Fibroblast growth factor 23 is pumping iron: C-terminal-fibroblast growth factor 23 cleaved peptide and its function in iron metabolism

Abstract

Purpose of review

Iron deficiency regulates the production of the bone-derived phosphaturic hormone fibroblast growth factor 23 (FGF23) but also its cleavage, to generate both intact (iFGF23) and C-terminal (Cter)-FGF23 peptides. Novel studies demonstrate that independently of the phosphaturic effects of iFGF23, Cter-FGF23 peptides play an important role in the regulation of systemic iron homeostasis. This review describes the complex interplay between iron metabolism and FGF23 biology.

Recent findings

C-terminal (Cter) FGF23 peptides antagonize inflammation-induced hypoferremia to maintain a pool of bioavailable iron in the circulation. A key mechanism proposed is the down-regulation of the iron-regulating hormone hepcidin by Cter-FGF23.

Summary

In this manuscript, we discuss how FGF23 is produced and cleaved in response to iron deficiency, and the principal functions of cleaved C-terminal FGF23 peptides. We also review possible implications anemia of chronic kidney disease (CKD).

INTRODUCTION

Iron is an essential element for multiple cellular and systemic processes. Iron plays a central role in erythropoiesis, including recruitment and differentiation of erythroid progenitors to mature erythrocytes, as well as subsequent oxygen transport. Iron deficiency is the most frequent nutritional deficiency worldwide and a leading cause of impaired erythropoiesis and anemia [1,2]. Iron also serves as an essential cofactor in many biological processes. Thus, iron deficiency will virtually impair every cell in the organism. ‘True iron deficiency’ is mainly caused by a nutritional deficit in iron intake and is characterized by low circulating iron levels coupled to depleted iron stores. A reduction of circulating iron levels termed ‘functional iron deficiency’ (FID) also occurs as a consequence of inflammation in which iron sequestration in the reticuloendothelial system decreases the amount of iron available for erythropoiesis despite adequate total body iron stores. Indeed, in response to inflammation, several cytokines such as interleukin (IL)-6 stimulate bone morphogenetic proteins (BMP) signaling via BMP receptors to regulate the production and secretion of the hepatic iron regulatory hormone hepcidin. Hepcidin binds the only iron exporter, ferroportin, leading to its internalization, preventing iron intake from enterocytes and iron release from the reticulo-endothelial system [3–5].

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that is essential for maintaining normal phosphate and vitamin D homeostasis. FGF23 is regulated by local bone factors that modulate turnover and mineralization and systemic factors that control mineral metabolism. Iron is also a more recent regulator of FGF23 production. In both animal and humans, iron deficiency stimulates Fgf23 transcription but also increases cleavage of newly synthesized FGF23, resulting in high circulating concentrations of FGF23 derived peptides. As a consequence, normal serum phosphate levels remain normal in response to iron deficiency given the lack of increase in levels of the intact, phosphaturic FGF23 that can be detected with intact FGF23 assays (iFGF23). Similarly, and independently of iron status, pro-inflammatory cytokines such as IL-6, IL-1β, and tumor necrosis factor (TNF) also lead to proportional increases in Fgf23 transcription and FGF23 cleavage and consequently to dramatic elevations in the levels of FGF23-derived peptides, but not iFGF23 [6–11].

The current consensus is that iFGF23 is the FGF23 bioactive peptide and that proteolytic cleavage deactivates iFGF23. Thus, the reason for increased FGF23-derived peptides concomitant with relatively normal iFGF23 levels remains unclear, if these peptides are devoid of a biologically relevant function. Recently, we have found that cleaved C-terminal FGF23 peptides (Cter-FGF23) secreted from osteoblasts and osteocytes in response to functional iron deficiency minimize hypoferremia in a hepcidin-dependent mechanism [12^▪▪]. In mice, reduction in cleaved Cter-FGF23 peptides aggravated hypoferremia while exogenous administration and/or transgenic overexpression of these peptides reduced inflammation-induced functional iron deficiency. Here, we will discuss this novel regulatory pathway and infer how such mechanism is relevant to FID in chronic inflammatory diseases such as chronic kidney disease (CKD).

