FGF23 Signaling Research Articles

Phosphorous Dysregulation Induced by MEK Small Molecule Inhibitors in the Rat Involves Blockade of FGF-23 Signaling in the Kidney

MEK, a kinase downstream of Ras and Raf oncogenes, constitutes a high priority target in oncology research. MEK small molecule inhibitors cause soft tissue mineralization in rats secondary to serum inorganic phosphorus (iP) elevation, but the molecular mechanism for this toxicity remains undetermined. We performed investigative studies with structurally distinct MEK inhibitors GEN-A and PD325901 (PD-901) in Sprague-Dawley rats. Our data support a mechanism that involves FGF-23 signal blockade in the rat kidney, causing transcriptional upregulation of 25-hydroxyvitamin D(3) 1-alpha-hydroxylase (Cyp27b1), the rate-limiting enzyme in vitamin D activation, and downregulation of 1,25-dihydroxyvitamin D(3) 24-hydroxylase (Cyp24a1), the enzyme that initiates the degradation of the active form of vitamin D. These transcriptional changes increase serum vitamin D levels, which in turn drive the increase in serum iP, leading to soft tissue mineralization in the rat.

Vitamin D receptor controls expression of the anti-aging klotho gene in mouse and human renal cells

Isoforms of the mammalian klotho protein serve as membrane co-receptors that regulate renal phosphate and calcium reabsorption. Phosphaturic effects of klotho are mediated in cooperation with fibroblast growth factor receptor-1 and its FGF23 ligand. The vitamin D receptor and its 1,25-dihydroxyvitamin D3 ligand are also crucial for calcium and phosphate regulation at the kidney and participate in a feedback loop with FGF23 signaling. Herein we characterize vitamin D receptor-mediated regulation of klotho mRNA expression, including the identification of vitamin D responsive elements (VDREs) in the vicinity of both the mouse and human klotho genes. In keeping with other recent studies of vitamin D-regulated genes, multiple VDREs control klotho expression, with the most active elements located at some distance (−31 to −46kb) from the klotho transcriptional start site. We therefore postulate that the mammalian klotho gene is up-regulated by liganded VDR via multiple remote VDREs. The phosphatemic actions of 1,25-dihydroxyvitamin D3 are thus opposed via the combined phosphaturic effects of FGF23 and klotho, both of which are upregulated by the liganded vitamin D receptor.

KL1 Internal Repeat Mediates Klotho Tumor Suppressor Activities and Inhibits bFGF and IGF-I Signaling in Pancreatic Cancer

Klotho is a transmembrane protein which can be shed, act as a circulating hormone and modulate the insulin-like growth factor (IGF)-I and the fibroblast growth factor (FGF) pathways. We have recently identified klotho as a tumor suppressor in breast cancer. Klotho is expressed in the normal pancreas and both the IGF-I and FGF pathways are involved in pancreatic cancer development. We, therefore, undertook to study the expression and activity of klotho in pancreatic cancer. Klotho expression was studied using immunohistochemistry and quantitative RT-PCR. Effects of klotho on cell growth were assessed in the pancreatic cancer cells Panc1, MiaPaCa2, and Colo357, using colony and MTT assays and xenograft models. Signaling pathway activity was measured by Western blotting. Klotho expression is downregulated in pancreatic adenocarcinoma. Overexpression of klotho, or treatment with soluble klotho, reduced growth of pancreatic cancer cells in vitro and in vivo, and inhibited activation of the IGF-I and the bFGF pathways. KL1 is a klotho subdomain formed by cleavage or alternative splicing. Compared with the full-length protein, KL1 showed similar growth inhibitory activity but did not promote FGF23 signaling. Thus, its administration to mice showed favorable safety profile. These studies indicate klotho as a potential tumor suppressor in pancreatic cancer, and suggest, for the first time, that klotho tumor suppressive activities are mediated through its KL1 domain. These results suggest the use of klotho or KL1 as potential strategy for the development of novel therapeutic interventions for pancreatic cancer.

