Both FGF23 and extracellular phosphate activate Raf/MEK/ERK pathway via FGF receptors in HEK293 cells

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced by bone and exerts its function in the target organs by binding the FGF receptor (FGFR) and Klotho. Since recent studies suggested that extracellular inorganic phosphate (Pi) itself triggers signal transduction and regulates gene expression in some cell types, we tested the notion that extracellular Pi induces signal transduction in the target cells of FGF23 also and influences its signaling, utilizing a human embryonic kidney cell line HEK293. HEK293 cells expressed low levels of klotho, and treatment with a recombinant FGF23[R179Q], a proteolysis‐resistant mutant of FGF23, resulted in phosphorylation of ERK1/2 and induction of early growth response‐1 (EGR1) expression. Interestingly, increased extracellular Pi resulted in activation of the Raf/MEK/ERK pathway and expression of EGR1, which involved type III sodium/phosphate (Na⁺/Pi) cotransporter PiT‐1. Since the effects of an inhibitor of Na⁺/Pi cotransporter on FGF23 signaling suggested that the signaling triggered by increased extracellular Pi shares the same downstream cascade as FGF23 signaling, we further investigated their convergence point. Increasing the extracellular Pi concentration resulted in the phosphorylation of FGF receptor substrate 2α (FRS2α), as did treatment with FGF23. Knockdown of FGFR1 expression diminished the phosphorylation of both FRS2α and ERK1/2 induced by the Pi. Moreover, overexpression of FGFR1 rescued the decrease in Pi‐induced phosphorylation of ERK1/2 in the cells where the expression of PiT‐1 was knocked down. These results suggest that increased extracellular Pi triggers signal transduction via PiT‐1 and FGFR and influences FGF23 signaling in HEK293 cells. J. Cell. Biochem. 111: 1210–1221, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of a second Klotho interaction site in the C terminus of FGF23

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Hormone-like fibroblast growth factors and metabolic regulation

The family of fibroblast growth factors (FGFs) consisting now of 22 members is generally considered to control a wide range of biological functions such as development, differentiation and survival. However, research during the past decade provided substantial evidence that a so called “hormone-like” subgroup of FGFs, comprised of FGF19, FGF21 and FGF23, is involved in the regulation of diverse metabolic pathways to control glucose, lipid, bile acid, phosphate and vitamin D metabolism. The unique properties of these FGFs include predominant production of the factors in selective tissues, their abundance in the blood due to the lack of extracellular heparin-mediated sequestration, and highly specific tissue-targeted action via engagement of their respective co-receptors. The important metabolic context of FGF19, FGF21, and FGF23 actions has revealed important novel roles for FGFs and provided significant means to explore an opportunity for therapeutic targeting of these factors and their corresponding pathways.     

Phosphate toxicity and tumorigenesis

In this article, we briefly summarized evidence that cellular phosphate burden from phosphate toxicity is a pathophysiological determinant of cancer cell growth. Tumor cells express more phosphate cotransporters and store more inorganic phosphate than normal cells, and dysregulated phosphate homeostasis is associated with the genesis of various human tumors. High dietary phosphate consumption causes the growth of lung and skin tumors in experimental animal models. Additional studies show that excessive phosphate burden induces growth-promoting cell signaling, stimulates neovascularization, and is associated with chromosome instability and metastasis. Studies have also shown phosphate is a mitogenic factor that affects various tumor cell growth. Among epidemiological evidence linking phosphate and tumor formation, the Health Professionals Follow-Up Study found that high dietary phosphate levels were independently associated with lethal and high-grade prostate cancer. Further research is needed to determine how excessive dietary phosphate consumption influences initiation and promotion of tumorigenesis, and to elucidate prognostic benefits of reducing phosphate burden to decrease tumor cell growth and delay metastatic progression. The results of such studies could provide the basis for therapeutic modulation of phosphate metabolism for improved patient outcome.     

The role of an intracellular cysteine stretch in the sorting of the type II Na/phosphate cotransporter

