Division of Nephrology, Department of Medicine, The University of Tennessee Health Science Center, Memphis, TN 38165, USAUnited States
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During the past decade a lot of work has been done to better understand the roles of fibroblast growth factor-23 (FGF-23); a relatively newly discovered endocrine hormone, in multiple organ systems in the body. This review focuses on expressions of FGF-23, coexpressions of α-Klotho and FGF receptors, and FGF-23 mediated end-organ effects in the physiological and pathological conditions. We also discuss the controversial reports regarding α-Klotho-dependent and α-Klotho-independent functions of FGF-23.
Keywords: controversial; coexpressions; fibroblast
Volume 1 : Issue 3 Article Ref. #: 1000NPOJ1110 During the past decade a lot of work has been done to better understand the roles of fibroblast growth factor-23 (FGF-23); a relatively newly discovered endocrine hormone, Article History in multiple organ systems in the body. This review focuses on expressions of FGF-23, co- th Received: March 10th, 2016 expressions of α-Klotho and FGF receptors, and FGF-23 mediated end-organ effects in the st Accepted: March 21st, 2016 physiological and pathological conditions. We also discuss the controversial reports regarding th Published: April 4th, 2016 α-Klotho-dependent and α-Klotho-independent functions of FGF-23. EXPRESSION OF FGF-23, α-KLOTHO, AND FGF RECEPTORS Citation Nayani SS, Xiao Z. Recent advances in fibroblast growth factor-23 func- FGF-23 tions. Nephrol Open J. 2016; 1(3): 51-58. doi: 10.17140/NPOJ-1-110 FGF-23 is a family member of 22 fibroblast growth factors (FGFs) including FGF-1- FGF-23, all of which are not only structurally but also evolutionarily related proteins. These FGFs have been classified as being paracrine (15 FGFs), endocrine (3 FGFs), or intracrine (4 FGFs)1 and using a mammalian (murine) model the endocrine FGFs, especially FGF-23 an ap- proximately 32 kD protein,2 have been thoroughly investigated in recent years. FGF-23 is expressed in multiple cells in the body. It has been conclusively shown to be secreted in bone by cells of the osteoblast lineage and osteocytes, as a part of the lacuna- canalicular system, under the influence of phosphate.3 Local factors derived from bone itself also regulate its expression as seen in cases of inactivating mutations of PHEX which results in increased transcription and circulating levels of FGF-23.4 It is also expressed in the pericyte- 5 2 like endothelial cells surrounding the venous sinusoids in bone marrow5 and in the thymus.2 FGF-23 also plays a part in the body’s immune response since its expression is induced in activated dendritic cells and macrophages in response to inoculation with E. coli and S. aureus via nuclear factor-kappa B (NF-κB) signaling.6 FGF-23 is found to be expressed in the ventero- lateral (VL) thalamic nucleus as well. In addition, FGF-23 is also expressed in heart by cardiac 7,8 interstitial fibroblasts.7,8 Apart from these tissues, there is also FGF-23 expression seen in the muscle, spleen, skin, lung, testes, kidney, and liver to a much lesser extent.7 α-Klotho α-Klotho is a unique molecule that establishes a regulatory system of calcium ho- meostasis by affecting transepithelial transport of calcium, parathyroid hormone secretion, and FGF-23 signal transduction.9 Evolutionarily the FGF-23-α-Klotho system is part of a major Copyright milestone in vertebrate evolution that started in the ocean when the early piscine ancestors ©2016 Xiao Z. This is an open ac- acquired the bony endoskeleton.10 α-Klotho has been demonstrated to enhance FGF-23 activity cess article distributed under the 11 over 10-fold and has also been identified as a necessary co-receptor for FGF-23 binding due Creative Commons Attribution 4.0 to the phenotypic similarity observed in α-Klotho and FGF-23 knockout mice, i.e.; hyperphos- International License (CC BY 4.0), 11 which permits unrestricted use, phatemia and hypercalcemia. distribution, and reproduction in any medium, provided the original α-Klotho is expressed in high levels in kidney, parathyroidgland (PTG), testis, ovary, work is properly cited.
