Erica L. Clinkenbeard

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.

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.

Osteocyte regulation of phosphate homeostasis and bone mineralization underlies the pathophysiology of the heritable disorders of rickets and osteomalacia

Although recent studies have established that osteocytes function as secretory cells that regulate phosphate metabolism, the biomolecular mechanism(s) underlying these effects remain incompletely defined. However, investigations focusing on the pathogenesis of X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR), and autosomal recessive hypophosphatemic rickets (ARHR), heritable disorders characterized by abnormal renal phosphate wasting and bone mineralization, have clearly implicated FGF23 as a central factor in osteocytes underlying renal phosphate wasting, documented new molecular pathways regulating FGF23 production, and revealed complementary abnormalities in osteocytes that regulate bone mineralization. The seminal observations leading to these discoveries were the following: 1) mutations in FGF23 cause ADHR by limiting cleavage of the bioactive intact molecule, at a subtilisin-like protein convertase (SPC) site, resulting in increased circulating FGF23 levels and hypophosphatemia; 2) mutations in DMP1 cause ARHR, not only by increasing serum FGF23, albeit by enhanced production and not limited cleavage, but also by limiting production of the active DMP1 component, the C-terminal fragment, resulting in dysregulated production of DKK1 and β-catenin, which contributes to impaired bone mineralization; and 3) mutations in PHEX cause XLH both by altering FGF23 proteolysis and production and causing dysregulated production of DKK1 and β-catenin, similar to abnormalities in ADHR and ARHR, but secondary to different central pathophysiological events. These discoveries indicate that ADHR, XLH, and ARHR represent three related heritable hypophosphatemic diseases that arise from mutations in, or dysregulation of, a single common gene product, FGF23 and, in ARHR and XLH, complimentary DMP1 and PHEX directed events that contribute to abnormal bone mineralization.

Conditional Deletion of Murine Fgf23: Interruption of the Normal Skeletal Responses to Phosphate Challenge and Rescue of Genetic Hypophosphatemia.

The transgenic and knockout (KO) animals involving Fgf23 have been highly informative in defining novel aspects of mineral metabolism, but are limited by shortened lifespan, inability of spatial/temporal FGF23 control, and infertility of the global KO. To more finely test the role of systemic and genetic influences in FGF23 production, a mouse was developed that carried a floxed (“f”)‐Fgf23 allele (exon 2 floxed) which demonstrated in vivo recombination when bred to global‐Cre transgenic mice (eIIa‐cre). Mice homozygous for the recombined allele (“Δ”) had undetectable serum intact FGF23, elevated serum phosphate (p < 0.05), and increased kidney Cyp27b1 mRNA (p < 0.05), similar to global Fgf23‐KO mice. To isolate cellular FGF23 responses during phosphate challenge, Fgf23Δ/f mice were mated with early osteoblast type Iα1 collagen 2.3‐kb promoter‐cre mice (Col2.3‐cre) and the late osteoblast/early osteocyte Dentin matrix protein‐1‐cre (Dmp1‐cre). Fgf23Δ/f/Col2.3‐cre+ and Fgf23Δ/f/Dmp1‐cre+ exhibited reduced baseline serum intact FGF23 versus controls. After challenge with high‐phosphate diet Cre– mice had 2.1‐fold to 2.5‐fold increased serum FGF23 (p < 0.01), but Col2.3‐cre+ mice had no significant increase, and Dmp1‐cre+ mice had only a 37% increase (p < 0.01) despite prevailing hyperphosphatemia in both models. The Fgf23Δ/f/Col2.3‐cre was bred onto the Hyp (murine X‐linked hypophosphatemia [XLH] model) genetic background to test the contribution of osteoblasts and osteocytes to elevated FGF23 and Hyp disease phenotypes. Whereas Hyp mice maintained inappropriately elevated FGF23 considering their marked hypophosphatemia, Hyp/Fgf23Δ/f/Col2.3‐cre+ mice had serum FGF23 <4% of Hyp (p < 0.01), and this targeted restriction normalized serum phosphorus and ricketic bone disease. In summary, deleting FGF23 within early osteoblasts and osteocytes demonstrated that both cell types contribute to baseline circulating FGF23 concentrations, and that targeting osteoblasts/osteocytes for FGF23 production can modify systemic responses to changes in serum phosphate concentrations and rescue the Hyp genetic syndrome.

