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Molecular mechanisms transducing physical forces in the bone microenvironment to regulate bone mass are poorly understood. Here, we used mouse genetics, mechanical loading, and pharmacological approaches to test the possibility that polycystin-1 and TAZ have interdependent mechanosensing functions in osteoblasts. We created and compared the skeletal phenotypes of control Pkd1flox/+;TAZflox/+, single Pkd1Oc-cKO, single TAZOc-cKO, and double Pkd1/TAZOc-cKO mice to investigate genetic interactions. Consistent with an interaction between polycystins and TAZ in bone in vivo, double Pkd1/TAZOc-cKO mice exhibited greater reductions of BMD and periosteal MAR than either single TAZOc-cKO or Pkd1Oc-cKO mice. Micro-CT 3D image analysis indicated that the reduction in bone mass was due to greater loss in both trabecular bone volume and cortical bone thickness in double Pkd1/TAZOc-cKO mice compared to either single Pkd1Oc-cKO or TAZOc-cKO mice. Double Pkd1/TAZOc-cKO mice also displayed additive reductions in mechanosensing and osteogenic gene expression profiles in bone compared to single Pkd1Oc-cKO or TAZOc-cKO mice. Moreover, we found that double Pkd1/TAZOc-cKO mice exhibited impaired responses to tibia mechanical loading in vivo and attenuation of load-induced mechanosensing gene expression compared to control mice. Finally, control mice treated with a small molecule mechanomimetic MS2 had marked increases in femoral BMD and periosteal MAR compared to vehicle control. In contrast, double Pkd1/TAZOc-cKO mice were resistant to the anabolic effects of MS2 that activates the polycystin signaling complex. These findings suggest that PC1 and TAZ form an anabolic mechanotransduction signaling complex that responds to mechanical loading and serve as a potential novel therapeutic target for treating osteoporosis.

Plasma membrane sodium–hydrogen exchangers (NHE) transport Na+ into cells in exchange for H+. While there are nine isoforms of NHE in humans, this review focuses on the NHE3 isoform, which is abundantly expressed in the gastrointestinal tract, where it plays a key role in acid–base balance and water homeostasis. NHE3 inhibition in the small intestine results in luminal sodium and water retention, leading to a general decrease in paracellular water flux and diffusional driving force, reduced intestinal sodium absorption, and increased stool sodium excretion. The resulting softer and more frequent stools are the rationale for the development of tenapanor as a novel, first-in-class NHE3 inhibitor to treat irritable bowel syndrome with constipation. NHE3 also has additional therapeutic implications in nephrology. Inhibition of intestinal NHE3 also lowers blood pressure by reducing intestinal sodium absorption. Perhaps, the most novel effect is its ability to decrease intestinal phosphate absorption by inhibiting the paracellular phosphate absorption pathway. Therefore, selective pharmacological inhibition of NHE3 could be a potential therapeutic strategy to treat not only heart failure and hypertension but also hyperphosphatemia. This review presents an overview of the molecular and physiological functions of NHE3 and discusses how these functions translate to potential clinical applications in nephrology.

Abstract:: Osteoporosis is a pathological loss of bone mass due to an imbalance in bone remodeling where osteoclast-mediated bone resorption exceeds osteoblast-mediated bone formation resulting in skeletal fragility and fractures. Anti-resorptive agents, such as bisphosphonates and SERMs, and anabolic drugs that stimulate bone formation, including PTH analogues and sclerostin inhibitors, are current treatments for osteoporosis. Despite their efficacy, severe side effects and loss of potency may limit the long term usage of a single drug. Sequential and combinational use of current drugs, such as switching from an anabolic to an anti-resorptive agent, may provide an alternative approach. Moreover, there are novel drugs being developed against emerging new targets such as Cathepsin K and 17β-HSD2 that may have less side effects. This review will summarize the molecular mechanisms of osteoporosis, current drugs for osteoporosis treatment, and new drug development strategies.

GPRC6A is a member of the Family C G-protein coupled receptors that is activated by cations, L-amino acids, the osteocalcin (Ocn) peptide, and testosterone. GPRC6A functions as a master regulator of energy metabolism and sex hormone production. Based on homology to the related receptors mGluR5 and CaSR, GPRC6A’s multiple ligand specificity is likely based on an orthosteric ligand binding site in the bilobed Venus fly trap (VFT) domain together with two positive allosteric modulator (PAM) sites, one in the VFT and the other in the 7TM domain. Here, we show that Ocn acts as a PAM for GPRC6A by binding to a site in the VFT that is distinct from the orthosteric site for calcium and L-amino acids. In agreement with this finding, alternatively spliced GPRC6A isoforms 2 and 3, which lack regions of the VFT, and mutations in the predicted Ocn binding site, K352E and H355P, prevent Ocn activation of GPRC6A. These observations provide a structural framework for understanding the ability of multiple distinct classes of compounds to activate GPRC6A and set the stage to develop novel small molecules to activate and inhibit this receptor.

There is controversy regarding whether excess FGF23 causes left ventricular hypertrophy (LVH) directly through activation of fibroblast growth factor receptor 4 (FGFR4) in cardiomyocytes or indirectly through reductions in soluble Klotho (sK). We investigated the respective roles of myocardial FGFR4 and sKL in mediating FGF23-induced LVH using mouse genetic and pharmacological approaches. To investigate a direct role of myocardial FGFR4 in mediating the cardiotoxic effects of excess circulating FGF23, we administered rFGF23 to mice with cardiac-specific loss of FGFR4 (FGFR4 heart-cKO). We tested a model of sKL deficiency, hypertension and LVH created by the conditional deletion of FGFR1 in the renal distal tubule (FGFR1DT cKO mice). The cardioprotective effects of sKL in both mouse models was assessed by the systemic administration of recombinant sKL. We confirmed that FGF23 treatment activates PLCγ in the heart and induces LVH in the absence of membrane α-Klotho. Conditional deletion of FGFR4 in the myocardium prevented rFGF23-induced LVH in mice, establishing direct cardiotoxicity of FGF23 through activation of FGFR4. Recombinant sKL administration prevented LVH, but not HTN, in FGFR1DT cKO mice, consistent with direct cardioprotective effects. Co-administration of recombinant sKL with FGF23 in culture inhibited rFGF23-induced p-PLCγ signaling. Thus, FGF23 ability to include LVH represents a balance between FGF23 direct cardiac activation of FGFR4 and the modulating effects of circulating sKL to alter FGF23-dependent myocardial signaling pathways.

