2024 年 71 巻 4 号 p. 335-343
Bone secrets the hormone, fibroblast growth factor 23 (FGF23), as an endocrine organ to regulate blood phosphate level. Phosphate is an essential mineral for the human body, and around 85% of phosphate is present in bone as a constituent of hydroxyapatite, Ca10(PO4)6(OH)2. Because hypophosphatemia induces rickets/osteomalacia, and hyperphosphatemia results in ectopic calcification, blood phosphate (inorganic form) level must be regulated in a narrow range (2.5 mg/dL to 4.5 me/dL in adults). However, as yet it is unknown how bone senses changes in blood phosphate level, and how bone regulates the production of FGF23. Our previous data indicated that high extracellular phosphate phosphorylates FGF receptor 1 (FGFR1) in an unliganded manner, and its downstream intracellular signaling pathway regulates the expression of GALNT3. Furthermore, the post-translational modification of FGF23 protein via a gene product of GALNT3 is the main regulatory mechanism of enhanced FGF23 production due to high dietary phosphate. Therefore, our research group proposes that FGFR1 works as a phosphate-sensing receptor at least in the regulation of FGF23 production and blood phosphate level, and phosphate behaves as a first messenger. Phosphate is involved in various effects, such as stimulation of parathyroid hormone (PTH) synthesis, vascular calcification, and renal dysfunction. Several of these responses to phosphate are considered as phosphate toxicity. However, it is not clear whether FGFR1 is involved in these responses to phosphate. The elucidation of phosphate-sensing mechanisms may lead to the identification of treatment strategies for patients with abnormal phosphate metabolism.
Bone works as an endocrine organ [1]. Certainly, bone contributes to supporting the body, protecting the central nervous system and other organs, hematopoiesis, the homeostasis of calcium (Ca) via parathyroid hormone (PTH), and assisting in respiration. In addition to these important functions, it has been recently revealed that bone secretes several hormones, and systemically regulates the functions of other organs [2]. Fibroblast growth factor 23 (FGF23) is the first bone-derived hormone to be reported. FGF23 was identified as a gene or a humoral factor responsible for hypophosphatemic rickets/osteomalacia [3, 4]. After that, FGF23 was established as the principal hormone in regulating blood phosphate level [5]. Inorganic phosphate, but not organic phosphate, is clinically measured, and hypophosphatemia and hyperphosphatemia are defined as low and high blood levels of inorganic phosphate, respectively. Phosphate is an essential mineral for the human body. Around 85% of phosphate is present in bone as one of constituents of hydroxyapatite, Ca10(PO4)6(OH)2 [6]. Hypophosphatemia induces rickets/osteomalacia, whereas hyperphosphatemia results in ectopic calcification [7]. Therefore, blood phosphate level must be regulated in a narrow range (generally 2.5 mg/dL to 4.5 mg/dL in adults). Since FGF23 is produced by bone, especially by osteoblasts and osteocytes, bone controls phosphate as an endocrine organ [8, 9]. Phosphate is a mineral with several characteristics. First, phosphate is present in large amounts in the human body, but not in seawater, although the composition of the human body and seawater is quite similar [10]. Second, blood phosphate levels were reported to negatively associate with life spans in mammals [11]. Generally, it is proposed that phosphate accelerates aging. Third, phosphate is ingested as a nutrient from almost all foods. In addition, phosphate is contained in large amounts in food additives [12]. Therefore, controlling dietary phosphate is quite difficult in the context of a modern lifestyle. From these facts, it could be assumed that the human body has some mechanisms for phosphate-sensing. However, it has been unclear how we sense phosphate in order to regulate blood phosphate level via a hormonal function of FGF23. Regarding this issue, our previous data proposed that FGF receptor 1 (FGFR1) works as a phosphate-sensing receptor at least in the regulation of FGF23 production in bone and blood phosphate level [13, 14]. According to these results, our research group considers that phosphate behaves as a first messenger. However, it is not clear whether FGFR1 is involved in other responses to phosphate, such as stimulation of PTH synthesis, vascular calcification, and renal dysfunction. Several of these responses to phosphate in the human body are considered as phosphate toxicity [15]. Therefore, elucidating the mechanisms of phosphate-sensing may facilitate the optimization of treatment strategies for organ damage induced by phosphate toxicity. Here, recent findings concerning phosphate-sensing including the regulatory mechanism of FGF23 production and phosphate as a first messenger are reviewed.
