Biochemical, Genetic, and Molecular Interactions in Development - part 2 potx

Chondrocyte Cell Fate Determination 27
SRY-related HMG- Campomelic dysplasia, Short limb dwarfism, large
Targeted disruption. Perinatal lethality. Skeletal defects in
box gene 9; SOX9, autosomal dominant
anterior fontanelle, macrocephaly, all bones derived from endochondral ossification, include
17q24.3-q25.1 (114290)
micrognathia, cleft palate, hypo- cleft secondary palate, hypoplasia and bending. Skeletal
(608160)
plastic thoracic cage, missing
abnormalities similar to those found in campomelic dysplasia
twelfth pair of ribs, hypoplastic, patients. Skeletal patterning was not affected. Premature
poorly ossified cervical vertebrae, m
ineralization of skeletal elements, including craniofacial
small iliac wings, short phalanges
region and vertebral column. Hypertrophic zone of growth
for both hands and feet, anterior plate was thicker
(208).
bowing of tibia, short fibula,
mildly bowed femur, absent
ossification of proximal tibial,
and distal femoral epiphysis
T-box 5;
Holt-Oram syndrome, Vertebral anomalies, thoracic
Conditional knockout. Embryonic lethality because of mal-
TBX5, 12q24.1 autosomal dominant
scoliosis, absent or bifid thumb,
formed heart tube. Elongated phalangeal segments of first
(601620)
(142900)
triphalangeal thumb, carpal forelimb digit and hypoplastic falciformis bones in the
bone anomalies, upper extremity

wrist
were present in multiple heterozygous mutant
phocomelia, radial-ulnar
mice
(209).
anomalies
Transforming growth Camurati-Engelmann
Sclerosis of skull base, mandible Targeted disruption. Lethality around weaning due to
factor, beta-1; disease autosomal
involvement, sclerosis of pos- massive inflammation lesions and tissue necrosis in
TGFB1, 19q13.1 dominant (131300)
terior part of vertebrae, scoliosis, many organs
(210,211)
.
(190180)
progressive diaphyseal widening,
thickened cortices, narrowing of
medullary canal
Vitamin D3 receptor; Vitamin D-resistant rickets,
Rickets
Targeted disruption. Animals normal until after weaning.
VDR, 12q12-q14 autosomal recessive
By 7 wk, null mice develop alopecia, flat face and short
(601769)
(277440)
nose. Severe bone malformation leading to growth retarda-
tion and 40% loss of bone density. Early lethality around
15 wk
(212).
a

Human gene description includes gene name, symbol, corresponding OMIM number, and locus.
b
Human disease description includes disease name and corresponding OMIM number.
27
28 Shum et al.
Table 2
Human Genetic Disorders with As-Yet No Known Genetic Associations
Disorder name
OMIM number Gene location
Brief description of skeletal defects
Acrocallosal syndrome;
200990
12p13.3-p11.2
Macrocephaly, large anterior fontanel, prominent occiput and forehead, hypoplastic
ACLS
midface, cleft palate, tapered fingers, fifth finger clinodactyly, brachydactyly,
postaxial polydactyly, bifid terminal phalanges of thumbs, toe syndactyly,
duplicated halluces
Chondrocalcinosis 1; CCAL1
600668 8q
(CCAL1) CCAL1; chondrocalcinosis, severe degenerative osteoarthritis
Chondrocalcinosis 2; CCAL2
118600
5p15(CCAL2)
CCAL2; chondrocalcinosis, arthropathy, acute intermittent arthritis, ankylosis
Chondoma; CHDM
215400 7q33
Sacrococcygeal chordoma
Cohen syndrome; COH1
216550 8q22-q23

Microcephaly, maxillary hypoplasia, micrognathia, joint hyperextensibility,
narrow hands and feet, mild shortening of metacarpals and metatarsals
Craniometaphyseal dysplasia;
218400 6q21-q22
Cranial hyperostosis, facial palsy, prominent supraorbital ridges and mandible,
CMDR
square profile, diaphyseal sclerosis, metaphyseal dysplasia, metaphyseal broadening
Otopalatodigital syndrome,
304120
Xq28
Prominent forehead, severe micrognathia, midface hypoplasia, cleft palate, sclerotic
type II; OPD2
skull base, bowing of long bones, small to absent fibula, subluxed elbow, wrist,
and knee, flexed, overlapping fingers, short, broad thumbs, postaxial polydactyly,
syndactyly, second finger clinodactyly, hypoplastic, irregular metacarpals
Craniosynostosis,
600593 4p16
Craniosynostosis, coned epiphyses of hands and feet, distal and middle phalangeal
Adelaide type; CRSA
hypoplasia, carpal bone malsegmentation, phalangeal, tarsonavicular and calcaneo-
navicular foot fusions
FG syndrome; FGS1
305450
Xq12-q21.31
Macrocephaly, large anterior fontanel caused by delayed closure, plagiocephaly,
micrognathia, cleft palate, joint contractures, broad thumbs, clinodactyly, syndactyly,
broad halluces
Fibrodysplasia ossificans
135100 4q27-q31
Heterotopic ossification, especially of the neck, spine, and shoulder girdle,

progressiva; FOP
malformed cervical vertebrae, short broad femoral necks, malformed big toes,
monophalangic big toes, short thumbs, fifth finger clinodactyly, severely restricted
arm mobility
Larsen syndrome; LRS1
150250 3p21.1-p14.1 Cleft palate, flattened frontal bone, small skull base, shallow orbits, dysplastic
epiphyseal centers, cervical vertebrae hypoplasia, scoliosis, spondylolysis, short
metacarpals and metatarsals, multiple carpal and calcaneal ossification centers with
delayed coalescence
28
Chondrocyte Cell Fate Determination 29
Otopalatodigital syndrome,
311300
Xq28
Prominent occiput and supraorbital ridges, cleft palate, absent frontal and sphenoid
type I; OPD1
sinuses, thick frontal bone and skull base, delayed closure of anterior fontanel,
steep clivus, dense middle-ear ossicles, short, broad distal phalanges, especially
thumbs, short third, fourth, fifth metacarpals, supernumerary carpal bones, fusion
of hamate and capitate, toe syndactyly, anomalous fifth metatarsal, extracalcaneal
ossification center
Pituitary dwarfism II
262500 5p13-p12
Acrohypoplasia, short limbs, delayed bone age, markedly advanced osseous
maturation for height and age
Russell-Silver syndrome;
180860 7p11.2
Micrognathia, skeletal maturation retardation, craniofacial disproportion, delayed
RSS
fontanel closure, asymmetry of arms and/or legs, fifth finger clinodactyly, fifth