Chronic Hyperphosphatemia and Vascular Calcification Are Reduced by Stable Delivery of Soluble Klotho

αKlotho (αKL) regulates mineral metabolism, and diseases associated with αKL deficiency are characterized by hyperphosphatemia and vascular calcification (VC). αKL is expressed as a membrane-bound protein (mKL) and recognized as the coreceptor for fibroblast growth factor-23 (FGF23) and a circulating soluble form (cKL) created by endoproteolytic cleavage of mKL. The functions of cKL with regard to phosphate metabolism are unclear. We tested the ability of cKL to regulate pathways and phenotypes associated with hyperphosphatemia in a mouse model of CKD-mineral bone disorder and αKL-null mice. Stable delivery of adeno-associated virus (AAV) expressing cKL to diabetic endothelial nitric oxide synthase-deficient mice or αKL-null mice reduced serum phosphate levels. Acute injection of recombinant cKL downregulated the renal sodium-phosphate cotransporter Npt2a in αKL-null mice supporting direct actions of cKL in the absence of mKL. αKL-null mice with sustained AAV-cKL expression had a 74%-78% reduction in aorta mineral content and a 72%-77% reduction in mineral volume compared with control-treated counterparts (P<0.01). Treatment of UMR-106 osteoblastic cells with cKL + FGF23 increased the phosphorylation of extracellular signal-regulated kinase 1/2 and induced Fgf23 expression. CRISPR/Cas9-mediated deletion of fibroblast growth factor receptor 1 (FGFR1) or pretreatment with inhibitors of mitogen-activated kinase kinase 1 or FGFR ablated these responses. In summary, sustained cKL treatment reduced hyperphosphatemia in a mouse model of CKD-mineral bone disorder, and it reduced hyperphosphatemia and prevented VC in mice without endogenous αKL. Furthermore, cKL stimulated Fgf23 in an FGFR1-dependent manner in bone cells. Collectively, these findings indicate that cKL has mKL-independent activity and suggest the potential for enhancing cKL activity in diseases of hyperphosphatemia with associated VC.

Erythropoietin stimulates murine and human fibroblast growth factor-23, revealing novel roles for bone and bone marrow.

Early stages of chronic kidney disease (CKD) are characterized by development of progressive anemia as well as concurrent marked elevation of the phosphaturic hormone fibroblast growth factor 23 (FGF23). As kidney function declines, FGF23 further increases and anemia worsens, due to either inadequate production of renal erythropoietin (EPO) or incidence of hypoferremia. Moreover, in CKD, anemia and elevated FGF23 levels are associated with left ventricular hypertrophy (LVH), CKD progression, and mortality. Treatment of CKD-related anemia involves iron repletion and erythropoietin (EPO) administration. EPO is one of the most extensively used medications in CKD, but its administration is associated with increased risks of cardiovascular disease and mortality. Although FGF23 levels increase early in CKD, the pathophysiological regulation of FGF23 is still not completely understood. Phosphate, 1,25-dihydroxyvitamin D (1,25D), parathyroid hormone, and calcium affect FGF23 production; however, these factors are still within normal ranges when bone and circulating FGF23 increase. Recent studies demonstrate intriguing associations between hypoxia, iron deficiency, and FGF23 upregulation. Indeed, in the settings of normal and impaired kidney function, iron deficiency potently increases bone Fgf23 expression. However, other anemia-related factors, including EPO, could potentially contribute to elevated FGF23 production. As both EPO therapy and FGF23 are associated with adverse outcomes in CKD, we explored the hypothesis that EPO is a previously unrecognized regulator of this phosphaturic hormone. Collectively, our pre-clinical findings suggest that modulating EPO exposure in CKD patients may lower FGF23 and thereby decrease its adverse effects. To examine whether exogenous EPO stimulates FGF23 in vivo, wild-type C57BL/6 mice at 6-8 weeks of age were injected with increasing doses of recombinant human EPO (25-250 U/g of body weight). A 3-day regimen induced a dose-dependent, 40-fold maximal increase in whole bone Fgf23 mRNA expression (Figure 1A), paralleled by increased serum total FGF23 as measured with an ELISA that detects both C-terminal FGF23 fragments (‘cFGF23’) and bioactive intact FGF23 (‘iFGF23’) (Figure

Systemic Control of Bone Homeostasis by FGF23 Signaling

The regulation of phosphate metabolism as an influence on bone homeostasis is profound. Recent advances in understanding the systemic control of fibroblast growth factor-23 (FGF23) has uncovered novel effectors of endocrine feedback loops for calcium, phosphate, and vitamin D balance that interact with ‘traditional’ feedback loops for mineral metabolism. Not only are these findings reshaping research studying phosphate handling and skeletal interactions, but they have also provided new therapeutic interventions. Emerging data support that the control of FGF23 production in bone and its circulating concentrations is a multilayered process, with some influences affecting FGF23 transcription and some posttranslational modification of the secreted, bioactive protein. Additionally, the actions of FGF23 on its target tissues via its coreceptor α-Klotho, are subject to regulatory events just coming to light. The recent findings of systemic influences on circulating FGF23 and the downstream manifestations on bone homeostasis will be reviewed herein.