A new schema proposes that the bone-derived osteocalcin (Ocn) peptide hormone activates the G-protein–coupled receptor GPRC6A to directly regulate glucose and fat metabolism in liver, muscle, and fat, and to stimulate the release of metabolism-regulating hormones, including insulin, fibroblast growth factor 21, glucagon-like peptide 1, testosterone, and interleukin 6. Ocn/GPRC6A activation has also been implicated in cancer progression. GPRC6A is activated by cations, amino acids, and testosterone. The multiligand specificity, the regulation of energy metabolism in diverse tissues, and the coordinated release of metabolically active hormones make the GPRC6A endocrine networks unique. Recently, the significance of Ocn/GPRCA has been questioned. There is a lack of metabolic abnormalities in newly created genetically engineered Ocn- and Gprc6a-deficient mouse models. There are also paradoxical observations that GPRC6A may function as a tumor suppressor. In addition, discordant published studies have cast doubt on the function of the most prevalent uniquely human GPRC6A-KGKY polymorphism. Explanations for these divergent findings are elusive. We provide evidence that the metabolic susceptibility of genetically engineered Ocn- and Gprc6a-deficient mice is influenced by environmental challenges and genetic differences in mouse strains. In addition, the GPRC6A-KGKY polymorphism appears to be a gain-of-function variant. Finally, alternatively spliced isoforms of GPRC6A may alter ligand specificity and signaling that modulate oncogenic effects. Thus, genetic, post-translational and environmental factors likely account for the variable results regarding the functions of GPRC6A in animal models. Pending additional information, GPRC6A should remain a potential therapeutic target for regulating energy and fat metabolism, hormone production, and cancer progression.

Fibroblast growth factor 23 (FGF23) is a therapeutic target for treating hereditary and acquired hypophosphatemic disorders, such as X-linked hypophosphatemic (XLH) rickets and tumor-induced osteomalacia (TIO), respectively. FGF23-induced hypophosphatemia is mediated by signaling through a ternary complex formed by FGF23, FGF receptor (FGFR), and alpha-Klotho. Currently, disorders of excess FGF23 are treated with an FGF23-blocking antibody, Burosumab. Small-molecule drugs that disrupt protein:protein interactions necessary for the ternary complex formation offer an alternative to disrupt FGF23 signaling. In this study, the FGF23:alpha-Klotho interface was targeted to identify small-molecule protein:protein interaction inhibitors. We computationally identified “hot spots” in the FGF23:alpha-Klotho interface of the ternary complex and performed in silico docking of ~5.5 million compounds from the ZINC database to the interface region of alpha-Klotho from the ternary crystal structure. Following docking, 23 and 18 compounds were chosen based on the lowest binding free energies to alpha-Klotho and the largest number of contacts with Tyr433, a predicted hot spot, respectively. 5 compounds available were assessed experimentally by their FGF23-mediated extracellular signal-regulated kinase (ERK) activities in vitro, and two of these reduce activities significantly. Both these compounds have a favorable predicted binding affinity, but not a large number of contacts with the hot spot residues. ZINC12409120 was found experimentally to reduce FGF23-mediated ERK activities by 70% and have a half maximal inhibitory concentration (IC50) of 5.0 ± 0.23 uM. ZINC12409120 exhibits contacts with residues on KL1 and KL2 domains and on the linker between the two domains of alpha-Klotho in in silico binding poses, thereby possibly disrupting the regular function of alpha-Klotho and impeding FGF23 binding. ZINC12409120 is a candidate for lead optimization.

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that binds to binary FGFR/α-Klotho receptor complexes in the kidney tubules to inhibit phosphate reabsorption and 1,25(OH)2D production. Excess FGF23 causes X-linked hypophosphatemia (XLH) and tumor induced osteomalacia (TIO). Recently, Burosumab, an FGF23 blocking antibody, was approved for treating these hypophosphatemic disorders. A small molecule inhibitor that specifically binds to FGF23 to prevent activation of FGFR/α-Klotho complexes has potential advantages over a systemically administered FGF23 blocking antibody. We previously identified the small molecule ZINC13407541 (N-[[2-(2-phenylethenyl)cyclopenten-1-yl]methylidene]hydroxylamine) as a therapeutic lead FGF23 antagonist. Additional structure-activity studies developed a series of ZINC13407541 analogues with enhanced drug-like properties. In this study, we tested in a pre-clinicalHypmouse homologue of XLH a direct connect analogue (8n) [(E)-2-(4-(tert-butyl)phenyl)cyclopent-1-ene-1-carbaldehyde oxime] that exhibited the greatest stability in microsomal assays, and13a[(E)-2-((E)-4-methylstyryl)benzaldehyde oxime] that exhibited increasedin vitropotency. We found that pharmacological inhibition of FGF23 with either of these compounds blocked FGF23 signaling and significantly increased serum phosphate and 1,25(OH)2D concentrations inHypmice. Long-term parenteral treatment with8nor13aalso enhanced linear bone growth, increased mineralization of bone, and narrowed the growth plate inHypmice. The more potent13acompound showed greater therapeutic efficacy inHypmice. Further optimization of these FGF23 inhibitors may lead to versatile drugs to treat FGF23-mediated disorders.

GPRC6A is proposed to regulate energy metabolism in mice, but in humans a KGKY polymorphism in the third intracellular loop (ICL3) is proposed to result in intracellular retention and loss-of-function. To test physiological importance of this human polymorphism in vivo, we performed targeted genomic humanization of mice by using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system to replace the RKLP sequence in the ICL3 of the GPRC6A mouse gene with the uniquely human KGKY sequence to create Gprc6a-KGKY-knockin mice. Knock-in of a human KGKY sequence resulted in a reduction in basal blood glucose levels and increased circulating serum insulin and FGF-21 concentrations. Gprc6a-KGKY-knockin mice demonstrated improved glucose tolerance, despite impaired insulin sensitivity and enhanced pyruvate-mediated gluconeogenesis. Liver transcriptome analysis of Gprc6a-KGKY-knockin mice identified alterations in glucose, glycogen and fat metabolism pathways. Thus, the uniquely human GPRC6A-KGKY variant appears to be a gain-of-function polymorphism that positively regulates energy metabolism in mice.

GPRC6A is a widely expressed G-protein coupled receptor that regulates energy metabolism. Global deletion of Gprc6a in mice is reported to result in a metabolic syndrome-like phenotype and conditional deletion of Gprc6a in pancreatic β-cell and skeletal muscle respectively impair insulin secretion and glucose uptake. In the current study, we explore the hepatic functions of GPRC6A by conditionally deleting Gprc6a in hepatocytes by cross breeding Alb-Cre and Gprc6aflox/flox mice to obtain Gprc6aLiver-cko mice. Gprc6aLiver-cko mice on a normal diet showed excessive hepatic fat accumulation and glycogen depletion. These mice also exhibit impaired glucose and pyruvate tolerance, but normal insulin sensitivity. Decreased circulating FGF-21 levels and FGF-21 message expression in the liver were found in Gprc6aLiver-cko mice. Hepatic transcriptome analysis identified alterations in multiple pathways regulating glucose, fat and glycogen metabolism in Gprc6aLiver-cko mice. Taken together, our studies suggest that GPRC6A directly regulates hepatic metabolism as well as regulates the production and release of FGF-21 to control systemic energy homeostasis. GPRC6A’s unique regulation of β-cell, skeletal muscle and hepatic function may represent a new therapeutic target for treating disordered energy metabolism metabolic syndrome and type 2 diabetes.

