Circulating αKlotho influences phosphate handling by controlling FGF23 production.

The FGF23 coreceptor αKlotho (αKL) is expressed as a membrane-bound protein (mKL) that forms heteromeric complexes with FGF receptors (FGFRs) to initiate intracellular signaling. It also circulates as an endoproteolytic cleavage product of mKL (cKL). Previously, a patient with increased plasma cKL as the result of a translocation [t(9;13)] in the αKLOTHO (KL) gene presented with rickets and a complex endocrine profile, including paradoxically elevated plasma FGF23, despite hypophosphatemia. The goal of this study was to test whether cKL regulates phosphate handling through control of FGF23 expression. To increase cKL levels, mice were treated with an adeno-associated virus producing cKL. The treated groups exhibited dose-dependent hypophosphatemia and hypocalcemia, with markedly elevated FGF23 (38 to 456 fold). The animals also manifested fractures, reduced bone mineral content, expanded growth plates, and severe osteomalacia, with highly increased bone Fgf23 mRNA (>150 fold). cKL activity in vitro was specific for interactions with FGF23 and was FGFR dependent. These results demonstrate that cKL potently stimulates FGF23 production in vivo, which phenocopies the KL translocation patient and metabolic bone syndromes associated with elevated FGF23. These findings have important implications for the regulation of αKL and FGF23 in disorders of phosphate handling and biomineralization.

A nonsecosteroidal vitamin D receptor ligand with improved therapeutic window of bone efficacy over hypercalcemia.

Vitamin D(3) analogues were shown to be beneficial for osteoporosis and other indications, but their narrow therapeutic window between efficacy and hypercalcemia has limited their clinical utility. A nonsecosteroidal, tissue-selective, orally bioavailable, vitamin D receptor (VDR) ligand was ascertained to be efficacious in bone while having modest calcemic effects in vivo. This compound (VDRM2) potently induced Retinoid X Receptor alpha (RXR)-VDR heterodimerization (EC(50) = 7.1 +/- 1.6 nM) and induced osteocalcin promoter activity (EC(50) = 1.9 +/- 1.6 nM). VDRM2 was less potent in inducing Ca(2+) channel transient receptor potential cation channel, subfamily V, member 6 (TRPV6) expression (EC(50) = 37 +/- 12 nM). VDRM2 then was evaluated in osteopenic ovariectomized (OVX) rats and shown to dose-dependently restore vertebral bone mineral density (BMD) from OVX to sham levels at 0.08 microg/kg per day. Hypercalcemia was observed at a dose of 4.6 microg/kg per day of VDRM2, suggesting a safety margin of 57 [90% confidence interval (CI) 35-91]. 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D], ED71, and alfacalcidol restored BMD at 0.030, 0.0055, and 0.046 microg/kg per day, respectively, whereas hypercalcemia was observed at 0.22, 0.027, and 0.23 microg/kg per day, indicating a safety margin of 7.3, 4.9, and 5.0, respectively (90% CIs 4.1-13, 3.2-7.7, and 3.5-6.7, respectively). Histomorphometry showed that VDRM2 increased cortical bone area and stimulated the periosteal bone-formation rate relative to OVX at doses below the hypercalcemic dose. By contrast, ED71 increased the periosteal bone-formation rate only above the hypercalcemic dose. VDRM2 suppressed eroded surface on trabecular bone surfaces at normal serum calcium dosage levels, suggesting dual anabolic and antiresorptive activity. In summary, vitamin D analogues were more potent than VDRM2, but VDRM2 had a greater safety margin, suggesting possible therapeutic potential.

Novel and selective small molecule stimulators of osteoprotegerin expression inhibit bone resorption.

Osteoprotegerin (OPG), a secreted member of the tumor necrosis factor receptor superfamily, is a potent inhibitor of osteoclast formation and bone resorption. Because OPG functions physiologically as a locally generated (paracrine) factor, we used high-throughput screening to identify small molecules that enhance the activity of the promoter of the human OPG gene. We found three structurally unrelated compounds that selectively increased OPG gene transcription, OPG mRNA levels, and OPG protein production and release by osteoblastic cells. Structural analysis of one compound, a benzamide derivative, led to the identification of four related molecules, which are also OPG inducers. The most potent of these compounds, Cmpd 5 inhibited osteoclast formation and parathyroid hormone-induced calvarial bone resorption. In vivo, Cmpd 5 completely blocked resorptive activity (serum calcium, osteoclast number) in parathyroid hormone-treated rats. Furthermore, Cmpd 5 reduced the ability of a rat breast cancer to metastasize to bone. Finally, the compound also prevented bone loss in a rat adjuvant arthritis model. These results provide proof of the concept that low molecular weight compounds can enhance OPG production in ways that can result in effective therapies.