Bone has many functions in the human body. Bone secretes several systemic humoral factors, such as FGF23, osteocalcin, sclerostin, lipocalin 2, and interleukin-11 (IL-11) (Fig. 1) [3, 4, 16-20]. Bone can communicate with many other organs via these bone-derived hormones. In short, osteocalcin increases insulin secretion from the pancreas, increases adiponectin secretion from adipose tissue and reduces fat mass, increases testosterone secretion from the testes, increases glucagon-like peptide-1 (GLP-1) secretion from the intestine, increases exercise capacity in skeletal muscle, and increases cognitive function in the brain [17, 21-25]. In addition, sclerostin induces browning of white adipose tissue, and lipocalin 2 reduces appetite in the brain [18, 19]. Recently, it has been reported that bone-derived IL-11 controls osteogenesis and systemic adiposity in response to mechanical loading [20]. Among these hormones, FGF23 is the first bone-derived hormone to be reported, and is understood to be the principal hormone in regulating phosphate metabolism. FGF23 reduces blood phosphate level via the kidney [5].
Bone as an endocrine organ and bone-derived hormones
Bone secretes several systemic humoral factors, such as fibroblast growth factor 23 (FGF23), osteocalcin, sclerostin, lipocalin 2, and interleukin-11 (IL-11). Bone can communicate with many other organs via these bone-derived hormones.
Phosphate is an essential mineral for the human body. Phosphate is an important component of cell membrane, adenosine triphosphate (ATP), and nucleic acid [26]. Moreover, phosphate plays an essential role in intracellular signal transductions as substrates of kinases and phosphatases. However, around 85% of phosphate is present in bone and tooth as a constituent of hydroxyapatite, Ca10(PO4)6(OH)2 [6]. Thus, chronic hypophosphatemia is a cause of rickets/osteomalacia, which is characterized by impaired mineralization [7]. The daily transport of phosphate is described in Fig. 2. A healthy daily diet for a man with a body weight of 60 to 70 kg contains approximately 1,200 mg of phosphate, and 800 mg is absorbed through the intestine. Simultaneously, 300 mg of phosphate is incorporated daily in hydroxyapatite formation, and an almost equivalent amount is excreted as bone resorption. Furthermore, 6,000 mg of phosphate is filtered daily by glomeruli, and 5,200 mg of phosphate is reabsorbed at proximal tubules to balance intake and excretion of phosphate [27]. Therefore, the kidney is the most important and potent organ in regulating blood phosphate level.
Daily transport of phosphate
Around 85% of phosphate is present in bone and tooth as a constituent of hydroxyapatite, Ca10(PO4)6(OH)2. Phosphate is also a component of cell membrane, adenosine triphosphate (ATP), and nucleic acid. A healthy daily diet for a man with a body weight of 60 to 70 kg contains approximately 1,200 mg of phosphate, and 800 mg is absorbed through the intestine. Simultaneously, 300 mg of phosphate is incorporated daily in hydroxyapatite formation, and an almost equivalent amount is excreted as bone resorption. Furthermore, 6,000 mg of phosphate is filtered daily by glomeruli, and 5,200 mg of phosphate is reabsorbed at proximal tubules to balance intake and excretion of phosphate.
A variety of foods contain phosphate (Fig. 3) [12]. Phosphate is ingested both as a natural component and as a food additive. As a natural food component, phosphate is available as inorganic phosphate salts or as consisting of phospho-proteins. Around 60% of dietary phosphate is absorbed in the intestine as inorganic phosphate, while the bioavailability of plant-origin phosphate is low. On the other hand, the net gastrointestinal absorption of phosphate is almost 100% for phosphate salts as food preservatives [28]. Dietary phosphate control is difficult in a real-life setting. For instance, olive oil, albumen, sugar, fruits, vegetables, rice, bread, and beans contain less phosphate, while yolk, nuts, cheese, liver, salmon, shrimp, and squid contain a large amount of phosphate. Moreover, foods and drinks with phosphate-containing additives, such as processed meats like ham and bacon, cola beverages, and beer include very high levels of phosphate [12]. However, the Japanese government does not mandate food manufacturers and retailers to report per serving phosphate amounts on food labels.
Phosphate content of foods
A variety of foods contain phosphate. Phosphate is ingested both as a natural component and as a food additive. Olive oil, albumen, sugar, fruits, vegetables, rice, bread, and beans contain less phosphate, while yolk, nuts, cheese, liver, salmon, shrimp, and squid contain a large amount of phosphate. Foods and drinks with phosphate-containing additives, such as processed meats including ham and bacon, cola beverages, and beer include very high levels of phosphate.