Background GPRC6A is implicated in the pathogenesis of prostate cancer, but its role remains uncertain because of a purported tolerant gene variant created by substitution of a K..Y polymorphism in the 3rd intracellular loop (IL) that evolved in the majority of humans and replaces the ancestral RKLP present in 40% of humans of African descent and all other species. Methods We determined whether the K..Y polymorphism is present in human-derived prostate cancer cell lines by sequencing the region of the 3rd IL and assessed the cellular localization of a “humanized” mouse GPRC6A containing the K..Y sequence by immunofluorescence. We assessed functions of GPRC6A in PC-3 cells expressing endogenous GPRC6A and in GPRC6A-deficient PC-3 cells created using CRISPR/Cas9 technology. The effect of GPRC6A on basal and ligand stimulated cell proliferation and migration was evaluated in vitro in wild-type and PC-3-deficient cell lines. The effect of editing GPRC6A on prostate cancer growth and progression in vivo was assessed in a Xenograft mouse model implanted with wild-type and PC-3 deficient cells and treated with the GPRC6A ligand osteocalcin. Results We found that all of the human prostate cancer cell lines tested endogenously express the “K..Y” polymorphism in the 3rd IL. Comparison of mouse wild-type GPRC6A with a “humanized” mouse GPRC6A construct created by replacing the “RKLP” with the “K..Y” sequence, found that both receptors were predominantly expressed on the cell surface. The transfected “humanized” GPRC6A receptor, however, preferentially activated mTOR compared to ERK signaling in HEK-293 cells. In contrast, in PC-3 cells expressing the endogenous GPRC6A with the “K..Y” polymorphism, the ligand osteocalcin stimulated ERK, AKT and mTOR phosphorylation, promoted cell proliferation and migration, and upregulated genes regulating testosterone biosynthesis. Targeting GPRC6A in PC-3 cells by CRISPR/Cas9 significantly blocked these responses in vitro. In addition, GPRC6A deficient PC-3 xenografts exhibited significantly less growth and were resistant to osteocalcin-induced prostate cancer progression compared to control PC-3 cells expressing GPRC6A. Conclusions Human GPRC6A is a functional osteocalcin and testosterone sensing receptor that promotes prostate cancer progression. GPRC6A may contribute to racial disparities in prostate cancer, and is a potential therapeutic target to develop antagonists to treat prostate cancer. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0561-x) contains supplementary material, which is available to authorized users.

We show that GPRC6A, an osteocalcin-sensing G-protein coupled receptor GPRC6A, uniquely regulates glucose and fatty acid metabolism in the liver. Conditional deletion of GPRC6A in hepatocytes results in hepatic fat accumulation and glycogen depletion associated with impaired glucose and pyruvate tolerance and suppression of FGF-21 in mice. Conversely, knock-in of a human KGKY gain-of-function polymorphism in the 3rd intracellular loop of GPRC6A, in mice results in reduced blood glucose associated with improved glucose, insulin and pyruvate tolerance and increased FGF-21. Hepatic transcriptome analysis shows that GPRC6A has counter regulatory effects on fatty acid oxidation and synthesis and glycolysis and gluconeogenesis, and concordant effects on glycogenesis and glycogenolysis. Taken together, our studies suggest that the level of GPRC6A activation determines how glucose and FAs are utilized by the liver. GPRC6A may be a new therapeutic target for treating metabolic syndrome and its complications.

Fibroblast growth factor receptor (FGFR) and α-Klotho transduce FGF-23 signaling in renal tubules to maintain systemic phosphate/vitamin D homeostasis. Mice deficient for either the ligand, FGF-23, or the co-receptor, Klotho, are phenocopies with both showing rapid and premature development of multiple aging-like abnormalities. Such similarity in phenotype, suggests that FGF-23 and Klotho have co-dependent systemic functions. Recent reports revealed inverse central nervous system (CNS) effects of Klotho deficiency or Klotho overexpression on hippocampal synaptic, neurogenic, and cognitive functions. However, it is unknown whether FGF-23 deficiency effects function of the hippocampus. We report that, similar to Klotho-deficient mice, FGF-23-deficient mice develop dose-dependent, hippocampal-dependent cognitive impairment. However, FGF-23-deficient brains had no gross structural or developmental defects, no change in hippocampal synaptic plasticity, and only minor impairment to postnatal hippocampal neurogenesis. Together, these data provide evidence that FGF-23 deficiency impairs hippocampal-dependent cognition but otherwise results in a brain phenotype that is distinct from the KL-deficient mouse.

G protein-coupled receptor family C group 6 member A (GPRC6A) is activated by testosterone and modulates prostate cancer progression. Most humans have a GPRC6A variant that contains a recently evolved KGKY insertion/deletion in the third intracellular loop (ICL3) (designated as GPRC6A(ICL3_KGKY)) that replaces the ancestral KGRKLP sequence (GPRC6A(ICL3_RKLP)) present in all other species. In vitro assays purport that human GPRC6A(ICL3_KGKY) is retained intracellularly and lacks function. These findings contrast with ligand-dependent activation and coupling to mammalian target of rapamycin complex 1 (mTORC1) signaling of endogenous human GPRC6A(ICL3_KGKY) in PC-3 cells. To understand these discrepant results, we expressed mouse (mGPRC6A(ICL3_KGRKLP)), human (hGPRC6A(ICL3_KGKY)), and humanized mouse (mGPRC6A(ICL3_KGKY)) GPRC6A into human embryonic kidney 293 cells. Our results demonstrate that mGPRC6A(ICL3_KGRKLP) acts as a classic G protein–coupled receptor, which is expressed at the cell membrane and internalizes in response to ligand activation by testosterone. In contrast, hGPRC6A(ICL3_KGKY) and humanized mouse mGPRC6A(ICL3_KGKY) are retained intracellularly in ligand naive cells, yet exhibit β-arrestin–dependent signaling responses, mitogen-activated protein kinase [i.e., extracellular signal–regulated kinase (ERK)], and p70S6 kinase phosphorylation in response to testosterone, indicating that hGPRC6A(ICL3_KGKY) is functional. Indeed, testosterone stimulates time- and dose-dependent activation of ERK, protein kinase B, and mTORC1 signaling in wild-type PC-3 cells that express endogenous GPRC6A(ICL3_KGKY). In addition, testosterone stimulates GPRC6A-dependent cell proliferation in wild-type PC-3 cells and inhibits autophagy by activating mTORC1 effectors eukaryotic translation initiation factor 4E binding protein 1 and Unc-51 like autophagy activating kinase 1. Testosterone activation of GPRC6A has the obligate requirement for calcium in the incubation media. In contrast, in GPRC6A-deficient cells, the effect of testosterone to activate downstream signaling is abolished, indicating that human GPRC6A is required for mediating the effects of testosterone on cell proliferation and autophagy.

