Norio Amizuka

Identification and Characterization of a Novel Protein, Periostin, with Restricted Expression to Periosteum and Periodontal Ligament and Increased Expression by Transforming Growth Factor β

We had previously identified the cDNA for a novel protein called osteoblast‐specific factor 2 (OSF‐2) from an MC3T3‐E1 cDNA library using subtraction hybridization and differential screening techniques. Here we describe the localization, regulation, and potential function of this protein. Immunohistochemistry using specific antiserum revealed that in adult mice, the protein is preferentially expressed in periosteum and periodontal ligament, indicating its tissue specificity and a potential role in bone and tooth formation and maintenance of structure. Based on this observation and the fact that other proteins have been called OSF‐2, the protein was renamed “periostin.” Western blot analysis showed that periostin is a disulfide linked 90 kDa protein secreted by osteoblasts and osteoblast‐like cell lines. Nucleotide sequence revealed four periostin transcripts that differ in the length of the C‐terminal domain, possibly caused by alternative splicing events. Reverse transcription‐ polymerase chain reaction analysis revealed that these isoforms are not expressed uniformly but are differentially expressed in various cell lines. Both purified periostin protein and the periostin‐Fc recombinant protein supported attachment and spreading of MC3T3‐E1 cells, and this effect was impaired by antiperiostin antiserum, suggesting that periostin is involved in cell adhesion. The protein is highly homologous to βig‐h3, a molecule induced by transforming growth factor β (TGF‐β) that promotes the adhesion and spreading of fibroblasts. Because TGF‐β has dramatic effects on periosteal expansion and the recruitment of osteoblast precursors, this factor was tested for its effects on periostin expression. By Western blot analysis, TGF‐β increased periostin expression in primary osteoblast cells. Together, these data suggest that periostin may play a role in the recruitment and attachment of osteoblast precursors in the periosteum.

Osteoblast-derived PTHrP is a potent endogenous bone anabolic agent that modifies the therapeutic efficacy of administered PTH 1–34

Mice heterozygous for targeted disruption of Pthrp exhibit, by 3 months of age, diminished bone volume and skeletal microarchitectural changes indicative of advanced osteoporosis. Impaired bone formation arising from decreased BM precursor cell recruitment and increased apoptotic death of osteoblastic cells was identified as the underlying mechanism for low bone mass. The osteoporotic phenotype was recapitulated in mice with osteoblast-specific targeted disruption of Pthrp, generated using Cre-LoxP technology, and defective bone formation was reaffirmed as the underlying etiology. Daily administration of the 1-34 amino-terminal fragment of parathyroid hormone (PTH 1-34) to Pthrp+/- mice resulted in profound improvement in all parameters of skeletal microarchitecture, surpassing the improvement observed in treated WT littermates. These findings establish a pivotal role for osteoblast-derived PTH-related protein (PTHrP) as a potent endogenous bone anabolic factor that potentiates bone formation by altering osteoblast recruitment and survival and whose level of expression in the bone microenvironment influences the therapeutic efficacy of exogenous PTH 1-34.

Incorporation of Tenascin-C into the Extracellular Matrix by Periostin Underlies an Extracellular Meshwork Architecture

Extracellular matrix (ECM) underlies a complicated multicellular architecture that is subjected to significant forces from mechanical environment. Although various components of the ECM have been enumerated, mechanisms that evolve the sophisticated ECM architecture remain to be addressed. Here we show that periostin, a matricellular protein, promotes incorporation of tenascin-C into the ECM and organizes a meshwork architecture of the ECM. We found that both periostin null mice and tenascin-C null mice exhibited a similar phenotype, confined tibial periostitis, which possibly corresponds to medial tibial stress syndrome in human sports injuries. Periostin possessed adjacent domains that bind to tenascin-C and the other ECM protein: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin-C and the ECM, which increased deposition of tenascin-C on the ECM. The deposition of hexabrachions of tenascin-C may stabilize bifurcations of the ECM fibrils, which is integrated into the extracellular meshwork architecture. This study suggests a role for periostin in adaptation of the ECM architecture in the mechanical environment.

