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Endocrinology, doi:10.1210/en.2003-0158
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Endocrinology Vol. 144, No. 10 4659-4668
Copyright © 2003 by The Endocrine Society

Expression of Fibroblast Growth Factor Receptor-3 (FGFR3), Signal Transducer and Activator of Transcription-1, and Cyclin-Dependent Kinase Inhibitor p21 during Endochondral Ossification: Differential Role of FGFR3 in Skeletal Development and Fracture Repair

Arata Nakajima, Sumito Shimizu, Hideshige Moriya and Masashi Yamazaki

Department of Orthopaedic Surgery, Chiba University Graduate School of Medicine, Chuo-ku, Chiba 260-8677, Japan

Address all correspondence and requests for reprints to: Arata Nakajima, M.D., Ph.D., Department of Orthopaedic Surgery, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8677, Japan. E-mail: 98md0509{at}insei.m.chiba-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increasing evidence suggests that fibroblast growth factor receptor-3 (FGFR3) is a negative regulator of endochondral bone growth; however, its role during skeletal repair is unknown. Using a rat model of closed femoral fracture healing, we analyzed the spatial and temporal expression of FGFR3. To assess a possible role for FGFR3 during healing, we also analyzed the spatial and temporal expression of signal transducer and activator of transcription-1 (STAT1) and cyclin-dependent kinase inhibitor p21, important mediators of FGFR3 signaling. Before these experiments, we studied the spatial expression of FGFR3 during skeletal development in mouse embryos. At 16.5 and 19.5 d post coitum, FGFR3 mRNA was strongly expressed in resting and proliferating chondrocytes but weakly in hypertrophic chondrocytes and not in osteoblasts. In contrast, during fracture repair, it was strongly expressed in prehypertrophic chondrocytes, and the expression level reached a maximum on d 14. Immunoreactivity for STAT1 was detected in the cytoplasm of chondrocytes on d 4 and 7 and both in the cytoplasm and nucleus of hypertrophic chondrocytes on d 14. Furthermore, FGFR3, STAT1, and p21 exhibited a similar temporal expression profile, suggesting that FGFR3-mediated STAT1-p21 signaling plays a role in fracture repair. These results indicate a differential role of FGFR3 in skeletal development and fracture repair.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FIBROBLAST GROWTH FACTOR receptors (FGFRs) are a family of receptor protein tyrosine kinases that have been shown to mediate a variety of cellular events during skeletal development. Analyses of spatial expression of FGFRs during embryogenesis (1, 2, 3, 4) and searches for mutations in human FGFR genes associated with dysmorphic syndromes (5, 6, 7, 8, 9, 10, 11, 12, 13) have revealed essential roles of FGFRs in skeletal development; however, FGFRs also play crucial roles in the repair of skeletal tissues. We previously demonstrated that FGFR1 mRNA was predominantly expressed in osteogenic cells compared with chondrogenic cells, particularly in mature osteoblasts during fracture repair. In addition to its spatial expression in the fracture callus, we showed that its temporal expression is rapidly up-regulated after fracture and maintained until the later stages of healing, suggesting that FGFR1 signaling contributes to bone formation and callus remodeling (14). However, the roles of FGFRs other than FGFR1 during fracture repair have not been fully established.

FGFR3, as well as FGFR1, is one of four distinct membrane-spanning tyrosine kinases that serve as high-affinity receptors for a number of fibroblast growth factors. Although this family of proteins plays a major role in a variety of developmental processes, in situ hybridization analyses of developing mouse embryos demonstrate that FGFR3 mRNA is expressed at high levels in the cartilage rudiments of a wide variety of bones (2). This expression pattern suggests a role of FGFR3 in embryonic skeletal development. Regarding the role of FGFR3 in postnatal skeletal development, genetic analyses of human skeletal disorders have revealed that mutations of the FGFR3 gene cause achondroplasia and hypochondroplasia (6, 7, 8). Furthermore, analyses of FGFR3-deficient mice revealed a remarkable increase in the length of the vertebral column and long bones as a result of enhanced and prolonged bone growth (15, 16). In addition to this mutant phenotype, previous in situ hybridization studies have shown that FGFR3 is expressed in the proliferation zone of chondrocytes of postnatal mice, suggesting that FGFR3 is essential for restraining chondrocyte proliferation, thereby inhibiting bone growth (16).

