Exploring mechanisms of FGF signalling through the lens of structural biology.

Fibroblast growth factors (FGFs) mediate a broad range of functions in both the developing and adult organism. The accumulated wealth of structural information on the FGF signalling pathway has begun to unveil the underlying molecular mechanisms that modulate this system to generate a myriad of distinct biological outputs in development, tissue homeostasis and metabolism. At the ligand and receptor level, these mechanisms include alternative splicing of the ligand (FGF8 subfamily) and the receptor (FGFR1-FGFR3), ligand homodimerization (FGF9 subfamily), site-specific proteolytic cleavage of the ligand (FGF23), and interaction of the ligand and the receptor with heparan sulphate cofactor and Klotho co-receptor.

α-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling.

The ageing suppressor α-klotho binds to the fibroblast growth factor receptor (FGFR). This commits FGFR to respond to FGF23, a key hormone in the regulation of mineral ion and vitamin D homeostasis. The role and mechanism of this co-receptor are unknown. Here we present the atomic structure of a 1:1:1 ternary complex that consists of the shed extracellular domain of α-klotho, the FGFR1c ligand-binding domain, and FGF23. In this complex, α-klotho simultaneously tethers FGFR1c by its D3 domain and FGF23 by its C-terminal tail, thus implementing FGF23-FGFR1c proximity and conferring stability. Dimerization of the stabilized ternary complexes and receptor activation remain dependent on the binding of heparan sulfate, a mandatory cofactor of paracrine FGF signalling. The structure of α-klotho is incompatible with its purported glycosidase activity. Thus, shed α-klotho functions as an on-demand non-enzymatic scaffold protein that promotes FGF23 signalling.

Endocrinization of FGF1 produces a neomorphic and potent insulin sensitizer.

Fibroblast growth factor 1 (FGF1) is an autocrine/paracrine regulator whose binding to heparan sulphate proteoglycans effectively precludes its circulation. Although FGF1 is known as a mitogenic factor, FGF1 knockout mice develop insulin resistance when stressed by a high-fat diet, suggesting a potential role in nutrient homeostasis. Here we show that parenteral delivery of a single dose of recombinant FGF1 (rFGF1) results in potent, insulin-dependent lowering of glucose levels in diabetic mice that is dose-dependent but does not lead to hypoglycaemia. Chronic pharmacological treatment with rFGF1 increases insulin-dependent glucose uptake in skeletal muscle and suppresses the hepatic production of glucose to achieve whole-body insulin sensitization. The sustained glucose lowering and insulin sensitization attributed to rFGF1 are not accompanied by the side effects of weight gain, liver steatosis and bone loss associated with current insulin-sensitizing therapies. We also show that the glucose-lowering activity of FGF1 can be dissociated from its mitogenic activity and is mediated predominantly via FGF receptor 1 signalling. Thus we have uncovered an unexpected, neomorphic insulin-sensitizing action for exogenous non-mitogenic human FGF1 with therapeutic potential for the treatment of insulin resistance and type 2 diabetes.

Inhibition of fibroblast growth factor 23 (FGF23) signaling rescues renal anemia.

Severe anemia and iron deficiency are common complications in chronic kidney disease. The cause of renal anemia is multifactorial and includes decreased erythropoietin (Epo) production, iron deficiency, and inflammation, and it is currently treated with injections of synthetic Epo. However, the use of recombinant Epo has several adverse effects. We previously reported that high fibroblast growth factor 23 (FGF23) levels in mice are associated with decreased red blood cell production, whereas genetic inactivation of Fgf23 results in expansion of the erythroid lineage. The present study is the first to show that high FGF23 levels in a mouse model of renal failure contribute to renal anemia, and inhibiting FGF23 signaling stimulates erythropoiesis and abolishes anemia and iron deficiency. Moreover, we show that inhibition of FGF23 signaling significantly decreases erythroid cell apoptosis and influences the commitment of hematopoietic stem cells toward the erythroid linage. Furthermore, we show that blocking FGF23 signaling attenuates inflammation, resulting in increased serum iron and ferritin levels. Our data clearly demonstrate that elevated FGF23 is a causative factor in the development of renal anemia and iron deficiency, and importantly, blocking FGF23 signaling represents a novel approach to stimulate erythropoiesis and possibly improve survival for millions of chronic kidney disease patients worldwide.-Agoro, R., Montagna, A., Goetz, R., Aligbe, O., Singh, G., Coe, L. M., Mohammadi, M., Rivella, S., Sitara, D. Inhibition of fibroblast growth factor 23 (FGF23) signaling rescues renal anemia.