brain, pituitary, and apical plasma membrane of ependymal cells in the choroid plexus but not in bone, lung, liver, skin, spleen, small intestines, or adrenal conclusion that FGFR-1c is the most significant binding site for glands.12 The identification of these sites of expression is impor- FGF-23.
tant because these must be compared to the sites of expression of FGFRs, since FGF-23 has the majority of its effects through the END-ORGAN EFFECTS OF FGF-23 IN THE PHYSIOLOGICAL FGFRs α-Klotho complexes pathway.13 AND PATHOLOGICAL CONDITIONS Expression of FGF Receptors Aside from the issues discussed above, there are also some gaping holes in the research regarding FGF-23 that have There are four single pass transmembrane proteins not been investigated yet and could possibly lead to answers that called the fibroblast growth factor receptors (FGFRs 1-4),14 will not only link multiple effects and disorders caused by FGF- which are involved with important biological processes ranging 23 in the body but also provide more paradigm-shifting knowl- from cell division and maturation to formation of blood vessels, edge regarding the complex metabolic balance in our bodies and wound healing, and embryonic development.15 Local and sys- the hitherto unknown roles of multiple organs in this. These is- temic secreted FGFs bind FGFRs to dimerize them, followed sues are briefly discussed here according to the organs that they by activation of intracellular FGF signaling pathways including concern most directly.
RAS–RAF–MAPK, PI3K–AKT, STAT, and PLCγ pathways.1 Kidney FGFR has been shown to be highly expressed in the skin and heart with moderate expression in the ovary.16 Some de- FGF-23 acts via the α-Klotho:FGFR-1 complex in gree of expression has also been shown in the kidney and urinary the renal tubules. The highest expression of these complexes bladder. {Luqmani, 1992 #67}Other than these sites, FGFR-1 has been seen in the distal tubule indicating that this is where expression was noticed in low levels in the breast, lung spinal 18 the initial effect of FGF-23 takes place. However, FGF-23 was cord, adrenal, thyroid, ileum, colon, and stomach. FGFR-2 ex- found to inhibit sodium-dependent phosphate reabsorption via pression has also been detected in these tissues except for the decreased expression of NPT-2a and NPT-2c in the proximal tu- heart. A particularly high level of expression of FGFR-2 was 26,27 16,18 bule. FGF-23 also promotes calcium reabsorption in the dis- noted in stomach and in the thyroid. FGFR-3 shows very tal tubule via the TRPV-5 channel. It is also linked to vitamin little expression, if any, throughout the human body. However, 19 D3 levels since 1, 25(OH)2D administration stimulates FGF-23 high expression levels have been noted in the skin. FGFR-4 expression. Furthermore, FGF-23 inhibits 1 α-hydroxylase ex- shows moderate levels of expression in the lung and low levels pression in the proximal tubule while simultaneously increasing in the ovary, kidney, intestines, and the liver. Nonetheless, other 20 24-hydroxylase expression leading to formation of a less active than these tissues there is limited expression seen in the body. 5,30 form of vitamin D, setting up an effective feedback loop. Tissue specific FGFR expression and activation has been shown FGF-23 also decreases the expression of α-Klotho by the kidney, to be modulated by heparin, heparan sulfate, or other glycos- establishing a complete feedback loop regarding its effects in the aminoglycan chains. FGFRs 1-3 show an alternative splicing kidney. Meanwhile the serum levels of FGF-23 have also been pattern which leads to formation of ‘b’ and ‘c’ isoforms. The ‘b’ established as biomarkers for renal failure.