Recent advances in molecular biology have enabled investigators to better understand intracellular signal transduction. Activation of FGFRs results in the recruitment of two different Grb/Sos adapter complexes leading to the activation of the Ras-MAPK signaling pathway (17); then the following activation of the signal transducer and activator of transcription (STAT) pathway via the nuclear translocation of STAT1 has been also demonstrated in chondrocytes of thanatophoric dysplasia type II patients (18). In addition, cycline-dependent kinase inhibitor p21 inhibits cell cycle progression by regulating cyclin-cycline-dependent kinase complexes and proliferating cell nuclear antigen (19, 20, 21, 22). Su et al. (18) revealed that expression of constitutively activated FGFR3 signaling induces nuclear translocation of STAT1, expression of p21, and growth arrest of the cell. Thus, it is likely that constitutively activated FGFR3 signaling uses STAT1 and p21 as mediators of growth retardation in bone development. Based on these findings, it is hypothesized that, in fracture repair, activated FGFR3 signaling induces the expression of STAT1 and p21, and regulates chondrogenesis and chondrocyte maturation during endochondral ossification. To test this hypothesis, we analyzed the spatial expression of FGFR3 during embryonic development, then investigated the spatial and temporal expression of FGFR3, STAT1, and p21 in fracture repair, and assessed possible roles of FGFR3 during endochondral ossification.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse embryos
The upper limbs of 12 mouse embryos were collected between 16.5 and 19.5 d post coitum (p.c.) from C57BL/6J outbred females mated with C57BL/6J males. Noon on the day of finding a vaginal plug was designated as d 0.5. Six to eight embryos from two to three pregnant mice were fixed in 4% paraformaldehyde and 0.1 M PBS (pH 7.4) at 4 C for 24 h and dehydrated through graded ethanol before being embedded in paraffin. Midsagittal sections 6 µm thick were mounted on silane-coated slides.

Fracture model
Thirty-six 7-wk-old male Sprague-Dawley rats were used in this study. A standard closed middiaphysial fracture was produced in the right femur of each rat according to the method of Dr. T. A. Einhorn (Boston University Medical Center, Boston, MA) (23). Briefly, following anesthesia, a Kirschner wire (1.1 mm in diameter) was introduced into the medullary canal of the right femur, and a middiaphyseal fracture was created with an apparatus composed of a blunt guillotine driven by a dropped weight. These experimental procedures were approved by the Animal Care and Use Committee of Chiba University. The animals were killed at 4, 7, 14, and 21 d after operation by an intracardiac injection of 4% paraformaldehyde after anesthesia. Midsagittal paraffin sections 6 µm thick were prepared as described previously (14).

Preparation of probes
The plasmid containing the 0.43-kb fragment of rat FGFR3 (nucleotides 914-1342, GenBank AF277717) was generated by RT-PCR using total RNA from unfractured rat femora. The cDNA fragment was inserted into a pGEM-T easy vector (Promega, Madison, WI) by T/A cloning, and the sequence was verified by sequence analysis. This cDNA fragment consists of the extracellular and transmembrane domains that are relatively unconserved among members of the FGFR family. A 0.64-kb fragment of mouse pro-{alpha}1 (II) collagen (COL2A1) cDNA, and a 0.60-kb fragment of mouse pro-{alpha}1 (X) collagen (COL10A1) cDNA was kindly provided by Dr. K. Andrikopoulos (24) and Dr. S. Apte (25), respectively. The specificity of the probes was confirmed before initiating experiments. A cDNA fragment corresponding to rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (nucleotides 451–758, GenBank M17701) was used as described previously (14).

In situ hybridization
To detect cells expressing FGFR3 mRNA in mouse embryos and fracture calluses, sections were hybridized with a probe for FGFR3. Furthermore, to determine the chondrocyte differentiation in the fracture callus, sections were also hybridized with probes for COL2A1 and COL10A1. Digoxigenin (DIG)-11-uridine 5-triphosphate-labeled single-strand RNA probes (antisense and sense probes) for rat FGFR3 cDNA (0.43 kb), mouse COL10A1 cDNA (0.64 kb), and mouse COL10A1 cDNA (0.60 kb) were prepared. In situ hybridization was carried out as previously described (14, 26, 27, 28, 29, 30). The sections were hybridized with the antisense probes at 50 C for 16 h, and the signals were detected using the DIG detection kit (Roche Molecular Biochemicals, Indianapolis, IN). After signal detection, sections were counterstained with methylgreen. The sense probes were used to exclude the possibility of nonspecific signals.

Immunohistochemistry
To clarify cells expressing STAT1 protein in mouse embryos and fracture calluses, sections were reacted with a monoclonal antibody against STAT1 (BD PharMingen, San Diego, CA). Immunohistochemical staining was performed as previously described (14, 27, 29, 30). Signals were detected using diaminobenzidine. Counterstaining was performed with Mayer’s hematoxylin. For the negative control sections, the same procedures were used, but the primary antibody was replaced by nonimmune mouse IgG (Vectastain, Vector Laboratories, Burlingame, CA).

Detection of apoptotic cells
To determine whether apoptotic cells were in the fracture callus, sections were stained by means of terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) reaction using an in situ apoptosis detection kit (Apop Tag, Intergen, Purchase, NY). In brief, sections were digested with proteinase K (20 µg/ml) for 30 min at room temperature and incubated with terminal deoxynucleotidyl transferase and DIG-deoxyuridine 5-triphosphate for 1 h at 37 C. After repeated rinses in buffer, sections were reacted with an antibody for DIG-horseradish peroxidase conjugate. Signals were detected using diaminobenzidine. In each TUNEL-labeling assay, sections of weaned rat mammary tissue were used as a positive control. Negative controls were made by omitting the transferase.