The type II Na/phosphate cotransporters (NaPi-II) are critical for the control of plasma phosphate levels in vertebrates. NaPi-IIb mediates phosphate uptake from the small intestine followed by glomerular filtration and selective reabsorption from the renal proximal tubule by NaPi-IIa and NaPi-IIc. A C-terminal stretch of cysteine residues represents the hallmark of the NaPi-IIb isoforms. This motif is well conserved among NaPi-IIb type transporters but not found in other membrane proteins. To investigate the role of this motif we analyzed NaPi-II constructs in transiently and stably transfected MDCK cells. This cell line targets the NaPi-IIb isoforms from flounder and mouse to the apical membrane whereas the mouse IIa isoform shows no plasma membrane preference. Different parts of mouse NaPi-IIa and NaPi-IIb C-termini were fused to GFP-tagged flounder NaPi-II. The constructs showed strong staining of the plasma membrane with NaPi-IIb related constructs sorted predominantly apically, the IIa constructs localized apically and basolaterally with slight intracellular retention. When the cysteine stretch was inserted into the NaPi-IIa C-terminus, the construct was retained in a cytoplasmic compartment. 2-bromopalmitate, a specific palmitoylation inhibitor, released the transporter to apical and basolateral membranes. The drug also leads to a redistribution of the NaPi-IIb construct to both plasma membrane compartments. Immunoprecipitation of tagged NaPi-II constructs from [³H]-palmitate labeled MDCK cells indicated that the cysteine stretch is palmitoylated. Our results suggest that the modified cysteine motif prevents the constructs from basolateral sorting. Additional sorting determinants located downstream of the cysteine stretch may release the cargo to the apical compartment.     

FGF-23 transgenic mice demonstrate hypophosphatemic rickets with reduced expression of sodium phosphate cotransporter type IIa

Fibroblast growth factor (FGF)-23 was identified as a causative factor of tumor-induced osteomalacia and also as a responsible gene for autosomal dominant hypophosphatemic rickets. To clarify the pathophysiological roles of FGF-23 in these diseases, we generated its transgenic mice. The transgenic mice expressing human FGF-23 reproduced the common clinical features of these diseases such as hypophosphatemia probably due to increased renal phosphate wasting, inappropriately low serum 1,25-dihydroxyvitamin D level, and rachitic bone. The renal phosphate wasting in the transgenic mice was accompanied by the reduced expression of sodium phosphate cotransporter type IIa in renal proximal tubules. These results reinforce the notion that the excessive action of FGF-23 plays a causative role in the development of several hypophosphatemic rickets/osteomalacia.     

FGF23, alpha-Klotho and vitamin D mediated calcium-phosphate metabolism in haemodialysis patients

BackgroundKlotho is a prote˝in that acts as a co-receptor for FGF23. FGF23-Klotho axis has great importance regarding the regulation of mineral metabolism by kidneys. In this study, we analysed FGF23, Klotho, 1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D, parathormone, Calcium and Phosphate levels of haemodialysis patients in order to investigate the nature of the mineral metabolism disruption in chronic kidney diseases.MethodsSixty haemodialysis patients and 34 healthy controls were included in the study. Serum iFGF, cFGF, and soluble Klotho were analysed using ELISA kits. Moreover, 1,25-dihydroxyvitamin D3 was determined using LCMS/MS. Calcium, phosphate, iPTH and 25-hydroxyvitamin D were measured using autoanalyzers.ResultsIn haemodialysis patients, iFGF23, cFGF23, iPTH and P levels were significantly higher, and 1,25-dihydroxyvitamin D3, Klotho and Ca levels were significantly lower compared with the control group. There was no significant difference in the 25-hydroxyvitamin D levels.ConclusionsOur study showed that lack of sufficient amounts of Klotho is crucial for mineral metabolism disruptions seen as a complication of chronic kidney diseases. Despite the high levels of the hormone, FGF23 is unable to accomplish its function properly, likely due to deteriorated kidney function in haemodialysis patients.     

The bone and the kidney

Renal tubular diseases may present with osteopenia, osteoporosis or osteomalacia, as a result of significant derangements in body electrolytes. In case of insufficient synthesis of calcitriol, as in renal failure, the more complex picture of renal osteodystrophy may develop. Hypothetically, also disturbed renal production of BMP-7 and Klotho could cause bone disease. However, the acknowledgment that osteocytes are capable of producing FGF23, a phosphaturic hormone at the same time modulating renal synthesis of calcitriol, indicates that it is also bone that can influence renal function. Importantly, a feed-back mechanism exists between FGF23 and calcitriol synthesis, while Klotho, produced by the kidney, determines activity and selectivity of FGF23. Identification of human diseases linked to disturbed production of FGF23 and Klotho underlines the importance of this new bone-kidney axis. Kidney and bone communicate reciprocally to regulate the sophisticated machinery responsible for divalent ions homeostasis and for osseous or extraosseous mineralisation processes.     

Klotho and aging

The klotho gene encodes a single-pass transmembrane protein that forms a complex with multiple fibroblast growth factor (FGF) receptors and functions as an obligatory co-receptor for FGF23, a bone-derived hormone that induces negative phosphate balance. Defects in either Klotho or Fgf23 gene expression cause not only phosphate retention but also a premature-aging syndrome in mice, unveiling a potential link between phosphate metabolism and aging. In addition, the extracellular domain of Klotho protein is clipped on the cell surface and secreted into blood stream, potentially functioning as an endocrine factor. The secreted Klotho protein has a putative sialidase activity that modifies glycans on the cell surface, which may explain the ability of secreted Klotho protein to regulate activity of multiple ion channels and growth factors including insulin, IGF-1, and Wnt. Secreted Klotho protein also protects cells and tissues from oxidative stress through a mechanism yet to be identified. Thus, the transmembrane and secreted forms of Klotho protein have distinct functions, which may collectively affect aging processes in mammals.     

