Fibroblast Growth Factor 23 Cleavage Research Articles

Gα11 deficiency increases fibroblast growth factor 23 levels in a mouse model of familial hypocalciuric hypercalcemia.

Fibroblast growth factor 23 (FGF23) production has recently been shown to increase downstream of Gαq/11-PKC signaling in osteocytes. Inactivating mutations in the gene encoding Gα11 (GNA11) cause familial hypocalciuric hypercalcemia (FHH) due to impaired calcium-sensing receptor signaling. We explored the effect of Gα11 deficiency on FGF23 production in mice with heterozygous (Gna11+/-) or homozygous (Gna11-/-) ablation of Gna11. Both Gna11+/- and Gna11-/- mice demonstrated hypercalcemia and mildly raised parathyroid hormone levels, consistent with FHH. Strikingly, these mice also displayed increased serum levels of total and intact FGF23 and hypophosphatemia. Gna11-/- mice showed augmented Fgf23 mRNA levels in the liver and heart, but not in bone or bone marrow, and also showed evidence of systemic inflammation with elevated serum IL-1β levels. Furin gene expression was significantly increased in the Gna11-/- liver, suggesting enhanced FGF23 cleavage despite the observed rise in circulating intact FGF23 levels. Gna11-/- mice had normal renal function and reduced serum levels of glycerol-3-phosphate, excluding kidney injury as the primary cause of elevated intact FGF23 levels. Thus, Gα11 ablation caused systemic inflammation and excess serum FGF23 in mice, suggesting that patients with FHH - at least those with GNA11 mutations - may be at risk for these complications.

Associations between anemia and FGF23 in the CKiD study

BackgroundFibroblast growth factor 23 (FGF23) is a bone-derived hormone that plays a central role in chronic kidney disease-mineral bone disorder and is associated with CKD progression and cardiovascular morbidity. Factors related to CKD-associated anemia, including iron deficiency, can increase FGF23 production. This study aimed to assess whether anemia and/or iron deficiency are associated with increased circulating concentrations of FGF23 in the large, well-characterized Chronic Kidney Disease in Children (CKiD) study cohort.MethodsHemoglobin concentrations, iron parameters, C-terminal (total) FGF23, intact FGF23, and relevant covariables were measured in cross-sectional analysis of CKiD study subjects.ResultsIn 493 pediatric patients with CKD (median [interquartile range] age 13 [9, 16] years), the median estimated glomerular filtration rate was 48 [35, 61] ml/min/1.73 m2, and 103 patients (21%) were anemic. Anemic subjects had higher total FGF23 concentrations than non-anemic subjects (204 [124, 390] vs. 109 [77, 168] RU/ml, p < 0.001). In multivariable linear regression modeling, anemia was independently associated with higher total FGF23, after adjustment for demographic, kidney-related, mineral metabolism, and inflammatory covariables (standardized β (95% confidence interval) 0.10 (0.04, 0.17), p = 0.002). In the subset of subjects with available iron parameters (n = 191), iron deficiency was not associated with significantly higher total FGF23 concentrations. In the subgroup that had measurements of both total and intact FGF23 (n = 185), in fully adjusted models, anemia was significantly associated with higher total FGF23 (standardized β (95% CI) 0.16 (0.04, 0.27), p = 0.008) but not intact FGF23 (standardized β (95% CI) 0.02 (−0.12, 0.15), p = 0.81).ConclusionsIn this cohort of pediatric patients with CKD, anemia was associated with increased total FGF23 levels but was not independently associated with elevated intact FGF23, suggesting possible effects on both FGF23 production and cleavage. Further studies are warranted to investigate non-mineral factors affecting FGF23 production and metabolism in CKD.Graphical abstractA higher resolution version of the Graphical abstract is available as Supplementary information

Bone-derived C-terminal FGF23 cleaved peptides increase iron availability in acute inflammation