FGF23 promotes renal calcium reabsorption through the TRPV5 channel.

αKlotho is thought to activate the epithelial calcium channel Transient Receptor Potential Vanilloid-5 (TRPV5) in distal renal tubules through its putative glucuronidase/sialidase activity, thereby preventing renal calcium loss. However, αKlotho also functions as the obligatory co-receptor for fibroblast growth factor-23 (FGF23), a bone-derived phosphaturic hormone. Here, we show that renal calcium reabsorption and renal membrane abundance of TRPV5 are reduced in Fgf23 knockout mice, similar to what is seen in αKlotho knockout mice. We further demonstrate that αKlotho neither co-localizes with TRPV5 nor is regulated by FGF23. Rather, apical membrane abundance of TRPV5 in renal distal tubules and thus renal calcium reabsorption are regulated by FGF23, which binds the FGF receptor-αKlotho complex and activates a signaling cascade involving ERK1/2, SGK1, and WNK4. Our data thereby identify FGF23, not αKlotho, as a calcium-conserving hormone in the kidney.

Parathyroid-specific deletion of Klotho unravels a novel calcineurin-dependent FGF23 signaling pathway that regulates PTH secretion.

Klotho acts as a co-receptor for and dictates tissue specificity of circulating FGF23. FGF23 inhibits PTH secretion, and reduced Klotho abundance is considered a pathogenic factor in renal secondary hyperparathyroidism. To dissect the role of parathyroid gland resident Klotho in health and disease, we generated mice with a parathyroid-specific Klotho deletion (PTH-KL(-/-)). PTH-KL(-/-) mice had a normal gross phenotype and survival; normal serum PTH and calcium; unaltered expression of the PTH gene in parathyroid tissue; and preserved PTH response and sensitivity to acute changes in serum calcium. Their PTH response to intravenous FGF23 delivery or renal failure did not differ compared to their wild-type littermates despite disrupted FGF23-induced activation of the MAPK/ERK pathway. Importantly, calcineurin-NFAT signaling, defined by increased MCIP1 level and nuclear localization of NFATC2, was constitutively activated in PTH-KL(-/-) mice. Treatment with the calcineurin-inhibitor cyclosporine A abolished FGF23-mediated PTH suppression in PTH-KL(-/-) mice whereas wild-type mice remained responsive. Similar results were observed in thyro-parathyroid explants ex vivo. Collectively, we present genetic and functional evidence for a novel, Klotho-independent, calcineurin-mediated FGF23 signaling pathway in parathyroid glands that mediates suppression of PTH. The presence of Klotho-independent FGF23 effects in a Klotho-expressing target organ represents a paradigm shift in the conceptualization of FGF23 endocrine action.

The demonstration of αKlotho deficiency in human chronic kidney disease with a novel synthetic antibody.

BACKGROUND: αKlotho is the prototypic member of the Klotho family and is most highly expressed in the kidney. αKlotho has pleiotropic biologic effects, and in the kidney, its actions include regulation of ion transport, cytoprotection, anti-oxidation and anti-fibrosis. In rodent models of chronic kidney disease (CKD), αKlotho deficiency has been shown to be an early biomarker as well as a pathogenic factor. The database for αKlotho in human CKD remains controversial even after years of study. METHODS: We used a synthetic antibody library to identify a high-affinity human antigen-binding fragment that recognizes human, rat and mouse αKlotho primarily in its native, rather than denatured, form. RESULTS: Using an immunoprecipitation-immunoblot (IP-IB) assay, we measured both serum and urinary levels of full-length soluble αKlotho in humans and established that human CKD is associated with αKlotho deficiency in serum and urine. αKlotho levels were detectably lower in early CKD preceding disturbances in other parameters of mineral metabolism and progressively declined with CKD stages. We also found that exogenously added αKlotho is inherently unstable in the CKD milieu suggesting that decreased production may not be the sole reason for αKlotho deficiency. CONCLUSION: Synthetic antibody libraries harbor tremendous potential for a variety of biomedical and clinical applications. Using such a reagent, we furnish data in support of αKlotho deficiency in human CKD, and we set the foundation for the development of diagnostic and therapeutic applications of anti-αKlotho antibodies.

