ABSTRACT:Introduction: Fibroblast growth factor (FGF)23 is produced primarily in bone and acts on kidney as a systemic phosphaturic factor; high levels result in rickets and osteomalacia. However, it remains unclear whether FGF23 acts locally and directly on bone formation. Materials and Methods: We overexpressed human FGF23 in a stage-specific manner during osteoblast development in fetal rat calvaria (RC) cell cultures by using the adenoviral overexpression system and analyzed its effects on osteoprogenitor proliferation, osteoid nodule formation, and mineralization. Bone formation was also measured by calcein labeling in parietal bone organ cultures. Finally, we addressed the role of tyrosine phosphorylation of FGF receptor (FGFR) in mineralized nodule formation. Results: Nodule formation and mineralization, but not osteoprogenitor proliferation, were independently suppressed by overexpression of FGF23 in RC cells. Increased FGF23 levels also suppressed bone formation in the parietal bone organ culture model. FGF23 overexpression enhanced phosphorylation of FGFR, whereas the impairment of mineralized nodule formation by FGF23 overexpression was abrogated by SU5402, an inhibitor of FGFR1 tyrosine kinase activity. Conclusions: These studies suggest that FGF23 overexpression suppresses not only osteoblast differentiation but also matrix mineralization independently of its systemic effects on Pi homeostasis.

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The complex pathogenesis of mineralization defects seen in inherited and/or acquired hypophosphatemic disorders suggests that local inorganic phosphate (P i ) regulation by osteoblasts may be a rate-limiting step in physiological bone mineralization. To test whether an osteoblast autonomous phosphate regulatory system regulates mineralization, we manipulated well-established in vivo and in vitro models to study mineralization stages separately from cellular proliferation/differentiation stages of osteogenesis. Foscarnet, an inhibitor of NaP i transport, blocked mineralization of osteoid formation in osteoblast cultures and local mineralization after injection over the calvariae of newborn rats. Mineralization was also down-and upregulated, respectively, with under-and overexpression of the type III NaP i transporter Pit1 in osteoblast cultures. Among molecules expressed in osteoblasts and known to be related to P i handling, stanniocalcin 1 was identified as an early response gene after foscarnet treatment; it was also regulated by extracellular P i , and itself increased Pit1 accumulation in both osteoblast cultures and in vivo. These results provide new insights into the functional role of osteoblast autonomous P i handling in normal bone mineralization and the abnormalities seen in skeletal tissue in hypophosphatemic disorders.Multiple dynamic cellular events underlie de novo bone formation. Osteoblasts develop, synthesize, and deposit extracellular (osteoid) matrix comprising collagen and noncollagenous proteins and participate in osteoid mineralization. Although much has been learned about the cellular and molecular regulation of sequential stages in the bone formation process, e.g., cellular proliferation and differentiation, osteoid deposition, and mineralization, from in vitro (2) and in vivo (20) models, much remains obscure, including, for example, the contribution of systemic versus local regulatory controls.The control of systemic inorganic phosphate (P i ) levels is known to be indispensable for bone formation, especially for osteoid mineralization processes, but the parathyroid hormone (PTH) (decreasing serum P i levels)-vitamin D (increasing serum P i levels) axis does not fully explain systemic P i homeostasis (31). For example, fibroblast growth factor 23 (FGF23) (1) and secreted frizzled-related protein 4 (sFRP-4) (9) were identified and found to display the biological properties of the putative circulating phosphaturic factor "phosphatonin," which may be primarily responsible for a variety of hypophosphatemic disorders. In contrast, osteomalacia/rickets occurs in Hyp and Gy mice (murine homologues of X-linked hypophosphatemia) (18) but not in mice lacking the primary renal sodium-dependent phosphate (NaP i ) transporter, Npt2 (6). Analyses of several Hyp mouse models have also suggested that Hyp osteoblasts may have an intrinsic impairment in mineralization (for example, see reference 13). Moreover, the mineralization defects seen in Fgf23-null mice with hyperphosphatemia (34, 35) and FGF23 tr...

Anthocyanins, one of the flavonoid subtypes, are a large family of water-soluble phytopigments and have a wide range of health-promoting benefits. Recently, an anthocyanin-rich compound from blueberries was reported to possess protective property against bone loss in ovariectomized (OVX) animal models. However, the active ingredients in the anthocyanin compound have not been identified. Here we show that delphinidin, one of the major anthocyanidins in berries, is a potent active ingredient in anti-osteoporotic bone resorption through the suppression of osteoclast formation. In vitro examinations revealed that delphinidin treatment markedly inhibited the differentiation of RAW264.7 cells into osteoclasts compared with other anthocyanidins, cyanidin and peonidin. Oral administration of delphinidin significantly prevented bone loss in both RANKL-induced osteoporosis model mice and OVX model mice. We further provide evidence that delphinidin suppressed the activity of NF-κB, c-fos, and Nfatc1, master transcriptional factors for osteoclastogenesis. These results strongly suggest that delphinidin is the most potent inhibitor of osteoclast differentiation and will be an effective agent for preventing bone loss in postmenopausal osteoporosis.