RNA extraction
For RNA extraction, the rats were killed after anesthesia with sodium pentobarbital at 2, 4, 7, 14, and 21 d postoperatively, and fractured and unfractured femora were harvested. As a positive control for the mRNA expression of FGFR3, mouse embryos (16.5) were also analyzed. The tissues were frozen immediately in liquid nitrogen and stored at -80 C until used for RNA isolation. Total cellular RNA was extracted using TRIzol (Gibco BRL, Rockville, MD) according to the manufacturer’s instructions.

Ribonuclease protection assay (RPA)
RPA was carried out as described previously (14). Briefly, radiolabeled antisense RNA probes for rat FGFR3 and GAPDH were transcribed from linearized plasmid templates with 5'-triphosphate ({alpha}-32P) and T3 or SP6 polymerase (Roche Molecular Biochemicals). Following exposure, hybridization signals were estimated by an image analyzer (Image Gauge, Fujifilm, Tokyo, Japan), and each band density was normalized to the ratio of the internal standard GAPDH.

Semiquantitative RT-PCR
Experiments were performed to determine the level of relative expression of STAT1 and p21 in a semiquantitative manner. Total cellular RNA was used as a template for synthesis of the first-strand cDNA by reverse transcription. A reaction mixture containing oligo (dT), 1 µg total cellular RNA, deoxynucleotide triphosphates, and random primers in a total volume of 20 µl was heated at 80 C for 3 min. Reverse transcriptase (Supersprict II, Gibco BRL) and Rnase inhibitor were then added, and the mixtures were incubated at 37 C for 2 h. The tubes were stored at -20 C until used for PCR amplification. We also prepared a negative control sample which was produced by incubating the mixtures without the reverse transcriptase.

The cDNAs of STAT1 and p21 were amplified using 20 U of Taq polymerase (Ex Taq, TaKaRa, Tokyo, Japan), and the cDNA of GAPDH was amplified with the same enzyme as a control. The sequences of the primers and the expected sizes of the PCR products are shown in Table 1Go. The reaction mixtures were preheated at 94 C for 5 min (hot-start PCR) and then amplified 23 times for STAT1 and 30 times for p21 at 94 C for 30 sec, 61 C for 30 sec, and 72 C for 1 min. Finally, they were extended at 72 C for 7 min. To verify that each amplification cycle was finalized within a linear range, we further amplified five more times (28 times for STAT1 and 35 times for p21) and confirmed that each cycle was definitely finalized within a linear range. For GAPDH, cDNA was amplified for 20 cycles. Following verification of the sequences of the PCR products, they were electrophoresed on a 2% agarose gel containing ethidium bromide and visualized under UV light. Photographs of the stained gels were taken and analyzed quantitatively using an image analyzer (Image Gauge). The results were normalized by the amount of PCR products amplified from GAPDH.


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  		TABLE 1. Primers used in this study with their orientation, sequence, length of amplification (bp), and number of cycles used for RT-PCR

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of FGFR3 mRNA and STAT1 in developing long bones
We first studied the expression of FGFR3 mRNA and STAT1 during the development of cartilage and bone in mouse embryos.

At 16.5 d p.c., the cartilage in developing long bones had not yet calcified, and chondrocytes at the central part of developing bones were hypertrophied and surrounded by matrix (Fig. 1AGo). At this stage, a strong FGFR3 signal was detected in resting and proliferating chondrocytes at the ends of developing long bones, but a weak signal was seen in hypertrophic chondrocytes. The membrane surrounding the developing cartilage, perichondrium, was also positive for FGFR3 mRNA (Fig. 1BGo). Immunoreactivity for STAT1 was detectable in chondrocytes between proliferating and hypertrophic zone but not in resting and proliferating chondrocytes (Fig. 1Go, G and I). The staining was observed in the cytoplasm (Fig. 1Go, H and J). A negative control for STAT1 using nonimmune mouse IgG shows no significant signal (Fig. 1Go, K and L).



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  		FIG. 1. Expression of FGFR3 mRNA and STAT1 in developing long bones of mouse embryos. Upper limbs at 16.5 d p.c. (A, B, G, and H) and 19.5 d p.c. (C–F and I–L). An area in the box in A, C, and E is enlarged in B, D, and F, respectively. G, I, and K, Fields correspond to the area in the box in A, C, and E, respectively. An area in the box in G, I, and K is enlarged in H, J, and L, respectively. The FGFR3 signal was strongly detected in resting and proliferating chondrocytes and in the perichondrium, but weakly in hypertrophic chondrocytes and not in osteoblasts at the ossification center (A–D). A negative control using sense probe for FGFR3 shows no significant signal (E and F). Immunoreactivity for STAT1 was clearly detectable in chondrocytes between proliferating and hypertrophic zone (G and I), and the staining was observed in the cytoplasm (H and J). A negative control for STAT1 using nonimmune mouse IgG shows no significant signal (K and L). rc, Resting chondrocytes; pc, proliferating chondrocytes; hc, hypertrophic chondrocytes; oc, ossification center; p, perichondrium; po, periosteum. Scale bars, 500 µm (A, C, and E), 100 µm (B, D, F, G, I, and K), 20 µm (H, J, and L).

