FGFR3 promotes synchondrosis closure and fusion
of ossification centers through the MAPK pathway
Takehiko Matsushita1, William R. Wilcox3,4, Yuk Yu Chan1, Aya Kawanami1, Hu ¨lya Bu ¨ku ¨lmez2,5,
Gener Balmes6, Pavel Krejci3,7,8, Pertchoui B. Mekikian3, Kazuyuki Otani9, Isakichi Yamaura9,
Matthew L. Warman10, David Givol11and Shunichi Murakami1,2,?
1Department of Orthopaedics and2Department of Genetics, Case Western Reserve University, 10900 Euclid Avenue,
Cleveland, OH 44106, USA,3Medical Genetics Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd, Los
Angeles, CA 90048, USA,4Department of Pediatrics, UCLA School of Medicine, Los Angeles, CA 90095, USA,
5Department of Pediatrics, Pediatric Rheumatology, MetroHealth Medical Center, 2500 MetroHealth Drive, Cleveland,
OH 44109, USA,6Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, 1515
Holcombe Blvd, Houston, TX 77030, USA,7Institute of Experimental Biology, Masaryk University 61137, Brno, Czech
Republic,8Department of Cytokinetics, Institute of Biophysics ASCR 61265, Brno, Czech Republic,9Department of
Orthopaedic Surgery, Kudanzaka Hospital, 2-1-39 Kudanzakaminami, Chiyoda-ku, Tokyo 102-0071, Japan,
10Department of Orthopaedic Surgery, and the Howard Hughes Medical Institute at Children’s Hospital, Boston,
300 Longwood Avenue, Boston, MA 02115, USA and11Weizmann Institute of Science, Rehovot, Israel 76100
Received July 9, 2008; Revised October 6, 2008; Accepted October 13, 2008
Activating mutations in FGFR3 cause achondroplasia and thanatophoric dysplasia, the most common human
skeletal dysplasias. In these disorders, spinal canal and foramen magnum stenosis can cause serious neuro-
logic complications. Here, we provide evidence that FGFR3 and MAPK signaling in chondrocytes promote
synchondrosis closure and fusion of ossification centers. We observed premature synchondrosis closure
in the spine and cranial base in human cases of homozygous achondroplasia and thanatophoric dysplasia
as well as in mouse models of achondroplasia. In both species, premature synchondrosis closure was
associated with increased bone formation. Chondrocyte-specific activation of Fgfr3 in mice induced prema-
ture synchondrosis closure and enhanced osteoblast differentiation around synchondroses. FGF signaling
in chondrocytes increases Bmp ligand mRNA expression and decreases Bmp antagonist mRNA expression
in a MAPK-dependent manner, suggesting a role for Bmp signaling in the increased bone formation. The
enhanced bone formation would accelerate the fusion of ossification centers and limit the endochondral
bone growth. Spinal canal and foramen magnum stenosis in heterozygous achondroplasia patients, there-
fore, may occur through premature synchondrosis closure. If this is the case, then any growth-promoting
treatment for these complications of achondroplasia must precede the timing of the synchondrosis closure.
Longitudinal bone growth occurs through endochondral ossifi-
cation, in which chondrocytes progress through a series of pro-
liferation and differentiation processes. Chondrocytes in the
reserve zone of growth plates proliferate and then exit the
cell cycle to differentiate into hypertrophic chondrocytes.
The increase in the number of chondrocytes by proliferation,
the increase in the size of chondrocytes by hypertrophy and
the synthesis of extracellular matrix all contribute to linear
growth. Chondrocytes in growth plates are continuously sup-
plied by the differentiation and proliferation of chondrocytes
in the reserve and proliferative zones, while terminally
differentiated hypertrophic chondrocytes are removed at
the chondro-osseous junction by apoptotic cell death. The
balance between the addition and removal of chondrocytes
?To whom correspondence should be addressed at: Dept of Orthopaedics, Case Western Reserve University, 2109 Adelbert Road, BRB 329, Cleveland,
OH 44106, USA. Tel: þ1 2163681371; Fax: þ1 2163681332; Email: firstname.lastname@example.org
# The Author 2008. Published by Oxford University Press. All rights reserved.
For Permissions, please email: email@example.com
Human Molecular Genetics, 2009, Vol. 18, No. 2
Advance Access published on October 15, 2008
as well as matrix production and degradation determines the
height of the growth plates. In humans, the cessation of
linear growth usually coincides with the end of puberty
when growth plates become entirely replaced by bone.