isoform is expressed preferentially in epithelial tissues while ‘c’ is found in mesenchymal tissues.22 This, along with the different Gastrointestinal Tract sites of expression of different FGFs, renders specificity to the FGF signaling system.14 FGF-23 has been shown to inhibit expression of the intestinal phosphate transporter NTP-2b,33 thus leading to a de- Co-expression of FGFRS and α-klotho Utilized by FGF-23 crease in serum phosphate levels. As an indirect effect of FGF- 23 we also see that due to decreased activation of 1, 25(OH)2D As noted above, FGFR-1, FGFR-2, and FGFR-4 have from the kidney, there is decreased absorption of calcium and some sites of expression in common with α-Klotho, particularly phosphate from the intestine.34 the kidneys and the ovary. FGF-23 was found to act preferential- ly via FGFR-1c, FGFR-3c, and FGFR-4 since α-Klotho forms 11,14 Parathyroid Gland complexes with them. However, a single or double depletion of FGFR-3 and FGFR-4 does not lead to defects in phosphate homeostasis.23 A deletion of FGFR-1 however, is embryonical- α-Klotho and FGFR-1 are both expressed in high lev- ly lethal in mice24 and FGF-23 expression is notably increased els in the PTG.35 However, many different studies have reached when FGFR-1 is activated in rats with normal kidney function different conclusions regarding FGF-23 and its effects on the and in vitro in osteo blast-like cells derived from bone.25 This PTG. This has raised a lot of questions and will be discussed in increased expression also occurs in cases of activating mutations the following section. The one undisputed fact is that PTH acts 3 36 of FGFR-1 as evident in osteoglophonic dysplasia.3 At the same on bone to cause an increased expression of FGF-2336 and FGF- time a conditional deletion of FGFR-1 in the osteocytes of Hyp 23 decreases PTH expression,37 setting up a feedback inhibition mice showed decreased FGF-23 expression.5 This leads to the loop.
’ Bone α-Klotho also has receptors in the ear, FGF-23’s specific effects were determined by the fact that functional assessments of hear- The bone is the major origin of FGF-23. However, ing in FGF-23 null mice did not match the auditory phenotype α-Klotho is not expressed in bone,38 but all of FGF-23’s recep- of α-Klotho null mice. There is also an overlap of initiation of tors (FGFR-1c, FGFR-2c, and FGFR-3c) are expressed in osteo- FGF-23 activity and the development of the eustachian tube blasts.39 FGF-23’s importance as a vital hormone came to light during embryogenesis which could be the cause of middle ear when the FGF-23 gene was identified as the one linked to Au- malformations in FGF-23 deficiency. FGF-23 heterozygous tosomal Dominant Hypophosphatemic Rickets (ADHR).40 This knockout mice have been found to be deaf with close to normal discovery prompted further research and FGF-23 was also found morphology, while homozygous knockout mice have dysplastic to be the causative agent behind human tumor-induced osteoma- bulla and ossicles.47 There is a difference in auditory phenotype lacia which is a disease linked to hypophosphatemia due to renal between the FGF-23 null mice and α-Klotho null mice.47 Inves- phosphate wasting,41 thus leading to the discovery of its role as tigating this further can not only help us find out how FGF-23 ’ a part of a bone-kidney-parathyroid axis. Given FGF-23’s obvi- might be carrying out α-Klotho independent functions in the ear, ously important role in bone-related pathologies, and the work but also set a template for investigation into α-Klotho indepen- already done on the indirect effects it has on the bone via its ef- dent functions of FGF-23 in other organs.
fects on the other endocrine organs, much more work needs to be done on the direct effects FGF-23 has on bones.