End stage renal disease (ESRD) is characterized by complex metabolic abnormalities, yet the clinical relevance of specific biomarkers remains unclear. The development of multiplex diagnostic platforms is creating opportunities to develop novel diagnostic and therapeutic approaches. SOMAscan is an innovative multiplex proteomic platform which can measure &gt, 1300 proteins. In the present study, we performed SOMAscan analysis of plasma samples and validated the measurements by comparison with selected biomarkers. We compared concentrations of SOMAscan-measured prostate specific antigen (PSA) between males and females, and validated SOMAscan concentrations of fibroblast growth factor 23 (FGF23), FGF receptor 1 (FGFR1), and FGFR4 using Enzyme-Linked immunosorbent assay (ELISA). The median (25th and 75th percentile) SOMAscan PSA level in males and females was 4304.7 (1815.4 to 7259.5) and 547.8 (521.8 to 993.4) relative fluorescence units (p = 0.002), respectively, suggesting biological plausibility. Pearson correlation between SOMAscan and ELISA was high for FGF23 (R = 0.95, p &lt, 0.001) and FGFR4 (R = 0.69, p &lt, 0.001), indicating significant positive correlation, while a weak correlation was found for FGFR1 (R = 0.13, p = 0.16). In conclusion, there is a good to near-perfect correlation between SOMAscan and standard immunoassays for FGF23 and FGFR4, but not for FGFR1. This technology may be useful to simultaneously measure a large number of plasma proteins in ESRD, and identify clinically important prognostic markers to predict outcomes.

There is a strong association between elevated circulating fibroblast growth factor-23 (FGF23) levels and adverse outcomes in patients with chronic kidney disease (CKD) of all stages. Initially discovered as a regulator of phosphate and vitamin D homeostasis, FGF23 has now been implicated in several pathophysiological mechanisms that may negatively impact the cardiovascular and renal systems. FGF23 is purported to have direct (off-target) effects in the myocardium, as well as canonical effects on FGF receptor/α-klotho receptor complexes in the kidney to activate the renin-angiotensin-aldosterone system, modulate soluble α-klotho levels, and increase sodium retention, to cause left ventricular hypertrophy (LVH). Conversely, FGF23 could be an innocent bystander produced in response to chronic inflammation or other processes associated with CKD that cause LVH and adverse cardiovascular outcomes. Further exploration of these complex mechanisms is needed before modulation of FGF23 can become a legitimate clinical target in CKD.

Phenome-wide association is a novel reverse genetic strategy to analyze genome-to-phenome relations in human clinical cohorts. Here we test this approach using a large murine population segregating for ∼5 million sequence variants, and we compare our results to those extracted from a matched analysis of gene variants in a large human cohort. For the mouse cohort, we amassed a deep and broad open-access phenome consisting of ∼4,500 metabolic, physiological, pharmacological and behavioural traits, and more than 90 independent expression quantitative trait locus (QTL), transcriptome, proteome, metagenome and metabolome data sets—by far the largest coherent phenome for any experimental cohort (www.genenetwork.org). We tested downstream effects of subsets of variants and discovered several novel associations, including a missense mutation in fumarate hydratase that controls variation in the mitochondrial unfolded protein response in both mouse and Caenorhabditis elegans, and missense mutations in Col6a5 that underlies variation in bone mineral density in both mouse and human., Phenome-wide association is a novel method that links sequence variants to a spectrum of phenotypes and diseases. Here the authors generate detailed mouse genetic and phenome data which links their phenome-wide association study (PheWAS) of mouse to corresponding PheWAS in human.

G protein-coupled receptor (GPCR) family C group 6 member A (GPRC6A) is a multiligand GPCR that is activated by cations, L-amino acids, and osteocalcin. GPRC6A plays an important role in the regulation of testosterone (T) production and energy metabolism in mice. T has rapid, transcription-independent (nongenomic) effects that are mediated by a putative GPCR. We previously found that T can activate GPRC6A in vitro, but the possibility that T is a ligand for GPRC6A remains controversial. Here, we demonstrate direct T binding to GPRC6A and construct computational structural models of GPRC6A that are used to identify potential binding poses of T. Mutations of the predicted binding site residues were experimentally found to block T activation of GPRC6A, in agreement with the modeling. Using Gpr6ca−/− mice, we confirmed that loss of GPRC6A resulted in loss of T rapid signaling responses and elucidated several biological functions regulated by GPRC6A-dependent T rapid signaling, including T stimulation of insulin secretion in pancreatic islets and enzyme expression involved in the biosynthesis of T in Leydig cells. Finally, we identified a stereo-specific effect of an R-isomer of a selective androgen receptor modulator that is predicted to bind to and shown to activate GPRC6A but not androgen receptor. Together, our data show that GPRC6A directly mediates the rapid signaling response to T and uncovers previously unrecognized endocrine networks.

Both the activation of the renin angiotensin aldosterone system (RAAS) and elevations of circulating Fibroblast Growth Factor-23 (FGF-23) have been implicated in the pathogenesis of left ventricular hypertrophy (LVH) in chronic kidney disease. To investigate potential cross-talk between RAAS and FGF-23, we administered angiotensin II (Ang II) to wild-type rodents and the Hyp mouse model of excess FGF-23. Ang II administration for four weeks to wild-type rodents resulted in significant increases in systolic blood pressure and LVH. Unexpectedly, FGF-23 circulating levels were increased by 1.5–1.7 fold in Ang II treated animals. In addition, Ang II treatment increased expression of FGF-23 message levels in bone, the predominant tissue for FGF-23 production, and induced expression of FGF-23 and its co-receptor α-Klotho in the heart, which normally does not express FGF-23 or α-Klotho in physiologically relevant levels. Hyp mice with elevated FGF-23 exhibited increased blood pressure and LVH at baseline. Ang II administration to Hyp mice resulted further increments in blood pressure and left ventricular hypertrophy, consistent with additive cardiovascular effects. These findings suggest that FGF-23 may participate in unexpected systemic and paracrine networks regulating hemodynamic and myocardial responses.

