SPATIAL AND TEMPORAL GENE EXPRESSION FOR FIBROBLAST … · healing, FGFR1 is produced locally but...
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Session 41 - Fracture Repair II - Gateway Ballroom, Wed 8:00 AM - 10:00 AM 0256 47th Annual Meeting, Orthopaedic Research Society, February 25 - 28, 2001, San Francisco, California SPATIAL AND TEMPORAL GENE EXPRESSION FOR FIBROBLAST GROWTH FACTOR TYPE I RECEPTOR DURING FRACTURE HEALING IN THE RAT +*Nakajima, A; *Nakajima, F; *Shimizu, S; *Ogasawara, A; *Goto, K; **Wanaka, A; *Moriya, H; ***Einhorn, T A; *Yamazaki, M +*Department of Orthopaedic Surgery Chiba University School of Medicine, Chba, Japan. 1-8-1 Inohana Chuo-ku Chiba Japan, +81-226-2117, Fax: +81-226-2116, [email protected] Introduction: Previous studies have demonstrated that FGF-FGFR signal transduction plays a crucial role in skeletal development (1). Recent reports have shown that exogenous bFGF enlarges fracture calluses and accerelates the healing of osteotomized long bones (2, 3). Thus, it is possible that FGF ligand-receptor signals play important roles not only in embryogenesis but also in skeletal repair. In the processes of fracture healing, however, detailed expression of FGF ligands and receptors have not yet been fully established. In the present study, we employed a standardized rat model of fracture healing and analyzed the spatial and temporal gene expression for bFGF and FGFR1. Based on the results, we discuss a possible role for FGF-FGFR1 signaling in fracture repair. Materials and Methods: Fracture model: Closed middiaphyseal fractures were created in the right femora of SD rats (4). The rats were euthanized at 1, 4, 7, 14, 21 and 28 days postoperatively, and fractured femora were fixed, decalcified, and embedded in paraffin. In situ hybridization (ISH): Sections were hybridized with DIG-labeled cRNA probes for bFGF and FGFR1. To evaluate matrix production and bone remodeling, sections were also hybridized with probes for collagen types I and V, and osteocalcin. Tartarate resistent acid phosphate (TRAP) staining: For the identification of osteoclasts, TRAP staining was performed. Immunohistochemistry: To evaluate cell growth activity, sections were reacted with an antibody for PCNA. RNAse protection assay (RPA): After the isolation of total RNA from fracture calluses, RPA was carried out with 32 P-labeled cRNA probes for bFGF, FGFR1 and GAPDH. Results: ISH: In normal femoral shafts, a weak FGFR1 signal was seen in cells in the periosteum, endosteum and medullary cavity. On day 1 after fracture, a moderate or relatively strong signal for FGFR1 was detected in osteoprogenitor cells under the periosteum (Fig. 1A, arrows) and in round- shaped mesenchymal cells among the muscle layers (Fig. 1B, arrows). Such FGFR1-positive cells at this stage were PCNA-positive, but negative for collagen types I and V. From day 4 to day 7, periosteal hard callus was formed. At this stage, a strong FGFR1 signal was detected in cuboidal osteoblasts in the deep layer of the hard callus, but the signal was weak in the superficial layer. Similarly, osteocalcin gene expession preferentially occurred in the deep layer. From day 14, cartilage was replaced by bony tissues and endochondral ossification rapidly progressed. At the ossification front, TRAP- positive multinuclear cells (Fig. 1D, arrow), presumably mature osteoclasts, expressed a moderate signal for FGFR1 (Fig. 1C, arrow). At the central portion of the hard callus, TRAP-positive cells not attaching to the trabeculae, presumably immature osteoclasts, expressed FGFR1 gene, but the signal was very weak. During bone remodeling of the hard callus, a strong FGFR1 signal was detected in mature osteoblasts lining the trabeculae even in the later stages of fracture healing. In spite of such widespread gene expression for FGFR1, we could not detect any bFGF signals throughout the healing stages. RPA: By quantification, a small amount of FGFR1 mRNA was detected in normal femora. On day 1, FGFR1 mRNA levels were approximately 3-fold higher than in normal femora (Fig. 2). They reached a maximum level on day 14, and remained at relatively high levels even on day 28 (Fig. 2). In contrast to the enhanced expression for FGFR1 mRNA, bFGF mRNA expression was hardly detected (Fig. 2). Discussion: The present results demonstrate that gene expression