digit middle or distal phalangeal hypoplasia, syndactyly of second and third toes
Shwachman-diamond
260400
Costochondral thickening, irregular ossification at anterior rib ends, delayed skel
etal
syndrome
maturation, slipped capital
femoral epiphyses, metaphyseal chondrodysplasia of
long bones
Sotos syndrome
117550 5q35
Macrocephaly, frontal bossing, prognathism, advanced bone age, large hands and
feet, disharmonic maturation of phalanges and carpal bones
Spastic paraplegia 9; SPG9
601162
10q23.3-q24.1
Skeletal abnormalities, short fifth finger, clinodactyly, delayed bone age, shallow
acetabulum, small carpal bones, dysplastic skull base
Syndactyly, type I
185900 2q34-q36
Syndactyly, complete or partial webbing between third and fourth fingers, fusion
of third and fourth finger distal phalanges, complete or partial webbing between
the second and third toes
Velocardiofacial syndrome
192430 22q11
Microcephaly, Pierre Robin syndrome, cleft palate
29
30 Shum et al.
the many functions of BMPs is to induce cartilage, bone, and connective tissue formation in verte-
brates (24,25). This osteochondro-inductive capacity of BMPs is highly promising for orthopedic

applications, such as skeletal repair and regeneration, and in dental applications, such as the treat-
ment of periodontal diseases (26–30). Since the discovery of BMPs over three decades ago, their abil-
ity to induce ectopic bone and cartilage formation remains a topic of intense investigation. In particular,
the characterization of the molecular mechanisms of BMP functions was reignited after the cloning
of the activin receptor, the first TGF-` type receptor, in 1991 (31). Thereafter, the molecular pathways
to differentiation have been meticulously dissected and exposed.
BMP signals through heterodimeric serine–threonine kinase receptor complexes, containing type
I and type II receptors, each class having a number of subtypes (32,33). Both type I and type II recep-
tors are capable of low-affinity interaction with BMP but only when the ligand binds to both receptors
can result in high-affinity heteromeric ligand–receptor complex formation capable of BMP-depen-
dent signaling (34,35). Therefore, it is likely that the presence and number of different BMP receptors
determine the cellular responses to the many ligands. Evidence suggests that the subtype BMPR-IB
is essential for chondrogenesis for the entire developing skeletal system (36–39). However, target
deletion studies of the BMPR-IB receptor suggest otherwise (39,40). In these animals, the BMPR-IB
does appear to have an essential role to play during limb bud morphogenesis because the abnormali-
ties are located in the appendicular skeletal elements and not in the axial skeletal structures. Moreover,
in vitro studies show that BMPR-IB does not possess exclusive chondrogenic potential, suggesting
that other BMP type I receptors may exert redundant functions during chondrogenesis (41–43). Taken
together, the response to BMP signal is not solely defined by the identity of the type I receptor but
additionally by elements in the signal transduction pathways that lie downstream of the receptor. These
are the various cytoplasmic and nuclear transducers, both positive and negative.
Downstream from the receptors, Smads are the predominant effectors of TGF-`/BMP signaling (44,
45). An important issue for BMP-dependent signaling is the type of Smad proteins involved in chondro-
genic differentiation and whether the Smads alone are sufficient to direct differentiation. Smads func-
tion as dimeric complexes and belong to three classes: regulatory, inhibitory, and common. The receptor-
regulated Smads (R-Smads) are further subdivided into two groups. Smad1, Smad5, and Smad8 are
directly phosphorylated and activated by BMP type I receptors. Smad2 and Smad3 are mediators of
activin or TGF-` type I receptor signaling. A series of in vitro studies have shown that Smad1, Smad5,
and Smad8 may be involved in osteochondrogenic differentiation (46–49). These findings suggest that
different Smads or Smad combinations are engaged at different stages of mesenchymal cell differentia-

tion into osteoblasts and chondrocytes. However, in vivo manipulations of Smads have not resulted
in conclusive evidence because genetically engineered animal models targeted against Smads pro-
duce embryonic lethality (50). Nevertheless, a glimpse of in vivo Smad function can be observed in
Smad3 knockout animals, which manifest osteopenia and early onset osteoarthritis (51). The class of
inhibitory Smads (I-Smads) includes Smad6 and Smad7. They have been shown to inhibit the effect of
R-Smads by competing for binding to activated type I receptors (52–56). Indeed, I-Smads are potent
inhibitors of skeletogenic differentiation (48,57,58). The common Smad4 (Co-Smad) associates with
activated R-Smad complex, which translocates into the nucleus and participates in the regulation of
target genes (59). Smad4 functions as a tumor suppressor gene, and mutations of the human SMAD4
lead to pancreatic carcinoma and juvenile intestinal polyposis, further illustrating the significance of
TGF-` superfamily signaling and its regulation of cellular physiology (60).
BMP signaling can be channeled through Smad-independent pathways, such as the extracellular
signal-regulated kinase, Jun N-terminal kinase, Wnt, and p38 mitogen-activated protein kinase path-
ways (61–65). Therefore, crosstalk between the signaling pathways during chondrogenic differentia-
tion is inevitable. However, a detailed recount of these interactions is beyond the scope of this review.
Finally, BMP and other growth factor signaling can coactivate chondrogenic differentiation. For exam-
ple, fibroblast growth factor (FGF) signaling through mitogen-activated protein kinase promotes
Chondrocyte Cell Fate Determination 31
chondrogenesis by increasing the level of Sox9 expression as well as increases its binding affinity on
the type II collagen promoter (66). It is obvious that BMPs control of chondrogenesis is a highly regu-
lated developmental process that involves multiple pathways and checkpoints. This combinatorial
mode of signaling ensures fidelity in the patterning and timing of the cartilaginous template onto which
most of the bony skeleton is produced.
CRANIOFACIAL MORPHOGENESIS
AND CRANIAL NEURAL CREST CELLS (CNCCS)
CNCCs give rise to most of the craniofacial tissues (67–69). Interestingly, this cell population is
derived from the dorsal cephalic neural tube. During embryogenesis, the ectoderm at the midline over-
lying the notochord thickens to form the neural plate. Progressively, the flattened neural plate begins
to bend, creating elevations, called the neural folds, with a central depression the neural groove. As
neurulation proceeds, the bilateral neural folds oppose each other and fuse at the midline to form the