Fibroblast growth factor 21 promotes bone loss by potentiating the effects of peroxisome proliferator-activated receptor γ.

The endocrine hormone fibroblast growth factor 21 (FGF21) is a powerful modulator of glucose and lipid metabolism and a promising drug for type 2 diabetes. Here we identify FGF21 as a potent regulator of skeletal homeostasis. Both genetic and pharmacologic FGF21 gain of function lead to a striking decrease in bone mass. In contrast, FGF21 loss of function leads to a reciprocal high-bone-mass phenotype. Mechanistically, FGF21 inhibits osteoblastogenesis and stimulates adipogenesis from bone marrow mesenchymal stem cells by potentiating the activity of peroxisome proliferator-activated receptor γ (PPAR-γ). Consequently, FGF21 deletion prevents the deleterious bone loss side effect of the PPAR-γ agonist rosiglitazone. Therefore, FGF21 is a critical rheostat for bone turnover and a key integrator of bone and energy metabolism. These results reveal that skeletal fragility may be an undesirable consequence of chronic FGF21 administration.

Conversion of a paracrine fibroblast growth factor into an endocrine fibroblast growth factor.

FGFs 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. Here we show that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. In support of our model, we convert a paracrine FGF into an endocrine ligand by diminishing heparan sulfate-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor-binding site to home the ligand into the target tissue. In addition to serving as a proof of concept, the ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.

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.

Timing of Ketogenic Dietary Therapy (KDT) Introduction and Its Impact on Cognitive Profiles in Children with Glut1-DS-A Preliminary Study.

The aim of this research was to characterize cognitive abilities in patients with Glut1-Deficiency syndrome (Glut1DS) following ketogenic diet therapy (KDT). METHODS: The cognitive profiles of eight children were assessed using the Wechsler Intelligence Scale (WISC-IV). The effect of ketogenic diet therapy (KDT) on individual subareas of intelligence was analyzed considering the potential influence of speech motor impairments. RESULTS: Patients with Glut1DS showed a wide range of cognitive performance levels. Some participants showed statistically and clinically significant discrepancies between individual subdomains of intelligence. Both variables, KDT initiation as well as duration, had a positive effect on the overall IQ score. Significant correlations were partially found between the time of KDT initiation and the level of IQ scores, depending on the presence of expressive language test demands of the respective subtests of the WISC-IV. Accordingly, the participants benefited les in the linguistic cognitive domain. The discrepancies in cognitive performance profiles of patients with Glut1DS can be attributed to the possibility of a negative distortion of the results due to the influence of speech motor impairments. CONCLUSIONS: The individual access skills of test persons should be more strongly considered in test procedures for the assessment of intelligence to reduce the negative influence of motor deficits on test performance. Specific characterization and systematization of the speech disorder are indispensable for determining the severity of speech motor impairment in Glut1DS. Therefore, a stronger focus on dysarthria during diagnosis and therapy is necessary.

FGF21 induces PGC-1alpha and regulates carbohydrate and fatty acid metabolism during the adaptive starvation response.

The liver plays a crucial role in mobilizing energy during nutritional deprivation. During the early stages of fasting, hepatic glycogenolysis is a primary energy source. As fasting progresses and glycogen stores are depleted, hepatic gluconeogenesis and ketogenesis become major energy sources. Here, we show that fibroblast growth factor 21 (FGF21), a hormone that is induced in liver by fasting, induces hepatic expression of peroxisome proliferator-activated receptor gamma coactivator protein-1alpha (PGC-1alpha), a key transcriptional regulator of energy homeostasis, and causes corresponding increases in fatty acid oxidation, tricarboxylic acid cycle flux, and gluconeogenesis without increasing glycogenolysis. Mice lacking FGF21 fail to fully induce PGC-1alpha expression in response to a prolonged fast and have impaired gluconeogenesis and ketogenesis. These results reveal an unexpected relationship between FGF21 and PGC-1alpha and demonstrate an important role for FGF21 in coordinately regulating carbohydrate and fatty acid metabolism during the progression from fasting to starvation.