AbstractInflammation leads to functional iron deficiency by increasing the expression of the hepatic iron regulatory peptide hepcidin. Inflammation also stimulates fibroblast growth factor 23 (FGF23) production by increasing both Fgf23 transcription and FGF23 cleavage, which paradoxically leads to excess in C-terminal FGF23 peptides (Cter-FGF23), rather than intact FGF23 (iFGF23) hormone. We determined that the major source of Cter-FGF23 is osteocytes and investigated whether Cter-FGF23 peptides play a direct role in the regulation of hepcidin and iron metabolism in response to acute inflammation. Mice harboring an osteocyte-specific deletion of Fgf23 showed a ∼90% reduction in Cter-FGF23 levels during acute inflammation. Reduction in Cter-FGF23 led to a further decrease in circulating iron in inflamed mice owing to excessive hepcidin production. We observed similar results in mice showing impaired FGF23 cleavage owing to osteocyte-specific deletion of Furin. We next showed that Cter-FGF23 peptides bind members of the bone morphogenetic protein (BMP) family, BMP2 and BMP9, which are established inducers of hepcidin. Coadministration of Cter-FGF23 and BMP2 or BMP9 prevented the increase in Hamp messenger RNA and circulating hepcidin levels induced by BMP2/9, resulting in normal serum iron levels. Finally, injection of Cter-FGF23 in inflamed Fgf23KO mice and genetic overexpression of Cter-Fgf23 in wild type mice also resulted in lower hepcidin and higher circulating iron levels. In conclusion, during inflammation, bone is the major source of Cter-FGF23 secretion, and independently of iFGF23, Cter-FGF23 reduces BMP-induced hepcidin secretion in the liver.

MO551: Targeted Ablation of PTEN in Osteocytes Induces Severe Anemia and Increases FGF23 Production in Renal Insufficiency

Abstract BACKGROUND AND AIMS Renal disease patients present bone alterations due to an imbalance of mineral metabolism. Previous studies have demonstrated that mice with conditional ablation of the phosphatase and tensin homolog (PTEN) in osteoblasts show increases in bone volume. The aim of this study was to investigate whether PTEN ablation in osteocytes has potential benefits on bone and mineral metabolism in the context of renal insufficiency. METHOD A few 2-month-old mice with constitutive ablation of PTEN in osteocytes (Ocy-PTEN-cKO) underwent 5/6 nephrectomy (5/6Nx) and were fed a 0.9% phosphate diet. After 2 weeks, 24-h urine samples were collected and animals were sacrificed to obtain blood samples for biochemical studies and organs for histological and biochemical analyses. Bone histomorphometry and microcomputed tomography analyses were performed in femurs. Fibroblast growth factor 23 (FGF23) expression was quantified in bone lysates and intact and c-terminal FGF23 concentrations were measured in plasma samples. Animals with normal renal function and Cre-negative littermates were used as controls. In addition, the effects of PTEN silencing were assessed in vitro in osteocyte-like bone marrow mesenchymal stem cells and in the osteosarcoma cell line SAOS-2. RESULTS Ocy-PTEN-cKO mice showed increased bone volume, mainly in the cortical compartment which showed higher cortical thickness and lower porosity. Of interest, we observed lower haematocrit in the Ocy-PTEN-cKO mice that was much lower in those with 5/6Nx. Anaemia was not associated with a lower renal EPO expression. Bone mRNA and protein expression of FGF23 were increased in Ocy-PTEN-cKO mice with normal and reduced renal function. 5/6Nx resulted in higher plasma intact FGF23, which remained similar between both, Ocy-PTEN-cKO and control groups, due to increased FGF23 cleavage as indicated by the higher plasma c-terminal FGF23 levels in the Ocy-PTEN-cKO groups, resulting in increased plasma phosphate. In vitro, PTEN silencing in bone marrow mesenchymal stem cells differentiated into osteoblasts or in the osteoblastic cell line SAOS-2 did not show significant changes regarding FGF23 levels, indicating that the effects must be indirect. CONCLUSION PTEN ablation in osteocytes increases cortical bone volume and exacerbates anemia in mice with reduced renal function. Moreover, bone FGF23 production was increased but subsequent proteolysis resulted in higher plasma phosphate. These results demonstrate a major role of the osteocyte in the development of anaemia in renal disease, which could be associated with FGF23 metabolism.

Interconnections of fibroblast growth factor 23 and klotho with erythropoietin and hypoxia-inducible factor.

Bone marrow (BM) hematopoiesis is tightly regulated process and bone components such as osteoblasts, extracellular matrix, and minerals influence hematopoiesis via regulation of hematopoietic stem cell function. Erythropoietin (EPO) secreted mostly by renal EPO producing (REP) cells which employ the hypoxia-inducible factor (HIF) pathway. When tissue hypoxia occurs, HIFs bind to hypoxia response element in the EPO promoter and induce EPO production. EPO binds to the EPO receptor on red cell progenitors in the BM and triggers expansion of red cell mass. Fibroblast growth factor-23 (FGF23) which is secreted mostly by osteoblasts and less by BM impacts hematopoiesis by influencing EPO production. Reciprocally, increases of EPO (acute or chronic) influence both FG23 production and cleavage resulting in variation of c fragment FGF23 (cFGF23) and intact FGF23 (iFGF23) ratios. As HIFs stimulate EPO production, they indirectly affect FGF23. Direct stimulation of FGF23 synthesis by binding of HIF on FGF23 promoter is also suggested. FGF23 cleavage by furin is another potential mechanism affecting FGF23 levels. Klotho is present in membrane-bound (transmembrane) and free (circulating) forms. Transmembrane klotho is the co-receptor of FGF23 and forms complexes with FGF23 receptors in the membrane surface and required for FGF23 actions. Recent evidence showed that klotho is also associated with EPO and HIF production suggesting a complex relationship between FGF23, klotho, EPO, and HIF. In this review, we have summarized the connections between FGF23, klotho, HIF, and EPO and their reflections to hematopoiesis.