 
At 19.5 d p.c., the central part of the cartilage had degraded and calcified, and the ossification center appeared (Fig. 1CGo). At this stage, the same results were seen as for 16.5 days p.c.: A strong FGFR3 signal was detected in resting and proliferating chondrocytes at the ends of the developing bones, but a weak signal was seen in hypertrophic chondrocytes, and no signal in osteoblasts at the ossification center. A strong FGFR3 signal was also observed in the perichondrium, particularly in the membrane surrounding the resting zone of chondrocytes (Fig. 1DGo). A control section in which the sense probe was used did not show a significant signal (Fig. 1Go, E and F).

Spatial expression of FGFR3 mRNA and STAT1 in the fracture callus
We next investigated the spatial expression of FGFR3 mRNA and STAT1 during fracture repair.

On d 4 after fracture, the thickness of the periosteum near the fracture site increased, and woven bone was formed by intramembranous ossification (hard callus). Near the fracture site, granulation tissue covered the fracture gap (soft callus) (Fig. 2AGo). In the soft callus, a faint signal for FGFR3 mRNA was seen in some proliferating chondrocytes (Fig. 2BGo, arrowheads), which also expressed COL2A1 mRNA (Fig. 2DGo); however, no signal was detected in undifferentiated mesenchymal cells that have the potential to differentiate into both osteoblasts and chondrocytes (Fig. 2BGo). These FGFR3-positive proliferating chondrocytes were also weakly immunoreactive for STAT1, and the staining was observed in the cytoplasm (Fig. 2EGo, arrowheads). At this stage, no significant signal for FGFR3 mRNA was detectable in cells in the hard callus (Fig. 2FGo). A control section in which the sense probe for FGFR3 was used did not show a significant signal (Fig. 2CGo).



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  		FIG. 2. Expression of FGFR3 mRNA and STAT1 on d 4 after fracture. A, Hematoxylin and eosin staining. (B–E, Fields correspond to the area in the left box in A. B–E are sequential sections. F, A field corresponds to the area in the right box in A. A faint signal for FGFR3 mRNA is seen in proliferating chondrocytes (B, arrowheads), which are also positive for COL2A1 mRNA (D). No signal is detectable in undifferentiated mesenchymal cells (B) and cells in the hard callus (F). FGFR3-positive proliferating chondrocytes are also weakly immunoreactive for STAT1, and the staining is observed in the cytoplasm (E, arrowheads). A negative control using sense probe for FGFR3 shows no significant signal (C). m, Mesenchymal cells; po, periosteum; cb, cortical bone; tb, trabecular bone. Asterisks show the fracture site. Scale bars, 800 µm (A), 100 µm (B–F).

 
On d 7, the thickness of the hard callus increased as intramembranous ossification proceeded. In the soft callus, abundant cartilaginous tissue was seen adjacent to the hard callus (Fig. 3AGo). At this stage, a weak signal for FGFR3 mRNA was detectable in prehypertrophic chondrocytes (Fig. 3BGo), which also expressed COL10A1 mRNA (Fig. 3CGo). Immunoreactivity for STAT1 was clearly detected in chondrocytes between proliferating and hypertrophic zone, and the staining was observed in the cytoplasm (Fig. 3DGo, arrowheads). At this stage, as on d 4, no significant signal for FGFR3 mRNA was detectable in cells in the hard callus (Fig. 3FGo).



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  		FIG. 3. Expression of FGFR3 mRNA and STAT1 on d 7 after fracture. A, Hematoxylin and eosin staining. B–E, Fields correspond to the area in the left box in A. B–E are sequential sections. F, A field corresponds to the area in the right box in A. A weak signal for FGFR3 mRNA is detectable in prehypertrophic chondrocytes (B), which are also positive for COL10A1 mRNA (C). No signal is detectable in cells in the hard callus (F). Immunoreactivity for STAT1 is also clearly detected in chondrocytes between proliferating and hypertrophic zone, and the staining is observed in the cytoplasm (D, arrowheads). A negative control for STAT1 using nonimmune mouse IgG shows no significant signal (E). pc, Proliferating chondrocytes; hc, hypertrophic chondrocytes; po, periosteum; tb, trabecular bone. Asterisk shows the fracture site. Scale bars, 800 µm (A), 50 µm (B–F).

 
From d 14, the cartilaginous tissue was replaced by woven trabecular bone in a process consistent with endochondral ossification. This was followed by bone remodeling, which produced mature trabeculae (Fig. 4AGo). In the soft callus on d 14, as on d 7, a signal for FGFR3 mRNA was detected in prehypertrophic chondrocytes (Fig. 4BGo), which also expressed COL10A1 mRNA (Fig. 4EGo), but the signal became more evident, compared with that of d 7. Immunoreactivity for STAT1 was also detectable in chondrocytes between proliferating and hypertrophic zone (Fig. 4DGo); however, at this stage, the staining was observed both in the cytoplasm (Fig. 4FGo, arrows) and in the nucleus (Fig. 4FGo, arrowheads) of hypertrophic chondrocytes. As on d 4 and 7, no significant signal for FGFR3 mRNA was detectable in cells in the hard callus (Fig. 4HGo).



