Cellular ATP synthesis mediated by type III sodium-dependent phosphate transporter Pit-1 is critical to chondrogenesis.

Disturbed endochondral ossification in X-linked hypophosphatemia indicates an involvement of P(i) in chondrogenesis. We studied the role of the sodium-dependent P(i) cotransporters (NPT), which are a widely recognized regulator of cellular P(i) homeostasis, and the downstream events in chondrogenesis using Hyp mice, the murine homolog of human X-linked hypophosphatemia. Hyp mice showed reduced apoptosis and mineralization in hypertrophic cartilage. Hyp chondrocytes in culture displayed decreased apoptosis and mineralization compared with WT chondrocytes, whereas glycosaminoglycan synthesis, an early event in chondrogenesis, was not altered. Expression of the type III NPT Pit-1 and P(i) uptake were diminished, and intracellular ATP levels were also reduced in parallel with decreased caspase-9 and caspase-3 activity in Hyp chondrocytes. The competitive NPT inhibitor phosphonoformic acid and ATP synthesis inhibitor 3-bromopyruvate disturbed endochondral ossification with reduced apoptosis in vivo and suppressed apoptosis and mineralization in conjunction with reduced P(i) uptake and ATP synthesis in WT chondrocytes. Overexpression of Pit-1 in Hyp chondrocytes reversed P(i) uptake and ATP synthesis and restored apoptosis and mineralization. Our results suggest that cellular ATP synthesis consequent to P(i) uptake via Pit-1 plays an important role in chondrocyte apoptosis and mineralization, and that chondrogenesis is ATP-dependent.

Regulation of osteoclast differentiation and function by phosphate: potential role of osteoclasts in the skeletal abnormalities in hypophosphatemic conditions.

UNLABELLED: Mice fed with a low Pi diet exhibited decreased osteoclast number. Hyp mice also showed decreased osteoclasts, and high Pi reversed it. Low Pi reduced osteoclast formation and bone resorption in vitro. Hypophosphatemia may suppress osteoclast differentiation/function, leading to skeletal abnormalities. INTRODUCTION: Skeletal abnormalities seen in hypophosphatemic disorders indicate a critical role of phosphate (Pi) in skeletogenesis. However, the role of osteoclasts in the pathogenesis of the disturbed skeletogenesis is unclear. MATERIALS AND METHODS: Mice fed with a low-Pi diet and Hyp mice that are characterized by hypophosphatemia and impaired osteogenesis were studied. Effects of Pi on osteoclast formation and bone resorption were also examined in vitro. RESULTS: Histomorphometric examination showed that mice on a low-Pi diet exhibited decreased osteoclast number. Furthermore, osteoclast number in Hyp mice was also decreased compared with wildtype (WT) mice. Of note, feeding of Hyp mice with high-Pi diet significantly reversed hypophosphatemia, improved disturbed osteogenesis, and increased osteoclast number. Osteoclast-like cell (OLC) formation and bone resorption in Hyp bone marrow cells was not different from WT bone marrow cells. On the other hand, OLC formation and bone resorption were decreased in conjunction with reduced mRNA expression of RANKL in WT bone marrow cells cultured in the medium containing low Pi (0.5 mM). Recombinant human matrix extracellular phosphoglycoprotein (MEPE), a candidate for phosphatonin, also decreased osteoclast formation, whereas fibroblast growth factor 23 (FGF23), another phosphatonin candidate, showed no effects. CONCLUSIONS: Our results suggest that Pi controls the differentiation and function of osteoclasts. These actions of Pi on osteoclasts may be associated with the pathogenesis of the skeletal abnormalities in hypophosphatemic disorders.

[Role of the type 3 sodium-dependent phosphate transporter in the calcification of growth plate chondrocytes].

Phosphate is a second most abundant mineral next to calcium. The facts that hypophosphatemia is associated with the retardation of skeletal development and phosphate levels increase during endochondral ossification suggest that phosphate plays a role in cartilage differentiation. The type 3 sodium-dependent phosphate transporter (NPT3) expressed in growth plate chondrocytes transports extracellular phosphates into the cells. These phosphates are utilized for ATP synthesis, which in turn promotes apoptosis of growth plate chondrocytes through activation of the caspase signal pathways. Subsequently, matrix vesicles released from apoptotic chondrocytes accelerate calcification of chondrocytes. Our results suggest that phosphate plays a critical role in terminal differentiation of chondrocytes.

Distribution of 10 periodontal bacteria in saliva samples from Japanese children and their mothers.