The effects of iron deficiency on bone

Multiple studies have reported the sensitivity of bone cells to inflammation including osteoclasts, osteoblasts and osteocytes, which respond via prompt immediate transcriptomic responses leading to shifts in cell activity, and eventually turning into altered bone turnover, mineralization defect, and loss of tissue volume [13–17]. The direct role of iron on the bone tissue is more cryptic, although it is commonly accepted that iron deficiency leads to a decreased bone mass and bone turnover. However, the reported effects of iron deficiency on bone formation and bone resorption vary [18–21] and converging mechanisms downstream of iron deficiency and inflammation on bone cells are poorly described. Hypoxia inducible factor 1α (HIF1α) has been reported as a central regulator mediating the response of bone cells in response to iron deficiency, inflammation as well as anemia and hypoxia. Recent studies showed that stabilization of the hypoxia inducible factor (HIF) induced by HIF prolyl hydroxylase inhibitors alters the transcriptomic signature of osteocytes in vitro, with decreased expression in Dmp1 and Phex mRNA, and increased expression of FGF23 [6,22▪▪]. Among the multiple ‘osseous’ genes directly regulated by HIF1α, Fgf23 bears a hypoxia response element (HRE) on its proximal promoter sequence [23]. HIF1α is also a potent activator of FGF23 cleavage, and explains abnormal circulating levels of FGF23 peptides in inflammation and iron deficiency [6]. Interestingly, a kinetics study of FGF23 regulations in a model of functional iron deficiency induced by i.p. injection of IL-1β revealed a dramatic increase in FGF23 stimulation 1h postinjection, and generation of cleaved Cter-FGF23 peptides in 2 h, reaching its peak before 6h following administration of the cytokine [12^▪▪]. Despite such a strong and rapid response to stimuli related to inflammation and iron, FGF23 excess also persists in chronic inflammation suggesting sustained elevations of this hormone in a context without perturbed phosphate metabolism [12^▪▪].

Regulation of fibroblast growth factor 23 production and cleavage

FGF23 is mainly produced by mature osteoblasts and osteocytes and regulates circulating phosphate levels by directly suppressing the synthesis of 1,25-vitamin D and reducing sodium-phosphate cotransporters in the kidney. In turn, FGF23 production is activated by 1,25-vitamin D and PTH, as well as phosphate and calcium [24,25]. However, beyond these classical mineral metabolism factors, multiple circulating and local factors also contribute to FGF23 excess, such as IL-1β, IL-6, TNF, Lipocalin-2/NGAL, erythropoietin (EPO), granulocyte colony-stimulating factor, have been shown to control both FGF23 transcription and posttranslational processing [6,26–30].

FGF23 transcription and translation are generally considered to be tightly coupled. Circulating FGF23 levels are regulated by a balance between Fgf23 transcription and FGF23 cleavage. Cleavage of biologically active intact FGF23 (iFGF23) occurs at a conserved RXXR recognition motif. Furin cleaves iFGF23 into a small carboxy terminal (Cter−) and a larger amino terminal (Nter−) FGF23 peptides, and GalNAc transferase 3 (GALNT3) protects newly synthesized iFGF23 from cleavage, by initiating the first steps of O-glycosylation at the cleavage site. The C-terminal domain is unique among all FGFs, and allows iFGF23 to bind to its only co-receptor Klotho. The N-terminal domain, consists in the conserved heparin-binding, FGF homology domain, and allows iFGF23 to activate FGF receptor signaling [31,32].

Numerous studies have investigated the binding of iFGF23 to FGF receptors and alpha-Klotho coreceptors, and it is widely acknowledged that the canonical effects of FGF23, including the regulation of phosphate reabsorption and the synthesis of 1,25-vitamin D requires iFGF23. Indeed, FGF23 cleaved peptides commonly referred as Nter-FGF23 and Cter-FGF23 peptides (155-aa and 72-aa, respectively) or mutated domains throughout FGF23 all fail to recruit FGFR1/alpha-Klotho complexes [33–36]. Since the direct injection of Nter-FGF23 peptide or Cter-FGF23 peptide does not affect phosphatemia and phosphaturia in mice it was initially proposed that cleaved peptides do not exert any biological function [37].

Two types of enzyme immuno-assays (EIA) have been developed to detect FGF23 and are commercially available [38▪]. The iFGF23 EIA is based on antibody recognition of epitopes spanning the Arginine-176 to Arginine-179 cleavage site and measures only the intact form of the hormone. The cFGF23 EIA (also referred to as tFGF23 for ‘total FGF23’) is based on epitopes restricted to the C-tail of the hormone and detects both the intact and cleaved Cter-FGF23 peptide. The ratio of iFGF23/cFGF23 provides the estimate of the cleavage ratio and is often used as a surrogate marker for FGF23 processing. However, several limitations might confound this interpretation. First, all assays do not exhibit the same sensitivity to different forms of FGF23. Second, in addition to these technical limitations the secreted Cter-FGF23 and iFGF23 in the blood do not always reflect production and cleavage by the same cells. Indeed, while a number of studies reports Fgf23 RNA in nonosseous tissues (by RT-qPCR, in situ hybridization, and transcriptomics) we do not know whether all these sources contribute to circulating levels of FGF23. Thus, more studies are warranted to demonstrate the cellular processing of FGF23 at homeostasis and during stress/disease, to identify whether nonosseous sources contribute to circulating levels of FGF23, and to refine and harmonize antibody-based methods available to measure FGF23 or develop competitive methods.

