Department of Molecular Biology and Pharmacology, Washington University Medical School, Campus Box 8103, 660 S. Euclid Avenue, St Louis, MO 63110, USA
*Author for correspondence (e-mail: dornitz{at}molecool.wustl.edu)
Accepted July 12, 2001
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SUMMARY |
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Key words: Fibroblast growth factor, FGF, FGF receptor, Achondroplasia, Mouse
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INTRODUCTION |
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Gain-of-function mutations in FGFR3 inhibit endochondral bone growth and cause the diseases hypochondroplasia, achondroplasia and thanatophoric dysplasia. By contrast, mutations in FGFR2, and a single mutation in FGFR1, are associated with the craniosynostosis syndromes, some of which also include phenotypes affecting the appendicular skeleton (Burke et al., 1998; Naski and Ornitz, 1998; Wilke et al., 1997). The phenotype of each syndrome correlates with a specific FGFR mutation, and with the spatial expression pattern of FGFRs in mesenchymal condensations and in developing endochondral and membranous bone (Delezoide et al., 1998; Iseki et al., 1999; Johnson et al., 2000; Orr-Urtreger et al., 1991; Peters et al., 1992).
In growing long bones with established growth plates, FGFR3 is highly expressed in proliferating chondrocytes and acts to inhibit proliferation (Colvin et al., 1996; Deng et al., 1996; Naski et al., 1998; Naski and Ornitz, 1998; Naski et al., 1996; Peters et al., 1992; Webster and Donoghue, 1996; Webster and Donoghue, 1997b). This activity of FGFR3 is remarkable considering the classical view that FGFs and their receptors transmit mitogenic signals. This raises the question of whether the inhibition of chondrocyte proliferation is a unique property of FGFR3 or a unique property of the chondrocyte.
In vitro, activated FGFR3 inhibits proliferation of several cell types. In 293T cells, constitutively active FGFR3 (containing the activation loop mutation K650E) specifically activated the transcription factor STAT1, which upregulates p21 expression, a known cell cycle inhibitor (Su et al., 1997). This observation was supported by the study of Stat1-/- bone explants, in which treatment with FGF was unable to inhibit longitudinal growth (Sahni et al., 1999). In CFK2 chondrocytes, FGFR3 (containing the weakly activating transmembrane domain mutation G380R), inhibited cell growth (Henderson et al., 2000). In contrast to these data, intracellular domains of FGFR1, FGFR3 or FGFR4 containing the constitutive activation loop mutation K650E and a plasma membrane targeting myristylation signal, all induced a transformed phenotype in NIH3T3 cells (Hart et al., 2000; Webster and Donoghue, 1997a). Furthermore, chromosomal translocations involving FGFR3 and constitutive activating mutations have been implicated as the etiological agent of some bladder carcinomas and some forms of myeloma (Cappellen et al., 1999; Chesi et al., 1997; Plowright et al., 2000; Richelda et al., 1997). These data demonstrate that constitutively activated FGFR3 can be mitogenic for some cell types.
In contrast to Fgfr3, which is expressed in proliferating chondrocytes, Fgfr1 is expressed in the adjacent hypertrophic chondrocytes and in articular chondrocytes. Fgfr1 and Fgfr2 are expressed in the perichondrium (Delezoide et al., 1998; Orr-Urtreger et al., 1991; Peters et al., 1993; Peters et al., 1992). The function of FGFR1 and FGFR2 in endochondral bone growth is not known; however, the non-overlapping expression patterns of FGFR1-FGFR3 suggest that these receptors have unique functions, mediated by differences in their ligand-binding specificity and/or downstream signaling.
The FGFR intracellular region contains a juxta-membrane domain, a bipartite tyrosine kinase domain and a kinase insert sequence, and is responsible for signal transduction. The intracellular regions of FGFR1 and FGFR3 share 73% amino acid sequence identity. Several studies have demonstrated differences in FGFR signaling potency in a variety of in vitro assays. In BaF3 cells, FGFR1 and FGFR2 elicit a strong mitogenic response whereas FGFR3 and FGFR4 fail to maintain cell proliferation, even in the presence of saturating ligand concentrations (Naski et al., 1996; Ornitz et al., 1996; Wang et al., 1994). Similarly, in PC12 cells, FGFR1 elicits a potent neurite outgrowth response, while FGFR3 shows less activity (Lin et al., 1996; Raffioni et al., 1999). However, when the intracellular domain of FGFR3 or FGFR4 is replaced with the intracellular domain of FGFR1, activity is restored in both cases (Naski et al., 1996; Wang et al., 1994). This suggests that sequence differences between intracellular regions of FGFR1 and FGFR3/FGFR4 have different signaling abilities. Additionally, mapping studies have localized a region in the juxta-transmembrane domain of FGFR1 that is required for neurite outgrowth in PC12 cells but not for mitogenesis in BaF3 cells (Lin et al., 1998). Thus, biochemical evidence supports the hypothesis that sequence differences between the FGFR1 and FGFR3 intracellular domains account for differences in signal transduction.