Fibroblast growth factor-23 (FGF-23) is a bone-derived hormone that activates FGFR/α-Klotho binary complexes in the kidney renal tubules to regulate phosphate reabsorption and vitamin D metabolism. The objective of this review is to discuss the emerging data that show that FGF-23 has functions beyond regulation of mineral metabolism, including roles in innate immune and hemodynamic responses. Excess FGF-23 is associated with inflammation and adverse infectious outcomes, as well as increased morbidity and mortality, particularly in patients with chronic kidney disease. Enhancer elements in the FGF-23 promoter have been identified that mediate the effects of inflammatory cytokines to stimulate FGF-23 gene transcription in bone. In addition, inflammation induces ectopic expression of FGF-23 and α-Klotho in macrophages that do not normally express FGF-23 or its binary receptor complexes. These observations suggest that FGF-23 may play an important role in regulating innate immunity through multiple potential mechanisms. Circulating FGF-23 acts as a counter-regulatory hormone to suppress 1,25D production in the proximal tubule of the kidney. Since vitamin D deficiency may predispose infectious and cardiovascular diseases, FGF-23 effects on innate immune responses may be due to suppression of 1,25D production. Alternatively, systemic and locally produced FGF-23 may modulate immune functions through direct interactions with myeloid cells, including macrophages and polymorphonuclear leukocytes to impair immune cell functions. Short-acting small molecules that reversibly inhibit FGF-23 offer the potential to block pro-inflammatory and cardiotoxic effects of FGF-23 with less side effects compared with FGF-23 blocking antibodies that have the potential to cause hyperphosphatemia and soft tissue calcifications in animal models. In conclusion, there are several mechanisms by which FGF-23 impacts the innate immune system and further investigation is critical for the development of therapies to treat diseases associated with elevated FGF-23.

Introduction: Abnormal phosphorus homeostasis develops early in chronic kidney disease (CKD). It is unclear if its correction results in improved clinical outcomes in non–dialysis dependent CKD. Methods: We conducted a randomized controlled, parallel design clinical trial in 120 patients with estimated glomerular filtration rate 15 to 59 ml/min per 1.73 m2 and abnormal phosphorus homeostasis (serum phosphorus >4.6 mg/dl, parathyroid hormone [PTH] >70 pg/ml or tubular reabsorption of phosphorus [TRP]

The optimal circulating concentration of 25(OH) vitamin D is controversial.The aim was to investigate if FGF-23 and 24,25(OH)2D can guide cholecalciferol replacement.Oral cholecalciferol (10,000 IU weekly) administered to subjects with 25(OH)D levels20 ηg/mL and eGFR60 mL/min/1.73 m(2) (n = 25), chronic kidney disease (CKD) (n = 27), or end stage renal disease (ESRD) (n = 14).The study was conducted at the Veterans Affairs clinics.Serum FGF-23, PTH, 25(OH)D, 1,25(OH)2D, 24,25(OH)2D, calcium, and phosphorous concentrations, and urinary excretion of calcium and phosphorus at baseline and after 8 weeks of treatment.Cholecalciferol treatment increased concentrations of serum 25(OH)D by (19.3 ± 8 ηg/mL, P = .001; 12.2 ± 9 ηg/mL, P = .0001) and 24,25(OH)2D (1.14 ± 0.89 ηg/mL, P = .0024; 1.0 ± 0.72 ηg/mL P = .0002), and reduced serum PTH (-11 ± 21 pg/mL, P = .0292; -42 ± 68 pg/mL, P = .0494) in normal and CKD subjects, respectively. Cholecalciferol increased serum FGF-23 levels only in normal subjects (44 ± 57 ηg/mL, P = .01). Increments in serum 25(OH)D positively correlated with serum FGF-23 and 24,25(OH)2D and negatively correlated with PTH. In ESRD, cholecalciferol administration increased 25(OH)D by (16.6 ± 6.6 ηg/mL P ≤ .05) without changing 24,25(OH)2D, FGF-23 or PTH levels.Modest elevations of serum 25(OH)D levels after cholecalciferol treatment are sufficient to induce compensatory degradative pathways in patients with sufficient renal reserves, suggesting that optimal circulating 25(OH)D levels are approximately 20 ηg/mL. In addition, catabolism of 25(OH)D may also contribute to the low circulating vitamin D levels in CKD, since elevations of FGF-23 in CKD are associated with increased 24,25(OH)2D after cholecalciferol administration.

The bone-derived hormone fibroblast growth factor-23 (FGF-23) activates complexes composed of FGF receptors (FGFRs), including FGFR1, and α-Klotho in the kidney distal tubule (DT), leading to increased sodium retention and hypertension. However, the role of FGFR1 in regulating renal processes linked to hypertension is unclear. Here, we investigated the effects of selective FGFR1 loss in the DT. Conditional knockout (cKO) of FGFR1 in the DT (FGFR1DT-cKO mice) resulted in left ventricular hypertrophy (LVH) and decreased kidney expression of α-Klotho in association with enhanced BP, decreased expression of angiotensin converting enzyme 2, and increased expression of the Na+-K+-2Cl- cotransporter. Notably, recombinant FGF-23 administration similarly decreased the kidney expression of α-Klotho and induced LVH in mice. Pharmacologic activation of FGFR1 with a monoclonal anti-FGFR1 antibody (R1MAb1) normalized BP and significantly attenuated LVH in the Hyp mouse model of excess FGF-23, but did not induce a response in FGFR1DT-cKO mice. The hearts of FGFR1DT-cKO mice showed increased expression of the transient receptor potential cation channel, subfamily C, member 6 (TRPC6), consistent with cardiac effects of soluble Klotho deficiency. Moreover, administration of recombinant soluble Klotho lowered BP in the Hyp mice. Thus, FGFR1 in the DT regulates systemic hemodynamic responses opposite to those predicted by the actions of FGF-23. These cardiovascular effects appear to be mediated by paracrine FGF control of kidney FGFR1 and subsequent regulation of soluble Klotho and TRPC6. FGFR1 in the kidney may provide a new molecular target for treating hypertension.

The molecular mechanisms that transduce the osteoblast response to physical forces in the bone microenvironment are poorly understood. Here, we used genetic and pharmacological experiments to determine whether the polycystins PC1 and PC2 (encoded by Pkd1 and Pkd2) and the transcriptional coactivator TAZ form a mechanosensing complex in osteoblasts. Compound-heterozygous mice lacking 1 copy of Pkd1 and Taz exhibited additive decrements in bone mass, impaired osteoblast-mediated bone formation, and enhanced bone marrow fat accumulation. Bone marrow stromal cells and osteoblasts derived from these mice showed impaired osteoblastogenesis and enhanced adipogenesis. Increased extracellular matrix stiffness and application of mechanical stretch to multipotent mesenchymal cells stimulated the nuclear translocation of the PC1 C-terminal tail/TAZ (PC1-CTT/TAZ) complex, leading to increased runt-related transcription factor 2-mediated (Runx2-mediated) osteogenic and decreased PPARγ-dependent adipogenic gene expression. Using structure-based virtual screening, we identified a compound predicted to bind to PC2 in the PC1:PC2 C-terminal tail region with helix:helix interaction. This molecule stimulated polycystin- and TAZ-dependent osteoblastogenesis and inhibited adipogenesis. Thus, we show that polycystins and TAZ integrate at the molecular level to reciprocally regulate osteoblast and adipocyte differentiation, indicating that the polycystins/TAZ complex may be a potential therapeutic target to increase bone mass.