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  		FIG. 4. Expression of FGFR3 mRNA and STAT1 on d 14 after fracture. A, Hematoxylin and eosin staining. B–E, Fields correspond to the area in the left box in A. B–E are sequential sections. F is a higher-magnification view of hypertrophic zone of chondrocytes in D. G is a similar region to F. H, A field corresponds to the area in the right box in A. A signal for FGFR3 mRNA is clearly detected in prehypertrophic chondrocytes (B), which are also positive for COL10A1 mRNA (E). No signal is detectable in cells in the hard callus (H). Immunoreactivity for STAT1 is also detectable in chondrocytes between proliferating and hypertrophic zone (D); however, the staining is observed both in the cytoplasm (F, arrows) and nucleus (F, arrowheads) of hypertrophic chondrocytes. Some hypertrophic chondrocytes near the chondro-osseous junction clearly show TUNEL-positive staining (G, arrowheads). A negative control using sense probe for FGFR3 shows no significant signal (C). pc, Proliferating chondrocytes; hc, hypertrophic chondrocytes; po, periosteum; tb, trabecular bone. Asterisk shows the fracture site. Scale bars, 800 µm (A), 50 µm (B–E and H), 20 µm (F and G).

 
Apoptotic cells in the fracture callus
The TUNEL staining showed a positive signal in hypertrophic chondrocytes in the soft callus. In particular, a strong signal was detected in some hypertrophic chondrocytes near the chondro-osseous junction, in which the nuclear localization of STAT1 was also detected (Fig. 4GGo, arrowheads). In the hard callus, osteocytes in the trabeculae were positively stained (data not shown). A positive control showed a consistent staining pattern in thymocytes undergoing spontaneous apoptosis in the rat thymus as well as hypertrophic chondrocytes in the growth plate from the bones containing the fracture callus (data not shown).

Temporal expression of FGFR3, STAT1, and p21 mRNAs in the fracture callus
Figure 5BGo shows changes in the amount of FGFR3 mRNA relative to the level of GAPDH mRNA. A small amount of FGFR3 mRNA was detectable in the unfractured femur. After fracture, sequential changes in the expression of FGFR3 mRNA were observed (Fig. 5AGo). The expression gradually increased between d 2 and 7, followed by the largest increase from d 7–14, with the maximum expression on d 14. From d 14–21, the expression rapidly declined, but was still elevated in comparison to the level in unfractured femur. The fold increase relative to the expression level in the unfractured femur at d 2, 4, 7, 14, and 21 was 1.4-, 1.7-, 1.7-, 2.2-, and 1.2-fold, respectively.



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  		FIG. 5. RPA for quantifying FGFR3 mRNA during fracture healing. RPA was performed on 20 µg total RNA isolated from unfractured femora and fracture calluses on d 2, 4, 7, 14, and 21. GAPDH was used as an internal standard for the amount and integrity of the RNA preparation. Embryos at 16.5 d p.c. were used as a positive control for the expression of FGFR3 mRNA. A, A representative autoradiographic image of the probe and the protective probe for FGFR3. B, Changes in the relative expression level of FGFR3 mRNA. Three samples were analyzed at each time point. The graph shows a mean value ± SD. UF, Unfractured femora.

 
Figure 6BGo shows changes in the amount of STAT1 and p21 mRNAs relative to the level of GAPDH mRNA. As shown in Fig. 6Go, A and B, STAT1 and p21 exhibited a similar expression profile throughout the healing process and were hardly detectable in the unfractured femur. After fracture, sequential changes in the expression of STAT1 and p21 mRNAs were also detected. The expression of these mRNAs gradually increased between d 4 and 14; in particular, p21 exhibited the largest increase ratio from d 7–14. Both mRNAs reached maximum levels of expression on d 14. From d 14–21, they rapidly declined together. Interestingly, the expression of STAT1 and p21 almost paralleled that of FGFR3 as was shown by RPA. A negative control sample did not show any specific bands for STAT1, p21, and GAPDH, indicating that the reverse transcribed products we used were not contaminated with genomic DNA (data not shown). It should be noted that because of the inherent limitations of semiquantitative PCR, the relative amounts of these mRNAs as presented in Fig. 6BGo should be considered approximate.