In Vivo Analysis of the Contribution of Proprotein Convertases to the Processing of FGF23.

Fibroblast growth factor 23 (FGF23) is a hormone secreted from fully differentiated osteoblasts and osteocytes that inhibits phosphate reabsorption by kidney proximal tubules. The full-length (i.e., intact) protein mediates FGF23 endocrine functions, while endoproteolytic cleavage at a consensus cleavage sequence for the proprotein convertases (PCs) inactivates FGF23. Two PCs, furin and PC5, were shown to cleave FGF23 in vitro at RHTR179↓, but whether they are fulfilling this function in vivo is currently unknown. To address this question, we used here mice lacking either or both furin and PC5 in cell-specific manners and mice lacking the paired basic amino acid-cleaving enzyme 4 (PACE4) in all cells. Our analysis shows that furin inactivation in osteoblasts and osteocytes results in a 25% increase in circulating intact FGF23, without any significant impact on serum phosphate levels, whether mice are maintained on a normal or a low phosphate diet. Under conditions of iron deficiency, FGF23 is normally processed in control mice, but its processing is impaired in mice lacking furin in osteoblasts and osteocytes. In contrast, FGF23 is normally cleaved following erythropoietin or IL-1β injections in mice lacking furin or both furin and PC5, and in PACE4-deficient mice. Altogether, these studies suggest that furin is only partially responsible for FGF23 cleavage under certain conditions in vivo. The processing of FGF23 may therefore involve the redundant action of multiple PCs or of other peptidases in osteoblasts, osteocytes and hematopoietic cells.

Glycerol-3-phosphate and fibroblast growth factor 23 regulation.

Both classical and nonclassical factors regulate fibroblast growth factor 23 (FGF23), with impacts on gene expression and proteolytic cleavage. Here, we review recent publications that extend current knowledge on these factors. Emerging nonclassical FGF23 regulators such as erythropoietin cause a balanced increase in FGF23 expression and cleavage, with minimal or no increase in biologically active intact FGF23 (iFGF23) in blood. However, circulating FGF23 profiles may not reflect the bone marrow microenvironment. For example, granulocyte colony-stimulating factor increases local marrow iFGF23 levels without impacting circulating iFGF23 levels. The view that phosphate does not increase bone FGF23 production also warrants reconsideration, as phosphate can reduce iFGF23 cleavage and phosphate-containing calciprotein particles increase FGF23 expression. Finally, a screen of renal venous plasma identifies glycerol-3-phosphate as a kidney-derived molecule that circulates to bone and bone marrow, where it is converted to lysophosphatidic acid and signals through a G-protein coupled receptor to increase FGF23 synthesis. FGF23 regulation is complex, requiring consideration of known and emerging stimuli, expression and cleavage, and circulating and local levels. Recent work identifies glycerol-3-phosphate as an FGF23 regulator derived from the injured kidney; whether it participates in FGF23 production downstream of classical or nonclassical factors requires further study.

Systemic Jak1 activation provokes hepatic inflammation and imbalanced FGF23 production and cleavage.

Fibroblast growth factor 23 (FGF23) is a main regulator of mineral homeostasis. Low and high circulating FGF23 levels are associated with bone, renal, cardiovascular diseases, and increased mortality. Understanding the factors and signaling pathways affecting FGF23 levels is crucial for the management of these diseases and their complications. Here, we show that activation of the Jak1/Stat3 signaling pathway leads to inflammation in liver and to an increase in hepatic FGF23 synthesis, a key hormone in mineral metabolism. This increased synthesis leads to massive C-terminal FGF23 circulating levels, the inactive C-terminal fragment, and increased intact FGF23 levels, the active form, resulting in imbalanced production and cleavage. Liver inflammation does not lead to activation of the calcineurin-NFAT pathway, and no signs of systemic inflammation could be observed. Despite the increase of active intact FGF23, excessive C-terminal FGF23 levels block the phosphaturic activity of FGF23. Therefore, kidney function and renal αKlotho expression are normal and no activation of the MAPK pathway was detected. In addition, activation of the Jak1/Stat3 signaling pathway leads to high calcitriol levels and low parathyroid hormone production. Thus, JAK1 is a central regulator of mineral homeostasis. Moreover, this study also shows that in order to assess the impact of high FGF23 levels on disease and kidney function, the source and the balance in FGF23 production and cleavage are critical.

