BoneKEy-Osteovision | Commentary
Bone talk: Klotho and FGF23 signaling
Gordon J Strewler
DOI:10.1138/20060242
Commentary on: Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Okawa K, Fujita T, Fukumoto S, Yamashita T. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature. 2006 Dec 7;444(7120):770-4.
Fibroblast growth factor-23 (FGF23) was identified as a phosphate wasting factor, or phosphatonin, in 2000 (), when the FGF23 gene was shown to harbor gain-of-function mutations that cause autosomal dominant hypophosphatemic rickets () and FGF23 was determined to be a phosphate-wasting signal elaborated by tumors in oncogenous osteomalacia (). FGFs and their receptors are ubiquitous, but the phosphaturic signal is specific: the clinical syndrome of FGF23 excess consists of phosphate wasting and impaired synthesis of the active vitamin D metabolite 1,25(OH)2D, both functions of the proximal convoluted tubule of the kidney. Urakawa et al. () have now reported a startling explanation for the paradox of FGF23 specificity: the principal FGF23 receptor, FGFR1(IIIc), responds to FGF23 only when the Klotho protein is present as a coreceptor, and Klotho is present only in the kidney, parathyroid and pituitary. The klotho phenotype, though it was originally described as a form of premature aging, is the result of FGF23 resistance.
To address the renal specificity of FGF23 action, Urakawa et al. () used gene array analysis to pick out Egr-1 as a kidney gene whose expression is rapidly upregulated by injection of FGF23. Although Egr-1 is ubiquitous, FGF23 activated Egr-1 only in kidney, pituitary and parathyroid, and not in other mouse tissues. To identify a renal molecule that might account for this tissue distribution, Urakawa and colleagues used FGF23 affinity chromatography to identify Klotho as the major FGF23-binding protein in renal homogenates. Cells exposed to FGF23 underwent ERK phosphorylation and increased their abundance of Egr-1 protein only when transfected with Klotho, similar to other recently reported results (). Klotho is expressed in all three tissues that have an Egr-1 response to FGF23 – kidney, pituitary and parathyroid – but not in other mouse or rat tissues.
Klotho was characterized as an aging gene () because one of the mouse strains created in a program of random insertional mutagenesis had a reduced lifespan, atherosclerosis, osteopenia, skin atrophy, impaired sexual maturation and pulmonary emphysema (). Since these traits were viewed as evidence of premature aging, the mutation was named klotho, after one of the three Fates who spins the thread of life. The klotho gene that was disrupted by insertional mutagenesis was shown to encode a cell surface protein with a short cytoplasmic tail, whose extracellular domain consists of tandem duplicated copies of a ß–glucosidase-like sequence, which can be released as a soluble form of Klotho.
FGF23 deficiency causes hyperphosphatemia and increased 1,25(OH)2D synthesis, which leads to hypercalcemia and eventually to tissue damage and nephrocalcinosis (). The biochemical profile of klotho mice is identical to this (), but in contrast to FGF23(-/-) mice, klotho mice have markedly increased FGF23 levels, consistent with resistance to FGF23 action as their underlying disorder (). These genetic data make a strong case that the Klotho protein is required for FGF23 action. Moreover, the short lifespan, infertility, osteoporosis, emphysema and skin changes of the klotho mouse are also present in Fgf23(-/-) mice and can be explained as the consequences of hyperphosphatemia and increased 1,25(OH)2D levels, since the Fgf23(-/-) phenotype can be rescued by removal of either the Cyp27B gene, which encodes the vitamin D 1α–hydroxylase (), or the vitamin D receptor ().
In further studies, a neutralizing monoclonal antibody against the extracellular domain of Klotho was shown to inhibit FGF23 action in Klotho-expressing CHO cells. Injection of the antibody into mice caused a sharp increase in the serum concentration of 1,25(OH)2D, with subsequent increases in the serum concentration of phosphate and FGF23. This independently confirms the requirement of Klotho for successful FGF23 signaling.
To determine the role of canonical FGF receptors (FGFR) in FGF23 signaling, individual receptors and splice variants were used to complement Klotho in L6 cells, which are deficient in native FGF receptors. Only FGFR1(IIIc) in combination with Klotho was able to support significant FGF23 signaling in these experiments. In contrast to FGF23, basic FGF, a universal ligand for FGF receptors, was able to signal without coexpression of Klotho. Thus, Klotho selectively converts FGFR1(IIIc) into a specific FGF23 receptor.
Bone is the predominant source of FGF23, and recent studies point to the osteocyte as the site of FGF23 synthesis in another hereditary phosphate-wasting disorder, the Hyp mutation (). In a BoneKEy Commentary in this issue (), Caroline Silve discusses two recent papers implicating the bone matrix protein DMP1 in the FGF23 signaling pathway (). These papers raise the possibility that osteocytes sample the state of bone matrix through DMP1 and then signal the kidney to dump phosphate and produce 1,25(OH)2D. FGF23 is thus a specific messenger from bone to kidney and perhaps other tissues and can be regarded as the first osteokine.
FGFs are heparin-binding molecules typically associated with the extracellular matrix (ECM). As a circulating cytokine, FGF23 is unusual among FGFs, but it is not unique. Other members of the same FGF subfamily – including FGF15, its human ortholog FGF19, and FGF21 – also seem to be able to deliver signals at a distance, circulating either in blood or lymph (see Table 1). What molecular adaptations are required for these FGFs to circulate? FGF19 has markedly reduced heparin binding, and structural studies of FGF19 show that ordered structure is not detected in a loop that includes strand 11, the principal heparin-binding domain of FGFs (). Other members of the subfamily, including FGF23, are predicted to have similar structures; it may well be that all are adapted to circulate by reduced binding to the heparan sulfate proteoglycans of the ECM.
The FGF19 subfamily may have another adaptation as well. Mice lacking β-Klotho, a second member of the Klotho family, have a marked increase in the synthesis and secretion of bile acids (), much of which can be accounted for by impaired bile acid suppression of cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid biosynthesis. This phenotype is quite similar to that resulting from removal of the gene encoding FGF15 (which is secreted by enterocytes and is the main feedback mechanism to regulate bile acid secretion by the liver ()) or removal of the gene for FGFR4, the liver receptor for FGF15/19 (). In addition, β-klotho(-/-) and FGFR4(-/-) mice both exhibit small gallbladders. It thus seems likely that Klotho and β-Klotho are both coreceptors for different FGFs, raising the possibility that the use of Klotho family members as coreceptors is another distinguishing feature of the FGF 15/19/21/23 subfamily (). FGF21 does not bind to extracellular domains of FGFRs, though it activates FGFR1 and FGFR2 in adipocytes, suggesting that it also requires a binding partner (). In addition to Klotho and β-Klotho, the family contains a third, more distantly related member, Klotho LPH-related protein (), whose function and relationship to FGF signaling are unknown.
This beautiful work opens up many vistas. Is FGF23 signaling restricted to parathyroid, pituitary and kidney (where Klotho is predominantly expressed in distal rather than proximal tubule, leaving open a critical question about how the signal reaches the proximal tubule)? Or can FGF23 use other Klotho family members or soluble Klotho to signal in other tissues, such as bone itself? If FGFs 15, 19, 21, and 23 are all circulating signals, how is specificity maintained; are there specific partnerships between a given Klotho and FGFR or does the FGFR determine the tissue specificity of signaling? Finally, if the fatality of klotho mice is attributable to the consequences of FGF23 deficiency, such as hyperphosphatemia and hypercalcemia, then why is the mouse lifespan considerably increased by overexpression of FGF23 ()?
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