We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE͞RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.

Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF–dependent bone marrow macrophages (M-BMMφ) appeared within 3 d. Tartrate-resistant acid phosphatase–positive osteoclasts were also formed when M-BMMφ were further cultured for 3 d with mouse tumor necrosis factor α (TNF-α) in the presence of M-CSF. Osteoclast formation induced by TNF-α was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti–RANK (ODF/RANKL receptor) antibody. Experiments using M-BMMφ prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-α. Osteoclasts induced by TNF-α formed resorption pits on dentine slices only in the presence of IL-1α. These results demonstrate that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system. TNF-α together with IL-1α may play an important role in bone resorption of inflammatory bone diseases.

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The human fibroblast growth factor 23 (hFGF23) and its autosomal dominant hypophosphatemic rickets (ADHR) mutant genes were incorporated into animals by naked DNA injection to investigate the action on phosphate homeostasis in vivo. The hFGF23 mutants (R176Q, R179Q, and R179W) markedly reduced serum phosphorus (6.2-6.9 mg/dl) compared with the plasmid MOCK (8.5 mg/dl). However, native hFGF23 did not affect serum phosphorus (8.6 mg/dl). Both hFGF23 and hFGF23R179Q mRNAs were expressed more than 100-fold in the liver 4 days after injection, however, the C-terminal portion of hFGF23 was detected only in the serum from hFGF23R179Q-injected animals (1109 pg/ ml). hFGF23R179Q mutant was secreted as a 32-kDa protein, whereas, native hFGF23 was detected as a 20-kDa protein in the cell-conditioned media. These results suggest the hFGF23R179Q protein is resistant to intracellular proteolytic processing. The hFGF23R179Q suppressed Na/P i co-transport activities both in kidney and in small intestine by 45 and 30%, respectively, as well as serum 1␣,25-dihydroxyvitamin D 3 to less than 15 pg/ml. However, it had little effect on serum parathyroid hormone (PTH). Infusion of hFGF23R179Q protein normalized serum phosphorus in thyroparathyroidectomized rats without affecting serum calcium. Taken together, the FGF23 mutants reduce both phosphate uptake in intestine and phosphate reabsorption in kidney, independent of PTH action.The FGF23 protein is a novel, secreted protein that consists of 251 amino acids, including a putative N-terminal signal peptide (residues 1-24) (1). Patients with autosomal dominant hypophosphatemic rickets (ADHR), 1 a rare renal phosphatewasting disorder, have been found to have three missense mutations in two arginine residues, R176Q, R179Q, and R179W in the FGF23 gene (2, 3). Also, the FGF23 was identified as a causative factor of oncogenic osteomalacia (OHO) (4). It was highly expressed in the tumor isolated from OHO patients (5), whereas, it was expressed at a very low level in normal tissues. Patients with OHO share similar manifestations with ADHR such as hypophosphatemia, decreased or inappropriately normal 1␣,25-dihydroxyvitamin D concentrations, and osteomalacia. Recent studies indicated administration of the recombinant FGF23 protein reduced serum phosphate without affecting serum calcium, as well as increasing renal phosphate excretion in mice. Mice bearing FGF23-expressing CHO cells showed a suppressed 25-hydroxyvitamin D 1␣-hydroxylase mRNA expression in the kidney (4). These results suggested FGF23 might inhibit renal sodium-dependent phosphate (Na/Pi) co-transporter activity. However, according to the results from the in vitro studies using opossum kidney cells, the FGF23 effects on Na/P i co-transporter are still controversial (4, 6). Yamashita et al. reported that heparin was required to activate FGF23⅐FGFR3c complex and phosphate reabsorption in opossum kidney cells. Also, the mitogen-activating protein kinase signal pathway was a major pathway for the FGF23 signaling (7). Shimada et al. a...