closed neural tube. At the time of neural tube closure and at the junction of where the thickened neuro-
ectoderm meets the non-thickened surface ectoderm, epithelial cells delaminate and emerge as mes-
enchymal cells into the underlying space. These are the neural crest cells (70). Neural crest cells are
formed along the entire length of the primary neural tube. CNCCs are formed from the neural tube at
the level of the forebrain, midbrain, and hindbrain.
Neural crest cells are multipotential, and they give rise to a number of cell lineages (71,72). Those
arising from the cranial region have different sets of potentials when compared with those arising in
the trunk. For example, trunk neural crest cells do not normally produce cartilage. However, recent
evidence from lineage tracing and transplantation strategies suggest that some trunk crest cells are
capable of differentiating into cranial cartilages when transplanted into the cranial region (73,74).
From a number of studies using various lineage tracing approaches, we have learned that neural crest
cells from the forebrain and midbrain contribute to the frontonasal mesenchyme for the formation of
the upper and midface structures, including part of the cranial base, nasal, and otic capsules (75–77).
CNCCs in the branchial arches are destined for skeletal, odontogenic, myogenic, neuronal, and con-
nective tissue lineages of the lower face and neck regions. Following the cartilage lineage in particular,
CNCCs in the first branchial arch contribute to form Meckel’s cartilage and the temporomandibular
joint cartilage. The hyoid is derived from CNCCs in both the second and third arches, and the fourth
and sixth arches in combination give rise to the thyroid, cricoid, arytenoid, corniculate, and cunei-
form cartilage (68,72,75,78–80).
BMP REGULATION OF CRANIOFACIAL
CARTILAGE DEVELOPMENT AND APOPTOSIS
The hindbrain is a segmented structure, each segment called a rhombomere (Fig. 1). In the verte-
brate head, there are eight pairs of rhombomeres and each gives rise to segment-specific CNCCs.
During the migratory phase of CNCC development, CNCCs converge into three major streams directed
toward the branchial arches in an orderly and patterned manner (81,82). Therefore, an early step in
the regulation of craniofacial cartilage differentiation is CNCC production and patterning within the
hindbrain. Similar to setting up the overall body plan, the hindbrain is patterned by a series of homeo-
box (Hox)- and homeobox-containing genes (83). The production of CNCCs from these rhombomeres
is in part regulated by their Hox genes. In addition, cell fate determination in the CNCCs is an orches-
trated process (84–86). CNCCs exert a “community effect” among themselves and cell–cell and/or

cell–matrix signaling in the group can maintain their segmental identity (87,88). In addition to this
“community” effect, it is also discovered that the isthmus, a region between the midbrain and hind-
brain, serves as a patterning center for the rhombomeres and the CNCC derivatives. The isthmus
expresses high levels of FGF8 that regulates the expression of the Hox genes in the rhombomeres.
32 Shum et al.
Transplantation experiments that include or exclude the isthmus yield different outcomes. The inclu-
sion of the isthmus during grafting allows the rhombomeres and CNCCs to maintain their original iden-
tity, whereas the exclusion of the isthmus renders CNCCs responsive to environmental cues (89). In
addition to the isthmus, CNCCs can be patterned by signals from the endoderm to give rise to distinct
pieces of craniofacial cartilages. Interestingly, this is only limited to CNCCs above the level of the
second rhombomere, the so-called Hox-negative cells. CNCCs expressing Hox genes are not respon-
sive to endodermal induction (90).
Although each rhombomere can give rise to CNCCs, it is observed that those of rhombomeres 3
and 5 contribute to a minority of the population. A large number of CNCCs within the rhombomere
undergo apoptosis, and only a small population migrate out. These cells join the major streams and,
thus, lateral to rhombomeres 3 and 5, the area appears relatively free of CNCCs (91–98). This may
serve to gauge the number of CNCCs being produced and to better delimit the migratory streams and
their eventual destination. Evidence suggests that CNCC apoptosis is regulated by BMP and Wnt
signaling. BMP4 is expressed coincidentally within rhombomeres 3 and 5. BMP4 induces the expres-
sion of Msx2 in these rhombomeres, and ectopic expression of Msx2 increases the number of apopto-
tic CNCCs (5,13,93,99). The lack of BMP signaling in even-numbered rhombomeres may be attributed
to the presence of the BMP antagonist, noggin (100).Taken together, these experiments suggest that
CNCC apoptosis is regulated by signals from BMP4 and is mediated by Msx2. Wnt signaling is signif-
icant in this cascade because of the expression of cSFRP2 in rhombomeres that have limited apoptosis.
cSFRP2 is an antagonist of the Wnt signaling and overexpression of cSFRP2 inhibits BMP4 expression
and rescues CNCC from apoptotic elimination. Consistently, inhibition of cSFRP2 or overexpression
of Wnt1 results in ectopic CNCC apoptosis (101). However, another Wnt family member; Wnt6, has
been recently shown to be necessary and sufficient for the induction of neural crest formation (102).
The use of different Wnt genes in combination that regulate CNCC formation is an elegant example
of the complexity of the system.

As CNCCs migrate from the neural tube towards the forming face, they converge into major streams,
migrating toward the respective branchial arches. Migration is largely governed by adhesive proper-
ties between cells and substrate, and a number of factors have defining roles in this developmental
event (103). During migration, the cells remain in an undifferentiated state such that they are allowed
to reach their destination before they expand further and undergo overt differentiation. Localization
studies reveal that premigratory CNCCs and a subpopulation of migrating CNCCs may already be par-
tially committed to the cartilage lineage by virtue of their expression of the key cartilage transcription
factor, Sox9 (104,105). However, these cells do not differentiate yet. Differentiation of these cells may
be suppressed by the coexpression of Msx2 in the Sox9-expressing cells. Msx2 may serve to main-
tain these cells in an undifferentiated state until migration is completed. Overexpression of dominant-
negative forms of Msx2 in these migratory cells inhibits normal Msx2 functions and leads to preco-
cious cartilage differentiation (105).
The mandible and maxilla arise from the anterior and posterior processes of the first branchial arch,
respectively. These structures receive extensive contributions of CNCCs from the posterior midbrain
and rhombomeres 1 and 2 of the anterior hindbrain. In addition to the lineages found in the other
branchial arches, CNCCs in the first arch also differentiate into tooth structures that are unique to this
arch (106,107).
Meckel’s cartilage formed within the mandibular process has a unique pattern. It consists of an
anterior, triangular piece at the midline, bilateral rod-shaped pieces that regress to form the sphenom-
andibular ligament, and posterior pieces that give rise to the malleus, incus, and temporomandibular
joint cartilage. The formation of Meckel’s cartilage is regulated by the mandibular epithelium through
epithelial-mesenchymal interactions (108,109). The instructive signal from the epithelium can be
substituted by epidermal growth factor (EGF), which sustains mesenchymal proliferation and delays
chondrocyte differentiation (110,111). Removal of the epithelium results in increased but dysmorphic
cartilage formation (112,113). Indeed, EGF and EGF receptors are endogenous to the mandibular
Chondrocyte Cell Fate Determination 33
process (114,115). Antisense oligonucleotide inhibition of EGF in the mandibular process results in
ectopic cartilage formation. In contrast, exogenous EGF reduces and disrupts cartilage formation
(46,115). Furthermore, targeted disruption of EGF receptor in the mouse results in Meckel’s carti-
lage deficiency as well (116). These defects are attributed to changes in matrix metalloproteinases