Erythropoiesis stimulating agents are associated with serum fibroblast growth factor 23 metabolism in patients on hemodialysis.

BackgroundThis study aimed to determine associations among short- and long-acting erythropoiesis stimulating agents (ESAs), changes in serum fibroblast growth factor 23 (FGF23) and biomarkers of iron metabolism.MethodsAmong 108 patients on hemodialysis (HD), 44 received every 2 weeks or monthly doses of continuous erythropoiesis receptor activator (CERA), 31 received weekly doses of darbepoetin-α, 24 received three doses per week of epoetin-β and 9 were not treated with an ESA. Intact and C-terminal FGF23 and transferrin saturation (TSAT), ferritin, erythroferrone and hepcidin 25 were measured in blood samples collected before the HD session at the end of the dialysis week (baseline, Day 0) and on Days 3, 5, 7 and 14 thereafter.ResultsLevels of ferritin, hepcidin 25 and erythroferrone as well as TSAT were significantly decreased or elevated in patients treated with CERA compared with other types of ESAs. Levels of C-terminal FGF23 increased in all groups during the observation period. Levels of intact FGF23 and ratios of intact FGF23 to C-terminal FGF23 gradually decreased between Days 3 and 7 in the CERA but not in the other groups. Multivariate models associated changes in hepcidin 25 and phosphate with those of intact FGF23.ConclusionThe long-acting ESA CERA might influence levels of intact FGF23 by increasing FGF23 cleavage in patients on HD in association with prolonged hepcidin 25 suppression.

Erythropoietin Is Associated with a Decline in the iFGF23/cFGF23 Ratio in Patients with Various Hereditary Hemolytic Anemias

Background Recently, erythropoietin (EPO) was identified as an important regulator of production and cleavage of fibroblast growth factor 23 (FGF23). Since erythroid progenitor cells express high levels of FGF23 and carry the FGF receptor, they are involved in the FGF23 metabolic pathway. FGF23 is a bone-derived hormone, a key player in phosphate and vitamin D metabolism and regulator of bone mineralization. FGF23 is formed as intact, biologically active protein (iFGF23). Proteolytic cleavage results in formation of an allegedly assumed inactive C-terminal tail FGF23 (cFGF23). FGF23-knockouts or iFGF23 blocking peptides increase erythropoiesis, reduce erythroid cell apoptosis and increase EPO mRNA and circulating EPO. By competitive inhibition, increase in cFGF23 relative to iFGF23 leads to suppression of FGF receptor signaling. (Recombinant) EPO increases the amount of circulating FGF23 (iFGF23 plus cFGF23) and, more importantly, alters the iFGF23/cFGF23 ratio in favor of cFGF23, thereby overcoming suppression of erythropoiesis by iFGF23. As recently discussed, (van Vuren et al. Front. Physiol. 2019) we hypothesize that EPO-FGF23 signaling plays a role in hereditary hemolytic anemias and is herein related to iron metabolism, inflammation and bone health. We explored this concept further in the current study. Methods FGF23 was measured in 90 patients and 10 healthy controls of the ZEbRA trial, an observational study at the UMC Utrecht on clinical sequelae and pathophysiology of rare congenital hemolytic (Netherlands Trial Register [NL5189]). An overview of relevant patient characteristics is provided in the Table. Two FGF23 assays were used: one that detects the C-terminal and therefore quantifies cFGF23 and (full-length) iFGF23 (total FGF23; Immunotopics/Quidel) and one that only detects iFGF23 (Kainos Laboratories). Chemokines/cytokines were measured simultaneously with a Luminex assay. Results We observed a strong correlation between levels of total FGF23 (cFGF23 and iFGF23) and EPO in patients with various hemolytic anemias (r=0.64, 95% confidence interval [0.09; 0.89]) (Figure). As expected, there was neither a correlation between iFGF23 and EPO values nor between iFGF23 and total FGF23. These data are consistent with a decline of the iFGF23/cFGF23 ratio in response to EPO. iFGF23 and total FGF23 were not correlated to serum calcium, phosphate, creatinine, parathyroid hormone and 25-OH vitamin D (p&gt;0.05). We did not identify a correlation of iFGF23 and total FGF23 with bone health (hip and spine T-scores). EPO was associated with transferrin saturation (r=0.45, 95% CI [0.28; 0.63]) and ferritin (r=0.47, 95% CI [0.06; 0.79]), whereas total FGF23 did not correlate with iron status. The influence of inflammation on the EPO-FGF23 axis was clearly illustrated by higher mean total FGF23 in SCD patients (549 RU/L) compared to other patients (172 RU/L) (95% CI [87; 747]), without a difference in EPO or iFGF23. We identified a correlation of cFGF23 with CCL4 (r=0.49, 95% CI [0.15; 0.72]) and with sICAM (r=0.54, 95% CI [0.16; 0.75]), markers of endothelial dysfunction and immune players in pulmonary hypertension. (Kato et al. Br J Haematol. 2005; Sweatt et al. Circ Res. 2019) Conclusion We show for the first time the importance of EPO in FGF23 signaling in hemolytic anemias and investigated its relation with iron, inflammation and bone health. Absence of a relation between iron and FGF23 suggests that EPO is not simply an intermediary in FGF23 regulation by iron. FGF23 data of SCD patients suggest that inflammation is associated with an additional increase in FGF23 cleavage. Absence of a correlation of FGF23 with bone health might be attributable to treatment for osteoporosis in many patients. Alternatively, systemic FGF23 might underestimate paracrine signaling in the bone. Additionally, we hypothesize on a contribution of decline in the iFGF23/cFGF23 ratio to vasculopathic complications. Our hypothesis aligns with previous observations that too little or too much FGF signaling is related to endothelial dysregulation and endothelial-to-mesenchymal transition, which contributes to development of pulmonary hypertension. (Chen et al. Cell. Rep. 2012; Morrell et al. J. Am. Coll. 2009) Current findings open a new area for research and treatment in hemolytic anemias. Therapeutic targeting of the FGF23 pathway might provide an opportunity to intervene in the development of complications. Disclosures Glenthøj: Agios Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Alexion: Research Funding; Novartis: Consultancy; Novo Nordisk: Honoraria. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding. van Beers:Novartis: Consultancy, Research Funding; Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding.