Abstract 2192: KL1 internal repeat mediates klotho tumor suppressor activities and inhibits bFGF and IGF-1 signaling in pancreatic cancer

Abstract Klotho is a transmembranal protein, which can be cleaved, shed and act as a circulating hormone. Klotho deficient mice present a syndrome of early aging, while overexpression of klotho extends lifespan. Klotho is a potent inhibitor of the insulin growth factor (IGF)-1 and the basic fibroblast growth factor (FGF) pathways. Klotho is also an essential co-factor for the activity of FGF23 and thus serves as a major regulator of phosphate homeostasis. We have recently identified klotho as a tumor suppressor in breast cancer. As klotho is expressed in the normal pancreas and as the IGF-1 and FGF pathways are involved in pancreatic cancer development, we undertook to study the expression and activity of klotho in pancreatic cancer. Immunohistochemistry analysis revealed significantly reduced expression of klotho in pancreatic cancer compared to normal pancreas, and low levels of klotho mRNA were noted in the pancreatic cancer cells Panc1, Colo357 and MiaPaCa2 compared to normal pancreas cells. Further studies revealed that klotho silencing is mediated by epigenetic mechanisms. Overexpression of klotho or treatment with soluble klotho (8pM) reduced clonal growth of pancreatic cancer cell lines, and a synergistic effect was observed in combination with chemotherapeutic drugs. Daily injections of klotho (10μg/kg) inhibited tumor formation of Panc1 cells in athymic mice. Klotho inhibited activation of the IGF-1 pathway in pancreatic cancer cells. As klotho further enhanced growth inhibition by the IGF-1R inhibitor AG1024, modulation of additional signaling pathways by klotho was suspected. Indeed, klotho overexpression inhibited the bFGF pathway in these cells. The extracellular region of klotho is composed of two internal repeats, KL1 and KL2, and each of them can be cleaved and act separately. While both domains share homology to glycosidase, a structural model suggested that only KL1 retains enzymatic activity. While the KL2 domain did not show growth inhibitory activities, KL1 effectively slowed growth of pancreatic cancer cells, both in vitro and in vivo and was found to be a potent inhibitor of the IGF-1 and bFGF pathways. Importantly, KL1 did not promote FGF23 signaling and therefore, in contrary to the full-length protein, its administration to mice did not affect phosphate homeostasis. In conclusion, these studies indicate klotho as a potent tumor suppressor in pancreatic cancer and suggest, for the first time, that klotho growth inhibitory activities are mediated by the KL1 domain, possibly due to its putative enzymatic activities. As the KL1 domain is not involved in regulation of phosphate homeostasis, its administration may serve as a potential safe and effective novel strategy for the treatment of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2192. doi:10.1158/1538-7445.AM2011-2192

Hormone-like (endocrine) Fgfs: their evolutionary history and roles in development, metabolism, and disease

Fibroblast growth factors (Fgfs) are proteins with diverse functions in development, repair, and metabolism. The human Fgf gene family with 22 members can be classified into three groups, canonical, intracellular, and hormone-like Fgf genes. In contrast to canonical and intracellular Fgfs identified in invertebrates and vertebrates, hormone-like Fgfs, Fgf15/19, Fgf21, and Fgf23, are vertebrate-specific. The ancestral gene of hormone-like Fgfs was generated from the ancestral gene of canonical Fgfs by gene duplication early in vertebrate evolution. Later, Fgf15/19, Fgf21, and Fgf23 were generated from the ancestral gene by genome duplication events. Canonical Fgfs act as autocrine/paracrine factors in an Fgf receptor (Fgfr)-dependent manner. In contrast, hormone-like Fgfs act as endocrine factors in an Fgfr-dependent manner. Canonical Fgfs have a heparin-binding site necessary for the stable binding of Fgfrs and local signaling. In contrast, hormone-like Fgfs acquired endocrine functions by reducing their heparin-binding affinity during their evolution. Fgf15/19 and Fgf23 require βKlotho and αKlotho as cofactors, respectively. However, Fgf21 might physiologically require neither. Hormone-like Fgfs play roles in metabolism at postnatal stages, although they also play roles in development at embryonic stages. Fgf15/19 regulates bile acid metabolism in the liver. Fgf21 regulates lipid metabolism in the white adipose tissue. Fgf23 regulates serum phosphate and active vitamin D levels. Fgf23 signaling disorders caused by hereditary diseases or tumors result in metabolic disorders. In addition, serum Fgf19 or Fgf21 levels are significantly increased by metabolic disorders. Hormone-like Fgfs are newly emerging and quite unique in their evolution and function.