Similar to the appendicular skeleton, in the vertebrae,
sternum and cranial base, bone growth occurs at synchon-
droses—cartilaginous structures consisting of two opposed
growth plates with a common zone of resting chondrocytes.
As with endochondral growth plates, synchondroses also
become replaced by bone. The regulation of growth plate
and synchondrosis closure is still not entirely understood.
Endochondral ossification is controlled by multiple regulat-
ory factors (1,2). An essential regulator of endochondral bone
growth is fibroblast growth factor receptor 3 (Fgfr3). Fgfr3 is
preferentially expressed in proliferating and prehypertrophic
chondrocytes in epiphyseal growth plates (3,4). Activating
mutations in FGFR3 cause autosomal dominant human skel-
etal disorders, achondroplasia, thanatophoric dysplasia and
hypochondroplasia (5–9). Thanatophoric dysplasia is the
most common lethal skeletal dysplasia, and achondroplasia
is the most common non-lethal form of dwarfism. Despite
its non-lethality, common and serious complications in achon-
droplasia are a small foramen magnum and spinal stenosis
(10,11) (Fig. 1). Stenosis of the foramen magnum, the
orifice in the occipital bone through which passes the spinal
cord from the medulla oblongata, has been associated with
hydrocephalus and sudden death in infancy (12–14) as well
as headaches in older children (15). Currently, surgical enlar-
gement of very small foramen magnum is recommended for
,10% of children with achondroplasia (11,16). Narrowing
of the spinal canal, which contains the spinal cord and cauda
equina, is a common complication in adults with achondropla-
sia and can cause neurologic deficits including myelopathy,
radiculopathy and neurogenic claudication. In addition, insuf-
ficient growth of the cranial base causes midface hypoplasia,
which leads to obstructive sleep apnea, otitis media and
Inadequate growth of the spinal canal, foramen magnum
and cranial base in patients with FGFR3 mutations could be
due to deficient cell proliferation, hypertrophy and/or matrix
production, and/or due to premature closure of synchondroses.
Support for the latter mechanism comes from computed
tomography (CT) studies in patients with achondroplasia
where premature closure of occipital bone synchondroses
was observed (17). To explore the developmental mechanisms
that contribute to these complications, we examined synchon-
droses of the spine and cranial base in human specimens from
children who died from homozygous achondroplasia and
thanatophoric dysplasia, and we studied the timing of synch-
ondrosis closure in mice with the Fgfr3 mutation G374R,
which corresponds to the common human achondroplasia
mutation, and in mice that express a constitutively active
form of MEK1, a downstream effector of Fgfr3 signaling.
In humans and in mice, we observed premature closure of
multiple synchondroses. Our results indicate that Fgfr3 and
MAPK signaling in chondrocytes regulate synchondrosis
closure, osteoblast differentiation and bone formation, provid-
ing novel insights into the developmental mechanisms of
spinal canal stenosis, foramen magnum stenosis and midface
hypoplasia in achondroplasia. If premature synchondrosis
closure accounts for spinal canal stenosis, foramen magnum
promoting treatment for these complications must start
before the synchondroses close.
Human specimens from homozygous achondroplasia and
thanatophoric dysplasia were examined at the International
Skeletal Dysplasia Registry at Cedars-Sinai Medical Center.