Fibroblast growth factor-23 (FGF-23) interacts with a binary receptor complex composed of α-Klotho (α-KL) and FGF receptors (FGFRs) to regulate phosphate and vitamin D metabolism in the kidney. Excess FGF-23 production, which causes hypophosphatemia, is genetically inherited or occurs with chronic kidney disease. Among other symptoms, hypophosphatemia causes vitamin D deficiency and the bone-softening disorder rickets. Current therapeutics that target the receptor complex have limited utility clinically. Using a computationally driven, structure-based, ensemble docking and virtual high-throughput screening approach, we identified four novel compounds predicted to selectively inhibit FGF-23-induced activation of the FGFR/α-KL complex. Additional modeling and functional analysis found that Zinc13407541 bound to FGF-23 and disrupted its interaction with the FGFR1/α-KL complex; experiments in a heterologous cell expression system showed that Zinc13407541 selectivity inhibited α-KL-dependent FGF-23 signaling. Zinc13407541 also inhibited FGF-23 signaling in isolated renal tubules ex vivo and partially reversed the hypophosphatemic effects of excess FGF-23 in a mouse model. These chemical probes provide a platform to develop lead compounds to treat disorders caused by excess FGF-23.

Serum levels of fibroblast growth factor 23 (FGF23) markedly increase with renal impairment, with FGF23 levels correlating with the presence of left ventricular hypertrophy (LVH) and mortality in patients with chronic kidney disease (CKD). FGF23 activates calcineurin/nuclear factor of activated T cell (NFAT) signaling and induces hypertrophy in murine cardiomyocytes. X-linked hypophosphatemia (XLH) is characterized by high circulating levels of FGF23 but, in contrast to CKD, is associated with hypophosphatemia. The cardiac effects of high circulating levels of FGF23 in XLH are not well defined. Thus, studies were undertaken to define the cardiac phenotype in the mouse model of XLH (Hyp mice). Echocardiographic and histological analyses demonstrated that Hyp left ventricles (LVs) are smaller than those of wild-type mice. Messenger RNA expression of cardiac hypertrophy markers was not altered in the LV or right ventricle of Hyp mice. However, the Hyp LVs had increased expression of the NFAT target genes NFATc1 and RCAN1. To determine whether phosphate alone can induce markers of hypertrophy, differentiated C2C12 myocytes were treated with phosphate. Phosphate treatment increased expression of cardiac hypertrophy markers, supporting a primary role for phosphate in inducing LVH. Although previous studies showed that increased circulating FGF23 and phosphate levels are associated with LVH, our results demonstrated that in XLH, high circulating levels of FGF23 in the setting of hypophosphatemia do not induce cardiac hypertrophy.

Calcium has recently been shown to regulate fibroblast growth factor 23 (FGF-23), a bone-derived phosphate and vitamin D-regulating hormone. To better understand the regulation of FGF-23 by calcium, phosphorus, 1,25 dihydroxyvitamin D3 [1,25(OH)2D], and PTH, we examined FGF-23 expression under basal conditions and in response to PTH, doxercalciferol, or high-calcium diet treatment in Gcm2−/− and Cyp27b1−/− mutant mice. Gcm2−/− mice exhibited low serum PTH and 1,25(OH)2D concentrations, hypocalcemia, and hyperphosphatemia, whereas Cyp27b1−/− mice had high PTH, undetectable 1,25(OH)2D, hypocalcemia, and hypophosphatemia. Serum FGF-23 levels were decreased in both mutant models. Doxercalciferol administration increased serum FGF-23 levels in both mutant models. PTH administration to Gcm2−/− mice also increased serum FGF-23 levels, in association with an increase in both 1,25(OH)2D and calcium concentrations. Multiple regression analysis of pooled data indicated that changes in FGF-23 were positively correlated with serum calcium and 1,25(OH)2D but not related to changes in serum phosphate concentrations. A high-calcium diet also increased serum FGF-23 concentrations in Cyp27b1−/− mice in the absence of 1,25(OH)2D and in Gcm2−/− mice with low PTH. The addition of calcium to the culture media also stimulated FGF-23 message expression in MC3T3-E1 osteoblasts. In addition, FGF-23 promoter activity in cultured osteoblasts was inhibited by the L-calcium-channel inhibitor nifedipine and stimulated by calcium ionophores. The effects of chronic low calcium to prevent 1,25(OH)2D and PTH stimulation of FGF-23 in these mutant mouse models suggest that suppression of FGF-23 plays an important physiological adaptive response to hypocalcemia.

25-Hydroxyvitamin D [25(OH)D] is a marker of nutritional status; however, chronic kidney disease (CKD) results in alterations in vitamin D metabolism, including the loss of vitamin D-binding proteins and alterations in CYP27B1 and CYP24 enzymes that metabolize 25(OH)D. This study was designed to determine the predictors of responsiveness to correction of vitamin D deficiency with oral vitamin D2 (ergocalciferol) in adults.A retrospective study of 183 veterans with 25(OH)D level30 ng/mL, who were treated with 50,000 IU per week of vitamin D2, was performed. Logistic regression models were developed to determine the factors predicting the response to treatment, defined as either the change in serum 25(OH)D level/1000 IU of vitamin D2 or the number of vitamin D2 doses (50,000 IU per dose) administered.The mean age of the patients was 63 ± 12 years. About 87% were men and 51% diabetic, and 29% had an estimated glomerular filtration rate of60 mL/min/1.73 m. The average number of vitamin D2 doses was 10.91 ± 5.95; the average increase in 25(OH)D level was 18 ± 10.80 ng/mL. 25(OH)D levels remained30 ng/mL in 61 patients after treatment. A low estimated glomerular filtration rate and the presence of diabetes mellitus were significant independent predictors for inadequate response to vitamin D2 treatment in logistic regression models. Patients with CKD required greater amounts of vitamin D2 to achieve similar increases in 25(OH)D levels, versus non-CKD patients.The presence of CKD and diabetes mellitus is associated with resistance to correction of 25(OH)D deficiency with vitamin D2 therapy. The underlying mechanism needs to be evaluated in prospective studies.

There are emerging data that the skeleton is connected to systemic biological functions through the release of two osteoblast-/osteocyte-derived hormones, fibroblastic growth factor 23 (FGF23) and undercarboxylated osteocalcin (Ocn). FGF23 is important in the regulation of phosphate and vitamin D metabolism, whereas Ocn participates in endocrine networks, coordinating bone and fat mass, energy metabolism, and sex hormone production. Bone remodeling and mineralization per se, along with the hormones leptin, insulin, glucocorticoids, PTH, and 1,25(OH)2D, regulate the release of FGF23 and Ocn, leading to complex cross-talk and coordination between endocrine networks previously thought to be distinct. These pathways are particularly important in chronic kidney disease, in which both FGF23 and Ocn are increased. Although these hormones initially serve an adaptive role, with progressive loss of renal function they show maladaptive effects, particularly on the cardiovascular system, through multiple mechanisms, including possible cross-talk with the renin angiotensin system. The complex interconnections between the various endocrine networks in chronic kidney disease may account for the difficulty in treating the uremic state.