Prostate Thalamus, Choroid Plexus, and Pituitary Gland FGF-23 is heavily linked with prostate cancer. It has been shown that it is not only expressed as an autocrine factor The venterolateral (VL) thalamic nucleus is related to in prostate cancer cells but also enhances proliferation, invasion the motor system and is an important relay for deep cerebellar and anchorage when given exogenously. FGF-23 knock down nuclei to the motor cortex which is in fact where, at the time of was shown to decrease in vivo tumor growth.48 Single nucleotide FGF-23’s discovery, it was thought it might have its effects.2 polymorphisms in FGF-23 have also been linked to a risk of Despite that, not much research has been carried out to assess prostate cancer.49 ’ FGF-23’s function there. The choroid plexus and pituitary gland both exhibit high levels of α-Klotho expression and thus are Vasculature potential targets for FGF-23.12 Following FGF-23 injections in mice in both of these organs, there is induction of early growth The role of FGF-23 in causing vascular calcification has response-1 (EGR-1) expression as well as phosphorylation of also been studied recently. Nevertheless, the scientific commu- the extracellular signal-regulated kinase (ERK).42 The choroid nity has reached a consensus neither regarding the exact effect plexus and the pituitary gland are both sites of α-Klotho expres- of FGF-23 on vasculature nor regarding the mechanism of such sion 12 but FGF-23’s function in these organs is still unknown. an effect. This issue is also outlined in the following sections. The choroid plexus is involved in the composition of the cere- Meanwhile the serum levels of FGF-23 have been definitely brospinal fluid (CSF) and the pituitary gland is a major endo- established as biomarkers for stroke. While previous studies crine gland. As such, the knowledge of how FGF-23 is involved have shown that ablation of FGF-23 leads to increased serum in the functioning of both these organs is of vital importance if phosphate levels and thus vascular calcification and death, a we are to understand completely the diverse effects it can cause new study states that FGF-23 enhances phosphate induced cal- physiologically as well as in certain pathologies. cification in the aortic rings of rats by promotion of osteoblastic differentiation involving the ERK1/2 pathway.52 However, there Heart are other studies which do not support α-Klotho mediated effects of FGF-23 in the vasculature.53 Studies looking into this could Cardiac tissue is one of the sites of FGF-23 expres- not only help understand the link between FGF-23 and cardio- 8 43 sion,8 but there is no α-Klotho expression in the heart tissues.43 ’ vascular abnormalities in the body but also FGF-23 s link with Single nucleotide polymorphisms (SNPs) in FGF-23 have been strokes.
identified as potential risk factors for cardiac abnormalities in Kawasaki disease,44 and the serum levels of FGF-23 have been Immune System (Macrophages) recognized as biomarkers for cardiovascular failure.45 FGF-23 had also been linked to greater risks of left ventricular hypertro- FGFR-1c is expressed in macrophages and FGF-23 has phy in patients with CKD. However, no conclusion has been been shown to not only increase the number of macrophages but reached whether this effect is via α-klotho independent path- also to induce TNF* expression in the macrophages. FGF-23 ways or indirectly due to the renal effects of FGF-23. This is might play a key role in inflammation via its effects on macro- also discussed in the next section.
phages as discussed above. However much more work needs to be done in this respect, since inflammation could also account Ear for, or at least play a major role in, many different complications associated with disease states with elevated FGF-23 levels. 57 ’ Indirect Effects via The RAAS Pathway via ERK-1/2 and SGK-1.57 This suggests that FGF-23’s actions in the kidney are all reliant on α-Klotho-dependent stimulation FGF-23 has been shown to stimulate the rennin-angio- of FGFR’s, in the proximal as well as the distal tubule, with no tensin-aldosterone-system (RAAS) by suppression of ACE-2 observable α-Klotho-independent actions. However the above expression in renal tissue, independent of other bone-mineral observation can possibly be confounded by the use of FGF-23 disorder abnormalities.54 Renin expression in the kidney also amounts high enough to activate α-Klotho-independent recep- increases indirectly due to the inhibition of 1, 25(OH)2D by tors of FGF-23, and by the authenticity of the proximal tubular FGF-23.34 Through this stimulation of RAAS, FGF-23 leads to phenotype in the cell culture model which could have been ei- numerous adverse effects like hypertension, diabetic nephropa- ther contaminated with distal tubular cells or have undergone thy, baroreceptor dysfunction, activation of sympathetic system, dedifferentiation. Thus where we already know of the effects endothelial dysfunction, atherosclerotic progression, and fibri- down-stream of α-Klotho-dependent stimulation by FGF-23 at nolytic system inhibition.55 This also helps link FGF-23 to many the distal tubular cells, further research should focus on possible of its observed adverse effects.
α-Klotho-independent receptor stimulation of FGF-23 at proxi- mal tubular cells.