Does the downregulation of the FGF23 signaling pathway in hyperplastic parathyroid glands contribute to refractory secondary hyperparathyroidism in CKD patients?

Compared to normal tissue, hyperplastic parathyroid glands of patients with chronic kidney disease on dialysis express lower levels of FGFR1 and Klotho proteins. Similar findings are reported in uremic rats with advanced chronic kidney disease. Moreover, in these animals, FGF23 administration fails to reduce PTH serum levels in vivo and to transmit downstream signals in parathyroid cells ex vivo. These findings may explain, at least partly, the concomitant elevation of both FGF23 and PTH serum levels in chronic kidney disease secondary hyperparathyroidism.

Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23-FGFR-Klotho complex formation

Fibroblast growth factor (FGF) 23 inhibits renal phosphate reabsorption by activating FGF receptor (FGFR) 1c in a Klotho-dependent fashion. The phosphaturic activity of FGF23 is abrogated by proteolytic cleavage at the RXXR motif that lies at the boundary between the FGF core homology domain and the 72-residue-long C-terminal tail of FGF23. Here, we show that the soluble ectodomains of FGFR1c and Klotho are sufficient to form a ternary complex with FGF23 in vitro. The C-terminal tail of FGF23 mediates binding of FGF23 to a de novo site generated at the composite FGFR1c-Klotho interface. Consistent with this finding, the isolated 72-residue-long C-terminal tail of FGF23 impairs FGF23 signaling by competing with full-length ligand for binding to the binary FGFR-Klotho complex. Injection of the FGF23 C-terminal tail peptide into healthy rats inhibits renal phosphate excretion and induces hyperphosphatemia. In a mouse model of renal phosphate wasting attributable to high FGF23, the FGF23 C-terminal peptide reduces phosphate excretion, leading to an increase in serum phosphate concentration. Our data indicate that proteolytic cleavage at the RXXR motif abrogates FGF23 activity by a dual mechanism: by removing the binding site for the binary FGFR-Klotho complex that resides in the C-terminal region of FGF23, and by generating an endogenous inhibitor of FGF23. We propose that peptides derived from the C-terminal tail of FGF23 or peptidomimetics and small-molecule organomimetics of the C-terminal tail can be used as therapeutics to treat renal phosphate wasting.

Evidence against a direct role of klotho in insulin resistance

The klotho gene may be involved in the aging process. Klotho is a coactivator of FGF23, a regulator of phosphate and vitamin D metabolism. It has also been reported to be downregulated in insulin resistance syndromes and paradoxically to directly inhibit IGF-1 and insulin signaling. Our aim was to study klotho's regulation and effects on insulin and IGF-1 signaling to unravel this paradox. We studied klotho tissue distribution and expression by quantitative real-time polymerase chain reaction and Western blotting in obese Zucker rats and high-fat fed Wistar rats, two models of insulin resistance. Klotho was expressed in kidneys but at much lower levels (<1.5%) in liver, muscle, brain, and adipose tissue. There were no significant differences between insulin resistant and control animals. We next produced human recombinant soluble klotho protein (KLEC) and studied its effects on insulin and IGF-1 signaling in cultured cells. In HEK293 cells, FGF23 signaling (judged by FRS2-alpha and ERK1/2 phosphorylation) was activated by conditioned media from KLEC-producing cells (CM-KLEC); however, IGF-1 signaling was unaffected. CM-KLEC did not inhibit IGF-1 and insulin signaling in L6 and Hep G2 cells, as judged by Akt and ERK1/2 phosphorylation. We conclude that decreased klotho expression is not a general feature of rodent models of insulin resistance. Further, the soluble klotho protein does not inhibit IGF-1 and/or insulin signaling in HEK293, L6, and HepG2 cells, arguing against a direct role of klotho in insulin signaling. However, the hypothesis that klotho indirectly regulates insulin sensitivity via FGF23 activation remains to be investigated.