To test the hypothesis that inhibition of proliferating chondrocytes is a unique property of FGFR3 signaling, the regulatory elements of the gene for type II collagen were used to over express either a weakly activated FGFR3 (G380R) (Naski et al., 1998) or a chimeric FGFR (FGFR31, containing the extracellular and transmembrane domains of FGFR3 (G380R) and the intracellular domain of FGFR1) in the growth plate. Comparison of the phenotypes of mice expressing these transgenes demonstrated that activation of FGFR1 signaling pathways mimicked the effect of FGFR3 in proliferating chondrocytes. These data support the alternative hypothesis that FGFR1 and FGFR3 intracellular domains have similar signaling properties in proliferating chondrocytes and that unique properties of proliferating chondrocytes predisposes to growth arrest in response to an FGF signal. Interestingly, we also observed that over-activation of FGFR signaling in cartilage tissue prevented proximal joint formation and distally caused a reduction in the number of phalangeal elements. This observation suggests a role for FGF signaling in the transition from condensed mesenchyme into cartilage tissue and in defining the boundaries of skeletal elements.
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MATERIALS AND METHODS |
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Genotyping
Transgenic mice were generated and maintained in an FVB/N genetic background. Heterozygous mice were identified by PCR amplification of hGH cDNA in the 3' untranslated region of the transgene as described previously (Naski et al., 1998). Homozygous mice were identified by DNA blotting of BglII restricted tail DNA hybridized to a Fgfr3-specific probe that recognizes both transgene and endogenous FGFR3 fragments. Homozygous mice were determined by the intensity ratio of the endogenous FGFR3 fragments to the transgene band.
In situ hybridization
The probes detected hGH exon V (T. Simon, St Louis, MO), type II collagen (Y. Yamada, Rockville, MD, USA) and GDF5 (D. Kingsley, Palo Alto, CA). In situ hybridization was carried out as described previously (Naski et al., 1998).
Histology
Tissues were fixed in 4% paraformaldehyde or 10% formalin. The skeletal tissues were decalcified in 14% EDTA (pH 8.0), embedded in paraffin and stained with Hematoxylin and Eosin.
BrdU labeling
Mice received an intraperitoneal injection of bromodeoxyuridine (BrdU) at a dose of 100µg/g body weight and were sacrificed after 1 hour. Tissues were processed as described above and immunostained for BrdU as described previously (Naski et al., 1998).
Skeletal preparation and bone morphometry
Skeletons were prepared as described previously (Colvin et al., 1996). Bone length was determined using Foster-Findley image analysis software.
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RESULTS |
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FGFR1 signaling negatively regulates long bone growth
Both ColII-FGFR31ach transgenic lines exhibited a dwarfism phenotype with characteristics that are strikingly similar to ColII-FGFR3ach transgenic mice. Both types of mice display shortened long bones and a domed-shaped skull, probably owing to craniofacial hypoplasia (Fig. 1C). The severity of the observed dwarfism phenotype correlated with transgene expression level. The body weight of line 1 mice was 85% of littermate controls and line 2 mice was 60-70% of controls (Table 1A). Morphometric comparison of ColII-FGFR31ach line 2 mice and ColII-FGFR3ach mice demonstrated a similar degree of dwarfism (Table 1B). In both lines of mice, the length of the tibia was reduced by 13-14% at postnatal day 24-26. Both body weight comparisons and tibial length comparisons showed no statistically significant differences between ColII-FGFR31ach line 2 mice and ColII-FGFR3ach mice. Similarly, histological examination of the growth plate revealed strikingly similar features in both lines of mice (Fig. 2A-C). Both the hypertrophic and proliferating zones were significantly decreased in length compared with wild-type littermates, and the formation of secondary ossification centers was delayed in both transgenic lines. Consistent with histological observations and previous data, cell proliferation in the proliferating zone chondrocytes was similarly decreased in both ColII-FGFR31ach line 2 mice and ColII-FGFR3ach mice (Fig. 2D-G).
