Faculty Bibliography

Genetic ablation of fibroblast growth factor 23 from mice (Fgf-23(-/-)) results in a short lifespan with numerous abnormal biochemical and morphological features. Such features include kyphosis, hypogonadism and associated infertility, osteopenia, pulmonary emphysema, severe vascular and soft tissue calcifications, and generalized atrophy of various tissues. To determine whether these widespread anomalies in Fgf-23(-/-) mice can be ameliorated by genetically restoring the systemic actions of FGF-23, we generated Fgf-23(-/-) mice expressing the human FGF-23 transgene in osteoblasts under the control of the 2.3 kb alpha1(I) collagen promoter (Fgf-23(-/-) /hFGF-23-Tg double mutants). This novel mouse model is completely void of all endogenous Fgf-23 activity, but produces human FGF-23 in bone cells that is subsequently released into the circulation. Our results suggest that lack of Fgf-23 activities results in extensive premature ageing-like features and early mortality of Fgf-23(-/-) mice, while restoring the systemic effects of FGF-23 significantly ameliorates these phenotypes, with the resultant effect being improved growth, restored fertility, and significantly prolonged survival of double mutants. With regard to their serum biochemistry, double mutants reversed the severe hyperphosphataemia, hypercalcaemia, and hypervitaminosis D found in Fgf-23(-/-) littermates; rather, double mutants show hypophosphataemia and normal serum 1,25-dihydroxyvitamin D(3) levels similar to pure FGF-23 Tg mice. These changes were associated with reduced renal expression of NaPi2a and 1 alpha-hydroxylase, compared to Fgf-23(-/-) mice. FGF-23 acts to prevent widespread abnormal features by acting systemically to regulate phosphate homeostasis and vitamin D metabolism. This novel mouse model provides us with an in vivo tool to study the systemic effects of FGF-23 in regulating mineral ion metabolism and preventing multiple abnormal phenotypes without the interference of native Fgf-23.

Maintenance of physiologic phosphate balance is of crucial biological importance, as it is fundamental to cellular function, energy metabolism, and skeletal mineralization. Fibroblast growth factor-23 (FGF-23) is a master regulator of phosphate homeostasis, but the molecular mechanism of such regulation is not yet completely understood. Targeted disruption of the Fgf-23 gene in mice (Fgf-23-/-) elicits hyperphosphatemia, and an increase in renal sodium/phosphate co-transporter 2a (NaPi2a) protein abundance. To elucidate the pathophysiological role of augmented renal proximal tubular expression of NaPi2a in Fgf-23-/- mice and to examine serum phosphate-independent functions of Fgf23 in bone, we generated a new mouse line deficient in both Fgf-23 and NaPi2a genes, and determined the effect of genomic ablation of NaPi2a from Fgf-23-/- mice on phosphate homeostasis and skeletal mineralization. Fgf-23-/-/NaPi2a-/- double mutant mice are viable and exhibit normal physical activities when compared to Fgf-23-/- animals. Biochemical analyses show that ablation of NaPi2a from Fgf-23-/- mice reversed hyperphosphatemia to hypophosphatemia by 6 weeks of age. Surprisingly, despite the complete reversal of serum phosphate levels in Fgf-23-/-/NaPi2a-/-, their skeletal phenotype still resembles the one of Fgf23-/- animals. The results of this study provide the first genetic evidence of an in vivo pathologic role of NaPi2a in regulating abnormal phosphate homeostasis in Fgf-23-/- mice by deletion of both NaPi2a and Fgf-23 genes in the same animal. The persistence of the skeletal anomalies in double mutants suggests that Fgf-23 affects bone mineralization independently of systemic phosphate homeostasis. Finally, our data support (1) that regulation of phosphate homeostasis is a systemic effect of Fgf-23, while (2) skeletal mineralization and chondrocyte differentiation appear to be effects of Fgf-23 that are independent of phosphate homeostasis.

Phosphate homeostasis is mostly regulated through humoral factors exerting direct or indirect effects on transporter proteins located in the intestine and kidney. Fibroblast growth factor 23 (FGF-23) is a major phosphate-regulating molecule, which can affect both renal and intestinal phosphate uptake to influence overall mineral ion homeostasis. We have found that Fgf-23 gene knockout mice (Fgf-23(-/-)) develop hyperphosphatemia that consequently leads to abnormal bone mineralization, and severe soft tissue calcifications. On the contrary, FGF-23 transgenic mice develop hypophosphatemia and produce rickets-like features in the mutant bone. Further studies using our Fgf-23(-/-) mice have identified an inverse correlation between Fgf-23, and vitamin D or NaPi2a; genomic elimination of either vitamin D or NaPi2a activities from Fgf-23(-/-) mice could reverse severe hyperphosphatemia to hypophosphatemia, and consequently could alter skeletal mineralization, suggesting that regulation of phosphate homeostasis in Fgf-23(-/-) mice is vitamin D- and NaPi2a-mediated process.

