Numerous mutations in FGFR1-3 have been found to cause at least 9 human skeletal dysplasias (2
). These mutations are thought to result in increased FGF/FGFR signals, as several studies showed ligand-independent activation of these mutant FGF receptors in vitro (25
). Consistently, the overexpression of FGFs in mice has resulted in skeletal conditions that mimic human disease (30
). FGF receptors normally exist as inactive monomers and become activated upon ligand-triggered dimerization. Because many mutations involved either gain or loss of a cysteine in the extracellular domain of FGF receptors, it has been postulated that the unpaired cysteine might promote intermolecular disulfide bond formation, leading to dimerization of the mutant receptors (32
). Our observation that both Gly375Cys and Ser371Cys mutations caused ligand-independent dimerization directly supports this hypothesis.
The effects of activated FGFR3 signals on bone growth have been tested in vivo recently by using transgenic (12
), and cDNA knock-in (23
) models. The former used the type II collagen promoter and enhancer to control Fgfr3
Gly380Arg transgene expression, and the latter knocked a FGFR3 cDNA containing the Lys644Glu mutation, which results in TDII in human, into the endogenous Fgfr3
locus. Mice with retarded long bone growth were generated by both studies, although the mutant alleles of FGFR3 were not expressed faithfully. The ectopic expression of the Fgfr3
Gly380Arg transgene resulted in bone defects that were not observed in human achondroplasia (12
), and the reduced expression of the Fgfr3
Lys644Glu allele did not cause the expected neonatal lethality seen in human patients with TD (23
). Moreover, the existence of multiple splicing variants in the Fgfr3
) makes it impossible to mimic the endogenous situation, as only 1 isoform of FGFR3 was mutated by the approaches described here. To overcome these difficulties, we introduced a point mutation into the Fgfr3
locus using a cotransfer approach (20
) followed by the excision of the neo
gene (Figure ). The mutant allele was expressed faithfully after removal of the neo
gene. A similar approach was used by a more recent study to introduce the Gly380Arg mutation into FGFR3 (11
). Although this study showed that the Gly380Arg mutation results in dwarf mice, the molecular mechanism through which the mutation retards bone growth remains unclear. Of note, mutant mice displayed much more severe skeletal dysplasia than that exhibited by both Gly369Cys mice and human Gly380Arg patients. It is not known whether such a discrepancy between the Gly380Arg mice and Gly380Arg patients reflects an intrinsic difference between 2 species or is due to other unknown factors.
Our analysis of the mutant mice indicated that the Gly369Cys mutation affects chondrogenesis by restraining chondrocyte proliferation. It has been suggested that FGFR3 is a negative regulator of bone growth (15
). This is primarily based on observations that loss of FGFR3 results in faster and prolonged bone growth (15
). One mechanism by which FGFR3 controls bone growth is by regulating signal transduction pathways through Stats, as demonstrated recently by the finding that FGFR3 carrying a TD II mutation caused activation of Stats and upregulation of cell-cycle inhibitors both in vitro and in vivo (22
). The observation that an achondroplasia mutation also causes activation of Stats and cell-cycle inhibitors suggests that the involvement of Stats and cell cycle proteins is a general phenomenon.
The markedly reduced sizes of mature and hypertrophic zones in mutant mice resemble phenotypes displayed by PTHrP
knockout mice (34
constitute a reciprocal regulation loop that controls chondrocyte proliferation and differentiation (36
). We found that the expression domains and intensities of both PTHrP-R
were decreased in the Fgfr3369/369
growth plates (data not shown). This is consistent with recent studies showing that activation (23
) or overexpression (12
) of Fgfr3
results in downregulation of Ihh
, supporting the notion that FGFR3 functions upstream of Ihh
and negatively regulates its activity.
In our ACH mice, retarded bone growth is accompanied by advanced ossification as manifested by premature closure of cranial synchondroses and early onset of the bone collar flanking long bone growth plates. Because chondrogenesis and osteogenesis are coupled processes during endochondral bone formation, we have considered the possibility that the advanced ossification is secondary to the decreased chondrogenesis in mutant mice. However, this option is difficult to reconcile with some observations. For example, the advanced bone collar was observed in P1 (Figure , d–f) and older mutant mice, whereas the decreased proliferation of growth plate chondrocytes became obvious only after P7, arguing that the advanced bone collar is not merely a consequence of the decreased chondrogenesis. The advanced ossification can result in bone dysplasia because it does not allow sufficient time for chondrocytes and osteoblasts to proliferate. Our molecular analysis of mutant mice revealed increased expression of osteopontin
, and osteocalcin
, which are associated with the mature osteoblast differentiation stage (37
), indicating that the Gly369Cys mutation enhances differentiation of osteoblasts. Enhanced differentiation is correlated with diminished osteoblast proliferation (41
), which was observed in our mice. Osteopontin is likely to play a role in the early formative stages of osteogenesis. Phosphorylated osteopontin enhances attachment of osteoclasts to the bone matrix during bone resorption (42
). Thus, the elevated expression of osteopontin
may be 1 factor causing increased activity of osteoclasts in mutant growth plates. Osteocalcin is an osteoblast-specific protein expressed mainly in cortical and trabecular bone (43
). In mutant mice, however, osteocalcin
was abnormally expressed in all prehypertrophic chondrocytes.
The consequence of the ectopic expression of osteocalcin
in the prehypertrophic zone of chondrocytes is not clear. Expression of osteocalcin
is associated with osteoblast differentiation (41
); thus, the ectopic expression of osteocalcin
may presage abnormal differentiation of prehypertrophic chondrocytes into osteoblastlike cells, a process termed transdifferentiation that is sometimes observed in chicken (44
). Osteocalcin is inferred to be a negative regulator of bone formation, based on the increased bone mass and accelerated bone growth caused by the null mutation (45
). Because the ACH mice exhibited retarded bone growth and reduced bone density, we believe that increased and ectopic expression of osteocalcin
by the activated FGFR3 signals is partially responsible for this phenotype. The reduced bone density, which is reflected by the reduced number and size of trabeculae, could also be caused by increased osteoclast activity (TRAP, Figure a) and/or decreased chondrogenesis. The mutant growth plate contains fewer clones of hypertrophic cells and fewer longitudinal septa between these clones. As the ossification front advances, fewer subchondral trabeculae will be left behind.
In summary, we have shown that the Gly375Cys mutation activates FGFR3 by inducing ligand-independent dimerization of the receptor. When this mutation is introduced into mouse, it results in dwarfism sharing similar phenotypes with human achondroplasia. Molecular analysis reveals activation of Stat proteins and cell-cycle inhibitors, which may be responsible for the decreased proliferation of mutant chondrocytes. The activation of FGFR3 also results in downregulation of Ihh and PTHrP-R and upregulation of osteoblast differentiation markers, as evidence for both abnormal chondrogenesis and osteogenesis. These data reveal an essential role of FGF/FGFR3 signals in both chondrogenesis and osteogenesis during endochondral ossification. The dwarf mouse model offers a valuable tool for further exploration of the signaling pathways that regulate normal bone growth.