Generation of mouse conditional and null alleles of the type III sodium‐dependent phosphate cotransporter PiT‐1

Accelerated vascular calcification occurs in several human diseases including diabetes and chronic kidney disease (CKD). In patients with CKD, vascular calcification is highly correlated with elevated serum phosphate levels. In vitro, elevated concentrations of phosphate induced vascular smooth muscle cell matrix mineralization, and the inorganic phosphate transporter‐1 (PiT‐1), was shown to be required. To determine the in vivo role of PiT‐1, mouse conditional and null alleles were generated. Here we show that the conditional allele, PiT‐1^flox, which has loxP sites flanking exons 3 and 4, is homozygous viable. Cre‐mediated recombination resulted in a null allele that is homozygous lethal. Examination of early embryonic development revealed that the PiT‐1^{Δe3,4/Δe3,4} embryos displayed anemia, a defect in yolk sac vasculature, and arrested growth. Thus, conditional and null PiT‐1 mouse alleles have been successfully generated and PiT‐1 has a necessary, nonredundant role in embryonic development. genesis 47:858–863, 2009. © 2009 Wiley‐Liss, Inc.     

Inorganic phosphate determination in the presence of a labile organic phosphate: assay for carbamyl phosphate phosphatase activity

A periodate-resorcinol method for bound sialic acid using the Technicon Autoanalyzer II is presented. It has a sensitivity similar to the manual method, is linear between 5 and 65 nmol/ml, requires less than 0.2 ml of sample, and can be run at the rate of 70 samples/h. Little cross-reaction with common matrix and cell components was found. The method is compatible with many commonly used volatile and nonvolatile chromatographic buffers. The use of a bound sialic acid standard such as N-acetylneuraminyl lactose is recommended.     

Studies on the distribution, re-translocation and homeostasis of inorganic phosphate in barley leaves

Changes in inorganic phosphate (P_i) concentrations in barley leaves during growth of plants with sufficient or deficient supplies of P_i were studied. Measurements of the P_i distribution from subcellular levels to the leaf tissue level under the same experimental conditions allowed us to analyse the relationship between the P_i homeostasis of various compartments and P_i re-translocation in the whole plant. Under P_i deficiency, the finding of growth-dependent changes in the P_i concentrations of whole leaves established that P_i was re-translocated from the older leaves to the young leaves. Translocation of ³²P_i was also confirmed with an ‘imaging plate’ system, which made it possible to follow P_i movement in the same plantlet. To analyse the mechanism of P_i re-translocation, the P_i distribution amongst various compartments of the leaves was measured. Under P_i deficiency, the cytoplasmic P_i concentration of the first leaf remained constant until 16d after sowing, while vacuolar P_i was completely exhausted after 8 to 10d. Exhaustion of vacuolar P_i in the first leaf coincided with the appearance of the second leaf. The P_i concentration in the apoplast changed similarly to that of the whole leaf. However, the apoplastic P_i concentration was affected to some extent by the vacuolar P_i concentration and the growth of the younger leaf, because the main change in apoplastic P_i concentration coincided with the time of the disappearance of the vacuolar P_i and the appearance of the younger leaf. The P_i concentration in the apoplast was about 0.1 to I molm^?3, even in the absence of P_i, which was much higher than that in the usual soil environment (a few mmolin^?3). This suggests that the P_i absorbed by root cells is concentrated in the transport process from the root to the leaf apoplast. The content of P_i in the xylem exudate was constant irrespective of growth culture conditions. The root may be functioning as the constant P_i supplier to the above tissues.     

Inorganic phosphate assay with malachite green: An improvement and evaluation

An improvement over existing procedures for the determination of nanomole quantities of inorganic phosphate (P_i) is described. The protocol is simplified, and the effective concentration range of P_i in which the assay may be used is increased to 60 nmol/ml. Many of the substances commonly used in association with P_i assays (i.e. phosphohydrolase studies) are shown not to interface with the measurement of P_i by this method. The effects of detergents and protein on the assay also were investigated, and methods for avoiding interferences by them are described.     