The phosphaturic activity of intact, full-length, fibroblast growth factor-23 (FGF-23) is well documented. FGF-23 circulates as the intact protein and as fragments generated as the result of proteolysis of the full-length protein. To assess whether short fragments of FGF-23 are phosphaturic, we compared the effect of acute, equimolar infusions of full-length FGF-23 and various FGF-23 fragments carboxyl-terminal to amino acid 176. In rats, intravenous infusions of full-length FGF-23 and FGF-23 176-251 significantly and equivalently increased fractional phosphate excretion (FE Pi) from 14 +/- 3 to 32 +/- 5% and 15 +/- 2 to 33 +/- 2% (p < 0.001), respectively. Chronic administration of FGF-23 176-251 reduced serum Pi and serum concentrations of 1alpha,25-dihydroxyvitamin D. Shorter forms of FGF-23 (FGF-23 180-251 and FGF-23 184-251) retained phosphaturic activity. Further shortening of the FGF-23 carboxyl-terminal domain, however, abolished phosphaturic activity, as infusion of FGF-23 206-251 did not increase urinary phosphate excretion. Infusion of a short fragment of the FGF-23 molecule, FGF-23 180-205, significantly increased FE Pi in rats and reduced serum Pi in hyperphosphatemic Fgf-23 ( -/- ) knockout mice. The activity of FGF-23 180-251 was confirmed in opossum kidney cells in which the peptide reduced Na(+)-dependent Pi uptake and enhanced internalization of the Na(+)-Pi IIa co-transporter. We conclude that carboxyl terminal fragments of FGF-23 are phosphaturic and that a short, 26-amino acid fragment of FGF-23 retains significant phosphaturic activity.

Fibroblast growth factor-23 (FGF-23) is one of the circulating phosphaturic factors associated with renal phosphate wasting. Fgf-23-/- animals show extremely high serum levels of phosphate and 1,25-dihydroxyvitamin D3, along with abnormal bone mineralization and soft tissue calcifications. To determine the role of vitamin D in mediating altered phosphate homeostasis and skeletogenesis in the Fgf-23-/- mice, we generated mice lacking both the Fgf-23 and 1alpha-hydroxylase genes (Fgf-23-/-/1alpha(OH)ase-/-). In the current study, we have identified the cellular source of Fgf-23 in adult mice. In addition, loss of vitamin D activities from Fgf-23-/- mice reverses the severe hyperphosphatemia to hypophosphatemia, attributable to increased urinary phosphate wasting in Fgf-23-/-/1alpha(OH)ase-/- mice, possibly as a consequence of decreased expression of NaPi2a. Ablation of vitamin D from Fgf-23-/- mice resulted in further reduction of total bone mineral content and bone mineral density and reversed ectopic calcification of skeleton and soft tissues, suggesting that abnormal mineral ion homeostasis and impaired skeletogenesis in Fgf-23-/- mice are mediated through enhanced vitamin D activities. In conclusion, using genetic manipulation studies, we have provided evidence for an in vivo inverse correlation between Fgf-23 and vitamin D activities and for the severe skeletal and soft tissue abnormalities of Fgf-23-/- mice being mediated through vitamin D.

Fibroblast growth factor 23 null mice (Fgf-23-/-) have a short lifespan and show numerous biochemical and morphological features consistent with premature aging-like phenotypes, including kyphosis, severe muscle wasting, hypogonadism, osteopenia, emphysema, uncoordinated movement, T cell dysregulation, and atrophy of the intestinal villi, skin, thymus, and spleen. Furthermore, increased vitamin D activities in homozygous mutants are associated with severe atherosclerosis and widespread soft tissue calcifications; ablation of vitamin D activity from Fgf-23-/- mice, by genetically deleting the 1alpha(OH)ase gene, eliminates atherosclerosis and ectopic calcifications and significantly rescues premature aging-like features of Fgf-23-/- mice, resulting in prolonged survival of Fgf-23-/-/1alpha(OH)ase-/- double mutants. Our results indicate a novel role of Fgf-23 in developing premature aging-like features through regulating vitamin D homeostasis. Finally, our data support a new model of interactions among Fgf-23, vitamin D, and klotho, a gene described as being associated with premature aging process.

Fibroblast growth factor-23 (FGF-23), a recently identified molecule that is mutated in patients with autosomal dominant hypophosphatemic rickets (ADHR), appears to be involved in the regulation of phosphate homeostasis. Although increased levels of circulating FGF-23 were detected in patients with different phosphate-wasting disorders such as oncogenic osteomalacia (OOM) and X-linked hypophosphatemia (XLH), it is not yet clear whether FGF-23 is directly responsible for the abnormal regulation of mineral ion homeostasis and consequently bone development. To address some of these unresolved questions, we generated a mouse model, in which the entire Fgf-23 gene was replaced with the lacZ gene. Fgf-23 null (Fgf-23-/-) mice showed signs of growth retardation by day 17, developed severe hyperphosphatemia with elevated serum 1,25(OH)2D3 levels, and died by 13 weeks of age. Hyperphosphatemia in Fgf-23-/- mice was accompanied by skeletal abnormalities, as demonstrated by histological, molecular, and various other morphometric analyses. Fgf-23-/-) mice had increased total-body bone mineral content (BMC) but decreased bone mineral density (BMD) of the limbs. Overall, Fgf-23-/- mice exhibited increased mineralization, but also accumulation of unmineralized osteoid leading to marked limb deformities. Moreover, Fgf-23-/- mice showed excessive mineralization in soft tissues, including heart and kidney. To further expand our understanding regarding the role of Fgf-23 in phosphate homeostasis and skeletal mineralization, we crossed Fgf-23-/- animals with Hyp mice, the murine equivalent of XLH. Interestingly, Hyp males lacking both Fgf-23 alleles were indistinguishable from Fgf-23/-/ mice, both in terms of serum phosphate levels and skeletal changes, suggesting that Fgf-23 is upstream of the phosphate regulating gene with homologies to endopeptidases on the X chromosome (Phex) and that the increased plasma Fgf-23 levels in Hyp mice (and in XLH patients) may be at least partially responsible for the phosphate imbalance in this disorder.

We have identified a single nucleotide polymorphism in the 5' region of the human interleukin-1 receptor type I (IL-1RI) gene, a C-->A transversion at position 52 in exon 1C (GenBank accession number AF172151) which creates a Bsr BI restriction endonuclease site. Allele frequencies in a Caucasian population were 0.72 (C allele) and 0.28 (A allele).