The regulation of the phosphaturic factor fibroblast growth factor 23 (FGF23) is not well understood. It was found that administration of 1,25-dihydroxyvitamin D 3 (1,25[OH] 2 D 3 ) to mice rapidly increased serum FGF23 concentrations from a basal level of 90.6 ؎ 8.1 to 213.8 ؎ 14.6 pg/ml at 8 h (mean ؎ SEM; P < 0.01) and resulted in a four-fold increase in FGF23 transcripts in bone, the predominate site of FGF23 expression. In the Hyp-mouse homologue of X-linked hypophosphatemic rickets, administration of 1,25(OH) 2 D 3 further increased circulating FGF23 levels. In Gcm2 null mice, low 1,25(OH) 2 D 3 levels were associated with a three-fold reduction in FGF23 levels that were increased by administration of 1,25 ( FGF23 also suppresses 1␣ hydroxylase activity in the proximal renal tubule, leading to reduced circulating levels of 1,25(OH) 2 D 3 (2,10,14,15). The significance of FGF23 regulation of 1,25(OH) 2 D 3 production is not clear, but the findings that FGF23 is produced predominantly by osteoblasts in bone and that FGF23 regulates phosphate reabsorption and 1,25(OH) 2 D 3 production by the kidney raise the possibility that FGF23 may be involved in a bone-kidney axis that controls phosphate and vitamin D homeostasis (16,17). How FGF23 is integrated with the vitamin D-parathyroid hormone (PTH) axis, which plays a central role in calcium homeostasis, skeletal development, and mineralization (18), however, is not clear. Understanding the effects of 1,25(OH) 2 D 3 on FGF23 production is important, because vitamin D therapy often is used to treat FGF23-mediated hypophosphatemic disorders, such as XLH (19).In an effort to understand more fully the regulation of FGF23 expression in osteoblasts and bone, we assessed the effect of 1,25(OH) 2 D 3 administration on circulating levels of FGF23 in wild-type Gcm2 null (20) and Hyp mice (21), as well as the effects of 1,25(OH) 2 D 3 on the FGF23 transcripts in bone. In addition, we investigated the ability of 1,25(OH) 2 D 3 to regulate endogenous FGF23 transcripts and the activity of a transfected murine FGF23 promoter luciferase reporter in osteoblasts. Our findings demonstrate the importance of bone as a target for vitamin D-mediated increments in FGF23 production and suggest that FGF23 production serves as a counterregulatory hormone to enhance renal phosphate clearance in response to vitamin D-mediated increments in gastrointestinal phosphate absorption and decrements in the phosphaturic hormone PTH. Materials and Methods 1,25(OH) 2 D 3 and PTH AdministrationBoth Hyp mice (21) and C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Male and female Gcm2 ϩ/Ϫ mice were mated to generate homozygous Gcm2 null mice that lacked parathyroid glands (22). All mice were maintained and used in accordance with recommendations in

The C family G-protein-coupled receptors contain members that sense amino acid and extracellular cations, of which calcium-sensing receptor (CASR) is the prototypic extracellular calcium-sensing receptor. Some cells, such as osteoblasts in bone, retain responsiveness to extracellular calcium in CASR-deficient mice, consistent with the existence of another calcium-sensing receptor. We examined the calcium-sensing properties of GPRC6A, a newly identified member of this family. Alignment of GPRC6A with CASR revealed conservation of both calcium and calcimimetic binding sites. In addition, calcium, magnesium, strontium, aluminum, gadolinium, and the calcimimetic NPS 568 resulted in a dose-dependent stimulation of GPRC6A overexpressed in human embryonic kidney cells 293 cells. Also, osteocalcin, a calcium-binding protein highly expressed in bone, dose-dependently stimulated GPRC6A activity in the presence of calcium but inhibited the calcium-dependent activation of CASR. Coexpression of ␤-arrestins 1 and 2, regulators of G-protein signaling RGS2 or RGS4, the RhoA inhibitor C3 toxin, the dominant negative G␣ q -(305-359) minigene, and pretreatment with pertussis toxin inhibited activation of GPRC6A by extracellular cations. Reverse transcription-PCR analyses showed that mouse GPRC6A is widely expressed in mouse tissues, including bone, calvaria, and the osteoblastic cell line MC3T3-E1. These data suggest that in addition to sensing amino acids, GPRC6A is a cation-, calcimimetic-, and osteocalcin-sensing receptor and a candidate for mediating extracellular calcium-sensing responses in osteoblasts and possibly other tissues.

BackgroundGPRC6A is a widely expressed orphan G-protein coupled receptor that senses extracellular amino acids, osteocalcin and divalent cations in vitro. The physiological functions of GPRC6A are unknown.Methods/Principal FindingsIn this study, we created and characterized the phenotype of GPRC6A −/− mice. We observed complex metabolic abnormalities in GPRC6A −/− mice involving multiple organ systems that express GPRC6A, including bone, kidney, testes, and liver. GPRC6A −/− mice exhibited hepatic steatosis, hyperglycemia, glucose intolerance, and insulin resistance. In addition, we observed high expression of GPRC6A in Leydig cells in the testis. Ablation of GPRC6A resulted in feminization of male GPRC6A −/− mice in association with decreased lean body mass, increased fat mass, increased circulating levels of estradiol, and reduced levels of testosterone. GPRC6A was also highly expressed in kidney proximal and distal tubules, and GPRC6A−/− mice exhibited increments in urine Ca/Cr and PO4/Cr ratios as well as low molecular weight proteinuria. Finally, GPRC6A −/− mice exhibited a decrease in bone mineral density (BMD) in association with impaired mineralization of bone.Conclusions/Significance GPRC6A−/− mice have a metabolic syndrome characterized by defective osteoblast-mediated bone mineralization, abnormal renal handling of calcium and phosphorus, fatty liver, glucose intolerance and disordered steroidogenesis. These findings suggest the overall function of GPRC6A may be to coordinate the anabolic responses of multiple tissues through the sensing of extracellular amino acids, osteocalcin and divalent cations.