expression and its regulation of cartilage morphogenesis. Expression of matrix metalloproteinases is
regulated by EGF, and they function in multiple tissue morphogenesis, including that of the anterior
segment of the developing Meckel’s cartilage (117).
Within the mesenchyme, cartilage formation is further delimited by the expression of the tran-
scription factor Msx2, which is excluded from regions with chondrogenic potential (118). Antisense
oligonucleotide inhibition of Msx2 expression in the mandible results in disruption of Meckel’s car-
tilage formation (119). Furthermore, adenoviral expression of ectopic Msx2 also abrogates cartilage
formation (120). Interestingly, endogenous Msx2 expression is regulated by BMP expression and
that ectopic BMP signaling can alter Msx2 expression domain, leading to cartilage dysmorphogenesis
(120–122). Msx2 can also inhibit ectopic cartilage formation that is induced by BMP4 as a feedback
reaction. However, the competence of the mesenchyme to respond to BMP4 is dependent on local
signals and the key cartilage transcription factor Sox9, functions in antagonistic combination with
Msx2 to regulate cartilage formation (120).
LIMB MORPHOGENESIS AND LIMB MESENCHYME
The limb cartilage develops from paired primordial buds that appear on the embryo’s lateral sur-
face at specific levels along its anterior posterior body axis. At the early stages of limb development,
the buds exhibit a paddle shape and consist of undifferentiated mesenchymal cells derived from the
lateral plate and somitic mesoderm, and overlying ectoderm. At the distal tip of the bud, the ectoderm
forms a specialized thickened epithelial structure, known as the apical ectodermal ridge (AER). Pat-
terning along the proximal–distal axis depends in part on signaling molecules from the AER (123,
124). Instrumental to this process is the family of FGFs (125–130). The classic model of limb pat-
terning involves the determination of positional values along the proximal–distal axis specified by
instructive signaling from the AER to the subridge mesenchyme, known as the progress zone (131).
However, recent revolutionary interpretation of limb patterning describes the specification of distinct
proximal–distal segments of the limb early in development, with subsequent development involving
expansion of these mesenchymal progenitor before differentiation (128,132). The anterior–posterior
axis of the limb is patterned by the zone of polarizing activity (ZPA), which is located at the posterior
margin of the limb (124,133,134). The major morphogen from this organizing center is the sonic hedge-
hog (Shh) gene (135), which maintains anterior–posterior patterning in conjunction with other gene
products, such as the HoxD gene (136), and participate in regulatory feedback signaling with the AER

(137). Dorsal–ventral patterning is governed by ectodermally expressed Wnt7a and engrailed-1 pro-
teins and their coregulation of Lmx1b gene expression at the dorsal mesenchyme (138,139). Therefore,
patterning along the three axes is interlinked with each other.
The limb cartilage elements form in a temporal proximal-to-distal sequence but are initially con-
tiguous (36). Through the gradual recruitment of cells, the primary condensation of the stylopod
(humorous/femur) forms first, the zeugopod (radius-ulna/tibia-fibula) forms second, and the autopod
(carpals/tarsals and phalanges) forms last. There is considerable mixing of cells along the proximal–
distal axis within each future segment but not between segments. Positional information is expressed
by determinants of the Hox family of genes. The first part of the limb in which a subset of Hoxa and
Hoxd genes are activated is the posterior limb (140,141). Subsequently, the expression domains extend
anteriorly, in the distal part. In the final stage of limb morphogenesis, the mesenchyme in the distal
region of the limb bud (autopod) can have two different fates, chondrogenesis or apoptosis, depend-
ing on whether they are incorporated into the digital ray or into the interdigital regions. There is now
considerable evidence to indicate that BMPs are essential mediators in specifying mesenchymal cells
34 Shum et al.
undergoing either apoptosis or chondrogenesis and in the determination of digit identity (3,6,142–144).
This point will be elaborated further in the next section. Finally, in regions of the mesenchymal conden-
sation where joints from, condensed chondroprogenitors do not differentiate into chondrocytes but
instead become tightly packed and adopt a fate of apoptosis as part of the normal program (25,145,146).
Therefore, the orchestration of the apoptotic and chondrogenic response results in the formation and
delineation of the limb cartilaginous template. Failure of either process results in limb malformations
such as syndactyly or polydactyly of soft or hard tissues.
BMP REGULATION OF LIMB
CARTILAGE DEVELOPMENT AND APOPTOSIS
BMPs are instrumental to the formation of the limb and are intimately involved in multiple stages
of limb development, including patterning, outgrowth, AER regression, digit formation, digit identity,
and interdigital apoptosis. To function in multiple developmental events, BMPs engage in signaling
networks during limb morphogenesis and operate in concert with other key morphoregulatory factors,
such as FGFs and Shh. Furthermore, several BMPs are already present during early development.
BMP2, BMP4, and BMP7 are expressed in the limb mesenchyme in overlapping patterns before the

formation of precartilagenous condensation (20). The specificity of BMPs for multistep action during
limb morphogenesis is also reflected by different expression profile of the receptor subtypes trans-
ducing the BMP signal (38). BMPs at an early stage regulate mesenchymal condensation into carti-
lage nodules, as well as the induction of the AER (147). At later stages, BMPs are responsible for the
maturation of limb cartilage and the regression of the AER (148). In vitro evidence supports that exog-
enous BMP enhances chondrogenesis in limb mesenchyme after the condensation step (149). Through
their function in the maintenance of the AER and consequential regulation of limb outgrowth along
the proximal–distal axis, BMPs also relay information and participate in interdependent developmen-
tal processes, such as patterning along the dorsal–ventral axis (150). There is little genetic evidence to
support the role of BMPs in limb development because target mutation in animal studies of BMPs
and their receptors result in early lethality or lack of phenotype directly related to cartilage formation
(14,20,151). However, experiments using retroviral-mediated misexpression to simulate loss of func-
tion result in limbs that show a lack of Alcian blue stain cartilage elements (37,42). However, infec-
tion of the chick limb with retrovirus encoding BMP2, BMP4, or constitutively active receptor type
I to simulate BMP gain of function results in fusion and hyperplasia of the cartilage elements (37,152).
Mouse models show that BMP receptor type IB appears to be the necessary mediator of BMP-induced
chondrogenesis (39,40), although overexpression of the receptor or constitutive activation of the
receptor can also cause apoptosis (37,153).
In addition to driving chondrogenesis, BMPs are key regulators of interdigital apoptosis that leads
to the delineation of the digits. Among them, BMP2, BMP4, and BMP7 are expressed in the inter-
digital regions before and during the occurrence of apoptosis, suggesting a role in cell death (20).
Implantation of BMP4-soaked beads in interdigital regions accelerates interdigital cell death. In addi-
tion, BMP4 can also cause ectopic cell death when applied at the tip of the developing digit pad (154).
The apoptotic effect of BMP4 can be antagonized by FGF2 (155). Similarly, BMP2 and BMP7 are
potent apoptotic signals for the undifferentiated limb mesenchyme but not for the ectoderm or the
differentiating chondrogenic cells (156). Perturbations of BMP signaling through manipulation of
BMP receptors also result in aberrations in interdigital apoptosis. For example, overexpressing domi-
nant-negative BMP receptors in chick leg bud via replication-competent retrovirus to block endog-
enous BMP signals results in inhibition of apoptosis in the interdigital mesenchyme, which leads to
webbed chick feet (37).Taken together, these results indicate that BMP signaling is necessary for the