Sources of fibroblast growth factor 23 in iron deficiency

Early studies investigating the production and function of FGF23 in conditions of disturbed iron metabolism showed that reduction of iron in bone cells led to increased FGF23 production: Fgf23 mRNA expression was reported increased in the total bones of mice fed a low iron diet [39], and later in the bone cortical fraction of wild-type (WT) mice fed a low iron diet and mice injected with IL-1β [6]. In vitro, treatment with the iron-chelator deferoxamine or IL-1β increased Fgf23 mRNA expression in osteogenic cell lines UMR-106 and MC3T3-E1, and in primary differentiated osteoblasts [39,6]. Later studies proposed that indirect stimulation models such as EPO injection and blood loss would also trigger Fgf23 mRNA expression in the bone marrow [27,28].

The respective amount of Cter-FGF23 peptides released in the circulation varies according to disease states and the source of FGF23 production. Intact FGF23 is mainly secreted from osteocytes and osteoblasts in bone [40–43]. In our recent study, we also report that IL-1β directly enhances Fgf23 promoter activity [12▪▪], and stimulates Fgf23 transcription in bone in response to both acute and chronic inflammation. Among several tissues expressing increased Fgf23 mRNA, bone and especially cortical bone was the major contributor in terms of RNA quantities and fold increase. The conditional deletion of Fgf23 in late osteoblasts and osteocytes corrected the excess of both intact FGF23 and Cter-FGF23 peptides in inflammation in vivo, and secretion of FGF23 in vitro [12▪▪]. In such conditions, over 90% of FGF23 is cleaved [6], by Furin, since deletion of Furin in late osteoblasts and osteocytes in mice led to increased stabilization of intact FGF23. This suggests that late osteoblasts and osteocytes are able to cleave intact FGF23 and secrete Cter-FGF23 peptides directly into the circulation [12▪▪].

C-terminal fibroblast growth factor 23 is a novel bioactive peptide that regulates systemic iron metabolism

Multiple studies have suggested that circulating iFGF23 might be a potent contributor to iron deficiency and consequent anemia in mice and humans [39,44–48]. We show that animals lacking Fgf23 in osteoblasts and osteocytes (Dmp1-Cre Fgf23 conditional-knockout mice) fail to secrete FGF23 in response to inflammatory stimuli such as IL-1β. In sharp contrast with prior studies, we further show a more severe reduction in iron levels in these animals lacking FGF23 postinjection of IL-1β, due to higher hepatic production of hepcidin [12▪▪]. Although this could be explained either by the decrease of intact FGF23 or Cter-FGF23 peptides, mice lacking Furin in DMP1-expressing cells, with higher intact FGF23 and higher iFGF23/cFGF23 ratio, exhibit a similar phenotype. This suggests that Cter-FGF23 peptides actively participate in the regulation of systemic iron metabolism, at least during inflammatory conditions. FGF23 cleavage has long been considered as a deactivating mechanism of the intact hormone, however, recent published evidence shows that FGF23 derived peptides might not be devoid of biological activity [12▪▪,49,50]. In par with those results, several studies in patients established better associations between cFGF23 and iron and hepcidin, than iFGF23 which tends to associate with kidney function and phosphate [51▪▪,52]. In turn, inflammation markers such as IL-6 and CRP predicted increased cFGF23 [51▪▪,52,53▪▪]. In a recent study in children with CKD, hepcidin levels were predicted by both iFGF23 and cFGF23, but after adjustments to iron parameters and iron treatment, only cFGF23 retained its relationship to hepcidin [53▪▪]. Those studies suggest that Cter-FGF23 peptides always associate better to parameters related to iron metabolism.

We previously called ‘mineralostat’ the process of sensing and regulating minerals, to maintain homeostasis or return to homeostasis following stress [54]. This mineralostat is highly relevant and of paramount interest in medical research because it contains the elusive mechanism that fails in chronic diseases such as CKD leading to profound alterations in mineral metabolism and eventually skeletal abnormalities [54,55]. Thus, iron metabolism could be part of the ‘mineralostat’ designation of the mechanisms leading to increased FGF23 production and opens a number of questions to understand how and why phosphate loading and iron deficiency would both regulate the same hormone, if in turn this hormone regulates only phosphate excretion and vitamin D metabolism [56–58].