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  		FIG. 6. Semiquantitative RT-PCR analysis for quantifying STAT1 and p21 mRNAs during fracture healing. A, Expression of STAT1 and p21 mRNAs in the unfractured femur and the fracture callus at different time points. Note that each amplification cycle (23 times for STAT1 and 30 times for p21) was finalized within a linear range. B, Changes in the relative expression level of STAT1 and p21 mRNAs. The expression was quantified by comparing amplification of STAT1 or p21 with that of GAPDH. Three samples were evaluated at each time point, and each sample was evaluated in duplicate. The graph shows mean values ± SD. UF, Unfractured femora.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Bone formation is a continuous process that begins during fetal development and persists throughout life as a remodeling process. In the event of injury, bones heal by generating new bone rather than scar tissue; thus, it can accurately be described as a regenerative process (31). This indicates that some functional proteins during fetal development and skeletal repair are overlapping. However, skeletal repair does not necessarily seem to be a recapitulation of skeletal development because it is a unique biological process accompanied by a cascade of events that begins with an inflammatory reaction. We therefore suggest that some functional proteins play differential roles in skeletal development and fracture repair.

In this study, we showed a differential expression pattern of FGFR3 mRNA between skeletal development and fracture repair and that it was highly expressed during endochondral ossification in both processes. During embryonic endochondral ossification, FGFR3 mRNA was strongly expressed in resting and proliferating chondrocytes. In previous analyses using organ cultures of rat bones, FGFR3 mRNA was strongly expressed in chondrocytes but not in osteoblasts (32). In addition, Deng et al. (16) demonstrated that FGFR3 mRNA was expressed in the proliferation zone of chondrocytes of postnatal mice. These results are consistent with our data. Previous analyses of FGFR3-deficient mice revealed a remarkable increase in the length of the vertebral column and long bones as a result of enhanced and prolonged bone growth (15, 16). On the other hand, analyses of FGFR3-transgenic mice showed endochondral growth inhibition with restrained chondrocyte proliferation and maturation, penetration of ossification tufts, and aberrant vascularization (33, 34, 35). These observations indicate that FGFR3 is a negative regulator of chondrocyte proliferation and that FGFR3-mediated restrain of chondrocyte proliferation is required to maintain homeostatic endochondral development of long bones. During fracture repair, however, FGFR3 mRNA was strongly expressed in prehypertrophic chondrocytes and was scarcely expressed in proliferating chondrocytes, suggesting that FGFR3 plays some important roles other than the regulation of chondrocyte proliferation.

To determine the role of FGFR3 during endochondral ossification, we focused on the downstream molecules of FGFR3 intracellular signal transduction, STAT1 and p21. At first, we investigated the spatial expression patterns of FGFR3 mRNA and STAT1 in mouse embryos and the fracture callus. In developing long bones, FGFR3 mRNA was clearly detected in resting and proliferating chondrocytes and STAT1 in chondrocytes between proliferating and hypertrophic zone. This suggests that FGFR3 is not correlated with STAT1 during skeletal development. On the other hand, in the fracture callus, both FGFR3 mRNA and STAT1 were strongly detected in prehypertrophic chondrocytes and their distribution was well correlated. We next investigated the temporal expression profiles of FGFR3, STAT1, and p21 mRNAs and demonstrated that the expression profiles of STAT1 and p21 mRNAs were similar to that of FGFR3 mRNA. These data show that FGFR3-mediated STAT1-p21 signaling plays a role during fracture repair. In the present study, we showed the temporal expression of STAT1 and p21 by not RPA but RT-PCR because of their low expression levels. However, we speculate that there might be enough STAT1 and p21 proteins in the cytoplasm of chondrocytes in the callus to respond immediately to activated FGFR3 signaling, which is followed by their nuclear translocation. Thus, we suppose that the magnitudes of STAT1 and p21 expression might relate more specifically to that of FGFR3 expression in protein levels rather than in mRNA levels.

With regard to FGFR3 intracellular signal transduction in chondrocytes, Su et al. (18) have shown that constitutively activated FGFR3 signaling specifically activates STAT1 and induces nuclear translocation of STAT1, expression of p21, and growth arrest of chondrocytes. During endochondral ossification and in fracture repair, apoptosis of hypertrophic chondrocytes is considered as an essential process for replacement of cartilage with bone. We therefore hypothesized that FGFR3 intracellular signaling induces apoptosis of hypertrophic chondrocytes during fracture repair and carried out a TUNEL assay. The results showed that TUNEL-positive apoptotic cells were localized to hypertrophic chondrocytes, in which the expression of FGFR3 mRNA and the nuclear staining of STAT1 were observed together. Previous reports have shown the presence of TUNEL-positive apoptotic cells in hypertrophic chondrocytes in the fracture callus (36), supporting our TUNEL staining results. An in vitro study using chondrocytes derived from thanatophoric dysplasia fetuses demonstrated that activated FGFR3 signaling does not hamper chondrocyte proliferation but rather alters their differentiation by triggering premature apoptosis through activation of a STAT signaling pathway (37). Taking these findings into account, we speculated that the role of FGFR3 during fracture repair, which differs from that in skeletal development, is to induce apoptosis of hypertrophic chondrocytes, thereby promoting endochondral ossification. However, functional studies using genetically relevant models such as transgenic or knockout mice are required to elucidate the correlation of FGFR3 signaling with chondrocyte apoptosis during fracture repair.