apoptotic cascade in the interdigital mesenchyme. Interestingly, in parallel with craniofacial apoptosis
as described in previous sections, Msx2 is also a mediator of BMP-induced interdigital apoptosis
Chondrocyte Cell Fate Determination 35
(157). However, it is still unclear as to how Msx2 expression is instructive or permissive to apoptosis.
Therefore, the totality of limb development and the emergence of its intricate design are dependent in
part on BMP signaling in the larger context of many other growth and transcription factor signaling
networks.
Of particular interest are the role of retinoic acid and its interactions with BMP signaling and their
coregulation of limb development. Retinoic acid is an endogenous morphogen at physiological levels
and a teratogen in excess. Endogenous retinoids serve to pattern the hindbrain and the limb bud (158).
Excessive retinoids lead to retinoic acid embryopathy characterized by craniofacial abnormalities
(159). There are three distinct aspects of how retinoic acid modulates BMP signals. First, retinoic acid
is well known for its ability to pattern the limb bud by virtue of its ability to substitute for the ZPA
and for its upregulation of Shh that is endogenous to the ZPA (160,161). In tandem, retinoic acid also
upregulates BMP expression that is needed for anterior–posterior patterning event (162,163). Second,
retinoic acid regulates interdigital apoptosis by activating BMP expression and activities (164). Third,
retinoic acid can also enhance chondrogenesis mediated by both BMP-dependent and BMP-indepen-
dent pathways (164,165). Therefore, the regulation of chondrogenesis and apoptosis by BMP may rest
on the ability of retinoic acid to divert BMP signaling to one pathway vs another, or the regulation by
retinoic acid on distinct cofactors of BMP signaling for different pathways.
SUMMARY AND FUTURE CHALLENGES
Studies from classical developmental models suggest that cell fate determination is a progressive
process that is dependent on combinatorial signaling of a repertoire of growth and differentiation fac-
tor networks. Signaling is modulated by restricted expression profiles of factors organized in precise
temporal and spatial arrays. Signaling is gauged by checkpoints where rate-limiting factors determine
the threshold for progression. Because BMPs are multifunctional factors, the challenge is to identify
the molecular basis for chondrogenic differentiation of mesenchymal cells. Functional studies should
establish the mechanisms of lineage commitment and diversification, and provide a platform for molec-
ular manipulations with predictable lineage outcomes. This knowledge will provide the molecular basis
for tissue engineering and biomimetics of mesenchymal cells.

ACKNOWLEDGMENTS
We are grateful to Dr. Rocky Tuan for his support and encouragement. We have benefited from
a long-standing scientific partnership with Dr. Glen Nuckolls. We have been blessed with outstand-
ing visiting and postdoctoral scientists who had contributed to our knowledge base. Finally, we are
indebted to Dr. Harold Slavkin, who continues to be an inspiration. This work was supported by NIH
funding Z01AR41114.
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Regulation of Chondrocyte Differentiation 43
43
From: The Skeleton: Biochemical, Genetic, and Molecular Interactions in Development and Homeostasis
Edited by: E. J. Massaro and J. M. Rogers © Humana Press Inc., Totowa, NJ
3
Regulation of Chondrocyte Differentiation
Andreia M. Ionescu, M. Hicham Drissi, and Regis J. O’Keefe
ENDOCHONDRAL OSSIFICATION: OVERVIEW
During the last decade, great progress has been made toward a better understanding of skeletal
development, cartilage, and bone formation. In particular, many mechanisms underlying a variety of
cellular and molecular processes that regulate growth and differentiation of chondrocytes, osteoblasts,
and osteoclasts have been elucidated. This chapter will review some of the molecular and genetic path-
ways known to regulate cartilage development. Skeletal formation occurs through both endochondral
and intramembraneous ossification. Flat bones and craniofacial bones are formed through intramem-
braneous ossification that relies on osteoblast differentiation directly from mesenchymal stem cells.
The axial and appendicular skeleton form through endochondral ossification, which requires the forma-
tion of a cartilage intermediate that forms a template for osteoid deposition and bone formation. During
endochondral bone formation, mesenchymal stem cells differentiate into both chondrocytes and osteo-
blasts. During development of the long bone, growth plates localize to either end of the skeletal element
and the region of cartilage is surrounded by a perichondrium that is composed of undifferentiated
mesenchymal cells. In the growth plates, chondrocytes undergo several stages of differentiation. One
of the important transitions is from proliferation to hypertrophy, an event that precedes mineraliza-

tion of the cartilage matrix (Fig. 1). Chondrocyte hypertrophy is characterized by profound physical
and biochemical changes, including a 5- to 10-fold increase in volume and expression of alkaline phos-
phatase, type X collagen, and MMP-13 (1,2). Type X collagen is a short-chain collagen found only in
the hypertrophic zone of the growth plate. Although its exact function remains unclear, mutations in
the colX gene have been found to cause Schmid metaphyseal chondrodysplasia (3), and transgenic
mice with disruption in the colX gene exhibit a mild alteration of the growth plate architecture (4).
Alkaline phosphatase is essential for calcification of the matrix and is present in high concentration
in matrix vesicles, which are small membrane vesicles released by budding from the surfaces of hyper-
trophic chondrocytes into the surrounding matrix (5). Matrix vesicles are the initial sites of mineral-
ization in the hypertrophic region of the growth plate and are critical components of the calcification
process. The calcified matrix subsequently serves as the template for primary bone formation. In paral-
lel, the perichondrium flanking the cartilage element differentiates into osteoblast-forming periosteum.
Primary bone formation is initiated at the center of the cartilage template and results in the subse-
quent formation of two separate regions of endochondral bone that develop at either end of the long bone.
The growth plate is responsible for longitudinal growth of bones. Both chondrocyte proliferation and
44 Ionescu et al.
hypertrophy contribute to lengthening of the limb (6,7). Because terminally differentiated hypertrophic
cartilage is continuously replaced by bone, the tight regulation of the various steps of chondrocyte dif-
ferentiation, particularly proliferation and hypertrophy, is critical for balancing the growth and ossifi-
cation of the skeletal elements.
Both local and systemic signaling molecules regulate endochondral ossification. Here we review
some of the factors that regulate chondrocyte maturation, including parathyroid hormone-related pep-
tide (PTHrP), Indian hedgehog (Ihh), transforming growth factor-` (TGF-`), and bone morphogenetic
proteins (BMPs). Although these factors are also involved in the early stages of endochondral ossifi-
cation, such as chondrogenesis and differentiation of precursor mesenchymal cells in chondrocyte, we
specifically address their role in the precise transition of chondrocytes from the proliferative phase to
the hypertrophic phase (Fig. 1).
IHH/PTHRP SIGNALING LOOP:
A CLASSIC MODEL FOR CHONDROCYTE DIFFERENTIATIONS
The paradigm for regulation of endochondral ossification involves the Ihh gene. Ihh expression