FGF23 at the crossroads of phosphate, iron economy and erythropoiesis.

Fibroblast growth factor 23 (FGF23) was initially characterized as an important regulator of phosphate and calcium homeostasis. New research advances demonstrate that FGF23 is also linked to iron economy, inflammation and erythropoiesis. These advances have been fuelled, in part, by the serendipitous development of two distinct FGF23 assays that can substitute for invasive bone biopsies to infer the activity of the three main steps of FGF23 regulation in bone: transcription, post-translational modification and peptide cleavage. This 'liquid bone biopsy for FGF23 dynamics' enables large-scale longitudinal studies of FGF23 regulation that would otherwise be impossible in humans. The balance between FGF23 production, post-translational modification and cleavage is maintained or perturbed in different hereditary monogenic conditions and in acquired conditions that mimic these genetic disorders, including iron deficiency, inflammation, treatment with ferric carboxymaltose and chronic kidney disease. Looking ahead, a deeper understanding of the relationships between FGF23 regulation, iron homeostasis and erythropoiesis can be leveraged to devise novel therapeutic targets for treatment of anaemia and states of FGF23 excess, including chronic kidney disease.

Crosstalk between fibroblast growth factor 23, iron, erythropoietin, and inflammation in kidney disease.

Recent research has revealed that regulation of the bone-secreted hormone fibroblast growth factor 23 (FGF23) is not limited to classical mineral factors. Specifically, bidirectional relationships have been described between FGF23 production and anemia, iron status, and inflammation. Here, we will review the latest published articles on the crosstalk between FGF23 and the aforementioned nonclassical factors. It has been recently reported that erythropoietin, iron deficiency, and inflammation increase FGF23 production and metabolism. Moreover, FGF23 promotes anemia and regulates inflammatory responses. These findings are particularly important in the setting of chronic kidney disease which is characterized by elevated FGF23 levels and several associated comorbidities. Regulation of FGF23 is complex and involves many bone and renal factors. More recently, erythropoietin, iron deficiency, and inflammation have been also shown to affect FGF23 transcription and cleavage. Importantly, FGF23 has emerged as a regulator of erythropoiesis, iron metabolism, and inflammation. These findings provide novel and important insights into the pathophysiologic mechanisms of chronic kidney disease and may present new opportunities for therapeutic clinical interventions.

1,25-Dihydroxyvitamin D Maintains Brush Border Membrane NaPi2a and Attenuates Phosphaturia in Hyp Mice.