Runx2 determines bone maturity and turnover rate in postnatal bone development and is involved in bone loss in estrogen deficiency

Runx2 is an essential transcription factor for osteoblast differentiation. However, the functions of Runx2 in postnatal bone development remain to be clarified. Introduction of dominant‐negative (dn)‐Runx2 did not inhibit Col1a1 and osteocalcin expression in mature osteoblastic cells. In transgenic mice that expressed dn‐Runx2 in osteoblasts, the trabecular bone had increased mineralization, increased volume, and features of compact bone, and the expression of major bone matrix protein genes was relatively maintained. After ovariectomy, neither osteolysis nor bone formation was enhanced and bone was relatively conserved. In wild‐type mice, Runx2 was strongly expressed in immature osteoblasts but downregulated during osteoblast maturation. These findings indicate that the maturity and turnover rate of bone are determined by the level of functional Runx2 and Runx2 is responsible for bone loss in estrogen deficiency, but that Runx2 is not essential for maintenance of the expression of major bone matrix protein genes in postnatal bone development and maintenance. Developmental Dynamics 236:1876–1890, 2007.

Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare autosomal recessively inherited disorder, characterized by hypophosphatemia, short stature, rickets and/or osteomalacia, and secondary absorptive hypercalciuria. HHRH is caused by a defect in the sodium-dependent phosphate transporter (NaPi-IIc/Npt2c/NPT2c), which was thought to have only a minor role in renal phosphate (P(i)) reabsorption in adult mice. In fact, mice that are null for Npt2c (Npt2c(-/-)) show no evidence for renal phosphate wasting when maintained on a diet with a normal phosphate content. To obtain insights and the relative importance of Npt2a and Npt2c, we now studied Npt2a(-/-)Npt2c(+/+), Npt2a(+/-)Npt2c(-/-), and Npt2a(-/-)Npt2c(-/-) double-knockout (DKO). DKO mice exhibited severe hypophosphatemia, hypercalciuria, and rickets. These findings are different from those in Npt2a KO mice that show only a mild phosphate and bone phenotype that improve over time and from the findings in Npt2c KO mice that show no apparent abnormality in the regulation of phosphate homeostasis. Because of the nonredundant roles of Npt2a and Npt2c, DKO animals showed a more pronounced reduction in P(i) transport activity in the brush-border membrane of renal tubular cells than that in the mice with the single-gene ablations. A high-P(i) diet after weaning rescued plasma phosphate levels and the bone phenotype in DKO mice. Our findings thus showed in mice that Npt2a and Npt2c have independent roles in the regulation of plasma P(i) and bone mineralization.

Cell‐Cell Interaction Mediated by Cadherin‐11 Directly Regulates the Differentiation of Mesenchymal Cells Into the Cells of the Osteo‐Lineage and the Chondro‐Lineage

We studied cadherin‐11 function in the differentiation of mesenchymal cells. Teratomas harboring the cadherin‐11 gene generated bone and cartilage preferentially. Cadherin‐11 transfectants of C2C12 cells and cadherin‐11 and/or N‐cadherin transfectants of L cells showed that cadherin‐11 together with N‐cadherin‐induced expression of ALP and FGF receptor 2. These results suggest that cadherin‐11 directly regulates the differentiation of mesenchymal cells into the cells of the osteo‐lineage and the chondro‐lineage in a different manner from N‐cadherin.

Type IIc Sodium–Dependent Phosphate Transporter Regulates Calcium Metabolism

Primary renal inorganic phosphate (Pi) wasting leads to hypophosphatemia, which is associated with skeletal mineralization defects. In humans, mutations in the gene encoding the type IIc sodium-dependent phosphate transporter lead to hereditary hypophophatemic rickets with hypercalciuria, but whether Pi wasting directly causes the bone disorder is unknown. Here, we generated Npt2c-null mice to define the contribution of Npt2c to Pi homeostasis and to bone abnormalities. Homozygous mutants (Npt2c(-/-)) exhibited hypercalcemia, hypercalciuria, and elevated plasma 1,25-dihydroxyvitamin D(3) levels, but they did not develop hypophosphatemia, hyperphosphaturia, renal calcification, rickets, or osteomalacia. The increased levels of 1,25-dihydroxyvitamin D(3) in Npt2c(-/-) mice compared with age-matched Npt2c(+/+) mice may be the result of reduced catabolism, because we observed significantly reduced expression of renal 25-hydroxyvitamin D-24-hydroxylase mRNA but no change in 1alpha-hydroxylase mRNA levels. Enhanced intestinal absorption of calcium (Ca) contributed to the hypercalcemia and increased urinary Ca excretion. Furthermore, plasma levels of the phosphaturic protein fibroblast growth factor 23 were significantly decreased in Npt2c(-/-) mice. Sodium-dependent Pi co-transport at the renal brush border membrane, however, was not different among Npt2c(+/+), Npt2c(+/-), and Npt2c(-/-) mice. In summary, these data suggest that Npt2c maintains normal Ca metabolism, in part by modulating the vitamin D/fibroblast growth factor 23 axis.