delineates the zone of prehypertrophic chondrocytes. During limb development, secreted Ihh binds
to receptors located on perichondrial cells and influences the expression of PTHrP by periarticular
chondrocytes. PTHrP subsequently signals back to the growth plate and regulates the rate of chondro-
cyte differentiation (Fig. 2). It is still unclear whether Ihh controls PTHrP expression directly or in-
directly through TGF-` superfamily members. Finally, it is possible that the role of Ihh in skeletal
Fig. 1. Regulation of chondrocyte maturation. Multiple factors control cell differentiation from mesenchymal
stem cells to hypertrophic chondrocytes, including members of the TGF-` superfamily and their downstream
SMAD mediators, the homeodomain proteins and AP-1/CREB/ATF/Runx2 transcription factors. Phenotypic genes
corresponding to each step of chondrocyte maturation are also indicated.
Regulation of Chondrocyte Differentiation 45
development is primarily during embryonic growth and is less important postnatally, as we subsequently
describe.
Hedgehog proteins are a conserved family of secreted molecules that provide key signals in embry-
onic patterning in many organisms. In vertebrates, there are three hedgehog genes: sonic hedgehog
(Shh), desert hedgehog (Dhh), and the aforementioned Ihh. Shh functions in embryonic development
by controlling the establishment of left–right and anterior–posterior limb axes, Dhh functions as a
spermatocyte survival factor in the testes, whereas Ihh is involved in endochondral ossification (8).
Hedgehog proteins signal through a transmembrane receptor called Patched (Ptc) and a transcription
factor called Gli. In vivo overexpression of Ihh in chicken limb bud through retrovirally mediated
infection leads to shorter and broader skeletal elements with a continuous cartilage core that lacks
hypertrophic chondrocytes (9). In contrast, Ihh-deficient mice exhibit premature hypertrophic differ-
entiation, reduced chondrocyte proliferation, and failure of osteoblast development (10).
The phenotype of Ihh misexpression in either chicken limb bud or in transgenic mice is similar
with the phenotype of PTHrP misexpression. Animals that overexpress PTHrP exhibit delay in chon-
drocyte terminal differentiation (11). Humans with an activating mutation in the PTH/PTHrP recep-
tor have Jansen’s metaphyseal chondrodysplasia, characterized by disorganization of the growth plate
and delayed chondrocyte terminal differentiation (12). In contrast, mice null for either PTHrP (13)
or its receptor (14) display accelerated chondrocyte differentiation and abnormal endochondral bone
formation.
Several lines of evidence indicate that Ihh and PTHrP interact in a negative feedback loop regulat-

ing the onset of hypertrophic differentiation. Ihh overexpression in the chicken limb bud leads to induc-
tion of PTHrP expression in the periarticular region (9), whereas lack of Ihh signaling in the Ihh-deficient
mice leads to lack of PTHrP expression (10). Additionally, limb explants of PTHrP knockout mice
treated with hedgehog protein in culture demonstrate that intact PTHrP signaling is required to mediate
the inhibitory effect of Ihh on chondrocyte differentiation (9). These findings established the following
Fig. 2. The role of Ihh/PTHrP in endochondral ossification. Chondrocyte cell differentiation is associated
with expression of specific genes involved in the regulation of chondrocyte maturation. Ihh is expressed in the pre-
hypertrophic chondrocytes and signals through TGF-
`
2 located in the perichondrium to enhance transcription of
the PTHrP gene in the periarticular region.
46 Ionescu et al.
mechanism for the Ihh/PTHrP regulation of chondrocyte differentiation: Ihh is produced by the pre-
hypertrophic chondrocytes, signals through Ptc and Gli in the adjacent perichondrium, and induces
expression of PTHrP in the periarticular region. In turn, PTHrP would diffuse across the growth plate,
bind to its receptor, which is expressed in the prehypertrophic chondrocyte, and subsequently delay
chondrocyte maturation.
One of the elusive aspects of this signaling pathway is the signals that are generated by Ihh-stim-
ulated perichondrium to influence PTHrP expression in the periarticular cartilage. Indeed, removal
of perichondrium causes inability of hedgehog signaling to delay chondrocyte hypertrophy (15). Other
studies have shown that removal of perichondrium results in an extended zone of cartilage expressing
colX and an extended zone of cartilage incorporating BrdU, indicating that perichondrium negatively
regulates both proliferation and hypertrophy of chondrocytes (16). Possible candidates as Ihh secon-
dary signals are the members of the TGF-` superfamily, namely the BMPs and TGF-` isoforms 1–3.
TGF-
``
``
` SIGNALING:
ROLE IN CHONDROCYTE DIFFERENTIATIONS
TGF-` family members are considered possible mediators of Ihh signaling on PTHrP expression.

TGF-` enhances PTHrP expression in both mouse metatarsal explants (17) and isolated chondrocytes
cultures (18,19). Similarly, intact PTHrP signaling appears to be required for of TGF-`-mediated effects
on chondrocyte hypertrophy (17). Furthermore, transgenic mice overexpressing a dominant-negative
TGF-` receptor exhibited a very similar phenotype to the PTHrP knockout mice. Both mouse models
present an accelerated chondrocyte differentiation (17,20). Finally, inhibition of TGF-` signaling in
the perichondrium, with a dominant-negative type II receptor, also inhibits the effects of hedgehog
on chondrocyte differentiation and on induction of PTHrP expression (15). The dominant-negative
type II receptor inhibits all three TGF-` isoforms.
Alvarez et al. recently showed that treatment of mouse metatarsals cultures with hedgehog protein
leads to upregulation of TGF-
`
2 and TGF-
`
3 isoforms but not TGF-
`
1 in the perichondrium. Fur-
thermore, the effects of hedgehog protein signaling are specifically dependent on TGF-`2 because cul-
tures from TGF-`3-null embryos respond to hedgehog protein signaling but cultures from TGF-`2-
null embryos do not (15). Altogether, the evidence suggests that TGF-`2 acts as a signal messenger
between Ihh and PTHrP in the regulation of cartilage hypertrophic differentiation in embryonic devel-
opment. Although the phenotype of the dominant-negative type II TGF-` receptor resembles the pheno-
types of Ihh and PTHrP knockout mice, the skeletal malformations are still less severe, suggesting
that, TGF-` may not be the only mediator of Ihh on PTHrP expression.
The role of Ihh/PTHrP signaling loop after embryonic development is not clearly established. It
has been suggested that this pathway is not operational in neonatal mice because of low or absent
levels of Ihh expression in the postnatal growth plate (21). Also for postnatal development, the physi-
cal distance between the periarticular region and the growth plate increases dramatically, and the
ability of PTHrP to diffuse to these tissues is questionable. A possible signaling scenario involving
TGF-` and PTHrP in postnatal development has been defined by our group (22). All of the necessary
elements for a signaling pathway involving TGF-` and PTHrP are existent in the growth plate, elimi-