Phosphate homeostasis is critical for many cellular processes and is tightly regulated. The sodium-dependent phosphate cotransporter, NaPi2a, is the major regulator of urinary phosphate reabsorption in the renal proximal tubule. Its activity is dependent upon its brush border localization that is regulated by fibroblast growth factor 23 (FGF23) and PTH. High levels of FGF23, as are seen in the Hyp mouse model of human X-linked hypophosphatemia, lead to renal phosphate wasting. Long-term treatment of Hyp mice with 1,25-dihydroxyvitamin D (1,25D) or 1,25D analogues has been shown to improve renal phosphate wasting in the setting of increased FGF23 mRNA expression. Studies were undertaken to define the cellular and molecular basis for this apparent FGF23 resistance. 1,25D increased FGF23 protein levels in the cortical bone and circulation of Hyp mice but did not impair FGF23 cleavage. 1,25D attenuated urinary phosphate wasting as early as one hour postadministration, without suppressing FGF23 receptor/coreceptor expression. Although 1,25D treatment induced expression of early growth response 1, an early FGF23 responsive gene required for its phosphaturic effects, it paradoxically enhanced renal phosphate reabsorption and NaPi2a protein expression in renal brush border membranes (BBMs) within one hour. The Na-H+ exchange regulatory factor 1 (NHERF1) is a scaffolding protein thought to anchor NaPi2a to the BBM. Although 1,25D did not alter NHERF1 protein levels acutely, it enhanced NHERF1-NaPi2a interactions in Hyp mice. 1,25D also prevented the decrease in NHERF1/NaPi2a interactions in PTH-treated wild-type mice. Thus, these investigations identify a novel role for 1,25D in the hormonal regulation of renal phosphate handling.

Regulation of Fibroblast Growth Factor 23 by Iron, EPO, and HIF.

Fibroblast growth factor-23 (FGF23) is the key hormone produced in bone critical for phosphate homeostasis. Elevated serum phosphorus and 1,25dihydroxyvitaminD stimulates FGF23 production to promote renal phosphate excretion and decrease 1,25dihydroxyvitaminD synthesis. Thus completing the feedback loop and suppressing FGF23. Unexpectedly, studies of common and rare heritable disorders of phosphate handling identified links between iron and FGF23 demonstrating novel regulation outside the phosphate pathway. Iron deficiency combined with an FGF23 cleavage mutation was found to induce the autosomal dominant hypophosphatemic rickets phenotype. Physiological responses to iron deficiency, such as erythropoietin production as well as hypoxia inducible factor activation, have been indicated in regulating FGF23. Additionally, specific iron formulations, used to treat iron deficiency, alter post-translational processing thereby shifting FGF23 protein secretion. Molecular and clinical studies revealed that iron deficiency, through several mechanisms, alters FGF23 at the transcriptional and post-translational level. This review will focus upon the novel discoveries elucidated between iron, its regulators, and their influence on FGF23 bioactivity.

Sleep disordered breathing and fibroblast growth factor 23 in the Hispanic Community Health Study/Study of Latinos.

Preclinical data suggest that hypoxia stimulates fibroblast growth factor 23 (FGF23) transcription and cleavage in osteocytes, resulting in elevated circulating c-terminal (cFGF23) levels but normal intact FGF23 (iFGF23) levels. We conducted a case-control study within the Hispanic Community Health Study/Study of Latinos to investigate whether sleep disordered breathing, as a model of hypoxemia, is independently associated with elevated cFGF23 levels in the general population and with elevated cFGF23 and iFGF23 levels in patients with chronic kidney disease (CKD), in whom FGF23 cleavage may be impaired. Cases (n = 602) had severe sleep disordered breathing defined as an apnea/hypopnea index (AHI) of ≥30. Controls without severe sleep disordered breathing (n = 602) were matched for sex and CKD stage. The median AHI in the cases was 45.8 (IQR 35.5-62.5) compared to 2.6 (IQR 0.6-8.2) in the controls. Cases had higher cFGF23 levels than controls (66.2 RU/mL, IQR 52.8-98.4 vs. 61.2 RU/mL, IQR 49.5-80.1, p value <0.001). There were no differences in iFGF23 levels between cases and controls. In adjusted linear regression and multinomial regression analyses, body mass index attenuated the relationship between severe sleep disordered breathing and cFGF23 levels. No significant relationships were seen in analyses of severe sleep disordered breathing and iFGF23 levels or in analyses of iFGF23 and cFGF23 stratified by CKD status. Additional studies using other models of intermittent and chronic hypoxia are needed to confirm whether hypoxia stimulates FGF23 transcription in humans and to determine the impact on iFGF23 levels in CKD.

