Spatial and Temporal Expression of Periostin, a Novel Gene Detected by Complementarydna Microarray during Fracture Healing

Introduction Recent advances in molecular biological techniques have enabled investigators to determine the spatial and temporal expression of genes during fracture healing. Thus far, however, a limited number of genes have been identified, and details of the healing processes have not been fully established. In the previous study, we have used a quantitative cDNA microarray technique, and comprehensively analyzed the gene expression during normal fracture healing (1). Among 2304 embryorelated genes, six genes were up-regulated 3 days after fracture, and 3 of 6 genes, periostin, calumenin and FHL-1 were identified as novel candidate genes involved in fracture repair (Table 1). In the present study, we focused on periostin since its up-regulation ratio was highest. The purpose of this study is to reveal the spatial and temporal expression of periostin and to assess its possible role in fracture repair. Material and methods Fracture model: Eight to 9 week old Balb/c mice were used for this study (13 animals per time point). Closed, transverse, middiaphyseal fractures were created in bilateral tibiae as previously described (2). The mice were euthanized at 3, 7 and 14 days after fracture, and fractured tibiae were harvested. For controls, diaphyses of unfractured tibiae were also harvested. These experimental procedures were approved by the Animal Care and Use Committee of Chiba University. In situ hybridization: To investigate the localization of cells expressing periostin, in situ hybridization was performed. Tibiae were fixed with 4% paraformaldehyde, decalcified with 20% EDTA/ 0.05M Tris-HCl (pH 7.4), and embedded in paraffin. Digoxigenin-11-UTPlabeled single-strand RNA probes (antisense and sense probe) for mouse periostin cDNA containing a 2.6-kbp (596-3193) fragment were prepared as previously described (3). Paraffin sections were hybridized with the antisense or sense probes at 50C for 16 h. The signals were detected with a DIG detection kit (Roche Molecular Biochemicals). Northern blotting: To confirm the quantitative changes of periostin gene expression, Northern analysis was performed. Total cellular RNA was extracted from harvested tibiae using TRIZOL (GIBCO BRL). Approximately 30 μg of RNA from each sample was separated on a 1% agarose-formaldehyde gel and transferred to a nylon membrane (Hybond-XL, Amersham). The nylon membrane was hybridized at 65C for 2 h with the P-labeled mouse periostin cDNA probe. Hybridization was also performed with a P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe to evaluate the mRNA sample loading. Results In situ hybridization: In the diaphysis of intact tibiae, a moderate signal for periostin was localized to preosteoblastic cells in the periosteum. On day 3 after fracture, a strong signal for periostin was detected in undifferentiated mesenchymal cells near the fracture site (Figure 1a, b). A moderate signal was detected in proliferating preosteoblastic cells in the periosteum and cells among muscle layers. On day 7, a moderate signal for periostin was detected in proliferating preosteoblastic cells in the periosteum. No signal was found in osteocytes or osteoblasts in subperiosteal woven bone. In the soft callus, the periostin signal that had been strongly detected on day 3 in undifferentiated mesenchymal cells was decreased, and the signal between muscle layers became faint. On day 14, the periostin signal that had been detected in preosteoblastic cells in the periosteum and undifferentiated mesenchymal cells in the soft callus on day 7 was almost depleted, and the signal was barely detectable in the fracture callus. No signal was observed in a negative control with sense probe. Northern blotting: Figure 2 shows the time course of periostin mRNA expression relative to that of GAPDH mRNA. A small amount of periostin mRNA was detected in normal tibial shafts. On day 3 after fracture, periostin mRNA expression reached a peak and the level increased 2.8-fold compared with that of the normal tibiae (Figure 2a, b). On day 7, periostin mRNA level was 2.6-fold higher than that of normal tibiae. On day 14, the mRNA level rapidly declined and showed a 1.8-fold expression increase compared to normal tibiae (Figure 2b). Discussion Periostin was originally identified as a secreted factor in an MC3T3E1 cDNA library screen (4). The amino acid sequence has homology with that of fasciclin-I, an adhesion molecule in insects, and previous studies suggested that periostin has a function as a homophilic adhesion molecule (5). The present results demonstrated that periostin mRNA was locally expressed in undifferentiated mesenchymal cells and immature osteoblasts, but not in mature osteoblasts and mature chondrocytes throughout the healing process. Furthermore, we revealed that the expression level was up-regulated after fracture, reached a peak on day 3, and rapidly decreased by day 14. These findings suggest that periostin is a specific marker for preosteoblasts and plays an important role in callus formation during the early stage of fracture healing.