A number of previous studies have implicated FGF23 and FGFRs in the regulation of phosphate metabolism; however, the identity of FGFR isoforms that are functionally important in phosphate regulation remains elusive. In the current study, we used selective antibody activators of FGFR1 to study the effects of systemic FGFR1 activation on phosphate homeostasis. Our results demonstrate that activation of FGFR1, but not other FGFRs, is sufficient to induce FGF23 expression and the resulting hypophosphatemia in adult mice. In addition, FGFR1 activation in cultured kidney epithelial cells is sufficient to induce FGF23 program. siRNA-mediated Fgfr1 knockdown induced the opposite effects. These results complement previous genetic studies with mice deficient for each FGFR isoform, and suggest the central role of FGFR1 in the FGF23 signaling network ().
Antibody-mediated activation of FGFR1 is potentially mimicking some aspect of osteoglophonic dysplasia (OGD), a rare genetic disorder characterized by a distinctive skeletal dysplasia caused by an activating FGFR1 germ line mutation 
. FGFR1 plays important roles during embryonic development, in particular in skeletal development 
. Individuals with OGD thus display mild to severe skeletal malformation including craniosynostosis and dwarfism 
. In addition, they also exhibit hypophosphatemia, which is likely to be independent of craniosynostosis, dwarfism, or other developmental problems. The mechanistic basis for hypophosphatemia in OGD is unclear, although one individual with hypophosphatemia has been found to have elevated circulating FGF23 levels 
. Together with our results, it is conceivable that genetic activation of FGFR1 in bones leads to elevated FGF23 in OGD, resulting in hypophosphatemia.
Typical OGD individuals also exhibit osteomalacia, which might be secondary to hypophosphatemia. Continuous production of recombinant FGF23 from implanted cell line was also shown to induce marked hypophosphatemia (~50% of normal) and osteomalacia in nude mice at day 45 after the implantation 
. Intriguingly, pharmacological activation of FGFR1 by R1MAb1 injection did not result in an apparent defect in bone mineral density in db/db
mice despite the ability of R1MAb1 to induce FGF23 production and hypophosphatemia. The method used for this study cannot directly assess the non-calcified osteoid volume, thus it is formally possible that R1MAbs affect osteoid formation independently of calcified bone volume in this timeframe. Also the lack of bone effect could be simply due to rather mild effects of R1MAb1 on phosphate regulation and short exposure. Further analysis is required to determine whether more frequent and long-term administration with R1MAb (or other FGFR1 activating agents) could significantly affect bone mineralization or relative osteoid volume.
In addition to FGF23, our gene expression studies in cultured differentiated osteoblasts identified a number of R1MAb target genes, which could also be altered in OGD patients. Many of these genes have been implicated in regulation of bone mineral density or function. For example, R1MAb2 induced Opg
, a gene encoding a secreted protein that inhibits RANKL and osteoclast differentiation 
. Dmp1 deficiency is implicated in hypophosphatemia and osteomalacia in humans 
, while overexpression promotes bone mineralization 
. In our assays, we found that R1MAb2 treatment upregulated both Opg
. Induction of these genes by FGFR1 activation in vivo
should promote bone mineralization. We also found that R1MAb2 treatment upregulated Ank
, whose induction in vivo
may promote bone loss. Ank
encodes multipass transmembrane protein that functions in pyrophosphate regulation 
. Loss of Ank
in humans leads to craniometaphyseal dysplasia characterized by progressive thickening of bones 
. As expected, R1MAb also induced negative feedback regulators of FGFR signaling, Spry2
. Thus, FGFR1 activation could perturb bone mineralization through multiple mechanisms.
Pharmacological modulation of FGFR1 activity has been explored as therapeutic strategy in several diseases. For example, FGFR1 overexpression or a somatic activating mutation in the kinase domain has been found in tumor cells, leading to the examination of FGFR1 as an anti-cancer target 
. Modulation of FGFR pathway has been implicated in major mood depressive and other psychiatric disorders 
. In addition, we recently provided evidence of FGFR1 activation in adipose tissues as a therapeutic target for the treatment of insulin resistance and type 2 diabetes 
. Not surprisingly, one common side effect of characterized FGFR modulators is an alteration in phosphate homeostasis. For example, pan-FGFR inhibitors PD173074 and PD176067 both induced hyperphosphatemia and changes in other FGF23-related parameters 
. Interestingly, both inhibitory PD173074 and the activating anti-FGFR1 antibodies described here increase serum FGF23 levels. PD173074 is proposed to act primarily in the kidney to increase serum vitamin D and phosphate, which in turn induces FGF23 production in bone through an FGFR-independent mechanism 
. We believe that R1MAbs function directly in the bone to induce FGF23 secretion. Thus, FGFR1 activation is sufficient, but not necessary, for skeletal production of FGF23 in vivo
In conclusion, the work presented here demonstrates that pharmacological FGFR1 activation is sufficient to increase FGF23 production and decrease serum phosphate levels in adult mice. These findings provide new insights into the mechanistic basis for human hypophosphatemic disorders such as OGD and hypophosphatemic rickets, associated with an increase in circulating FGF23 and the resulting urinal phosphate wasting. FGFR1 activation could affect phosphate homeostasis at least in two tissues: bone and kidney (). Thus, tissue specific modulation of FGFR1 is likely necessary for therapeutic intervention for metabolic diseases or psychiatric disorders, to gain efficacy without adverse side effects related to the FGF23-axis. In addition, tissue specific modulation of FGFR1 can conceivably counteract conditions associated with FGF23 resistance or chronic hyperphosphatemia, such as those seen in patients with chronic kidney disease 
. Further studies are warranted to elucidate the physiological pathways downstream of FGFR1 or other FGFRs.