Neonatal iron deficiency causes abnormal phosphate metabolism by elevating FGF23 in normal and ADHR mice.

Fibroblast growth factor 23 (FGF23) gain of function mutations can lead to autosomal dominant hypophosphatemic rickets (ADHR) disease onset at birth, or delayed onset following puberty or pregnancy. We previously demonstrated that the combination of iron deficiency and a knock-in R176Q FGF23 mutation in mature mice induced FGF23 expression and hypophosphatemia that paralleled the late-onset ADHR phenotype. Because anemia in pregnancy and in premature infants is common, the goal of this study was to test whether iron deficiency alters phosphate handling in neonatal life. Wild-type (WT) and ADHR female breeder mice were provided control or iron-deficient diets during pregnancy and nursing. Iron-deficient breeders were also made iron replete. Iron-deficient WT and ADHR pups were hypophosphatemic, with ADHR pups having significantly lower serum phosphate (p < 0.01) and widened growth plates. Both genotypes increased bone FGF23 mRNA (>50 fold; p < 0.01). WT and ADHR pups receiving low iron had elevated intact serum FGF23; ADHR mice were affected to a greater degree (p < 0.01). Iron-deficient mice also showed increased Cyp24a1 and reduced Cyp27b1, and low serum 1,25-dihydroxyvitamin D (1,25D). Iron repletion normalized most abnormalities. Because iron deficiency can induce tissue hypoxia, oxygen deprivation was tested as a regulator of FGF23, and was shown to stimulate FGF23 mRNA in vitro and serum C-terminal FGF23 in normal rats in vivo. These studies demonstrate that FGF23 is modulated by iron status in young WT and ADHR mice and that hypoxia independently controls FGF23 expression in situations of normal iron. Therefore, disturbed iron and oxygen metabolism in neonatal life may have important effects on skeletal function and structure through FGF23 activity on phosphate regulation.

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.

Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice.

Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 (176)RXXR(179)/S(180) proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene-environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR.

Altered renal FGF23-mediated activity involving MAPK and Wnt: effects of the Hyp mutation.

Fibroblast growth factor-23 (FGF23), a hormone central to renal phosphate handling, is elevated in multiple hypophosphatemic disorders. Initial FGF23-dependent Erk1/2 activity in the kidney localizes to the distal convoluted tubule (DCT) with the co-receptor α-Klotho (KL), distinct from Npt2a in proximal tubules (PT). The Hyp mouse model of X-linked hypophosphatemic rickets (XLH) is characterized by hypophosphatemia with increased Fgf23, and patients with XLH elevate FGF23 following combination therapy of phosphate and calcitriol. The molecular signaling underlying renal FGF23 activity, and whether these pathways are altered in hypophosphatemic disorders, is unknown. To examine Npt2a in vivo, mice were injected with FGF23. Initial p-Erk1/2 activity in the DCT occurred within 10 min; however, Npt2a protein was latently reduced in the PT at 30-60  min, and was independent of Npt2a mRNA changes. KL-null mice had no DCT p-Erk1/2 staining following FGF23 delivery. Under basal conditions in Hyp mice, c-Fos and Egr1, markers of renal Fgf23 activity, were increased; however, KL mRNA was reduced 60% (P<0.05). Despite the prevailing hypophosphatemia and elevated Fgf23, FGF23 injections into Hyp mice activated p-Erk1/2 in the DCT. FGF23 injection also resulted in phospho-ß-catenin (p-ß-cat) co-localization with KL in wild-type mice, and Hyp mice demonstrated strong p-ß-cat staining under basal conditions, indicating potential crosstalk between mitogen-activated protein kinase and Wnt signaling. Collectively, these studies refine the mechanisms for FGF23 bioactivity, and demonstrate novel suppression of Wnt signaling in a KL-dependent DCT-PT axis, which is likely altered in XLH. Finally, the current treatment of phosphate and calcitriol for hypophosphatemic disorders may increase FGF23 activity.

Initial FGF23-mediated signaling occurs in the distal convoluted tubule.

