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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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.

Endoprosthetic or biological reconstructions as limb salvage provided good functional outcome in skeletally immature children with a malignant bone tumor of the distal aspect of the femur despite a high rate of revisions and limb-lengthening procedures.