In summary, FGFR3, STAT1, and p21 exhibited a similar temporal expression profile during fracture repair. Additionally, STAT1 was localized to the nucleus in FGFR3-positive hypertrophic chondrocytes in the callus, which also showed TUNEL-positive staining, suggests that FGFR3-mediated STAT1-p21 signal transduction finally induces apoptosis of hypertrophic chondrocytes and modulates the replacement of cartilage with bone. These mechanisms may regulate chondrogenesis and promote endochondral ossification, contributing to the healing of fractured bones.


   Acknowledgments
 
We thank Drs. M. Tahara and S. Sano (Department of Orthopaedic Surgery, Chiba University Graduate School of Medicine) for their technical supports, and Dr. N. Amizuka (Faculty of Dentistry, Niigata University, Niigata, Japan) for reviewing this manuscript and providing useful suggestions.


   Footnotes
 
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

Abbreviations: DIG, Digoxigenin; FGFR, fibroblast growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p.c., post coitum; RPA, ribonuclease protection assay; STAT, signal transducer and activator of transcription; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.

Received January 31, 2003.

Accepted for publication June 26, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Peters K, Werner S, Chen G, Williams LT 1992 Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development 114:233–243[Abstract]
  2. Peters K, Ornitz D, Werner S, Williams LT 1993 Unique expression pattern of the FGF receptor 3 gene during mouse organogenesis. Dev Biol 155:423–430[CrossRef][Medline]
  3. Orr-Urtreger A, Givol G, Yayon A, Yarden Y, Lonai P 1991 Developmental expression of two murine fibroblast growth factor receptors, flg and bek. Development 113:1419–1434[Abstract]
  4. Stark K, McMahon J, McMahon A 1991 FGFR-4, a new member of the fibroblast growth factor family, expressed in the definitive endoderm and skeletal muscle lineages of the mouse. Development 113:641–651[Abstract]
  5. Park WJ, Bellus GA, Jabs EW 1995 Mutations in fibroblast growth factor receptors: phenotypic consequences during eukaryotic development. Am J Hum Genet 57:748–754[Medline]
  6. Shiang R, Thompson LM, Zhu Y, Church DM, Fielder TJ, Bocian M, Winokur ST, Wasmuth JJ 1994 Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 78:335–342[CrossRef][Medline]
  7. Rousseau F, Bonaventure J, Legeal-Mallet L, Pelet A, Rozet J, Maroteaux P, LeMerrer M, Munnich A 1994 Mutations in the gene encoding fibroblast growth factor receptor-3 in achondroplasia. Nature 371:252–254[CrossRef][Medline]
  8. Bellus GA, McIntosh I, Smith EA, Aylsworth AS, Kaitila I, Horton WA, Greenhaw GA, Hecht JT, Francomano CA 1995 A recurrent mutation in the tyrosine kinase domain of fibroblast growth factor receptor 3 causes hypochondroplasia. Nat Genet 10:357–359[CrossRef][Medline]
  9. Munke M, Schell U, Hehr A, Robin NH, Losken HW, Schinzel A, Pulleyn LJ, Rutland P, Reardon W, Malcolm S, Winter RM 1994 A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome. Nat Genet 8:269–274[CrossRef][Medline]
  10. Rutland P, Pulleyn LJ, Reardon W, Baraister M, Hayward R, Jones B, Malcolm S, Winter RM, Oldridge M, Slaney SF, Poole MD, Wilkie AO 1995 Identical mutations in the FGFR2 gene cause both Pfeiffer and Crouzon syndrome phenotypes. Nat Genet 9:173–176[CrossRef][Medline]
  11. Reardon W, Winter RM, Rutland P, Pulleyn LJ, Jones BM, Malcolm S 1994 Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nat Genet 8:98–103[CrossRef][Medline]
  12. Wilkie AO, Slaney SF, Oldridge M, Poole MD, Ashworth GJ, Hockley AD, Hayward RD, David DJ, Pulleyn L, Rutland P 1995 Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome. Nat Genet 9:165–172[CrossRef][Medline]
  13. Jabs EW, Li X, Scott AF, Meyers C, Chen W, Eccles M, Mao J, Charnas LR, Jackson CE, Jaye M 1994 Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nat Genet 8:275–279[CrossRef][Medline]
  14. Nakajima A, Nakajima F, Shimizu S, Ogasawara A, Wanaka A, Moriya H, Einhorn TA, Yamazaki M 2001 Spatial and temporal gene expression for fibroblast growth factor type I receptor (FGFR1) during fracture healing in the rat. Bone 29:458–466[Medline]
  15. Colvin JS, Bohne BA, Harding GW, McEwen DG, Ornitz DM 1996 Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat Genet 12:390–397[CrossRef][Medline]