Then why is FGF23 cleaved and why are there so many Cter-FGF23 peptides in iron deficiency? Cleavage of larger proteins into smaller, easier to degrade, peptides is usually the first step of protein catabolism [59]. But FGF23 is already a sufficiently small 32kDa protein suitable for a direct inactivation and removal from the circulation. Second, such a process could limit iFGF23 half-life in the circulation. However, the half-life of intact FGF23 is already shorter than many hormones and comparable to the half-life of a cytokine. Recorded half-life of iFGF23 and cFGF23 showed a nonsignificantly different time of clearance (under 1 h, typically 15–45 min in humans and rodents), suggesting that the Cter FGF23 peptides measured with the cFGF23 ELISA in the experiments did not improved clearance [60–62]. Third, cleavage could be a regulatory mechanism for cells that do not process FGF23; intact would be produced by osteocytes and secreted into the bloodstream whereas other tissues would only sporadically contribute to Cter-FGF23. However, osteocytes are entirely capable to cleave FGF23 and secrete cleaved peptides [12▪▪]. Thus, Cter-FGF23 might have a distinct biological function independent of these exerted by iFGF23 (Fig. 1). Many proteins among which several peptide hormones, are in this category in which synthesis of an intact form generates more peptidic forms with distinct roles [63–66]. In the kidneys, polycystin-1 comprises a long Nter domain and a shorter (15 kDa) Cter domain, like FGF23. The intact protein is constitutively degraded but stabilized during hypoxia (similar to HIFs). A recent study shows that its Cter mediates distinct function in lactate metabolism which contributes to ADPKD progression [66]. Many other hormones (such as calcitonin and propiomelanocortin processing) and nonhormone proteins (such as the complement cascade) follow posttranslational modification and generate distinct peptides with distinct functions [63,65]. Finally, in bone and mineralized tissues Dentin sialophosphoprotein (DSPP) lead to DSP, DPP, and DGP [64].

FIGURE 1.

FGF23 and hepcidin interplay through inflammation/functional iron deficiency stages. (a) Depicts homeostasis with normal levels of phosphate and iron. (b) Summarizes the past decade of research: during stress, the osteocytes produce intact FGF23 to rapidly decrease blood phosphate levels, while hepatocytes undergo a BMP-induced production of hepcidin to rapidly decrease blood iron levels. (c) Represents a novel mechanism in which FGF23 cleavage generates Cter-FGF23 peptides; Cter-FGF23 peptides bind to BMP to prevent BMP-induced hepcidin production, thus maintaining both iron and phosphate levels in the normal range. FGF23, fibroblast growth factor 23; BMP, bone morphogenetic proteins.

In mice, two studies have found that injection of Cter-FGF23 to WT mice at homeostasis and during inflammation increased iron levels by lowering hepcidin [12▪▪,49]. Interestingly, mice with conditional over-expression of Cter-FGF23 in DMP1-expressing cells show the same hepcidin-related signature, at homeostasis and during inflammation, further suggesting that Cter-FGF23 regulates hepcidin production [12▪▪]. These novel functions are not dependent on the presence or absence of intact FGF23 and Klotho. Indeed, the Cter-FGF23 peptide corrected iron levels in adenine-induced CKD mice [49], but interestingly the treatment with α-Klotho did not correct the iron and hemoglobin levels in this model [50]. Additionally, the injection of recombinant Cter-FGF23 peptide repressed hepcidin production in FGF23^null mice lacking all endogenous forms of FGF23 [12▪▪].

CONCLUSION

Associations between iron deficiency, inflammation, anemia and FGF23, capture novel regulatory loops of FGF23 regulation and function [6,8,27,28,39]. Anemia is often observed in settings of FGF23 excess and is a common complication of CKD that develops early and becomes nearly universal as CKD progresses. Both true iron deficiency and functional iron deficiency occur in CKD patients due to increased blood loss, poor oral iron absorption and impaired iron release from storage sites. In diseases of impaired FGF23 cleavage in bone, such as CKD, iron deficiency and inflammation contribute to disproportionately and extremely high iFGF23 levels compared to the levels of FGF23 cleaved peptides, leading to adverse outcomes [41,67–69]. Thus, inappropriately low Cter-FGF23 levels for the degree of FGF23 excess may contribute to iron deficiency and anemia of CKD. To date, the functions of Nter-FGF23 are unknown and there is currently no commercial assay to directly detect Nter-FGF23 in the circulation, and direct effective methods to detect Nter-FGF23 and Cter-FGF23 peptides, based on antibodies or mass-spectrometry, should be considered in the future.

CKD-MBD has for a long time brought nephrology experts and bone biology experts at the table, however new FGF23 regulations and functions in inflammation and FID combined with the canonical relation of FGF23 to the mineral metabolism, are bridging immunology and hematology, perhaps setting again FGF23 at the center of the whole organism physiology for the next decade of research.

KEY POINTS.

• Fibroblast growth factor 23 (FGF23) production and cleavage is highly dependent upon iron status.

• Circulating FGF23-derived peptides are not devoid of biological activity.

• C-terminal-FGF23 peptides reduce hepcidin production in inflammatory conditions.