Genotypic variation of rice in phosphorus acquisition from iron phosphate: Contributions of root morphology and phosphorus uptake kinetics

To elucidate the contributions of rice root morphology and phosphorus uptake kinetics to P uptake by rice from iron phosphate, a sand culture experiment with either sufficient P supply (control treatment, 10 mg P/l as NaH₂PO₄) or Fe-P as the only source of P (40 mg P/pot as FePO₄ × 4H₂O) and a solution culture experiment supplied with either sufficient P (10 mg P/l) or deficient P (0.5 mg P/l) were conducted. Eight rice cultivars, which differed in P uptake from Fe-P, were investigated. Plant P uptake, root morphology, and P uptake kinetics were determined. There were significant (P < 0.05) genotypic variations in both plant dry weight and P uptake per plant among eight rice (Oryza sativa L.) cultivars when supplied with Fe-P as the P source. The Fe-P treatment significantly (P < 0.05) decreased plant dry weight, P uptake per plant, and P concentration in plant dry matter of all cultivars in comparison with the control plants. In Fe-P treated plants, significant (P < 0.05) genotypic variation was shown in root morphology, including root length, surface area, volume, and number of lateral roots. The P uptake per plant from Fe-P by rice was significantly (P < 0.05) correlated with root surface area and root volume as well as with the number of lateral roots, suggesting that the ability of rice to absorb P from Fe-P was closely related to root morphology. Low P supply in solution significantly increased the I _max (P < 0.05), but significantly decreased the K _M (P < 0.05) for P absorption by all rice cultivars. We supposed that kinetic characteristics of root P uptake could not account for the ability of rice to absorb P from Fe-P. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 2, pp. 260–266. The text was submitted by the authors in English.     

Arabidopsis inositol phosphate kinases IPK1 and ITPK1 constitute a metabolic pathway in maintaining phosphate homeostasis

Emerging studies have suggested that there is a close link between inositol phosphate (InsP) metabolism and cellular phosphate (P_i) homeostasis in eukaryotes; however, whether a common InsP species is deployed as an evolutionarily conserved metabolic messenger to mediate P_i signaling remains unknown. Here, using genetics and InsP profiling combined with P_i‐starvation response (PSR) analysis in Arabidopsis thaliana, we showed that the kinase activity of inositol pentakisphosphate 2‐kinase (IPK1), an enzyme required for phytate (inositol hexakisphosphate; InsP₆) synthesis, is indispensable for maintaining P_i homeostasis under P_i‐replete conditions, and inositol 1,3,4‐trisphosphate 5/6‐kinase 1 (ITPK1) plays an equivalent role. Although both ipk1‐1 and itpk1 mutants exhibited decreased levels of InsP₆ and diphosphoinositol pentakisphosphate (PP‐InsP₅; InsP₇), disruption of another ITPK family enzyme, ITPK4, which correspondingly caused depletion of InsP₆ and InsP₇, did not display similar P_i‐related phenotypes, which precludes these InsP species from being effectors. Notably, the level of d /l ‐Ins(3,4,5,6)P₄ was concurrently elevated in both ipk1‐1 and itpk1 mutants, which showed a specific correlation with the misregulated P_i phenotypes. However, the level of d /l ‐Ins(3,4,5,6)P₄ is not responsive to P_i starvation that instead manifests a shoot‐specific increase in the InsP₇ level. This study demonstrates a more nuanced picture of the intersection of InsP metabolism and P_i homeostasis and PSRs than has previously been elaborated, and additionally establishes intermediate steps to phytate biosynthesis in plant vegetative tissues.     

High phosphate feeding induced arterial medial calcification in uremic rats: Roles of Lanthanum carbonate on protecting vasculature

Aims

High cardiovascular mortality in patients with end-stage renal disease is closely associated with arterial medial calcification (AMC) caused by hyperphosphatemia, the mechanism of which associated hormones (FGF-23, klotho) and osteochondrogenic events is unclear. We examined the effect of Lanthanum carbonate on AMC via regulating the abnormalities in phosphorus metabolism of uremic rats.

Main methods

45 healthy SD rats were randomly divided into 3 groups: Normal group (n = 15), CRF group (n = 15), CRF diet supplemented with 2% La (n = 15). AMC in great arteries were evaluated by VonKossa. Osteochondrogenic specific genes were analyzed by Immunohistochemistry and qRT-PCR. Serum FGF-23 and klotho levels were detected by ELISA kit.

Key findings

Serum phosphate was markedly increased in CRF group (6.94 ± 0.97 mmol/L) and 2%La group (5.12 ± 0.84 mmol/L) at week 4, while the latter became hypophosphatemic (2.92 ± 0.73 mmol/L vs CRF group, p < 0.01) at week 10. Inhibitory effect of 2%La on development of AMC was reflected by downregulated Runx2, Osterix, BSP, Osteocalcin and collagenII and a reduction of FGF-23 at week 4(vs CRF group, p < 0.01) but not week 10.

Significance

Beneficial effects of Lanthanum carbonate on progression of AMC in CRF could be mainly due to the decreased phosphate retention and FGF-23 in early stage and likewise a reduction of bone-associated proteins via osteochondrogenic pathway. Lanthanum carbonate has no effect on soluble klotho and serum FGF-23 in late stage of CRF.