Motohiko Sato

Maturational disturbance of chondrocytes in Cbfa1-deficient mice.

Cbfa1, a transcription factor that belongs to the runt‐domain gene family, plays an essential role in osteogenesis. Cbfa1‐deficient mice completely lacked both intramembranous and endochondral ossification, owing to the maturational arrest of osteoblasts, indicating that Cbfa1 has a fundamental role in osteoblast differentiation. However, Cbfa1 was also expressed in chondrocytes, and its expression was increased according to the maturation of chondrocytes. Terminal hypertrophic chondrocytes expressed Cbfa1 extensively. The significant expression of Cbfa1 in hypertrophic chondrocytes was first detected at embryonic day 13.5 (E13.5), and its expression in hypertrophic chondrocytes was most prominent at E14.5–16.5. In Cbfa1‐deficient mice, whose entire skeleton was composed of cartilage, the chondrocyte differentiation was disturbed. Calcification of cartilage occurred in the restricted parts of skeletons, including tibia, fibula, radius, and ulna. Type X collagen, BMP6, and Indian hedgehog were expressed in their hypertrophic chondrocytes. However, osteopontin, bone sialoprotein, and collagenase 3 were not expressed at all, indicating that they are directly regulated by Cbfa1 in the terminal hypertrophic chondrocytes. Chondrocyte differentiation was severely disturbed in the rest of the skeleton. The expression of PTH/PTHrP receptor, Indian hedgehog, type X collagen, and BMP6 was not detected in humerus and femur, indicating that chondrocyte differentiation was blocked before prehypertrophic chondrocytes. These findings demonstrate that Cbfa1 is an important factor for chondrocyte differentiation. Dev Dyn 1999;214:279–290.

Transcriptional regulation of osteopontin gene in vivo by PEBP2αA/CBFA1 and ETS1 in the skeletal tissues

Osteopontin (Opn) and polyoma enhancer-binding protein (PEBP) 2αA/core binding factor (CBFA) 1 have been suggested to play important roles in ossification. The overlapping localization of opn and PEBP2αA/CBFA1 mRNA, and the marked decrease of opn mRNA expression in PEBP2αA knockout mice, indicated that the transcription of opn gene was controlled by PEBP2αA. In the present study, we determined the direct regulation of PEBP2αA on the opn promoter activity. Opn promoter activity was markedly enhanced by PEBP2αA and ETS1 in a synergistic manner. The synergistic effect was diminished when either the PEBP2αA or ETS1 binding site was mutated, or the spatial arrangement of these sites was mutated by a 4-nt insertion. The distance between these sites was important for transactivation but not protein-DNA binding. The direct interaction between PEBP2αA and ETS1 was depended on protein-DNA binding. These results suggested that the specific spatial arrangement of both sites and direct interaction between PEBP2αA and ETS1, were essential for promoter function. Furthermore, endogenous opn mRNA was decreased with the introduction of dominant negative PEBP2αA to MC3T3/E1 cells expressing endogenous PEBP2αA, ETS1 and opn. These findings suggest that PEBP2αA and ETS1 cooperate in vivo to regulate expression of the opn gene in the skeletal tissue. Cell type-specific regulation of Opn gene expression will also be discussed.

Mechanical Tension-Stress Induces Expression of Bone Morphogenetic Protein (BMP)-2 and BMP-4, but Not BMP-6, BMP-7, and GDF-5 mRNA, During Distraction Osteogenesis