There is phosphate homeostasis via the principal phosphotropic hormone, FGF23. FGF23 is a member of the FGF19 subfamily, the constituents of which are known as endocrine FGFs together with FGF19 and FGF21 [29, 30]. FGF23 is produced by bone, especially by osteoblasts and osteocytes [8, 9]. FGF23 can bind to FGFR1c and α-Klotho complex, and can transduce hormonal signals into the cells [31-33]. Through these transduction signals, FGF23 suppresses the expression of type Ⅱa and Ⅱc sodium-phosphate cotransporters (NaPis) in renal proximal tubules. Simultaneously, FGF23 reduces blood 1,25-dihydroxyvitamin D [1,25(OH)2D] level not only by suppressing the expression of CYP27B1, which produces 25-hydroxyvitamin D-1α-hydroxylase, but also by enhancing that of CYP24A1, which encodes 25-hydroxyvitamin D-24-hydroxylase [5]. Therefore, FGF23 can reduce blood phosphate level by inhibiting both proximal tubular phosphate reabsorption and intestinal phosphate absorption via reducing blood 1,25(OH)2D level, because 1,25(OH)2D enhances intestinal phosphate absorption.
As mentioned above, FGF23 is the principal hormone in regulating blood phosphate level. However, it has been unclear how phosphate affects FGF23 production in bone. For instance, PTH is the principal hormone in regulating blood Ca level. PTH works to increase blood Ca level, and increased Ca suppresses the synthesis and secretion of PTH from the parathyroid glands by binding to and activating Ca-sensing receptor (CaSR) [34]. Therefore, there is a negative feedback loop in the regulation of blood Ca level.
Previously, it was reported that alternations of extracellular phosphate can transduce signals into cells to regulate gene expression and cell behavior [26]. These results indicate that phosphate is a signaling molecule. Several studies showed that high extracellular phosphate activates the extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway [35-39]. Our research group has also reported that high extracellular phosphate induced the phosphorylation of ERK, and induced the expression of several transcription factors downstream in the MEK/ERK pathway by transcriptome analysis in in vitro experiments [13]. On the other hand, upstream of the MEK/ERK pathway has not been observed. Because the MEK/ERK pathway is known to be activated by some receptor tyrosine kinases (RTKs), our research group attempted to examine whether high extracellular phosphate can activate some RTKs using the proteomic approach employed in a previous study [40]. Finally, proteome analysis identified FGFR1 as the only RTK phosphorylated by high extracellular phosphate [13]. It is known that the phosphorylation of the two tyrosine residues 653 and 654 dramatically increases tyrosine kinase activity of FGFR1 [30, 41, 42]. In our analysis, two types of FGFR1 peptides—one with phosphotyrosine 583 and 585, the other with phosphotyrosine 653 and 654—were identified in which tyrosine phosphorylation was induced by high extracellular phosphate [13]. Therefore, the amount of peptide with phosphotyrosine 653 and 654 was quantified via a parallel-reaction monitoring (PRM) method. As a result, the amount of this peptide increased about three-fold due to high extracellular phosphate [13]. These results indicate that FGFR1 is a potential candidate for phosphate-sensing in mammals.
FGF23 protein is produced as a peptide with 251 amino acids. After the cleavage of a signal peptide with 24 amino acids, FGF23 protein with 227 amino acids is secreted from bone [4]. A part of FGF23 protein is proteolytically cleaved into inactive fragments before or during the process of secretion. It has been revealed that FGF23 protein can be cleaved between arginine 179 and serine 180 by enzymes that recognize the arginine 176-X177-X178-arginine 179 motif [4]. Only full-length FGF23 protein is biologically active, while processed FGF23 protein fragments are inactive [43]. Previously, it was reported that O-glycosylation of threonine 178 in FGF23 protein by polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3), a gene product of GALNT3, inhibits this cleavage, and works to increase the level of active full-length FGF23 protein [44]. While there are 20 GALNT genes in humans—GALNT3 being one of them—, the gene product of GALNT3 is considered to be essential for the O-glycosylation of FGF23 protein, and other GALNT gene products cannot compensate for this post-translational modification of FGF23 protein [45, 46]. Therefore, there are indications that the hormonal activity of FGF23 is regulated not only by the transcription of the FGF23 gene but also by the post-translational modification of FGF23 protein. Our previous data demonstrated that a high phosphate diet increased both blood phosphate and FGF23 levels in wild-type mice; however, a high phosphate diet did not enhance the expression of Fgf23 in bone. On the contrary, a high phosphate diet enhanced the expression of Galnt3 instead of Fgf23 in bone. Furthermore, high extracellular phosphate increased the expression of Galnt3 in a dose-dependent manner and stimulated GalNAc-T3 protein expression in in vitro experiments using osteoblastic UMR106 cells [13]. Therefore, it is proposed that a high phosphate diet increases blood active full-length FGF23 level by enhancing the expression of GALNT3 gene. Together, our research group considers that GALNT3 is a phosphate-responsive gene, and the post-translational modification of FGF23 protein through the gene product of GALNT3 is the main mechanism responding to the high dietary phosphate.