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Both ColII-FGFR31ach and ColII-FGFR3ach heterozygous transgenic mice developed defects in synovial and non-synovial joints. The middle phalangeal joint of the second and third digits in ColII-FGFR31ach line 2 and the second digit of ColII-FGFR3ach mice failed to develop. Both forefeet and hindfeet were affected with 100% penetrance (n=15). The occurrence of this phenotype in both FGFR3ach and FGFR31ach transgenic lines indicated that this was not a consequence of insertional mutagenesis. Histological sections revealed that a cartilaginous remnant is present in the presumptive interphalangeal joint position with two opposing growth plates (Fig. 3B,D) suggesting that the initial specification of the presumptive joint has occurred normally. Additionally, the non-synovial joints of the sternum were also affected. The wild-type sternum is segmented into four sternebrae, and in both the FGFR3ach and FGFR31ach transgenic lines, the joints between S2, S3 and S4 failed to form (Fig. 3E-F, and data not shown). Movement is known to play a crucial role in joint cavitation. Because non-synovial joints that are rarely subjected to movement were also affected in FGFR3ach and FGFR31ach transgenic mice, the defect in joint development is unlikely to result from lack of movement.
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Gdf5 is one of the earliest known markers of the presumptive joint. Gdf5 is also expressed in perichondrium. Gdf5 expression was therefore examined in ColII-FGFR31ach line 2 transgenic limb tissue at E13.5 (Fig. 8). In wild-type tissue, type II collagen-negative regions expressed high levels of Gdf5. This is consistent with other studies, which localized Gdf5 expression in the presumptive joint space as early as E11.5 (Storm and Kingsley, 1999). In tissue from homozygous ColII-FGFR31ach line 2 transgenic limb tissue, the joint boundary can still be identified by a region of decreased type II collagen expression (Fig. 6B). Notably, Gdf5 expression was completely lost in the joint interzone and dramatically reduced in the perichondrium. Only a trace amount of GDF5 expression was observed at the dorsal edge (n=3 out of 3). This observation suggests that expanded chondrification initiates centrally and progresses laterally. Examination of transgene expression in heterozygous and homozygous limb tissue (Fig. 8A,B, inset) demonstrated that ColII-FGFR31ach expression was excluded from the presumptive joint space and was complementary to that of Gdf5. In homozygous ColII-FGFR31ach tissue, transgene expression expanded diffusely into the presumptive joint space.
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DISCUSSION |
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Inhibitory effect of FGF receptor signaling on cell proliferation is a unique property of proliferating chondrocyte
Activation of FGFRs results in auto-phosphorylation of tyrosine residues in the intracellular domain, which triggers mitogen-activated protein kinase, phospholipase C and PI-3 kinase signaling cascades. The cellular response to FGFR activation is complex and sometimes paradoxical, and includes proliferation, anti-proliferation, differentiation, migration or apoptosis. The extent to which this wide spectrum of responses results from differences between the four FGFR signaling domains was not known. The results of this study suggest that cellular context, and to a lesser extent differences in signaling capacity, are crucial determinants of the cellular response to FGF. This is consistent with increasing evidence that different FGFRs can activate largely overlapping downstream signaling pathways with varying potency (Raffioni et al., 1999). In chondrocytes, STAT1 has been implicated as a key signaling molecule that mediates the anti-proliferative activity of FGFR3 (Sahni et al., 1999). Interestingly, it has recently been demonstrated that activation of FGFR1 or FGFR3 can activate STAT family members in NIH3T3 and PC12 cells (Hart et al., 2000). This observation provides a possible molecular basis for the inhibitory effect of FGFR1 on chondrocyte proliferation.
