiotropic effects and may need to synergize
with other family members to achieve the desired biological response and
that the biological effect triggered by addition of a particular TGF-β family member will depend on growth factor concentration, species, cell type,
stage of differentiation, and local presence of additional growth factors.
A tempting task for the future will be to reveal optimal growth factor or
growth factor antagonist concentrations, combinations, delivery methods, and duration of growth factor and/or antagonist administration to
functionally restore the affected tissues in the various skeletal disorders
in which TGF-β family members are involved.
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Ahn K., Mishina Y., Hanks M.C., Behringer R.R., and Crenshaw E.B., 3rd. 2001. BMPR-IA
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Alliston T.N. and Derynck R. 2000. Transforming growth factor-β in skeletal development
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