Inflammation and functional iron deficiency regulate fibroblast growth factor 23 production.

Circulating levels of fibroblast growth factor 23 (FGF23) are elevated in patients with chronic kidney disease (CKD), but the mechanisms are poorly understood. Here we tested whether inflammation and iron deficiency regulate FGF23. In wild-type mice, acute inflammation induced by single injections of heat-killed Brucella abortus or interleukin-1β (IL-1β) decreased serum iron within 6 hours, and was accompanied by significant increases in osseous Fgf23 mRNA expression and serum levels of C-terminal FGF23, but no changes in intact FGF23. Chronic inflammation induced by repeated bacteria or IL-1β injections decreased serum iron, increased osseous Fgf23 mRNA and serum C-terminal FGF23, but modestly increased biologically active, intact FGF23 serum levels. Chronic iron deficiency mimicked chronic inflammation. Increased osseous FGF23 cleavage rather than a prolonged half-life of C-terminal FGF23 fragments accounted for the elevated C-terminal FGF23 but near-normal intact FGF23 levels in inflammation. IL-1β injection increased Fgf23 mRNA and C-terminal FGF23 levels similarly in wild-type and Col4a3KO mice with CKD, but markedly increased intact FGF23 levels only in the CKD mice. Inflammation increased Fgf23 transcription by activating Hif1α signaling. Thus, inflammation and iron deficiency stimulate FGF23 production. Simultaneous upregulation of FGF23 cleavage in osteocytes maintains near-normal levels of biologically active, intact circulating FGF23, whereas downregulated or impaired FGF23 cleavage may contribute to elevated intact serum FGF23 in CKD.

Coupling fibroblast growth factor 23 production and cleavage: iron deficiency, rickets, and kidney disease.

High levels of fibroblast growth factor 23 (FGF23) cause the rare disorders of hypophosphatemic rickets and are a risk factor for cardiovascular disease and death in patients with chronic kidney disease (CKD). Despite major advances in understanding FGF23 biology, fundamental aspects of FGF23 regulation in health and in CKD remain mostly unknown. Autosomal dominant hypophosphatemic rickets (ADHR) is caused by gain-of-function mutations in FGF23 that prevent its proteolytic cleavage, but affected individuals experience a waxing and waning course of phosphate wasting. This led to the discovery that iron deficiency is an environmental trigger that stimulates FGF23 expression and hypophosphatemia in ADHR. Unlike osteocytes in ADHR, normal osteocytes couple increased FGF23 production with commensurately increased FGF23 cleavage to ensure that normal phosphate homeostasis is maintained in the event of iron deficiency. Simultaneous measurement of FGF23 by intact and C-terminal assays supported these breakthroughs by providing minimally invasive insight into FGF23 production and cleavage in bone. These findings also suggest a novel mechanism of FGF23 elevation in patients with CKD, who are often iron deficient and demonstrate increased FGF23 production and decreased FGF23 cleavage, consistent with an acquired state that mimics the molecular pathophysiology of ADHR. Iron deficiency stimulates FGF23 production, but normal osteocytes couple increased FGF23 production with increased cleavage to maintain normal circulating levels of biologically active hormone. These findings uncover a second level of FGF23 regulation within osteocytes, failure of which culminates in elevated levels of biologically active FGF23 in ADHR and perhaps CKD.

Hexa-D-arginine treatment increases 7B2•PC2 activity in hyp-mouse osteoblasts and rescues the HYP phenotype

Inactivating mutations of the "phosphate regulating gene with homologies to endopeptidases on the X chromosome" (PHEX/Phex) underlie disease in patients with X-linked hypophosphatemia (XLH) and the hyp-mouse, a murine homologue of the human disorder. Although increased serum fibroblast growth factor 23 (FGF-23) underlies the HYP phenotype, the mechanism(s) by which PHEX mutations inhibit FGF-23 degradation and/or enhance production remains unknown. Here we show that treatment of wild-type mice with the proprotein convertase (PC) inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (Dec), increases serum FGF-23 and produces the HYP phenotype. Because PC2 is uniquely colocalized with PHEX in osteoblasts/bone, we examined if PC2 regulates PHEX-dependent FGF-23 cleavage and production. Transfection of murine osteoblasts with PC2 and its chaperone protein 7B2 cleaved FGF-23, whereas Signe1 (7B2) RNA interference (RNAi) transfection, which limited 7B2 protein production, decreased FGF-23 degradation and increased Fgf-23 mRNA and protein. The mechanism by which decreased 7B2•PC2 activity influences Fgf-23 mRNA was linked to reduced conversion of the precursor to bone morphogenetic protein 1 (proBMP1) to active BMP1, which resulted in limited cleavage of dentin matrix acidic phosphoprotein 1 (DMP1), and consequent increased Fgf-23 mRNA. The significance of decreased 7B2•PC2 activity in XLH was confirmed by studies of hyp-mouse bone, which revealed significantly decreased Sgne1 (7B2) mRNA and 7B2 protein, and limited cleavage of proPC2 to active PC2. The expected downstream effects of these changes included decreased FGF-23 cleavage and increased FGF-23 synthesis, secondary to decreased BMP1-mediated degradation of DMP1. Subsequent Hexa-D-Arginine treatment of hyp-mice enhanced bone 7B2•PC2 activity, normalized FGF-23 degradation and production, and rescued the HYP phenotype. These data suggest that decreased PHEX-dependent 7B2•PC2 activity is central to the pathogenesis of XLH.