for FGFR1 is rapidly upregulated after fracture, and maintained at relatively high levels even in the later stages of healing. In spite of such enhanced expression of FGFR1, little expression for bFGF mRNA was detected by our ISH and RPA analyses. Previous studies showed that a considerable amount of bFGF peptide is stored in bone matrix (5). Thus we suggest that, during fracture healing, FGFR1 is produced locally but its ligand (bFGF) is mainly released from the bone matrix. From the expression patterns of FGFR1, we suggest that FGF-FGFR1 signaling may preferentially contribute to cell proliferation during initial callus formation, and to bone remodeling in the later stages. In this study, we also revealed that FGFR1 mRNA was expressed not only in osteoblastic cells but also in mature osteoclasts in fracture calluses, suggesting that FGFs directly act on osteoclasts in vivo. When bFGF will be used for fracture treatment, it should be noted that role of FGF-FGFR1 signaling may not necessarily be consistent throughout healing, but may vary depending on the healing stage. (A) (B) (C) (D) Fig. 1: In situ hybridization for FGFR1 (A, B, C) and TRAP staining (D). (A) On day 1 after fracture, osteoblastic cells under the periosteum expressed FGFR1 mRNA (arrows). (B) On day 1, mesenchymal cells among the muscle layers were FGFR1-positive (arrows). (C, D) High-power magnification views of the endochondral ossification front on day 14. C and D are sequential sections. A TRAP-positive multinuclear cell (D, arrow) expressed FGFR1 mRNA (C, arrow). PO: periosteum, MS: muscle, HC: hypertrophic chondrocytes. Fig. 2: RNAse protection assay for FGFR1 mRNA and bFGF mRNA (BR: brain, NF: normal femoral shaft). A right graph shows relative expression for FGFR1 mRNA. References: (1) Passos-Bueno et al.: Human Mutat 14: 115, 1999. (2) Nakamura et al.: J Bone Miner Res 13: 942, 1998. (3) Radomsky et al.: J Orthop Res 17: 607, 1999. (4) Bonnarens and Einhorn: J Orthop Res 2: 97, 1984. (5) Globus: Endocrinol 124: 1539, 1989. **Department of Cell Science Institute of Biomedical Sciences Fukushima Medical University School of Medicine, Fukushima, Japan. ***Department of Orthopaedic Surgery Boston University School of Medicine, Boston, MA.
Transcript of SPATIAL AND TEMPORAL GENE EXPRESSION FOR FIBROBLAST … · healing, FGFR1 is produced locally but...
Session 41 - Fracture Repair II - Gateway Ballroom, Wed 8:00 AM - 10:00 AM0256 47th Annual Meeting, Orthopaedic Research Society, February 25 - 28, 2001, San Francisco, California
SPATIAL AND TEMPORAL GENE EXPRESSION FOR FIBROBLAST GROWTH FACTOR TYPE I RECEPTOR DURINGFRACTURE HEALING IN THE RAT
+*Nakajima, A; *Nakajima, F; *Shimizu, S; *Ogasawara, A; *Goto, K; **Wanaka, A; *Moriya, H; ***Einhorn, T A; *Yamazaki, M+*Department of Orthopaedic Surgery Chiba University School of Medicine, Chba, Japan. 1-8-1 Inohana Chuo-ku Chiba Japan, +81-226-2117, Fax: +81-226-2116,
Introduction: Previous studies have demonstrated that FGF-FGFR signaltransduction plays a crucial role in skeletal development (1). Recent reportshave shown that exogenous bFGF enlarges fracture calluses and accerelatesthe healing of osteotomized long bones (2, 3). Thus, it is possible that FGFligand-receptor signals play important roles not only in embryogenesis butalso in skeletal repair. In the processes of fracture healing, however, detailedexpression of FGF ligands and receptors have not yet been fully established.In the present study, we employed a standardized rat model of fracture healingand analyzed the spatial and temporal gene expression for bFGF and FGFR1.Based on the results, we discuss a possible role for FGF-FGFR1 signaling infracture repair.Materials and Methods: Fracture model: Closed middiaphyseal fractureswere created in the right femora of SD rats (4). The rats were euthanized at 1,4, 7, 14, 21 and 28 days postoperatively, and fractured femora were fixed,decalcified, and embedded in paraffin. In situ hybridization (ISH): Sectionswere hybridized with DIG-labeled cRNA probes for bFGF and FGFR1. Toevaluate matrix production and bone remodeling, sections were alsohybridized with probes for collagen types I and V, and osteocalcin. Tartarateresistent acid phosphate (TRAP) staining: For the identification of osteoclasts,TRAP staining was performed. Immunohistochemistry: To evaluate cellgrowth