Inactivation of klotho function induces hyperphosphatemia even in presence of high serum fibroblast growth factor 23 levels in a genetically engineered hypophosphatemic ( Hyp ) mouse model

Hyp mice possess a mutation that inactivates the phosphate-regulating gene, which is homologous to the endopeptidases of the X-chromosome (PHEX). The mutation is associated with severe hypophosphatemia due to excessive urinary phosphate wasting. Such urinary phosphate wasting in Hyp mice is associated with an increased serum accumulation of fibroblast growth factor (FGF) 23. We wanted to determine the biological significance of increased serum FGF23 levels and concomitant hypophosphatemia in Hyp mice and to evaluate whether FGF23 activity could be modified by manipulating klotho (a cofactor of FGF23 signaling). We generated Hyp and klotho double-mutant mice (Hyp/klotho(-/-)). Severe hypophosphatemia of Hyp mice was reversed to hyperphosphatemia in Hyp/klotho(-/-) double mutants, despite the fact that the double mutants showed significantly increased serum levels of FGF23. Hyperphosphatemia in Hyp/klotho(-/-) mice was associated with increased renal expression of sodium/phosphate cotransporter 2a (NaPi2a) protein. Exogenous injection of bioactive parathyroid hormone 1-34 down-regulated renal expression of NaPi2a and consequently reduced serum levels of phosphate in Hyp/klotho(-/-) mice. Moreover, in contrast to the Hyp mice, the Hyp/klotho(-/-) mice showed significantly higher serum levels of 1,25-dihydroxyvitamin D and developed extensive calcification in soft tissues and vascular walls. Furthermore, compared with the Hyp mice, Hyp/klotho(-/-) mice were smaller in size, showed features of generalized tissue atrophy, and generally died by 15-20 wk of age. Our in vivo studies provide genetic evidence for a pathological role of increased FGF23 activities in regulating abnormal phosphate homeostasis in Hyp mice. Moreover, these results suggest that even when serum levels of FGF23 are significantly high, in the absence of klotho, FGF23 is unable to regulate systemic phosphate homeostasis. Our in vivo observations have significant clinical implications in diseases associated with increased FGF23 activity and suggest that the functions of FGF23 can be therapeutically modulated by manipulating the effects of klotho.

Clinical aspect of recent progress in phosphate metabolism. FGF23; physiological action and molecular mechanism in the regulation of phosphate and vitamin D metabolism

Fibroblast growth factor (FGF) 23 is an endocrine hormone regulating the phosphate and vitamin D metabolism in the normal physiology. Recent studies have revealed that Klotho plays an essential role in FGF23 signaling. Klotho-deficient mice exhibit premature aging-like phenotypes, and therefore, this molecule has provided insights into the molecular mechanism of aging. However, it is suggested that the phenotype of the Klotho-deficient mice results from the disturbance in the phosphate and vitamin D metabolism. The importance of the FGF23/Klotho system thus is evident, but additional questions arose from this new concept.

In vivo genetic evidence for klotho‐dependent, fibroblast growth factor 23 (Fgf23) ‐mediated regulation of systemic phosphate homeostasis

A major breakthrough in systemic phosphate homeostasis regulation was achieved by the demonstration of strikingly similar physical, morphological, and biochemical phenotypes of fibroblast growth factor 23 (Fgf23) and klotho ablated mice, which led to identification of klotho as an Fgf23 signaling cofactor. Here, we generated Fgf23 and klotho double-knockout (Fgf23(-/-)/klotho(-/-)) mice to test the hypothesis whether Fgf23 has a klotho-independent function. Fgf23(-/-)/klotho(-/-) mice are viable and have high serum phosphate levels, similar to Fgf23(-/-) and klotho(-/-) single-knockout mice. In addition, the Fgf23(-/-)/klotho(-/-) mice have increased renal expression of the sodium/phosphate cotransporter NaP(i)2a and of 1- alpha-hydroxylase concomitant with increased serum levels of 1,25-dihydroxyvitamin-D, as also observed in the Fgf23(-/-) and klotho(-/-) mice. Moreover, Fgf23(-/-)/klotho(-/-) mice show soft tissue and vascular calcification, severe muscle wasting, hypogonadism, pulmonary emphysema, distention of intestinal wall, and skin atrophy, all of which are also seen in Fgf23(-/-) and klotho(-/-) mice. Notably, injection of bioactive FGF23 protein into Fgf23(-/-)/klotho(-/-) and klotho(-/-) mice does not lower serum phosphate, whereas in wild-type and Fgf23(-/-) mice, it reduces serum phosphate. Together, these results provide compelling evidence that Fgf23 does not have a klotho-independent role in the regulation of systemic phosphate and vitamin D homeostasis.