KLOTHO-DEPENDENT AND KLOTHO-INDEPENDENT FUNC- TIONS OF FGF-23 Bone As discussed above, FGF-23 is linked deeply with Despite being the site of greatest expression and pro- many of the organ systems in the physiological conditions to duction of the hormone, little is known about the direct effects contribute to the body’s overall endocrine homeostasis and is of FGF-23 on the bone itself. Though the absence of α-Klotho also responsible for diseases linked to disturbances in the patho- indicates a greatly reduced affinity of FGF-23 for its receptors, physiological conditions. So far the new focus of research re- studies have shown that FGF-23 might directly inhibit bone for- 39,58 garding FGF-23 is concerned with the identification of α-Klotho mation via weak activation of FGFR signaling. This could be dependent and α-Klotho independent effects of FGF-23. It has due to a α-Klotho-independent action of FGF-23 on bone cells. been reported that besides acting as a co-receptor of FGF-23 There have been defects in bone mineralization noted in FGF-23 binding to FGFRs, α-Klotho also acts as a molecular switch. null mice but these might be secondary to the elevated levels of Its presence or absence determines which intracellular signal- 1, 25(OH)2D in these mice since the deletion of the vitamin D ing pathways will be recruited downstream of the FGFRs. Thus receptor rescues the phenotype of FGF-23 null mice.59 Further disease states with α-Klotho deficiency may not involve global research in this aspect would serve to further the understanding FGF-23 resistance, but rather they may in fact promote a switch of the role that bone plays in the delicate endocrine axis and the of the FGF-23-induced signaling towards different cellular re- role of α-Klotho, if any, in this regard.
sponses and outcomes in cells which express FGFRs, but not α-Klotho.56 Parathyroid Gland Previously FGF-23 functions on major organs, directly In human disease conditions and in murine models, or indirectly, were considered only when α-Klotho was co-ex- FGF-23 has been linked to increased levels of PTH,60 while in pressed along with FGFRs in the target tissue,42 but with new more recent studies FGF-23 has been shown to decrease PTH studies that have been conducted exploring different mecha- expression and secretion from the PTG.37 However due to lack nisms of action of FGF-23, researchers need to reassess the role of research into FGF-23’s Klotho-independent signaling via FG- FGF-23 plays in the organs and how that is achieved. There is FRs in the PTG, it is as of yet unknown whether FGF-23 directly clearly a delicate balance between FGF-23’s α-Klotho depen- causes an increase in PTH, or if the increase in PTH might be a dent actions on the FGFRs and its α-Klotho independent actions misleading finding due to FGF-23 induced down regulation of on FGFR’s throughout the body that need to be considered si- α-Klotho in the PTG rendering FGF-23 unable to exert its sup- multaneously and further studied. We discuss these issues here pressive effects on PTH via a α-Klotho-dependent mechanism. It according to the target organ they impact.
is also possible, as discussed above, that the down regulation of α-Klotho in the PTG enables FGF-23 to activate different down- Kidney stream signaling pathways that lead to different effects than those seen in the presence of α-Klotho. Another theory is that What needs to be clarified regarding FGF-23’s role in only the extremely elevated levels of FGF-23 in disease states, the kidney by future studies is how exactly FGF-23 stimulation like chronic kidney disease (CKD), lead to a 1, 25(OH)2D level of the distal renal tubule leads to regulation of proximal tubule low enough to cause not only the release of PTG from the FGF- function, and which signaling pathways are involved in signal 23 induced inhibition, but also to cause a subtle hypocalcemia transduction from the distal to the proximal tubule. It has been which chronically stimulates the PTG to cause a secondary hy- shown that murine proximal tubular epithelium also expressed perparathyroidism.61 This is supported by the