The significance of FGFR1 for phosphate-sensing in in vivo experiments was also addressed. A selective ablation of Fgfr1 in bone using osteocalcin-Cre negated the increase of blood FGF23 level and Galnt3 upregulation in bone caused by a high phosphate diet [14]. Then, as a high phosphate diet induces the expression of Galnt3 through FGFR1, our research group wondered if a canonical FGFR ligand could also induce enhanced expression of Galnt3. In practice, a canonical FGFR ligand, FGF2, did not enhance the expression of Galnt3 in in vitro experiments [13]. The molecular basis in the differences of FGFR1 activation by high extracellular phosphate and conventional FGFR ligands was explored. Although FGFR1 activates the MEK/ERK pathway via FGFR substrate 2α (FRS2α), FGF2 phosphorylated both tyrosine residues, 196 and 436, of FRS2α. On the other hand, high extracellular phosphate phosphorylated only tyrosine 196. In addition, the phosphorylation of ERK by high extracellular phosphate was transient, while that by FGF2 was sustained [13]. Therefore, the signal transduction by high extracellular phosphate and conventional FGFR ligands is different.
In line with these results, our research group assumes that the unliganded FGFR1 activation is caused by high extracellular phosphate. There is the possibility of the involvement of type Ⅲ sodium-phosphate cotransporters, PiT1 and PiT2, encoded by SLC20A1 and SLC20A2, respectively [47]. Previously, administration of an inhibitor of these transporters, PiT1 and PiT2, was reported to block cellular uptake of phosphate and effects of phosphate on gene expression [35]. A previous study demonstrated that extracellular phosphate induces PiT1 and PiT2 heterodimerization, and mediates the activation of the MEK/ERK pathway independent of phosphate uptake [48]. As of today, however, it has not been elucidated whether type Ⅲ sodium-phosphate cotransporters, PiT1 and PiT2, are involved in the activation of unliganded FGFR1 by high extracellular phosphate.
In addition, our research group focused on 17 genes whose expression was simultaneously induced by high extracellular phosphate from our transcriptome analysis as mentioned above. Among them, the significance of two transcription factors in the MEK/ERK pathway in the induction of Galnt3 by high extracellular phosphate was confirmed: early growth response 1 (Egr1) and ETS variant 5 (Etv5). Although the importance of Egr1 and Etv5 in the upregulation of Galnt3 by high extracellular phosphate was shown, these two transcription factors could not induce Galnt3 expression by themselves [13]. These results indicate that EGR1 and ETV5 are not enough to enhance the expression of Galnt3, indicating that some unknown transcription activator is also required. Finally, the phosphate-sensing mechanisms in bone and regulatory mechanisms of FGF23 discussed in this chapter are described in Fig. 4.
The model of phosphate-sensing mechanisms in bone to regulate FGF23 production and blood phosphate level
High extracellular phosphate phosphorylates FGF receptor 1 (FGFR1), and its downstream intracellular signaling pathway regulates the expression of GALNT3. The post-translational modification of fibroblast growth factor 23 (FGF23) protein via a gene product of GALNT3 is the main regulatory mechanism of enhanced FGF23 production by high dietary phosphate.
EGR1: early growth response 1; ERK: extracellular signal-regulated kinase; ETV5: ETS variant 5; FRS2α: fibroblast growth factor receptor substrate 2α; GalNAc-T3: polypeptide N-acetylgalactosaminyltransferase 3; H: Histidine; PiT1/2: type Ⅲ sodium-phosphate cotransporter 1/2; R: arginine; S: serine; T: threonine; Y: tyrosine
FGFR1 works as a phosphate-sensing molecule at least in the regulation of FGF23 production in bone and blood phosphate level. Therefore, phosphate behaves as a first messenger, and a feedback system is present in the regulation of blood phosphate level involving FGFR1. However, it is not clear whether FGFR1 is involved in other responses to phosphate as a first messenger, such as stimulation of PTH synthesis, vascular calcification, and renal dysfunction (Table 1). Several of these responses to phosphate in the human body are considered as phosphate toxicity [15].