Bone lengthening with osteotomy and gradual distraction was achieved in 57 rats, and the effect of mechanical tension‐stress on gene expression of bone morphogenetic proteins (BMPs) was investigated by in situ hybridization and Northern blot analysis using probes of BMP‐2, BMP‐4, BMP‐6, BMP‐7, and growth/differentiation factor (GDF)‐5. There was a lag phase for 7 days after femoral osteotomy until gradual distraction was carried out for 21 days at a rate of 0.25 mm/12 h using a small external fixator. The signals of the above BMPs mRNA were not detected in the intact rat bone but they were induced after osteotomy except those for BMP‐7. By 4 days after osteotomy, BMP‐2 and BMP‐4 mRNAs were detected in chondrogenic precursor cells in the subperiosteal immature callus. BMP‐6 and GDF‐5 mRNA were detected in more differentiated cells in chondroid bone. By 7 days after osteotomy, cartilaginous external callus and bony endosteal callus were formed. Meanwhile, the signals of BMP‐2 and BMP‐4 mRNAs declined to preoperative levels, whereas the signals of BMP‐6 and GDF‐5 mRNAs were rather elevated. As distraction was started, the callus elongated and eventually separated into proximal and distal segments forming a fibrous interzone in the middle. Expression of BMP‐2 and BMP‐4 mRNAs was markedly induced at this stage. Their signals were detected widely among chondrogenic and osteogenic cells and their precursor cells sustaining mechanical tension‐stress at the fibrous interzone. BMP‐6 and GDF‐5 mRNAs were detected exclusively in chondrogenic cells at both ends of the fibrous interzone, where endochondral ossification occurred. But neither mRNA was detected in terminally differentiated hypertrophic chondrocytes. As distraction advanced, the cartilage was progressively resorbed from both ends and new bone was formed directly by intramembranous ossification. There was no new cartilage formation in the advanced stage of distraction. The signals of BMP‐6 and GDF‐5 mRNA declined by this stage, while those of BMP‐2 and BMP‐4 were maintained at high level for as long as distraction was continued. After completion of distraction, the fibrous interzone fused and the lengthened segment was consolidated. BMP‐2, BMP‐4, BMP‐6, nor GDF‐5 was expressed at this stage. The signals of BMP‐7 were not detected throughout the experiment. The present results suggest that excellent and uninterrupted bone formation during distraction osteogenesis owes to enhanced expression of BMP‐2 and BMP‐4 genes by mechanical tension‐stress. Abundant gene products of BMP‐2 and BMP‐4 could induce in situ bone formation by paracrine and autocrine mechanisms.

Role of Osteopontin in Bone Remodeling Caused by Mechanical Stress

Changes in the number and proportion of osteopontin mRNA (Opn) expressing osteocytes and osteoclasts caused by the mechanical stress applied during experimental tooth movement were examined in the present study. Opn expression was detected in the osteocytes on the pressure side at the early stage, and gradually spread to those on the tension side and also to the osteoblasts and bone‐lining cells in the alveolar bone. Only 3.3% of the osteocytes located on the pressure side expressed Opn in the interradicular septum of control rats; in contrast, the value was increased to 87.5% at 48 h after the initiation of tooth movement. These results indicate that these cells responded to mechanical stress loaded on the bone with expression of the osteopontin gene. Following the increased expression of Opn in these cells, a 17‐fold greater number of osteoclasts compared with the control and numerous resorption pits were observed on the pressure side of the alveolar bone. Injection of arginine‐glycine‐aspartic acid‐serine peptide but not that of arginine‐glycine‐glutamic acid‐serine peptide strongly inhibited the increase in the number of osteoclasts. Furthermore, an in vitro migration assay demonstrated the chemotactic activity of osteopontin (OPN) on the precursor of osteoclasts. Our study strongly suggests that OPN is an important factor triggering bone remodeling caused by mechanical stress.