Heritable and acquired disorders of phosphate metabolism: Etiologies involving FGF23 and current therapeutics

Phosphate is critical for many cellular processes and structural functions, including as a key molecule for nucleic acid synthesis and energy metabolism, as well as hydroxyapatite formation in bone. Therefore it is critical to maintain tight regulation of systemic phosphate levels. Based upon its broad biological importance, disruption of normal phosphate homeostasis has detrimental effects on skeletal integrity and overall health. Investigating heritable diseases of altered phosphate metabolism has led to key discoveries underlying the regulation and systemic actions of the phosphaturic hormone Fibroblast growth factor-23 (FGF23). Both molecular and clinical studies have revealed novel targets for the development and optimization of therapies for disorders of phosphate handling. This review will focus upon the bridge between genetic discoveries involving disorders of altered FGF23 bioactivity, as well as describe how these findings have translated into pharmacologic application.

The metabolic bone disease associated with the Hyp mutation is independent of osteoblastic HIF1α expression

Fibroblast growth factor-23 (FGF23) controls key responses to systemic phosphate increases through its phosphaturic actions on the kidney. In addition to stimulation by phosphate, FGF23 positively responds to iron deficiency anemia and hypoxia in rodent models and in humans. The disorder X-linked hypophosphatemia (XLH) is characterized by elevated FGF23 in concert with an intrinsic bone mineralization defect. Indeed, the Hyp mouse XLH model has disturbed osteoblast to osteocyte differentiation with altered expression of a wide variety of genes, including FGF23. The transcription factor Hypoxia inducible factor-1α (HIF1α) has been implicated in regulating FGF23 production and plays a key role in proper bone cell differentiation. Thus the goals of this study were to determine whether HIF1α activation could influence FGF23, and to test osteoblastic HIF1α production on the Hyp endocrine and skeletal phenotypes in vivo. Treatment of primary cultures of osteoblasts/osteocytes and UMR-106 cells with the HIF activator AG490 resulted in rapid HIF1α stabilization and increased Fgf23 mRNA (50–100 fold; p < 0.01–0.001) in a time- and dose-dependent manner. Next, the Phex gene deletion in the Hyp mouse was bred onto mice with a HIF1α/Osteocalcin (OCN)-Cre background. Although HIF1α effects on bone could be detected, FGF23-related phenotypes due to the Hyp mutation were independent of HIF1α in vivo. In summary, FGF23 can be driven by ectopic HIF1α activation under normal iron conditions in vitro, but factors independent of HIF1α activity after mature osteoblast formation are responsible for the disease phenotypes in Hyp mice in vivo.

Gene-by-diet interactions affect serum 1,25-dihydroxyvitamin D levels in male BXD recombinant inbred mice

1,25-Dihydroxyvitamin D (1,25[OH]2D) regulates calcium (Ca), phosphate, and bone metabolism. Serum 1,25(OH)2D levels are reduced by low vitamin D status and high fibroblast growth factor 23 (FGF23) levels and increased by low Ca intake and high PTH levels. Natural genetic variation controls serum 25-hydroxyvitamin D (25[OH]D) levels, but it is unclear how it controls serum 1,25(OH)2D or the response of serum 1,25(OH)2D levels to dietary Ca restriction (RCR). Male mice from 11 inbred lines and from 51 BXD recombinant inbred lines were fed diets with either 0.5% (basal) or 0.25% Ca from 4 to 12 weeks of age (n = 8 per line per diet). Significant variation among the lines was found in basal serum 1,25(OH)2D and in the RCR as well as basal serum 25(OH)D and FGF23 levels. 1,25(OH)2D was not correlated to 25(OH)D but was negatively correlated to FGF23 (r = -0.5). Narrow sense heritability of 1,25(OH)2D was 0.67 on the 0.5% Ca diet, 0.66 on the 0.25% Ca diet, and 0.59 for the RCR, indicating a strong genetic control of serum 1,25(OH)2D. Genetic mapping revealed many loci controlling 1,25(OH)2D (seven loci) and the RCR (three loci) as well as 25(OH)D (four loci) and FGF23 (two loci); a locus on chromosome 18 controlled both 1,25(OH)2D and FGF23. Candidate genes underlying loci include the following: Ets1 (1,25[OH]2D), Elac1 (FGF23 and 1,25[OH]2D), Tbc1d15 (RCR), Plekha8 and Lyplal1 (25[OH]D), and Trim35 (FGF23). This report is the first to reveal that serum 1,25(OH)2D levels are controlled by multiple genetic factors and that some of these genetic loci interact with the dietary environment.