Functions of phosphate as a first messenger
| Target organ | Function | Intermediate molecule |
|---|---|---|
| Physiological | ||
| Bone | FGF23 production to regulate blood phosphate level | FGFR1 |
| Parathyroid glands | PTH production to regulate blood phosphate level | CaSR |
| Pathological (phosphate toxicity) | ||
| Blood vessels | Vascular calcification resulting in cardiovascular disease | unknown |
| Kidney | Renal tubular damage and interstitial fibrosis resulting in CKD | unknown |
Abbreviations: FGF23, fibroblast growth factor 23; FGFR1, fibroblast growth factor receptor 1; PTH, parathyroid hormone; CaSR, calcium-sensing receptor; CKD, chronic kidney disease.
PTH can also affect blood phosphate level, while blood Ca level is mainly regulated by PTH. The increase in blood phosphate level by oral phosphate supplementation is counteracted within one to two hours via an increase in blood PTH level [49]. On the other hand, induction of hyperphosphatemia for six hours did not provoke an increase in blood FGF23 level in humans [50]. However, it was shown to take one to five days for FGF23 to respond to dietary phosphate intake [51, 52]. Therefore, chronic changes in blood phosphate level are regulated by bone-derived FGF23 through FGFR1, while acute changes in blood phosphate level seem to be amended by PTH. A previous study indicated that extracellular phosphate stimulates the secretion of PTH via CaSR from the parathyroid cells. It was reported that phosphate acts as a noncompetitive antagonist for CaSR, and phosphate-binding sites in the extracellular domain of CaSR were clarified [53]. These results indicate that several molecules other than FGFR1 can also work as phosphate-sensing mechanisms regulating cell-specific functions in the human body.
Cardiovascular disease is the major consequence of phosphate toxicity [15]. Clinical observational studies showed that high blood phosphate level is identified as an independent contributor to cardiovascular morbidity and mortality [54]. Hyperphosphatemia may facilitate the ectopic calcification of blood vessels [55]. It was demonstrated that high extracellular phosphate induces the expression of RUNX2, and causes osteoblastic transdifferentiation in cultured vascular smooth muscle cells (VSMCs) [56, 57]. Furthermore, the dietary phosphate load has been known to induce renal tubular damage and interstitial fibrosis in rats [58]. Elderly individuals were reported to be at high risk for developing acute kidney injury and subsequent chronic kidney disease (CKD) due to the gavage of phosphate, which is part of the colon-cleansing pretreatment for colonoscopy [59, 60]. However, it has not been elucidated whether FGFR1 is involved in these phosphate toxicity effects. Considering these phosphate functions as a first messenger, we should prevent excess phosphate intake, and give thought to the various nutrient needs of healthy adults. Thus, it is important that phosphate content is appropriately labelled, and that clinical research is conducted on accurate phosphate availability.
Previously, our data showed that phosphate activates FGFR1 to phosphorylate FRS2α, leading to the activation of the MEK/ERK pathway that induces the expression of GALNT3. These findings provide a molecular basis for phosphate-sensing in the regulation of FGF23 production and blood phosphate level, and uncover an unrecognized facet of FGFR1 function. In addition, it has been demonstrated that phosphate may contribute to various effects in the human body as a first messenger. On the other hand, several issues remain to be clarified. First, the precise mechanism of FGFR1 phosphorylation by phosphate is largely unknown. Second, it is not clear to what extent FGFR1 is involved in responses to phosphate. As the identification of CaSR has resulted in new drug discoveries for patients with primary and secondary hyperparathyroidism, elucidation of the phosphate-sensing mechanism may lead to the identification of novel drugable molecules for patients with abnormal phosphate metabolism.
The author thanks all members of our laboratories and all respected colleagues. This work was supported by the Japan Endocrine Society (JES) Grant for Promising Investigator, KAKENHI Grant-in-Aid for Young Scientists 20K17301 from the Japan Society for the Promotion of Science (JSPS), and KAKENHI Grant-in-Aid for Scientific Research 23K07712 from the JSPS.
The author declares no conflicts of interest associated with this research.