Role of the Klotho Gene in Bone and Mineral Metabolism

Klotho was originally identified when a random insertional mutation disrupting the mouse gene caused an abnormal phenotype with accelerated aging and shortened lifespan. Klotho appears to play an important role in the renal handling of calcium and phosphate, in part by interacting with the FGF23 signaling system. In general, klotho tends to decrease phosphate retention and 1,25-dihydroxyvitamin D3 synthesis. It directly stimulates calcium reabsorption in the kidney and tends to increases serum calcium, but may have opposite indirect effects related to the changes in vitamin D metabolism. Klotho may also influence the response of the parathyroids to hypocalcemia and may interact with other biological systems, including Wnt, OPG/RANKL, insulin/IGF-I, and sex steroids. The actual relevance of klotho in human physiology has been recently illustrated by the identification of some rare patients with loss-of-function and gain-of-function mutations of the Klotho gene. Several studies also suggest that certain polymorphisms of the gene may influence human aging and the development of age-related diseases, such as arteriosclerosis or osteoporosis, but further investigations are needed to elucidate their true importance.

Kidney as a regulating organ for calcium and phosphate homeostasis

Kidney is a central effector organ on calcium (Ca) and phosphate (P) homeostasis. Their homeostasis are mainly preserved by renal reabsorption and excretion, which often regulated through common hormonal signals, parathyroid hormone (PTH), vitamin D, and calcitonin. The hormones also involves phosphatonins as a circulating factor with potent phosphaturic activity and the key phosphatonin appears to be a fibroblast growth factor (FGF) 23. FGF23 is associated with vitamin D metabolism as well as tubular phosphate excretion. Moreover, recent study using animal models revealed the task of klotho protein, which gene functions as an aging-suppressor gene, for the regulation of FGF23 signaling, Ca(2+) absorption and vitamin D modulation in the kidney.

Premature aging in klotho mutant mice: Cause or consequence?

Suitable mammalian models for aging with a wide range of age-associated pathology are desirable to study molecular mechanisms of human aging. Recent studies have identified that fibroblast growth factor 23 ( Fgf-23) null mice and klotho hypomorphs could generate multiple premature aging-like features, including shortened lifespan, infertility, kyphosis, atherosclerosis, extensive soft tissue calcifications, skin atrophy, muscle wasting, T cell dysregulation, pulmonary emphysema, osteoporosis/osteopenia, abnormal mineral ion metabolism, and impaired vitamin-D homeostasis. The strikingly similar in vivo phenotypes of two separate genetically altered mouse lines implicate that the premature aging-like features may be partly regulated through a common signaling pathway involving both Fgf-23 and klotho; such speculation is experimentally supported by the observation that Fgf-23 requires klotho as a cofactor to exert its functions. Despite about 2000-fold higher serum levels of Fgf-23 in klotho mutants (compared to wild-type animals), these mice show physical, biochemical and morphological features similar to Fgf-23 null mice, but not as Fgf-23 transgenic mice; these observations suggest that widely encountered premature aging-like features in klotho mutant mice are due to the inability of Fgf-23 to exert its bioactivities in absence of klotho. The results of recent studies showing klotho as a cofactor in Fgf-23 signaling consequently explains that the premature aging-like features in klotho-deficient mice is not a primary cause, rather a consequence of lacking Fgf-23 activity. These understandings will help us to redefine the role of klotho as an aging factor.