Prostate cancer is the most commonly diagnosed cancer among men in the developed countries.(1) One in six males in the U.S.(2) and one in nine males in the U.K.(3) will develop the disease at some point during their lifetime. Despite advances in prostate cancer screening, more than a quarter of a million men die from the disease every year(1) due primarily to treatment-resistance and metastasis. Colloidal nanotechnologies can provide tremendous enhancements to existing targeting/treatment strategies for prostate cancer to which malignant cells are less sensitive. Here, we show that antiandrogen gold nanoparticles--multivalent analogues of antiandrogens currently used in clinical therapy for prostate cancer--selectively engage two distinct receptors, androgen receptor (AR), a target for the treatment of prostate cancer, as well as a novel G-protein coupled receptor, GPRC6A, that is also upregulated in prostate cancer. These nanoparticles selectively accumulated in hormone-insensitive and chemotherapy-resistant prostate cancer cells, bound androgen receptor with multivalent affinity, and exhibited greatly enhanced drug potency versus monovalent antiandrogens currently in clinical use. Further, antiandrogen gold nanoparticles selectively stimulated GPRC6A with multivalent affinity, demonstrating that the delivery of nanoscale antiandrogens can also be facilitated by the transmembrane receptor in order to realize increasingly selective, increasingly potent therapy for treatment-resistant prostate cancers.

The discovery of fibroblast growth factor 23 (FGF-23) has expanded our understanding of phosphate and vitamin D homeostasis and provided new insights into the pathogenesis of hereditary hypophosphatemic and hyperphosphatemic disorders, as well as acquired disorders of phosphate metabolism, such as chronic kidney disease. FGF-23 is secreted by osteoblasts and osteocytes in bone and principally targets the kidney to regulate the reabsorption of phosphate, the production and catabolism of 1,25-dihydroxyvitamin D and the expression of α-Klotho, an anti-ageing hormone. Secreted FGF-23 plays a central role in complex endocrine networks involving local bone-derived factors that regulate mineralization of extracellular matrix and systemic hormones involved in mineral metabolism. Inactivating mutations of PHEX, DMP1 and ENPP1, which cause hereditary hypophosphatemic disorders and primary defects in bone mineralization, stimulate FGF23 gene transcription in osteoblasts and osteocytes, at least in part, through canonical and intracrine FGF receptor pathways. These FGF-23 regulatory pathways may enable systemic phosphate and vitamin D homeostasis to be coordinated with bone mineralization. FGF-23 also functions as a counter-regulatory hormone for 1,25-dihydroxyvitamin D in a bone-kidney endocrine loop. FGF-23, through regulation of additional genes in the kidney and extrarenal tissues, probably has broader physiological functions beyond regulation of mineral metabolism that account for the association between FGF-23 and increased mortality and morbidity in chronic kidney disease.

Calcium (Ca2+) and phosphate (PO43−) homeostasis are coordinated by systemic and local factors that regulate intestinal absorption, influx and efflux from bone, and kidney excretion and reabsorption of these ions through a complex hormonal network. Traditionally, the parathyroid hormone (PTH)/vitamin D axis provided the conceptual framework to understand mineral metabolism. PTH secreted by the parathyroid gland in response to hypocalcemia functions to maintain serum Ca2+levels by increasing Ca2+reabsorption and 1,25-dihydroxyvitamin D [1,25(OH)2D] production by the kidney, enhancing Ca2+and PO43−intestinal absorption and increasing Ca2+and PO43−efflux from bone, while maintaining neutral phosphate balance through phosphaturic effects. FGF23 is a recently discovered hormone, predominately produced by osteoblasts/osteocytes, whose major functions are to inhibit renal tubular phosphate reabsorption and suppress circulating 1,25(OH)2D levels by decreasing Cyp27b1-mediated formation and stimulating Cyp24-mediated catabolism of 1,25(OH)2D. FGF23 participates in a new bone/kidney axis that protects the organism from excess vitamin D and coordinates renal PO43−handling with bone mineralization/turnover. Abnormalities of FGF23 production underlie many inherited and acquired disorders of phosphate homeostasis. This review discusses the known and emerging functions of FGF23, its regulation in response to systemic and local signals, as well as the implications of FGF23 in different pathological and physiological contexts.

A bone-pancreas endocrine loop has been identified recently that involves insulin secreted from β-cells in the pancreas stimulating insulin receptors in osteoblasts, leading to osteoblastic differentiation and increased secretion of osteocalcin (Ocn), a bone-derived hormone that regulates insulin secretion in β-cells. The identity of the Ocn-sensing receptor in β-cells is a missing component of this endocrine loop. The abnormalities in glucose homeostasis in Gprc6a null mice suggests that this pertussis toxin–sensitive G protein–coupled receptor is a candidate for mediating the effects of Ocn on insulin secretion in the pancreas. In support of this possibility, we found that transfection of non-Gprc6a-expressing HEK-293 cells with a full-length Gprc6a cDNA imparted a dose-dependent response to Ocn (5 to 60 ng/mL), as measured by PKD1 and ERK phosphorylation. In addition, Gprc6a is highly expressed in mouse pancreatic tissue and in the mouse TC-6 pancreatic β-cell line. Ocn also stimulated ERK activity in TC-6 pancreatic β-cells. Finally, intraperitoneal injection of Ocn stimulated ERK activity in the pancreas and increased serum insulin levels in wild-type mice, but these responses were markedly attenuated in Gprc6a−/− mice. These findings suggest that GPRC6A is a candidate for mediating the response to Ocn in the bone-pancreas endocrine loop regulating insulin signaling. © 2011 American Society for Bone and Mineral Research.