Regulation of serum 1,25(OH)2 vitamin D3 levels by fibroblast growth factor 23 is mediated by FGF receptors 3 and 4.

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone implicated in the pathogenesis of several hypophosphatemic disorders. FGF23 causes hypophosphatemia by decreasing the expression of sodium phosphate cotransporters (NaPi-2a and NaPi-2c) and decreasing serum 1,25(OH)(2)Vitamin D(3) levels. We previously showed that FGFR1 is the predominant receptor for the hypophosphatemic actions of FGF23 by decreasing renal NaPi-2a and 2c expression while the receptors regulating 1,25(OH)(2)Vitamin D(3) levels remained elusive. To determine the FGFRs regulating 1,25(OH)(2)Vitamin D(3) levels, we studied FGFR3(-/-)FGFR4(-/-) mice as these mice have shortened life span and are growth retarded similar to FGF23(-/-) and Klotho(-/-) mice. Baseline serum 1,25(OH)(2)Vitamin D(3) levels were elevated in the FGFR3(-/-)FGFR4(-/-) mice compared with wild-type mice (102.2 ± 14.8 vs. 266.0 ± 34.0 pmol/l; P = 0.001) as were the serum levels of FGF23. Administration of recombinant FGF23 had no effect on serum 1,25(OH)(2)Vitamin D(3) in the FGFR3(-/-)FGFR4(-/-) mice (173.4 ± 32.7 vs. 219.7 ± 56.5 pmol/l; vehicle vs. FGF23) while it reduced serum 1,25(OH)(2)Vitamin D(3) levels in wild-type mice. Administration of FGF23 to FGFR3(-/-)FGFR4(-/-) mice resulted in a decrease in serum parathyroid hormone (PTH) levels and an increase in serum phosphorus levels mediated by increased renal phosphate reabsorption. These data indicate that FGFR3 and 4 are the receptors that regulate serum 1,25(OH)(2)Vitamin D(3) levels in response to FGF23. In addition, when 1,25(OH)(2)Vitamin D(3) levels are not affected by FGF23, as in FGFR3(-/-)FGFR4(-/-) mice, a reduction in PTH can override the effects of FGF23 on renal phosphate transport.

A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis.

Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia due to inactivating mutations in FGF23 or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). Herein we report a homozygous missense mutation (H193R) in the KLOTHO (KL) gene of a 13-year-old girl who presented with severe tumoral calcinosis with dural and carotid artery calcifications. This patient exhibited defects in mineral ion homeostasis with marked hyperphosphatemia and hypercalcemia as well as elevated serum levels of parathyroid hormone and FGF23. Mapping of H193R mutation onto the crystal structure of myrosinase, a plant homolog of KL, revealed that this histidine residue was at the base of the deep catalytic cleft and mutation of this histidine to arginine should destabilize the putative glycosidase domain (KL1) of KL, thereby attenuating production of membrane-bound and secreted KL. Indeed, compared with wild-type KL, expression and secretion of H193R KL were markedly reduced in vitro, resulting in diminished ability of FGF23 to signal via its cognate FGF receptors. Taken together, our findings provide what we believe to be the first evidence that loss-of-function mutations in human KL impair FGF23 bioactivity, underscoring the essential role of KL in FGF23-mediated phosphate and vitamin D homeostasis in humans.

The parathyroid is a target organ for FGF23 in rats.

Phosphate homeostasis is maintained by a counterbalance between efflux from the kidney and influx from intestine and bone. FGF23 is a bone-derived phosphaturic hormone that acts on the kidney to increase phosphate excretion and suppress biosynthesis of vitamin D. FGF23 signals with highest efficacy through several FGF receptors (FGFRs) bound by the transmembrane protein Klotho as a coreceptor. Since most tissues express FGFR, expression of Klotho determines FGF23 target organs. Here we identify the parathyroid as a target organ for FGF23 in rats. We show that the parathyroid gland expressed Klotho and 2 FGFRs. The administration of recombinant FGF23 led to an increase in parathyroid Klotho levels. In addition, FGF23 activated the MAPK pathway in the parathyroid through ERK1/2 phosphorylation and increased early growth response 1 mRNA levels. Using both rats and in vitro rat parathyroid cultures, we show that FGF23 suppressed both parathyroid hormone (PTH) secretion and PTH gene expression. The FGF23-induced decrease in PTH secretion was prevented by a MAPK inhibitor. These data indicate that FGF23 acts directly on the parathyroid through the MAPK pathway to decrease serum PTH. This bone-parathyroid endocrine axis adds a new dimension to the understanding of mineral homeostasis.