Bone talk: Klotho and FGF23 signaling

Bone talk: Klotho and FGF23 signaling

Advances in endocrinology of aging research, 2005–2006

The purpose of this brief review is to highlight some of the more important advances in endocrinology of aging research over the past year. Four advances were chosen and briefly described. First, exploration of the early steps in the generation of the internal steroidal hormonal signal involved in lifespan extension via the insulin/IGF-like signaling pathway in the nematode by two research groups revealed that the product of cholestanoic acid derivatives metabolized by a cytochrome P-450-like protein activates a protein with homology to the mammalian nuclear receptor superfamily, a process strikingly similar to the steroid hormone signaling pathway documented in mammalian systems. Second is the discovery that sirtuins, proteins that regulate lifespan in model organisms, enhance pancreatic insulin secretion in mice following a glucose challenge, suggesting the potential to regulate mammalian lifespan through regulation of the insulin signaling pathway. Third, the newly discovered hormone klotho, which also plays a role in regulating lifespan, in this case in mice, is reported to not only negatively affect insulin sensitivity but, perhaps more importantly, significantly affects calcium and phosphate metabolism as a required cofactor of Fgf-23 signaling. Finally the gonadotropin FSH is shown to directly affect bone density in mice separate from any direct effect of estrogen, suggesting that reproductive hormones other than estrogen can directly impact menopause-associated pathophysiology in non-reproductive tissues.

Toward a Better Understanding of Klotho

klotho mutant mice were originally described as a short-lived mouse model with premature aging-like disorders. The klotho gene responsible for these phenotypes encodes a type I membrane protein with a considerable similarity to beta-glycosidase. klotho is predominantly expressed in tissues functioning in the regulation of calcium homeostasis. Suggested functions of Klotho are (i) a fundamental regulator of calcium homeostasis, namely, a cofactor for the fibroblast growth factor (FGF) receptor 1c in FGF23 signaling and a regulator of parathyroid hormone secretion; (ii) a hormone that interferes with the intracellular signaling of insulin and insulin-like growth factor-1; and (iii) a beta-glucuronidase that activates the transient receptor potential ion channel TRPV5 by trimming its sugar moiety. How can we reconcile these pleiotropic functions of Klotho? Is there any common mechanism? Further in vivo studies, and biochemical as well as physiological analyses, are required for a better understanding of the molecular aspects of Klotho.

Klotho is an essential molecule responsible for tissue specific FGF23 signaling

Klotho is an essential molecule responsible for tissue specific FGF23 signaling

Analysis of the Biochemical Mechanisms for the Endocrine Actions of Fibroblast Growth Factor-23

Fibroblast growth factor (FGF)-23 has emerged as an endocrine regulator of phosphate and of vitamin D metabolism. It is produced in bone and, unlike other FGFs, circulates in the bloodstream to ultimately regulate phosphate handling and vitamin D production in the kidney. Presently, it is unknown which of the seven principal FGF receptors (FGFRs) transmits FGF23 biological activity. Furthermore, the molecular basis for the endocrine mode of FGF23 action is unclear. Herein, we performed surface plasmon resonance and mitogenesis experiments to comprehensively characterize receptor binding specificity. Our data demonstrate that FGF23 binds and activates the c splice isoforms of FGFR1-3, as well as FGFR4, but not the b splice isoforms of FGFR1-3. Interestingly, highly sulfated and longer glycosaminoglycan (GAG) species were capable of promoting FGF23 mitogenic activity. We also show that FGF23 induces tyrosine phosphorylation and inhibits sodium-phosphate cotransporter Npt2a mRNA expression using opossum kidney cells, a model kidney proximal tubule cell line. Removal of cell surface GAGs abolishes the effects of FGF23, and exogenous highly sulfated GAG is capable of restoring FGF23 activity, suggesting that proximal tubule cells naturally express GAGs that are permissive for FGF23 action. We propose that FGF23 signals through multiple FGFRs and that the unique endocrine actions of FGF23 involve escape from FGF23-producing cells and circulation to the kidney, where highly sulfated GAGs most likely act as cofactors for FGF23 activity. Our biochemical findings provide important insights into the molecular mechanisms by which dysregulated FGF23 signaling leads to disorders of hyper- and hypophosphatemia.