Fibroblastic growth factor 23 (FGF23) is a circulating phosphaturic hormone. Inactivating mutations of the endopeptidase PHEX or the SIBLING protein DMP1 result in equivalent intrinsic bone mineralization defects and increased Fgf23 expression in osteocytes. The mechanisms whereby PHEX and DMP1 regulate Fgf23 expression are unknown. We examined the possibility that PHEX and DMP1 regulate Fgf23 through a common pathway by analyzing the phenotype of compound Phex and Dmp1 mutant mice (Hyp/Dmp1−/−). Compared to single-mutant littermates, compound-mutant Hyp/Dmp1−/− mice displayed nonadditive elevations of serum FGF23 (1912 ± 183, 1715 ± 178, and 1799 ± 181 pg/ml), hypophosphatemia (Pi: 6.0 ± 0.3, 5.8 ± 0.2, and 5.4 ± 0.1 mg/dl), and severity of rickets/osteomalacia (bone mineral density: −36, −36, and −30%). Microarray analysis of long bones identified gene expression profiles implicating common activation of the FGFR pathway in all the mutant groups. Furthermore, inhibiting FGFR signaling using SU5402 in Hyp- and Dmp1−/−-derived bone marrow stromal cells prevented the increase in Fgf23 mRNA expression (129- and 124-fold increase in Hyp and Dmp1−/− vs. 1.3-fold in Hyp+SU5402 and 2.5-fold in Dmp1−/−+SU5402, P

We investigated whether polycystin-1 is a bone mechanosensor. We conditionally deleted Pkd1 in mature osteoblasts/osteocytes by crossing Dmp1-Cre with Pkd1flox/m1Bei mice, in which the m1Bei allele is nonfunctional. We assessed in wild-type and Pkd1-deficient mice the response to mechanical loading in vivo by ulna loading and ex vivo by measuring the response of isolated osteoblasts to fluid shear stress. We found that conditional Pkd1 heterozygotes (Dmp1-Cre;Pkd1flox/+) and null mice (Pkd1Dmp1-cKO) exhibited a ∼40 and ∼90% decrease, respectively, in functional Pkd1 transcripts in bone. Femoral bone mineral density (12 vs. 27%), trabecular bone volume (32 vs. 48%), and cortical thickness (6 vs. 17%) were reduced proportionate to the reduction of Pkd1 gene dose, as were mineral apposition rate (MAR) and expression of Runx2-II, Osteocalcin, Dmp1, and Phex. Anabolic load-induced periosteal lamellar MAR (0.58±0.14; Pkd1Dmp1-cKO vs. 1.68±0.34 μm/d; control) and increases in Cox-2, c-Jun, Wnt10b, Axin2, and Runx2-II gene expression were significantly attenuated in Pkd1Dmp1-cKO mice compared with controls. Application of fluid shear stress to immortalized osteoblasts from Pkd1null/null and Pkd1m1Bei/m1Bei-derived osteoblasts failed to elicit the increments in cytosolic calcium observed in wild-type controls. These data indicate that polycystin-1 is essential for the anabolic response to skeletal loading in osteoblasts/osteocytes.—Xiao, Z., Dallas, M., Qiu, N., Nicolella, D., Cao, L., Johnson, M., Bonewald, L., Quarles, L. D. Conditional deletion of Pkd1 in osteocytes disrupts skeletal mechanosensing in mice.

Uncertainty exists regarding the physiologically relevant fibroblast growth factor (FGF) receptor (FGFR) for FGF23 in the kidney and the precise tubular segments that are targeted by FGF23. Current data suggest that FGF23 targets the FGFR1c-Klotho complex to coordinately regulate phosphate transport and 1,25-dihydroxyvitamin D [1,25(OH)2D] production in the proximal tubule. In studies using the Hyp mouse model, which displays FGF23-mediated hypophosphatemia and aberrant vitamin D, deletion of Fgfr3 or Fgfr4 alone failed to correct the Hyp phenotype. To determine whether FGFR1 is sufficient to mediate the renal effects of FGF23, we deleted Fgfr3 and Fgfr4 in Hyp mice, leaving intact the FGFR1 pathway by transferring compound Fgfr3/Fgfr4-null mice on the Hyp background to create wild-type (WT), Hyp, Fgfr3−/−/Fgfr4−/−, and Hyp/Fgfr3−/−/Fgfr4−/−mice. We found that deletion of Fgfr3 and Fgfr4 in Fgfr3−/−/Fgfr4−/−and Hyp/Fgfr3−/−/Fgfr4−/−mice induced an increase in 1,25(OH)2D. In Hyp/Fgfr3−/−/Fgfr4−/−mice, it partially corrected the hypophosphatemia (Pi= 9.4 ± 0.9, 6.1 ± 0.2, 9.1 ± 0.4, and 8.0 ± 0.5 mg/dl in WT, Hyp, Fgfr3−/−/Fgfr4−/−, and Hyp/Fgfr3−/−/Fgfr4−/−mice, respectively), increased Na-phosphate cotransporter Napi2a and Napi2c and Klotho mRNA expression in the kidney, and markedly increased serum FGF23 levels (107 ± 20, 3,680 ± 284, 167 ± 22, and 18,492 ± 1,547 pg/ml in WT, Hyp, Fgfr3−/−/Fgfr4−/−, and Hyp/Fgfr3−/−/Fgfr4−/−mice, respectively), consistent with a compensatory response to the induction of end-organ resistance. Fgfr1 expression was unchanged in Hyp/Fgfr3−/−/Fgfr4−/−mice and was not sufficient to transduce the full effects of FGF23 in Hyp/Fgfr3−/−/Fgfr4−/−mice. These studies suggest that FGFR1, FGFR3, and FGFR4 act in concert to mediate FGF23 effects on the kidney and that loss of FGFR function leads to feedback stimulation of Fgf23 expression in bone.

Runx2 is a transcription factor controlling skeletal development, and is also expressed in extraskeletal tissues where its function is not well understood. Existing Runx2 mutant and transgenic mouse models do not allow the necessary control of Runx2 expression to understand its functions in different tissues. We generated conditional, doxycyline-inducible, triple transgenic mice (CMV-Cre;ROSA26-neoflox/+-rtTA;Tet-O-Runx2) to investigate the effects of wide spread overexpression of Runx2. Osteoblasts isolated from CMV-Cre;ROSA26-neoflox/+-rtTA; Tet-O-Runx2 mice demonstrated a dose-dependent effect of doxycycline to stimulate Runx2 transgene expression. Doxycycline administration to CMV-Cre;ROSA26-neoflox/+-rtTA;Tet-O-Runx2 mice induced Runx2 transgene expression in all tissues tested, with the highest levels observed in kidney, ovary, and bone. Runx2 overexpression resulted in deceased body size and reduced viability. With regard to bone, Runx2 overexpressing mice paradoxically displayed profound osteopenia and diminished osteogenesis. Induced expression of Runx2 in extraskeletal tissues resulted in ectopic calcification and induction of the osteogenic program in a limited number of tissues, including lung and muscle. In addition, the triple transgenic mice showed evidence of a myeloproliferative disorder and an apparent inhibition of lymphocyte development. Thus, overexpression of Runx2 both within and outside of the skeleton can have diverse biological effects. Use of tissue specific Cre mice will allow this model to be used to conditionally and inducibly overexpress Runx2 in different tissues and provide a means to study the post-natal tissue- and cell context-dependent functions of Runx2. J. Cell. Biochem. 112: 653–665, 2011. © 2010 Wiley-Liss, Inc.