nating the need for diffusion. The growth plate makes large amounts of TGF-` and PTHrP expres-
sion is induced up to 20-fold in chondrocytes stimulated with TGF-` (18). Because TGF-` is secreted
in an inactive complex, a possible source for TGF-` could be from hypertrophic cartilage during
remodeling. Matrix metalloproteinase 13 (MMP-13) is highly expressed in the hypertrophic region
and has been shown to activate latent TGF-` (23). Another source is the zone of calcification as osteo-
clasts have been also been shown to activate TGF-` (24). During matrix catabolism, TGF-` is activated
from the latency-associated peptide (LAP) Activated TGF-` then acts in an autocrine/paracrine man-
ner to stimulate the expression of PTHrP in the growth plate (18). The elevated expression of PTHrP
then slows the rate of chondrocyte differentiation. This leads to a decrease in the terminal differentia-
Regulation of Chondrocyte Differentiation 47
tion and a fall in the activation of TGF-`, which results in a reacceleration in the rate of chondrocyte
differentiation, with a subsequent increase in the release of active TGF-` from the matrix. This positive
feedback loop between TGF-` and PTHrP in the growth plate results in a cycling of differentiation
from an on or off state, similar to the effect of the Ihh/PTHrP signaling pathway during development.
Interestingly, although animals overexpressing dominant-negative TGF-` receptors and the deletion
of Smad3, a critical transcription factor downstream of TGF-`, both develop premature maturation, the
phenotype only becomes evident postnatally (25). This suggests that unique signals might be present
during limb development and postnatal growth and that TGF-` might be particularly important in this
latter process.
BMP REGULATION OF CHONDROCYTE DIFFERENTIATION
Similar to the TGF-` family, BMPs are key regulators of organogenesis in early embryonic develop-
ment because they also play an active role in regulating cartilage formation and differentiation during
later stages. BMP-2, BMP-3, BMP-4, BMP-5, and BMP-7 all are expressed in the perichondrial cells
(26). Although BMP-4 and BMP-7 are expressed at low levels by chondrocytes undergoing maturation
(27), BMP-6 and BMP-7 are highly expressed by hypertrophic chondrocytes (9). Finally, BMP-2, BMP-4,
and BMP-7 are expressed in the precartilaginous mesenchyme (28) and were shown to enhance chondro-
genesis in vitro (29) and in vivo in chick limb buds (30). Regulation of BMP-2 and BMP-4 expression
by Ihh (31) further indicates that the interplay between these various signals in vivo increases the
intricacy by which chondrocyte growth and differentiation is regulated.
The role of BMP signaling in chondrocyte differentiation has been somewhat controversial because

of disparate effects that occur in vitro in isolated chondrocyte cell cultures and in in vivo in the chick
limb bud model. Cell culture studies in various models all demonstrate an induction in chondrocyte
maturation with gain of BMP signaling and a decrease in maturation with loss of function (32,33). In
contrast, in the chick limb bud, overexpression of activated type I BMP receptors inhibits chondro-
cyte maturation (26). However, our laboratory has recently demonstrated that activated BMP signal-
ing induces Ihh expression in chondrocytes, with a subsequent increase in PTHrP expression in the
periarticular region (34). This activation of the Ihh/PTHrP pathway likely explains the differential
effects of BMP signaling on isolated chondrocytes compared with the developing limb. Whereas BMPs
act to stimulate chondrocyte maturation directly, induction of Ihh/PTHrP signaling through paracrine-
mediated events has the opposite effect and inhibits maturation. The findings suggest that BMP sig-
naling is integrated into the Ihh/PTHrP signaling loop and that the ultimate effect is caused by a fine
balance of BMP signaling.
INTEGRATION OF MULTIPLE SIGNALING PATHWAYS:
COMBINATORIAL REGULATION OF CARTILAGE MATURATION
The growth factors and signaling molecules that control chondrocyte differentiation are intercon-
nected and center on interactions between the Ihh/PTHrP loop and the TGF-` superfamily members.
Although a significant amount of information about their role in skeletal development is available,
less is known about the mechanisms by which their signaling pathways affect potential targets in the
growth plate. Recently, we and others have characterized some of the transcriptional mechanisms down-
stream of these pathways (35–38). It is indeed necessary to define how interactions between these
signaling events result in progression toward the hypertrophic phenotype.
PTHrP Signaling
PTHrP signaling increases cAMP and calcium levels in chondrocytes, with subsequent activation
of protein kinases A and C (PKA and PKC; refs. 39–41). Potential downstream targets of PKA/PKC sig-
naling are the transcription factors cAMP response element binding protein (CREB) and activator pro-
tein 1 (AP-1) CREB is a member of the ATF/CREB family of transcription factors, which is activated
48 Ionescu et al.
in response to cAMP/PKA signals. CREB binds constitutively to DNA at a consensus cAMP response
element primarily as a homodimer via a leucine zipper domain (42). Activation occurs secondary to
a phosphorylation event at Ser133. This results in the recruitment of the coactivator CBP (CREB-

binding protein) and activation of the transcriptional machinery. The AP-1 complex is formed through
dimerization between Fos and Jun family members. Subsequently, AP-1 binds DNA response elements
known as TPA response elements.
Although PTHrP does not alter CREB protein levels or DNA binding, it stimulates kinases that
activate CREB by stimulating phosphorylation at Ser133 (35). In addition, PTHrP induces c-Fos pro-
tein production and enhances AP-1 binding to its consensus element. This stimulation of CREB and
AP-1 signaling is associated with an increase in gene transcription while inhibition of their signaling
leads to inhibition of PTHrP effects on both proliferation and maturation of chondrocytes (35). Thus,
the transcription factor CREB has a role in skeletal development through involvement in PTHrP sig-
naling and direct regulation of chondrocyte differentiation.
In addition to PTHrP signaling through cAMP, several other growth factors, including insulin-like
growth factor, epidermal growth factor, TGF-`, fibroblast growth factor, and platelet-derived growth
factor have also been shown to promote the phosphorylation of CREB family members (42,43),
suggesting a potential role for these factors in cell proliferation. Therefore, it is possible that CREB
can transduce signals of other signaling pathways and its role in skeletal development is more gener-
alized than just PTHrP signaling. This idea is supported by transgenic animal models exhibiting dis-
ruptions in the CREB gene. CREB-null mice targeting all isoforms (44) have been generated and are
smaller than their littermates and die immediately after birth from respiratory distress. This is similar
to the fate of PTHrP knockout animals, which are runted and also die in the neonatal period of respira-
tory distress. Nevertheless, the skeleton of the CREB-null mice has not been analyzed and the causes
for the observed dwarfism are not yet investigated. The function of CREB in skeletal formation also
was assessed through the generation of transgenic mice in which a dominant-negative form of CREB
(A-CREB; ref. 45) was driven by the cartilage-specific collagen type II promoter/enhancer in the growth
plate chondrocytes (46). A-CREB transgenic mice show short-limb dwarfism and a markedly reduced
rib cage that may underlie their perinatal lethality. Consistent with a pronounced defect in growth
plate development, tibias from transgenic embryos were bowed and exhibited asymmetric deposition
of cortical bone beneath perichondrium. The proposed cause for the severe dwarfism was inhibition of
proliferation. In agreement with previous studies, the expression of the dominant-negative CREB
inhibits proliferation, lowering the proportion of BrdU-positive cells in the growth plate and reducing
the height of the proliferative zone in developing limbs. However, contrary to the initial hypothesis,