Ameloblastin, the most abundant nonamelogenin enamel matrix protein, plays a role in ameloblast differentiation. Here, we found that ameloblastin was expressed in osteosarcoma cells; to explore the potential functions of ameloblastin in osteoblasts, we investigated whether this protein is involved in osteogenic differentiation and bone formation on the premise that CD63, a member of the transmembrane-4 glycoprotein superfamily, interacts with integrins in the presence of ameloblastin. Ameloblastin bound to CD63 and promoted CD63 binding to integrin ␤1. The interaction between CD63 and integrin ␤1 induced Src kinase inactivation via the binding of CD63 to Src. The reduction of Src activity and osteogenic differentiation mediated by ameloblastin were abrogated by treatment with anti-CD63 antibody and overexpression of constitutively active Src, respectively. Therefore, our results suggest that ameloblastin is expressed in osteoblasts and functions as a promoting factor for osteogenic differentiation via a novel pathway through the interaction between CD63 and integrin ␤1.Ameloblastin (AMBN), also known as sheathlin or amelin, is the most abundant nonamelogenin enamel matrix protein (4, 5, 14) and a member of the secretory calcium-binding phosphoprotein (SCPP) gene cluster of evolutionarily related molecules that regulate skeletal mineralization (13). Further, AMBN induces cell attachment, proliferation, and differentiation of periodontal ligament cells in vitro (34). In AMBN-null mice, ameloblasts are detached from the matrix, lose cell polarity, and resume proliferation. However, protein expression was not completely inactivated, and truncated RNA missing a portion of exons 5 and 6 is still translated in AMBN knockout (KO) mice (30). Therefore, it is conceivable that exons 5 and 6 of AMBN play a role in ameloblast differentiation (7,30). In this mouse model, structural change was shown in the alveolar bone (30): the alveolar bone exhibited more porosity in truncated-AMBN-expressing mice than in wild-type mice. The changes in alveolar bone in mice lacking exons 5 and 6 of AMBN (AMBN ⌬5-6 ) cannot be directly related to the protein as they could arise from other factors such as changes in occlusal forces in teeth without enamel (30). On the other hand, it has recently been reported that AMBN is expressed in osteoblasts during craniofacial development (25). Although AMBN may play a significant role in not only in tooth development but also bone formation, the role of AMBN in bone formation is still unclear.AMBN has been shown to interact with CD63 via a yeast two-hybrid assay (29). CD63 is a member of the transmembrane-4 glycoprotein superfamily, also known as the tetraspanin family (26,32). Most of these proteins are cell surface proteins that are characterized by the presence of four hydrophobic domains and two extracellular domains (26, 32). CD63 mediates signal transduction events in the regulation of cell survival, development, activation, growth, and motility (12,16,32). In particular, cell surface CD63 is ...

Stanniocalcin 1 (STC1) is a mammalian homolog of STC, the fish calcium/phosphate-regulating polypeptide whose functions are only beginning to be elucidated. Recently, we demonstrated that STC1 stimulates, in an autocrine/paracrine fashion, bone mineralization by increasing phosphate uptake in osteoblasts apparently via the functional activity of the sodium-dependent phosphate transporter, Pit1. We have now assessed the role of STC1 on osteoblast development in fetal rat calvaria (RC) cell cultures. STC1 mRNA and protein were differentially expressed over the time course of cultures, and dexamethasone, a potent stimulator of differentiation in this model, shifted peak STC1 expression levels to earlier times. Overexpression [recombinant human (rh) STC1] and underexpression (antisense oligonucleotides) of STC1 accelerated and retarded, respectively, osteogenic development as well as osteopontin and osteocalcin mRNA expression in mature osteoblast cultures, but not osteoprogenitor cell cultures. Dexamethasone shifted the effective doses required for these effects to higher and lower concentrations of antisense oligonucleotides and rhSTC1, respectively. Concomitantly, rhSTC1 increased both sodium-dependent phosphate uptake and Pit1 gene expression in nodule formation stages, but not in primitive progenitor stages of RC cell cultures. Thus, STC1 accelerates osteoblast development in an autocrine/paracrine manner in the RC cell culture model.

Of the four prostaglandin (PG) E receptor subtypes (EP1-EP4), EP2 and EP4 have been proposed to mediate the anabolic action of PGE(2) on bone formation but comparative evaluation studies of EPs on bone formation do not necessarily share a common mechanism, implying that their additional features including downstream MAPK pathways may be beneficial to resolve this issue. We systematically assessed the roles of EPs in the rat calvaria (RC) cell culture model by using four selective EP agonists (EPAs). Consistent with relative expression levels of the respective receptors, multiple phenotypic traits of bone formation in vitro, including proliferation of nodule-associated cells, osteoblast marker expression and mineralized nodule formation were upregulated not only by PGE(2) but equally by EP2A and EP4A, but not by EP1A and EP3A. EP2A and EP4A were effective when cells were treated chronically or pulse-treated during nascent nodule formation. EP2A and EP4A equally stimulated the endogenous PGE(2) production, while EP2A caused a greater increase in cAMP production and c-Fos gene expression compared to EP4A. EP2A and EP4A activated predominantly p38 MAPK and ERK respectively, while c-Jun N-terminal kinase (JNK) was equally activated by both agonists. SB203580 (p38 MAPK inhibitor) blocked the PGE(2) effect on mineralized nodule formation, while U0126 (ERK inhibitor) and dicumarol (JNK inhibitor) were less effective. PGE(2)-dependent phosphorylation of the MAPKs was affected not only by protein kinase (PK)A and PKC inhibitors but also by adenylate cyclase and PKC activators. Co-treatment of RC cells with EP2A or EP4A and bone morphogenetic protein (BMP)2, whose effects on bone nodule formation is known to be, in part, mediated through the PKA and p38 MAPK pathways, resulted in an additive effect on mineralized nodule formation. Further, PGE(2), EP2A and EP4A did not increase BMP2/4 mRNA levels in RC cells, and EP2-induced phosphorylation of p38 MAPK was not eliminated by Noggin. These results suggest that, in the RC cell model, the anabolic actions of PGE(2) on mineralized nodule formation are mediated at least in part by activation of the EP2 and EP4 receptor subtype-specific MAPK pathways, independently of BMP signaling, in cells associated with nascent bone nodules.

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