  16. Deng C, Wynshaw-Boris A, Zhou F, Kuo A, Leder P 1996 Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell 84:911–921[CrossRef][Medline]
  17. Kanai M, Goke M, Tsunekawa S, Podolsky DK 1997 Signal transduction pathway of human fibroblast growth factor receptor 3. Identification of a novel 66-kDa phosphoprotein. J Biol Chem 272:6621–6628[Abstract/Free Full Text]
  18. Su WC, Kitagawa M, Xue N, Xie B, Garofalo S, Cho J, Deng C, Horton WA, Fu XY 1997 Activation of stat1 by mutant fibroblast growth-factor receptor in thanatophoric dysplasia type II dwarfism. Nature 386:288–292[CrossRef][Medline]
  19. Waga S, Hannon GJ, Beach D, Stillman B 1994 The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 369:574–578[CrossRef][Medline]
  20. Chen J, Jackson PK, Kirschner MW, Dutta A 1995 Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature 374:386–388[CrossRef][Medline]
  21. Luo Y, Hurwitz J, Massague J 1995 Cell-cycle inhibition by independent CDK and PCNA binding domains in p21 Cip1. Nature 375:159–161[CrossRef][Medline]
  22. Sherr CJ, Roberts JM 1995 Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9:1149–1163[Free Full Text]
  23. Bonnarens F, Einhorn TA 1984 Production of a standard closed fracture in laboratory animal bone. J Orthop Res 2:97–101[CrossRef][Medline]
  24. Andrikopoulos K, Suzuki HR, Solursh M, Ramirez F 1992 Localization of pro-{alpha}2 (V) collagen transcripts in the tissues of the developing mouse embryo. Dev Dyn 195:113–120[Medline]
  25. Apte SS, Seldin MF, Hayashi M, Olsen BR 1992 Cloning of the human and mouse type X collagen gene to chromosome 10. Eur J Biochem 206:217–224[Medline]
  26. Yamazaki M, Majeska RJ, Yoshioka H, Moriya H, Einhorn TA 1997 Spatial and temporal expression of fibril-forming minor collagen genes (types V and XI) during fracture healing. J Orthop Res 15:757–764[CrossRef][Medline]
  27. Yamazaki M, Nakajima F, Ogasawara A, Moriya H, Majeska RJ, Einhorn TA 1999 Spatial and temporal distribution of CD44 and osteopontin in fracture callus. J Bone Joint Surg Br 81:508–515
  28. Suzuki H, Takahashi K, Yamagata M, Shimizu S, Moriya H, Yamazaki M 2001 Spatial and temporal collagen gene expression in lumbar intertransverse fusion in the rabbit. J Bone Joint Surg Br 83:760–766
  29. Nakajima F, Ogasawara A, Goto K, Moriya H, Ninomiya Y, Einhorn TA, Yamazaki M 2001 Spatial and temporal gene expression in chondrogenesis during fracture healing and the effects of basic fibroblast growth factor. J Orthop Res 19:935–944[CrossRef][Medline]
  30. Nakajima A, Shimoji N, Shiomi K, Shimizu S, Moriya H, Einhorn TA, Yamazaki M 2002 Mechanisms for the enhancement of fracture healing in rats treated with intermittent low dose human parathyroid hormone (1–34). J Bone Miner Res 17:2038–2047[CrossRef][Medline]
  31. Ferguson C, Alpern E, Miclau T, Helms JA 1999 Does adult fracture repair recapitulate embryonic skeletal formation? Mech Dev 87:57–66[CrossRef][Medline]
  32. Molteni A, Modrowski D, Hott M, Marie PJ 1999 Differential expression of fibroblast growth factor receptor-1, -2, -3 and syndecan-1, -2, and -4 in neonatal rat mandibular condyle and calvaria during osteogenic differentiation in vitro. Bone 24:337–347[Medline]
  33. Wang Y, Spatz MK, Kannan K, Hayk H, Avivi A, Gorivodsky M, Pines M, Yayon A, Lonai P, Givol D 1999 A model for achondroplasia produced by targeting fibroblast growth factor 3. Proc Natl Acad Sci USA 96:4455–4460[Abstract/Free Full Text]
  34. Chen L, Adar R, Yang X, Monsonego EO, Li C, Hauschka PV, Yayon A, Deng CX 1999 Gly369Cys mutation in mouse FGFR3 causes achondroplasia by affecting both chondrogenesis and osteogenesis. J Clin Invest 104:1517–1525[Medline]
  35. Segev O, Chumakov I, Nevo Z, Givol D, Madar-Shapiro L, Sheinin Y, Weinreb M, Yayon A 2000 Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3G380R transgenic mice. Human Mol Genet 9:249–258[Abstract/Free Full Text]
  36. Li G, White G, Connolly C, Marsh D 2002 Cell proliferation and apoptosis during fracture healing. J Bone Miner Res 17:791–799[CrossRef][Medline]
  37. Legeai-Mallet L, Benoist-Lasselin C, Delezoide A, Munnich A, Bonaventure J 1998 Fibroblast growth factor receptor 3 mutations promote apoptosis but do not alter chondrocyte proliferation in thanatophoric dysplasia. J Biol Chem 273:13007–13014[Abstract/Free Full Text]



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