THREE MODES OF OSSIFICATION DURING DISTRACTION OSTEOGENESIS IN THE RAT

We developed a rat model of limb lengthening to study the basic mechanism of distraction osteogenesis, using a small monolateral external fixator. In 11-week-old male rats we performed a subperiosteal osteotomy in the midshaft of the femur with distraction at 0.25 mm every 12 hours from seven days after operation. Radiological and histological examinations showed a growth zone of constant thickness in the middle of the lengthened segment, with formation of new bone at its proximal and distal ends. Osteogenic cells were arranged longitudinally along the tension vector showing the origin and the fate of individual cells in a single section. Typical endochondral bone formation was prominent in the early stage of distraction, but intramembraneous bone formation became the predominant mechanism of ossification at later stages. We also showed a third mechanism of ossification, transchondroid bone formation. Chondroid bone, a tissue intermediate between bone and cartilage, was formed directly by chondrocyte-like cells, with transition from fibrous tissue to bone occurring gradually and consecutively without capillary invasion. In situ hybridisation using digoxigenin-11-UTP-labelled complementary RNAs showed that the chondroid bone cells temporarily expressed type-II collagen mRNA. They did not show the classical morphological characteristics of chondrocytes, but were assumed to be young chondrocytes undergoing further differentiation into bone-forming cells. We found at least three different modes of ossification during bone lengthening by distraction osteogenesis. We believe that this is the first report of such a rat model, and have shown the validity of in situ hybridisation techniques for the study of the cellular and molecular mechanisms involved in distraction osteogenesis.

Expression of Bone Matrix Proteins mRNA During Distraction Osteogenesis

Distraction osteogenesis is a recently advanced principle of bone lengthening in which a bone separated by osteotomy is subjected to slow progressive distraction using an external fixation device. Appropriate mechanical tension‐stress is believed not to break the callus but rather to stimulate osteogenesis. To study the molecular features of this process, the expression and localization of the mRNAs encoding osteopontin (OPN), osteocalcin (OC), matrix Gla protein (MGP), osteonectin (ON), and collagen type I and II during distraction osteogenesis were examined by in situ hybridization and Northern blot analysis. The process can be divided into three distinct phases: the lag phase for 7 days between osteotomy and the beginning of distraction, the distraction phase for 21 days, and the consolidation phase for several weeks. The histologic and molecular events taking place during the lag phase were similar to those observed in fracture healing. The osteotomy site was surrounded by external callus consisting of hyaline cartilage. As distraction started at the rate of 0.25 mm/12 h, the cartilaginous callus was elongated, deformed, and eventually separated into proximal and distal segments. The chondrocytes were stretched along the tension vector and became fibroblast‐like in shape. Although morphologically these cells were distinguishable from osteogenic cells, they expressed OPN, OC, and alkaline phosphatase mRNAs. As distraction advanced, the cartilaginous callus was progressively replaced by bony callus by endochondral ossification and thereafter new bone was formed directly by intramembranous ossification. OPN mRNA was detected in preosteoblasts and osteoblasts at the boundary between fibrous tissue and new bone. ON, MGP, and OC mRNAs appeared early in the differentiation stage. The variety of cell types expressing mRNA encoding bone matrix proteins in distraction osteogenesis was much greater than that detected in the embryonic bone formation and fracture healing process. Moreover, the levels of OPN, ON, MGP, and OC mRNA expression markedly increased during the distraction phase. These results suggested that mechanical tension‐stress modulates cell shape and phenotype, and stimulates the expression of the mRNA for bone matrix proteins.

Mouse ATF-2 Null Mutants Display Features of a Severe Type of Meconium Aspiration Syndrome

Mouse null mutants of transcription factor ATF-2 were generated by the gene targeting method. They died shortly after birth and displayed symptoms of severe respiratory distress with lungs filled with meconium. These features are similar to those of a severe type of human meconium aspiration syndrome. The increased expression of the hypoxia inducible genes suggests that hypoxia occurs in the mutant embryos and that it may lead to strong gasping respiration with consequent aspiration of the amniotic fluid containing meconium. A reduced number of cytotrophoblast cells in the mutant placenta was found and may be responsible for an insufficient supply of oxygen prior to birth. Using the cDNA subtraction and microarray-based expression monitoring method, the expression level of the platelet-derived growth factor receptor α gene, which plays an important role in the proliferation of trophoblasts, was found to be low in the cytotrophoblasts of the mutant placenta. In addition, ATF-2 can trans-activate the PDGF receptor α gene promoter in the co-transfection assay. These results indicate the important role of ATF-2 in the formation of the placenta and the relationship between placental anomalies and neonatal respiratory distress. The ATF-2 null mutants should enhance our understanding of the mechanism of severe neonatal respiratory distress.