FGF receptors function in joint development
Expression studies and human genetic diseases suggest that FGFR signaling is required for normal joint development. FGFR2 is expressed early in prechondrogenic condensations and later in the perichondrium, periosteum and periarticular cartilage (Delezoide et al., 1998). In Apert syndrome, which results from mutations in Fgfr2, the proximal interphalangeal joints are absent at birth, and there is a gradual loss of distal phalangeal joints with age (Green, 1982; Holten et al., 1997). Cases of lateral cartilaginous fusion of the digits have also been documented (Cohen and Kreiborg, 1995). We show that both ColII-FGFR31ach and ColII-FGFR3ach transgenic mice develop phalangeal joint fusion that is suggestive of the phenotype seen in Apert syndrome. This probably results from transgene expression in resting chondrocytes near the presumptive articular surface. This region of type II collagen expression overlaps the endogenous Fgfr2 expression domain. In human embryos, FGFR2 is intensely expressed in the developing elbow joints and the adjacent cartilage tissues (Delezoide et al., 1998). Thus, the function of the ColII-FGFR transgenes in joint development may be similar to that of endogenous Fgfr2 in vivo. This supports the hypothesis that the cellular context is the predominant factor in determining the effect of FGFR signaling. Furthermore, these data suggest that morphogenesis of joints at different physical positions responds to a graded threshold of FGFR activation. Phalangeal joints are affected by lower levels of FGFR signaling compared with knee and elbow joints. This may explain why joint fusion in non-phalangeal joints has not been observed in individuals with activating mutations in FGFR2.
Interestingly, a mouse model that resembles Apert/Pfeiffer syndromes also develops a sternal joint fusion phenotype (Hajihosseini et al., 2001) similar to that of ColII-FGFR31ach and ColII-FGFR3ach transgenic mice. In this mouse mutant, altered FGFR2 splicing is thought to activate FGFR2 signaling inappropriately in developing mesenchymal condensations.
FGF receptors regulate chondrification and define the boundary of skeletal elements
FGFs have the ability to induce the transition from mesenchyme to chondrocytes in vitro (Richman and Crosby, 1990). However, the role of FGFR signaling in chondrification in vivo is poorly understood. Fgfr1 is expressed in loose mesenchyme and in condensed pre-cartilage mesenchyme. By contrast, Fgfr2 is expressed only in mesenchymal condensations. After condensed mesenchyme undergoes chondrification, Fgfr3 is expressed in proliferating growth plate chondrocytes, Fgfr1 is expressed in hypertrophic chondrocytes, and both Fgfr1 and Fgfr2 are expressed in the perichondrium and periosteum (Delezoide et al., 1998; Orr-Urtreger et al., 1991; Peters et al., 1992; Szebenyi et al., 1995).
We show that joint loss in ColII-FGFR3ach and ColII-FGFR31ach transgenic mice results from expanded chondrification, providing in vivo evidence that FGFR signaling promotes chondrocyte differentiation from condensed mesenchyme. The initial step in joint morphogenesis is the specification of the presumptive joint position in a continuous condensed mesenchymal rod. The presumptive joint position is marked by the appearance of a zone expressing low levels of type II collagen juxtaposed by cartilaginous elements expressing higher levels of type II collagen (Fig. 9). As joint development progresses, type II collagen expression is abolished in the joint space. This suggests that cells in the presumptive joint space may elaborate anti-chondrogenic signal(s) that antagonize the chondrification process. This hypothesis is supported by the identification of Wnt14 expression specifically in the joint interzone (Hartmann and Tabin, 2001). In chick, Wnt14 is sufficient to suppress chondrification in micromass mesenchymal cell culture. In ColII-FGFR3ach and ColII-FGFR31ach transgenic mice and in Apert syndrome, activation of FGFR signaling promotes chondrification. As a result of overactivation of FGFR signaling, the balance between the anti-chondrogenic signal (WNT14) and pro-chondrogenic signal (FGFR) becomes disrupted. It is likely that this antagonistic signaling in the chondrification process is a unique property of WNT14, as other WNT family members promote chondrification (Hartmann and Tabin, 2001). In addition, the BMP family member, GDF5, expressed in the presumptive joint space, could also restrict the FGF signaling domain. Antagonistic relationships between FGFs and BMP family members are well established in several systems (Neubuser et al., 1997; Weaver et al., 2000). The development of chondrocytes and the perichondrium are closely coupled. Previous studies have revealed that overactivating FGFR3 signaling in chondrocytes resulted in decreased proliferation and reduced Bmp4 expression in the perichondrium (Naski et al., 1998). Thus, it is not surprising to see loss of Gdf5 expression in the perichondrium as well. This model also predicts that chondrocytes surrounding the presumptive joint space elaborate a signal that negatively regulates either Wnt14 or Gdf5 in the joint space and Gdf5 in the perichondrium.
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ACKNOWLEDGMENTS |
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