fact that among α-Klotho and that FGF-23 acts directly on these proximal tu- the factors known as chronic kidney disease related mineral and bular cells to down regulate membrane expression of NPT-2a bone disorders (CKD-MBD), FGF-23 has also been noted to be the first to increase in concentration before changes in levels of 4. Liu S, Guo R, Simpson LG, Xiao Z-S, Burnham CE, Quarles 62,63 PTH, 1, 25, (OH)2D, or serum phosphate, pointing to it hav- LD. Regulation of fibroblastic growth factor 23 expression but ing a causal role in this disease. It is also possible that the hypo- not degradation by PHEX. J Biol Chem. 2003; 278(39): 37419- phosphatemia that results from elevated FGF-23 levels leads to a 37426. doi: 10.1074/jbc.M304544200 decreased PTH secretion, since phosphorus levels in vitro in rat PTGs have been linked directly to PTH secretion.64 Further stud- 5. Liu S, Zhou J, Tang W, Menard R, Feng JQ, Quarles LD. ies need to work specifically on this aspect of FGF-23’s actions Pathogenic role of FGF-23 in Dmp1-null mice. Am J Physiol since knowledge of how exactly the FGF-23-PTG axis is set up Endocrinol Metab. 2008; 295(2): E254-E261. doi: 10.1152/ and the role of α-Klotho-dependent and α-Klotho-independent ajpendo.90201.2008 receptors will help clarify the development and mechanism of many pathologies associated with variance in FGF-23 levels. 6. Masuda Y, Ohta H, Morita Y, et al. Expression of FGF-23 in activated dendritic cells and macrophages in response to immu- Heart nological stimuli in mice. Biol Pharm Bull. 2015; 38(5): 687- 693. doi: 10.1248/bpb.b14-00276 Left ventricular hypertrophy (LVH) is a serious mortal- ity causing condition linked to chronic kidney disease (CKD) 7. Liu S, Quarles LD. How fibroblast growth factor 23 works. and the elevated FGF-23 levels that it is associated with. Since J of the Am Soc of Nephrology. 2007; 18(6): 1637-1647. doi: there is a scarcity of research into the α-Klotho-dependent and 10.1681/ASN.2007010068 α-Klotho-independent actions of FGF-23 in the heart muscle, and since α-Klotho is absent in cardiomyocytes, the current 8. Bowman MH, Gardner B, Earley J, Rateri D, Daugherty A, thought is that LVH in CKD must be a complication due to the Yan L. Abstract 478: FGF-23 expression in cardiac fibroblasts indirect effects of FGF-23 on the kidneys.
is augmented by S100/Calgranulins-Mediated Inflammation and Associated With Cardiac Hypertrophy, but Not in Angiotensin However, studies have demonstrated the role of FGF- II-Induced Cardiac Hypertrophy. Arteriosclerosis, Thrombosis, 23 in causing pathological hypertrophy in isolated rat cardio- and Vascular Biology. 2015; 35(Suppl 1):A478. Web site. http:// myocytes via an α-Klotho independent, FGFR-4 dependent 56,65 atvb.ahajournals.org/content/35/Suppl_1/A478.abstract. Ac- activation of calcineurin-NFAT pathway. Activation of this cessed March 9, 2016.
FGFR-4/calcineurin/NFAT pathway has been shown to be enough to cause cardiac hypertrophy in mice while FGFR-4 9. Nabeshima Y, Imura H. alpha-Klotho: a regulator that inte- blockade, even with high serum FGF-23 levels, attenuates car- 56 grates calcium homeostasis. Am J Nephrol. 2008; 28(3): 455- diac hypertrophy in rats with CKD. In another study FGF- 464. doi: 10.1159/000112824 23 has been shown to induce cardiomyocyte hypertrophy via PLC-γ signaling, upstream of calcineurin/NFAT, independent 10. Hu MC, Shiizaki K, Kuro-o M, Moe OW. Fibroblast growth of Klotho. These studies bring to light questions regarding α-Klotho independent functions of FGF-23 in the heart that must factor 23 and klotho: Physiology and pathophysiology of an en- be further investigated. Another thing that needs to be clarified docrine network of mineral metabolism. Annu Rev Physiol. 2013; by future research is whether the FGF-23 of cardiac origin plays 75(1): 503-533. doi: 10.1146/annurev-physiol-030212-183727 a role in causing LVH in a paracrine manner.
11. Kurosu H, Ogawa Y, Miyoshi M, et al. Regulation of fibro- CONFLICTS OF INTEREST: None.
blast growth factor-23 signaling by klotho. J Biol Chem. 2006; 281(10): 6120-6123. doi: 10.1074/jbc.C500457200