activity, sections were reacted with an antibody for PCNA. RNAseprotection assay (RPA): After the isolation of total RNA from fracturecalluses, RPA was carried out with 32P-labeled cRNA probes for bFGF,FGFR1 and GAPDH.Results: ISH: In normal femoral shafts, a weak FGFR1 signal was seen incells in the periosteum, endosteum and medullary cavity. On day 1 afterfracture, a moderate or relatively strong signal for FGFR1 was detected inosteoprogenitor cells under the periosteum (Fig. 1A, arrows) and in round-shaped mesenchymal cells among the muscle layers (Fig. 1B, arrows). SuchFGFR1-positive cells at this stage were PCNA-positive, but negative forcollagen types I and V. From day 4 to day 7, periosteal hard callus wasformed. At this stage, a strong FGFR1 signal was detected in cuboidalosteoblasts in the deep layer of the hard callus, but the signal was weak in thesuperficial layer. Similarly, osteocalcin gene expession preferentially occurredin the deep layer. From day 14, cartilage was replaced by bony tissues andendochondral ossification rapidly progressed. At the ossification front, TRAP-positive multinuclear cells (Fig. 1D, arrow), presumably mature osteoclasts,expressed a moderate signal for FGFR1 (Fig. 1C, arrow). At the centralportion of the hard callus, TRAP-positive cells not attaching to the trabeculae,presumably immature osteoclasts, expressed FGFR1 gene, but the signal wasvery weak. During bone remodeling of the hard callus, a strong FGFR1 signalwas detected in mature osteoblasts lining the trabeculae even in the laterstages of fracture healing. In spite of such widespread gene expression forFGFR1, we could not detect any bFGF signals throughout the healing stages.RPA: By quantification, a small amount of FGFR1 mRNA was detected innormal femora. On day 1, FGFR1 mRNA levels were approximately 3-foldhigher than in normal femora (Fig. 2). They reached a maximum level on day14, and remained at relatively high levels even on day 28 (Fig. 2). In contrastto the enhanced expression for FGFR1 mRNA, bFGF mRNA expression washardly detected (Fig. 2).Discussion: The present results demonstrate that gene expression for FGFR1is rapidly upregulated after fracture, and maintained at relatively high levelseven in the later stages of healing. In spite of such enhanced expression ofFGFR1, little expression for bFGF mRNA was detected by our ISH and RPAanalyses. Previous studies showed that a considerable amount of bFGFpeptide is stored in bone matrix (5). Thus we suggest that, during fracturehealing, FGFR1 is produced locally but its ligand (bFGF) is mainly releasedfrom the bone matrix. From the expression patterns of FGFR1, we suggestthat FGF-FGFR1 signaling may preferentially contribute to cell proliferationduring initial callus formation, and to bone remodeling in the later stages. Inthis study, we also revealed that FGFR1 mRNA was expressed not only in
osteoblastic cells but also in mature osteoclasts in fracture calluses, suggestingthat FGFs directly act on osteoclasts in vivo. When bFGF will be used forfracture treatment, it should be noted that role of FGF-FGFR1 signaling maynot necessarily be consistent throughout healing, but may vary depending onthe healing stage.
(A) (B)
(C) (D)
Fig. 1: In situ hybridization for FGFR1 (A, B, C) and TRAP staining (D). (A)On day 1 after fracture, osteoblastic cells under the periosteum expressedFGFR1 mRNA (arrows). (B) On day 1, mesenchymal cells among the musclelayers were FGFR1-positive (arrows). (C, D) High-power magnification viewsof the endochondral ossification front on day 14. C and D are sequentialsections. A TRAP-positive multinuclear cell (D, arrow) expressed FGFR1mRNA (C, arrow). PO: periosteum, MS: muscle, HC: hypertrophicchondrocytes.
Fig. 2: RNAse protection assay for FGFR1 mRNA and bFGF mRNA (BR:brain, NF: normal femoral shaft). A right graph shows relative expression forFGFR1 mRNA.
References: (1) Passos-Bueno et al.: Human Mutat 14: 115, 1999. (2)Nakamura et al.: J Bone Miner Res 13: 942, 1998. (3) Radomsky et al.: JOrthop Res 17: 607, 1999. (4) Bonnarens and Einhorn: J Orthop Res 2: 97,1984. (5) Globus: Endocrinol 124: 1539, 1989.
**Department of Cell Science Institute of Biomedical Sciences FukushimaMedical University School of Medicine, Fukushima, Japan.***Department of Orthopaedic Surgery Boston University School ofMedicine, Boston, MA.