PEBP2αA/CBFA1 mutations in Japanese cleidocranial dysplasia patients

Cleidocranial dysplasia (CCD) is an autosomal dominant human bone disease whose genetic locus has been located on chromosome 6p21, where the PEBP2alphaA/CBFA1 gene essential for osteogenesis also maps. Previously, several heterozygous mutations in PEBP2alphaA/CBFA1 were found in CCD patients. In this study, we identified six different types of mutations in PEBP2alphaA/CBFA1 in Japanese CCD patients. Four cases were similar to those reported previously: two were nonsense mutations in the Runt domain, one was a hemizygous deletion, and the other was a missense mutation in the Runt domain which abolished the DNA-binding activity of Runx2/PEBP2alphaA/CBFA1. The remaining two mutations were novel: one had a heterozygous gt-to-tt mutation at the splice donor site (gt) between the exon3-intron junction, which resulted in abnormal exon3 skipping, and the other had a mutation in exon7, which led to the introduction of a translational stop codon in the middle of the transactivation domain. Thus, defects in either the DNA-binding domain or transactivation domain of Runx2/PEBP2alphaA/CBFA1 can cause CCD. The results not only provide a strong genetic evidence that mutations involving in PEBP2alphaA/CBFA1 contribute to CCD, but also provide a useful tool to study how Runx2/PEBP2alphaA/CBFA1 plays its pivotal role during osteoblastic differentiation.

Expression of bone matrix proteins in urolithiasis model rats

Abstract Urinary calcium stones are a pathological substance, and they show similarities to physiological mineralization and other pathological mineralizations. The expression of messenger (m) RNAs of osteopontin (OPN), matrix Gla protein (MGP), osteonectin (ON) and osteocalcin (OC) in bones and teeth has been described. We previously identified OPN as an important stone matrix protein. In addition, the spontaneous calcification of arteries and cartilage in mice lacking MGP was recently reported, a finding which indicates that MGP has a function as an inhibitor of mineralization. Here, we examined the mRNA expressions of OPN, MGP, ON, and OC in the kidneys of stone-forming model rats administered an oxalate precursor, ethylene glycol (EG) for up to 28 days. The Northern blotting showed that the mRNA expressions of OPN and MGP were markedly increased with the administration of EG, but their expression patterns differed. The OPN mRNA expression reached the maximal level at day 7 after the initiation of the EG treatment and showed no significant difference after 14 and 28 days, whereas the MGP mRNA expression rose gradually to day 28. The in situ hybridization demonstrated that the cell type expressing OPN mRNA was different from that expressing MGP. We suggest that OPN acts on calcification and MGP acts on suppression.

Synergy of PEBP2/CBF with mi transcription factor (MITF) for transactivation of mouse mast cell protease 6 gene

The mi locus encodes a member of the basicu2009–u2009helixu2009–u2009loopu2009–u2009helixu2009–u2009leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF). Although the bHLH-Zip family transcription factors generally recognize and bind CANNTG motifs, the expression of mouse mast cell protease 6 (MMCP-6) gene is regulated by MITF through the GACCTG motif in the promoter region. The GACCTG motif was partly overlapped the TGTGGTC sequence, which was bound by polyomavirus enhancer binding protein 2 (PEBP2). In the present study, the effect of PEBP2 on the expression of MMCP-6 gene was examined. PEBP2 that is composed of α and β subunits was expressed by mast cell lines and cultured mast cells derived from spleen. The overexpression of dominant negative PEBP2 cDNA reduced the expression of MMCP-6. Moreover, the simultaneous transfection of the plasmid containing MITF cDNA and the plasmid containing PEBP2 cDNA increased the MMCP-6 promoter activity. For the synergistic action of PEBP2 and MITF, the intact GACCTG and TGTGGTC motifs were prerequisite. The PEBP2αB1 mutant which lacked the region downstream from the Runt domain did not bind MITF and lost the synergistic function. These results indicated that PEBP2 and MITF synergistically transactivated the MMCP-6 gene and that the region downstream from the Runt domain of PEBP2αB1 was essential for the physical and functional interactions with MITF.