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.

Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members.

Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/betaKlotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between beta strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the beta1-beta2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/betaKlotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors.

FGF23 decreases renal NaPi-2a and NaPi-2c expression and induces hypophosphatemia in vivo predominantly via FGF receptor 1.

Fibroblast growth factor-23 (FGF23) is a phosphaturic hormone that contributes to several hypophosphatemic disorders by reducing the expression of the type II sodium-phosphate cotransporters (NaPi-2a and NaPi-2c) in the kidney proximal tubule and by reducing serum 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] levels. The FGF receptor(s) mediating the hypophosphatemic action of FGF23 in vivo have remained elusive. In this study, we show that proximal tubules express FGFR1, -3, and -4 but not FGFR2 mRNA. To determine which of these three FGFRs mediates FGF23's hypophosphatemic actions, we characterized phosphate homeostasis in FGFR3(-/-) and FGFR4(-/-) null mice, and in conditional FGFR1(-/-) mice, with targeted deletion of FGFR1 expression in the metanephric mesenchyme. Basal serum phosphorus levels and renal cortical brush-border membrane (BBM) NaPi-2a and NaPi-2c expression were comparable between FGFR1(-/-), FGFR3(-/-), and FGFR4(-/-) mice and their wild-type counterparts. Administration of FGF23 to FGFR3(-/-) mice induced hypophosphatemia in these mice (8.0 +/- 0.4 vs. 5.4 +/- 0.3 mg/dl; p < or = 0.001) and a decrease in renal BBM NaPi-2a and NaPi-2c protein expression. Similarly, in FGFR4(-/-) mice, administration of FGF23 caused a small but significant decrease in serum phosphorus levels (8.7 +/- 0.3 vs. 7.6 +/- 0.4 mg/dl; p < or = 0.001) and in renal BBM NaPi-2a and NaPi-2c protein abundance. In contrast, injection of FGF23 into FGFR1(-/-) mice had no effects on serum phosphorus levels (5.6 +/- 0.3 vs. 5.2 +/- 0.5 mg/dl) or BBM NaPi-2a and NaPi-2c expression. These data show that FGFR1 is the predominant receptor for the hypophosphatemic action of FGF23 in vivo, with FGFR4 likely playing a minor role.

A G protein-coupled, IP3/protein kinase C pathway controlling the synthesis of phosphaturic hormone FGF23.

Dysregulated actions of bone-derived phosphaturic hormone fibroblast growth factor 23 (FGF23) result in several inherited diseases, such as X-linked hypophosphatemia (XLH), and contribute substantially to the mortality in kidney failure. Mechanisms governing FGF23 production are poorly defined. We herein found that ablation of the Gq/11α-like, extralarge Gα subunit (XLαs), a product of GNAS, exhibits FGF23 deficiency and hyperphosphatemia in early postnatal mice (XLKO). FGF23 elevation in response to parathyroid hormone, a stimulator of FGF23 production via cAMP, was intact in XLKO mice, while skeletal levels of protein kinase C isoforms α and δ (PKCα and PKCδ) were diminished. XLαs ablation in osteocyte-like Ocy454 cells suppressed the levels of FGF23 mRNA, inositol 1,4,5-trisphosphate (IP3), and PKCα/PKCδ proteins. PKC activation in vivo via injecting phorbol myristate acetate (PMA) or by constitutively active Gqα-Q209L in osteocytes and osteoblasts promoted FGF23 production. Molecular studies showed that the PKC activation-induced FGF23 elevation was dependent on MAPK signaling. The baseline PKC activity was elevated in bones of Hyp mice, a model of XLH. XLαs ablation significantly, but modestly, reduced serum FGF23 and elevated serum phosphate in Hyp mice. These findings reveal a potentially hitherto-unknown mechanism of FGF23 synthesis involving a G protein-coupled IP3/PKC pathway, which may be targeted to fine-tune FGF23 levels.