the A-CREB transgenic mouse limbs exhibit delay of maturation accompanied by delay of vascular-
ization and bone formation (46). In contrast, in isolated chick chondrocytes cultures, the same domi-
nant-negative CREB accelerates the process of maturation and inhibits PTHrP (35). Similarly, inhibition
of PTHrP signaling through disruption of the hormone, its receptor, or Gs_ signaling leads to premature
hypertrophy in the growth plate (13,47,48). The discrepancy between the different studies may reside
in the difference between the two systems used. Studies performed in a cell culture system allow pertur-
bation of CREB signaling at later stages of chondrocyte development. In contrast, in the transgenic
mice, Long et al. (46) used a system in which the transgene is overexpressed at very early embryonic
stages. Therefore, the phenotype might reflect the effect of perturbation of CREB signaling at earlier
stages of skeletal development, such as chondrogenesis, cell aggregation, and nodule formation or tran-
sition from precursor (mesenchymal) cells to chondrocytes. The alterations in these steps may have
an overall negative impact on the normal progression of skeletal development by causing delay in the
cartilage anlage formation.
TGF-
``
``
`
Signaling
TGF-` receptor binding results in activation of the TGF-` type I receptor with phosphorylation
events that activate downstream signaling pathways, including the Smad family of transcription fac-
Regulation of Chondrocyte Differentiation 49
tors (49) and the mitogen-activated protein kinase (MAPK) family (Fig. 3; ref. 50) In chondrocytes,
overexpression of either Smad2 or Smad3 mimics TGF-` treatment, whereas the dominant negatives
are capable of diminishing TGF-` effects on cell differentiation (51). Targeted disruption of the Smad3
gene in mice causes a skeletal phenotype consistent with inhibition of TGF-` signaling, with pro-
gressive loss of articular cartilage resembling osteoarthritis and enhanced terminal differentiation of
epiphyseal chondrocytes (25).
Activation of the MAPK family (the extracellular signal-regulated kinase, the c-Jun N-terminal
kinase, and the p38 kinase) involves stimulation of transcription factors from the CREB/ATF and
AP-1 family (52–54). Not surprisingly, many promoters of the TGF-`-regulated genes contain either

AP-1 sites, such as the PAI-1 (55), TIMP-1 (56), TGF-`ß1 (57), c-Jun (58), and _2(I) collagen (59)
or CREB/ATF binding sites, such as fibronectin (60), cyclinD1 (37), and cyclin A (38).
The presence of multiple DNA binding sites for different transcription factors allows for transcrip-
tional crosstalk between Smads and the MAPKs during TGF-`-induced gene expression. Recent results
from our group demonstrate that both Smad3 and ATF-2 contribute to TGF-`-mediated effects on chon-
drocyte differentiation, and both participate in the induction of PTHrP in chondrocytes (36). TGF-`
is capable of phosphorylating and activating transcription factor ATF-2 and, subsequently, ATF-2
cooperates with Smad3 to regulate TGF-`-regulated gene transcription. Either ATF-2 or Smad3 over-
expressing chondrocytes exhibit delay of maturation with lower levels of colX but higher levels of
PTHrP. Similarly, overexpression of the dominant negatives ATF-2 and Smad3 either alone or together
block TGF-` inhibitory effects on chondrocyte maturation and colX. The role of ATF-2 seems to be
restricted to TGF-` but not BMP because overexpression of either ATF-2 or its dominant-negative does
not alter BMP signaling (36).
Fig. 3. Signal transduction pathway of TGF-` and BMP signaling. BMP signaling involves activation of
Smad1, 5, and 8 and coupling with the coactivator Smad4, whereas negative regulators, such as Smad6, Smurfs,
PKC, and ERK maintain a negative feedback loop. TGF-` signaling pathway involves activation of both Smad2
and 3 and the MAPK family members whereas Smad7 acts in an inhibitory manner.
50 Ionescu et al.
Although the total disruption of TGF-` signaling in vivo accelerates chondrocyte maturation (20),
the relative contribution of ATF-2 as mediator of TGF-` effects is not entirely understood. Disruption
of the ATF-2 gene results in a skeletal phenotype similar to hypochondrodysplasia (61). Although the
rate of survival is significantly diminished, the homozygotes reached adulthood and had a normal life
span despite uniform dwarfism and disorganization of the growth plate. In vivo labeling of cartilage
cells confirms that cartilage cell division is dramatically reduced, possibly as the result of reduced
levels of cyclin D1 and cyclin A (37,38).
However, further analysis of these mice demonstrated the presence of a mutant protein generated
through alternative splicing that allowed residual ATF-2 signaling. Subsequently, Maekawa et al.
designed a new ATF-2-null mouse that exhibits disruption in exon 10, corresponding to the DNA
binding domain, which inactivates all the isoforms (62). These mice die of meconium aspiration at
the time of birth, therefore not allowing one to determine whether absence of ATF-2 function and

signaling is associated with postnatal abnormalities in growth. Interestingly, however, these mice
have no apparent skeletal abnormalities at the time of birth. A similar situation has been encountered
for the Smad3 </< mouse, whose phenotype (accelerated chondrocyte maturation) becomes apparent
3 wk after birth (25).
BMP Signaling
BMP signaling involves direct transcriptional activation of receptor-regulated Smads 1, 5, and 8
with subsequent translocation to the nucleus and binding to specific DNA sequences (Fig. 3). This
event has been shown to induce the differentiation of isolated chondrocytes and there are numerous
points of control of these signaling events. Secreted extracellular molecules, including chordin, nog-
gin, follistatin, and others, bind to BMPs and prevent their interaction with cell surface receptors.
Our laboratory showed that chondrocytes express chordin and that overexpression of chordin in the
chick limb bud results in a delay of maturation (63). This suggests that BMP expression is necessary
for chondrocyte maturation to proceed. Smad6 is an intracellular signaling molecule that acts as a
negative regulator of BMP signaling. Smad6 blocks Smad1, 5, or 8 signaling by preventing receptor
activation (64,65) as well as binding to the coactivator Smad4 (66). Smad6 expression is induced by
BMP-2 (67), and we have shown that gain of Smad6 function inhibits chondrocyte maturation while
loss of function induces maturation (68).
Smad ubiquitination regulatory factor 1 (Smurf1), a member of the E3 class of ubiquitin ligases, is
another inhibitor of BMP signaling that targets the Smads. Smurf1 specifically targets Smad1 and
Smad5 for ubiquitination, leading to proteosomal degradation (69). The degradation of Smad1 and 5
by Smurf1 occurs independent of BMP receptor activation, indicating that Smurf1 does not function
downstream of activated Smads to turn off BMP signals, but may rather adjust the basal level of
Smads available for BMP signaling (69).
PTHrP blocks BMP effects on chondrocyte differentiation. Although work in our laboratory (70)
has shown that PKA/CREB is responsible for this effect, this is likely not the sole mechanism. Other
kinases, such as PKC and extracellular signal-regulated kinase, are activated by PTHrP and may
directly alter Smad1 signaling through phosphorylation at residues important in DNA binding (71) or
at residues involved in the translocation to the nucleus (72). In addition, virtually nothing is known
with respect to PTHrP interaction with Smurfs; therefore, future experiments will be needed for a
complete elucidation of PTHrP/BMP antagonism in chondrocyte.

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