Fibroblast growth factor-23 (FGF23), a hormone central to phosphate and vitamin D metabolism, reduces renal absorption of phosphate by downregulating the sodium-phosphate cotransporter Npt2a. However, the mechanisms of FGF23 action in the kidney are unclear, as Npt2a localizes to the proximal tubule (PT) and the FGF23 coreceptor alpha-Klotho (KL) localizes to the distal convoluted tubule (DCT). Immunofluorescent analyses following FGF23 injection in mice showed robust staining for phospho-ERK1/2, a marker of FGF23 bioactivity, only within the DCT in a subset of KL-positive cells. This activity colocalized with the FGF23 receptor FGFR1 and was present in DCT cells that were adjacent to Npt2a-expressing PT segments. Although KL is expressed as both secreted and membrane-bound isoforms, only the membrane-bound isoform was capable of mediating FGF23 bioactivity. These findings provide novel insight into the mechanisms of hormone-regulated phosphate metabolism by identifying an intrarenal signaling axis for FGF23.

Molecular analysis of DMP1 mutants causing autosomal recessive hypophosphatemic rickets.

We previously demonstrated that the mutations Met1Val (M1V) and the deletion of nucleotides 1484-1490 (1484-1490del) in Dentin matrix protein-1 (DMP1) cause the novel disorder autosomal recessive hypophosphatemic rickets (ARHR), which is associated with elevated fibroblast growth factor-23 (FGF23). To further understand the role of DMP1 in ARHR, we undertook molecular genetic and in vitro expression studies. First, we examined a kindred with a severe hypophosphatemic rickets phenotype and recessive inheritance. Analyses of this family demonstrated that the affected members had elevated serum FGF23 and carried a large, biallelic deletion that removed the majority of DMP1. At a minimum, this deletion encompassed 49 kb between DMP1 exon 3 and an intergenic region 5' to the next telomeric gene, integrin-binding sialoprotein (IBSP). We next performed immunofluorescent studies in cells to understand the effects of the known ARHR mutations on DMP1 cellular processing. These analyses showed that the M1V DMP1 mutant was not sorted to the trans-Golgi network (TGN) and secretory pathway, but filled the entire cytoplasm. In contrast, the 1484-1490del mutant localized to the TGN and was secreted, similar to wild type DMP1. The 1484-1490del mutation replaces the DMP1 18 C-terminal amino acids with 33 non-native residues. Truncation of wild type DMP1 by these native 18 residues followed by Western blot and confocal microscopic analyses demonstrated a wild type expression pattern when compared with the 1484-1490del mutant, indicating that the last 18 residues are not critical for cellular trafficking, but that the 33 additional residues arising from the 1484-1490del mutation likely compromise DMP1 processing. The relationship between DMP1 and FGF23 is unclear. To test endogenous DMP1 response to serum metabolites that also regulate FGF23, UMR-106 cells were treated with 1,25(OH)(2) vitamin D (1x10(-7) M) and showed a 12-fold increase in DMP1 mRNA and protein at 24 h. In summary, we have identified a novel DMP1 deletion as the cause of ARHR, as well as demonstrated that the ARHR mutations alter DMP1 cellular processing, and that DMP1 can be regulated by vitamin D. Taken together, this work expands our understanding of the genetic and molecular mechanisms associated with DMP1 alterations causing ARHR.

Disruption of ROCK1 gene attenuates cardiac dilation and improves contractile function in pathological cardiac hypertrophy.

The development of left ventricular cardiomyocyte hypertrophy in response to increased hemodynamic load and neurohormonal stress is initially a compensatory response. However, persistent stress eventually leads to dilated heart failure, which is a common cause of heart failure in human hypertensive and valvular heart disease. We have recently reported that Rho-associated coiled-coil containing protein kinase 1 (ROCK1) homozygous knockout mice exhibited reduced cardiac fibrosis and cardiomyocyte apoptosis, while displaying a preserved compensatory hypertrophic response to pressure overload. In this study, we have tested the effects of ROCK1 deficiency on cardiac hypertrophy, dilation, and dysfunction. We have shown that ROCK1 deletion attenuated left ventricular dilation and contractile dysfunction, but not hypertrophy, in a transgenic model of Galphaq overexpression-induced hypertrophy which represents a well-characterized and highly relevant genetic mouse model of pathological hypertrophy. Although the development of cardiomyocyte hypertrophy was not affected, ROCK1 deletion in Galphaq mice resulted in a concentric hypertrophic phenotype associated with reduced induction of hypertrophic markers indicating that ROCK1 deletion could favorably modify hypertrophy without inhibiting it. Furthermore, ROCK1 deletion also improved contractile response to beta-adrenergic stimulation in Galphaq transgenic mice. Consistent with this observation, ROCK1 deletion prevented down-regulation of type V/VI adenylyl cyclase expression, which is associated with the impaired beta-adrenergic signaling in Galphaq mice. The present study establishes for the first time a role for ROCK1 in cardiac dilation and contractile dysfunction.