The synchondroses in the cranial base and lumbar vertebrae
were examined in one case of homozygous achondroplasia,
four cases of thanatophoric dysplasia (three perinatal, one
fetal) and one control fetus (Table 1).
Premature synchondrosis closure in homozygous
Gross inspection and radiographic examination of the mid-
sagittal slice of the cranial base in an infant who died from
homozygous achondroplasia (Case 1) showed absence or pre-
mature closure of the spheno-occipital synchondrosis (Fig. 2B
and C), which normally closes between 11 and 25 years of age
(18–22). We also examined the neurocentral synchondroses of
the lumbar vertebrae that normally close between 3 and 14
years of age (23–26). X-ray examination of the third lumbar
spine showed complete fusion of the neurocentral synchondro-
sis on one side and partial fusion on the other side (Fig. 2D),
consistent with premature closure. Fusion was confirmed
histologically (data not shown).
Premature synchondrosis closure in thanatophoric
We also examined one 27-week thanatophoric dysplasia fetus
with an R248C mutation (Case 2) and one 26-week gestation
control without any signs of skeletal dysplasia (Case 3). Radio-
graphic examination showed narrowing of the foramen
magnum in the thanatophoric dysplasia fetus (Fig. 2E and F).
Although the intraoccipital synchondroses were still open
at this stage, this fetus had a bony bridge forming around
the anterior intraoccipital synchondroses (Fig. 2E) that was
Figure 1. Spinal canal stenosis in achondroplasia. Axial CT myelogram of the
fifth lumbar spine shows the narrowing of the spinal canal in a 46-year-old
female achondroplasia patient with FGFR3 G380R mutation, who presented
with paraparesis (right). The contrast medium injected into the subarachnoid
space was excluded in the achondroplasia patient due to severe spinal canal
stenosis. (Left) 41-year-old female patient with irrelevant spinal disorder.
White bars indicate 1 cm.
228Human Molecular Genetics, 2009, Vol. 18, No. 2
into double-stranded cDNA. cDNA was labeled with biotin
and fragmented according to the manufacturer’s protocol.
The fragmented cRNA was hybridized onto Affymetrix
mouse 4.30 chips. Data analysis was done using the Affyme-
trix GCOS software.
Supplementary Material is available at HMG Online.
We thank Chu-Xia Deng (National Institutes of Health,
Bethesda, MD, USA), Tamayuki Shinomura (Tokyo Medical
and Dental University, Tokyo, Japan), Ce ´line Colnot (Univer-
sity of California at San Francisco, CA, USA), Richard
Harland (University of California at Berkeley, CA, USA),
Yasuhide Furuta (U.T.M.D.
Houston, TX, USA), Andrew McMahon (Harvard University,
Cambridge, MA, USA), Jinkun Chen (U.T Health Science
Center at San Antonio, TX, USA) for probes, Yoshihiko
Yamada (National Institutes of Health, Bethesda, MD, USA)
for the transgenic expression vector, James Martin (Institute
of Biosciences and Technology, Houston, TX, USA) and
Center) for Prx1-Cre and Col1a1-LacZ transgenic mice. We
are grateful to Dr. Masahiro Kurosaka (Kobe University) for
his continuous support for this work. We also thank Drs.
Guang Zhou and Douglas Armstrong for scientific discussion
and Ms. Valerie Schmedlen for editorial assistance.
Conflict of Interest statement. None declared.
This work was supported by Arthritis Investigator Award
of the Arthritis Foundation, March of Dimes Birth Defects
Foundation [grant number #6-FY06-341], National Institutes
of Health [grant number R21DE017406] to S.M., National
Institutes of Health[grant
M01-RR00425], and Winnick family research scholar’s award
to W.R.W, Arthritis National Research Foundation and
National Institutes of Health [grant number 5K08AR53943-2]
to H.B., the Howard Hughes Medical Institute to M.L.W. The
Gene Expression and Genotyping Facility of the Case Com-
prehensive Cancer Center was supported by National Institutes
of Health [grant number P30 CA43703].
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