The Primary Calcification in Bones Follows Removal of Decorin and Fusion of Collagen Fibrils

To elucidate the mechanisms of primary calcification in bone, ultrastructural changes in collagen fibrils, as well as cytochemical alteration of proteoglycan, especially decorin, were investigated morphologically in 19‐day postcoitum embryonic rat calvariae. Below the osteoblast layer, calcification of the osteoid area increased in direct proportion to its distance from the osteoblasts. In the uncalcified osteoid area, collagen fibrils near matrix vesicles possessed sharp contours and were a uniform 50 nm in diameter. Immunoelectron microscopy revealed decorin to be abundantly localized in the vicinity of the collagen fibrils. In the osteoid area undergoing the process of calcification, collagen fibrils tended to fuse side by side. Where calcification was progressed, this fusion was even more so. Some very large fibrils exhibited complicated contours, 400 nm or more in diameter. Although the calcification at this stage affected areas both inside and outside of the collagen fibrils, the interior areas manifested a lower density of calcification. The immunolocalization of decorin was also much decreased around these fibrils. Thus, primary calcification in bone matrix follows the removal of decorin and fusion of collagen fibrils. This phenomenon may aid in the process of calcification and bone formation, because (1) inhibitors of calcification, such as decorin, are removed, (2) the fusion of collagen fibrils provides the room necessary for rapid growth of mineral crystals, and (3) the soft elastic bone matrix containing abundant fused collagen fibrils less subjective to calcification is safe for both maternal and embryonic bodies and is convenient for subsequent bone remodeling.

Immunolocalization of tissue non-specific alkaline phosphatase in mice

Abstract Immunolocalization of tissue non-specific alkaline phosphatase (TNAP) was examined in murine tissues, employing a specific antiserum to TNAP on frozen sections, 50-μm tissue slices, and paraffin sections. TNAP was detected at high levels in hard tissues including bone, cartilage, and tooth. In bone tissue, the TNAP immunoreactivity was localized on the entire cell surface of preosteoblasts, as well as the basolateral cell membrane of osteoblasts. It was also localized on some resting chondrocytes and most of the proliferative and hypertrophic cells in cartilage. In the incisor, cells of the stratum intermedium, the subodontoblastic layer, the proximal portion of secretory ameloblasts, and the basolateral portion of odontoblasts showed particularly strong immunoreactivity. Immunoreactivity was observed in other soft tissues, such as the brush borders of proximal renal tubules in kidney, on cell membrane of the biliary canalicula in liver and in trophoblasts in the placenta. These immunolocalizations were quite similar to enzyme histochemical localizations. However, neither the submandibular gland nor the intestine, which both exhibited alkaline phosphatase activity by enzyme histochemistry, revealed immunoreactivity for TNAP. Therefore, immunocytohistochemical studies for TNAP enabled us to localize the TNAP isozyme, thus distinguishing it from other isozymes.

Ultrastructural and cytochemical studies on cell death of osteoclasts induced by bisphosphonate treatment

The process of apoptosis and fate of osteoclasts are not well elucidated because dying osteoclasts are rarely seen in normal bone. Histological, cytochemical, and ultrastructural features of osteoclasts undergoing apoptosis were studied in the femur and tibia of rats treated with a third-generation bisphosphonate (disodium dihydrogen (cycloheptylamino)-methylene-1, 1-bisphosphonate). After the bisphosphonate administration, osteoclasts decreased significantly in number. Initially, they became devoid of ruffled borders and detached from the bone surface. In such osteoclasts, the Golgi apparatus was degraded, or dispersed in the cytoplasm. Later, osteoclasts revealed typical features of apoptosis, with pyknotic nuclei showing condensation and margination of heterochromatins and DNA fragmentation. They were often convoluted to give rise to apoptotic bodies. In addition, enlargement and fusion of nuclear envelopes and subsequent disruption leading to leakage of nuclear contents into the cytoplasm were observed in osteoclasts in the late stage of apoptosis. These osteoclasts as well as apoptotic bodies were surrounded by cytoplasmic processes of macrophages, which often contained degenerated cytoplasmic fragments of osteoclasts. Apoptotic osteoclasts migrating into or present in capillaries were also observed in some areas. In conclusion, bisphosphonate induces apoptosis of osteoclasts, which was characterized by ultrastructural changes of the nucleus typical of apoptosis accompanied by degradation of cell organelles. The majority of them are eliminated by macrophages, but there are some that escape into blood vessels.