OBJECTIVE: We analyzed the distribution of 10 periodontal bacteria species (Porphyromonas gingivalis, Tannerella forsythensis, Prevotella intermedia, Prevotella nigrescens, Campylobacter rectus, Eikenella corrodens, Actinobacillus actinomycetemcomitans, Capnocytophaga ochracea, Capnocytophaga sputigena, and Treponema denticola) in children, and then compared their distribution in those children and their mothers, with special attention given to three of the species known as the red complex (P. gingivalis, T. forsythensis, and T. denticola) whose presence has been shown to be associated with conditions related to periodontal diseases. METHODS: One hundred thirteen pairs of children and their mothers were randomly selected from patients treated at the Pedodontic Clinic of Osaka University Dental Hospital. Saliva samples were taken at the second visit prior to receiving professional tooth brushing instruction. Genomic DNA was extracted from each saliva sample, followed by a polymerase chain reaction assay with species-specific sets of primers. RESULTS: A. actinomycetemcomitans was the most frequently detected species in the mothers, followed by C. sputigena, P. gingivalis, and T. forsythensis, while C. sputigena had the highest detection rate, followed by A. actinomycetemcomitans and T. denticola in the children. The detection rate of the red complex species in children whose mothers possessed the same species was significantly higher than in those whose mothers did not possess them. CONCLUSIONS: Our results indicate a correlation between the presence of periodontal bacteria in children and their mothers, while the presence of red complex bacteria in children was highly associated with that in their mothers.

A synthetic peptide fragment of human MEPE stimulates new bone formation in vitro and in vivo.

UNLABELLED: Matrix extracellular phosphoglycoprotein (MEPE) was proposed as a candidate for the phosphaturic hormone phosphatonin. We found that a synthetic peptide fragment of MEPE containing the RGD and SGDG sequence stimulated new bone formation in vitro and in vivo. INTRODUCTION: Matrix extracellular phosphoglycoprotein (MEPE) was recently identified as a candidate for the phosphaturic hormone phosphatonin, which has been implicated in disturbed phosphate metabolism, rickets, and osteomalacia associated with X-linked hypophosphatemic rickets (XLH) and oncogenic hypophosphatemic osteomalacia (OHO). MEPE expression was predominantly found in osteoblasts, and mice deficient in a homolog of MEPE showed increased bone density, suggesting that MEPE produced in osteoblasts negatively regulates bone formation. In this study, we examined the effects of a synthetic 23mer peptide fragment of MEPE (AC-100, region 242-264) containing the RGD (integrin-binding) and SGDG (glycosaminoglycan-attachment) motif on bone formation in vitro and in vivo. MATERIALS AND METHODS: The osteogenic activity of AC-100 was examined in organ cultures of neonatal mouse calvariae and in vivo by injecting AC-100 onto the calvariae of mice. RESULTS: Histomorphometric examination showed that AC-100 stimulated new bone formation with increased numbers of osteoblasts in neonatal mouse calvariae in organ culture. In contrast, synthetic MEPE fragment peptides without either the RGD or SGDG motif failed to increase new bone formation. Repeated daily subcutaneous injections of AC-100 onto the calvariae in mice increased bone thickness and stimulated new bone formation as determined by the calcein double-labeling technique. However, peptides in which the RGD or SGDG sequence was scrambled did not stimulate new bone formation in vivo. AC-100 increased cell proliferation and alkaline phosphatase activity and activated focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) in human primary osteoblasts. CONCLUSION: Our results show that a synthetic peptide corresponding with the sequence of human MEPE fragment stimulates new bone formation with increased number of osteoblasts. The results also suggest that the RGD and SGDG motifs are critical to the osteogenic activity of AC-100, presumably through activating integrin signaling pathways in osteoblasts. The anabolic effects of AC-100 may be beneficial for bone diseases associated with decreased bone formation.

Clinico-pathological aspects of a residual natal tooth: a case report.

A Japanese girl was referred to Osaka University Dental Hospital for examination of a tooth-like structure that had erupted following spontaneous exfoliation of a natal tooth in the lower left primary central incisor region. The structure had erupted at 6 months of age, and radiographic and clinical examination showed composition of pulp and dentin, but no enamel. On histological examination, the majority of the dentin area had a tubular dentin-like appearance, while the outer area of the root appeared to be composed of an osteodentin-like substance. Most of the dentin was covered by cementum. These findings suggest that the structure had originated from a developing remnant of the extracted natal tooth, which must have remained in the gingival tissues. We termed this calcified structure a residual natal tooth.