Mechanism of FGF23 processing in fibrous dysplasia.

Fibroblast growth factor-23 (FGF23) is a phosphate- and vitamin D-regulating hormone derived from osteoblasts/osteocytes that circulates in both active (intact, iFGF23) and inactive (C-terminal, cFGF23) forms. O-glycosylation by O-glycosyl transferase N-acetylgalactosaminyltransferase 3 (ppGalNAcT3) and differential cleavage by furin have been shown to be involved in regulating the ratio of active to inactive FGF23. Elevated iFGF23 levels are observed in a number of hypophosphatemic disorders, such as X-linked, autosomal recessive, and autosomal dominant hypophosphatemic rickets, whereas low iFGF23 levels are found in the hyperphosphatemic disorder familial tumoral calcinosis/hyperphosphatemic hyperostosis syndrome. Fibrous dysplasia of bone (FD) is associated with increased total FGF23 levels (cFGF23 + iFGF23); however, classic hypophosphatemic rickets is uncommon. Our results suggest that it can be explained by increased FGF23 cleavage leading to an increase in inactive cFGF23 relative to active iFGF23. Given the fact that FD is caused by activating mutations in the small G-protein G(s) α that results in increased cyclic adenosine monophosphate (cAMP) levels, we postulated that there may be altered FGF23 cleavage in FD and that the mechanism may involve alterations in cAMP levels and ppGalNacT3 and furin activities. Analysis of blood specimens from patients with FD confirmed that the elevated total FGF23 levels are the result of proportionally increased cFGF23 levels, consistent with less glycosylation and enhanced cleavage by furin. Analysis of primary cell lines of normal and mutation-harboring bone marrow stromal cells (BMSCs) from patients with FD demonstrated that BMSCs harboring the causative G(s) α mutation had higher cAMP levels, lower ppGalNAcT3, and higher furin activity. These data support the model wherein glycosylation by ppGalNAcT3 inhibits FGF23 cleavage by furin and suggest that FGF23 processing is a regulated process that controls overall FGF23 activity in FD patients.

Reciprocal Control of 1,25-Dihydroxyvitamin D and FGF23 Formation Involving the FGF23/Klotho System

Fibroblast growth factor 23 (FGF23) is a circulating hormone that is synthesized by osteocytes and osteoblasts. This glycosylated peptide controls phosphate balance by modulating urinary phosphate excretion and indirectly intestinal phosphate absorption by reducing expression of the renal and intestinal sodium phosphate transporters. In a feedback loop, 1,25-dihydroxyvitamin D and phosphate intake control FGF23 production. FGF23 is inactivated by cleavage by a still unidentified enzyme. FGF23 cleavage occurs within cells and probably in the circulation. Klotho, a protein expressed at the cell surface of few organs, forms complexes with FGF receptors, which increases their affinity for FGF23. Klotho is also released into the plasma and urine by an enzymatic cleavage. FGF23 plays a central role in vitamin D metabolism: It inhibits calcitriol synthesis in the kidney and stimulates the catabolism of active vitamin D sterols. In turn, calcitriol stimulates FGF23 and Klotho expression. In chronic kidney diseases, FGF23 concentration increases as GFR declines, whereas Klotho tissue expression decreases. The modifications of FGF23 and Klotho expression are probably involved in the genesis of hyperparathyroidism and the resistance to vitamin D receptor (VDR) activation in chronic kidney disease. Low vitamin D, high FGF23 concentrations, and defects in VDR activation are associated with similar risks, which evoke the possibility that potential FGF23 toxicity might be partly mediated by FGF23-induced decrease in calcitriol or 25-hydroxyvitamin D. Conversely, VDR activators could be used to modulate Klotho or FGF23 expression.