Min Pi

Fibroblast Growth Factor 23 Is a Counter-Regulatory Phosphaturic Hormone for Vitamin D

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(OH)(2)D(3). In osteoblast cell cultures, 1,25(OH)(2)D(3) but not calcium, phosphate, or parathyroid hormone stimulated FGF23 mRNA levels and resulted in a dose-dependent increase in FGF23 promoter activity. Overexpression of a dominant negative vitamin D receptor inhibited 1,25(OH)(2)D(3) stimulation of FGF23 promoter activity, and mutagenesis of the FGF23 promoter identified a vitamin D-responsive element (-1180 GGAACTcagTAACCT -1156) that is responsible for the vitamin D effects. These data suggest that 1,25(OH)(2)D(3) is an important regulator of FGF23 production by osteoblasts in bone. The physiologic role of FGF23 may be to act as a counterregulatory phosphaturic hormone to maintain phosphate homeostasis in response to vitamin D.

Identification of a Novel Extracellular Cation-sensing G-protein-coupled Receptor

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 β-arrestins 1 and 2, regulators of G-protein signaling RGS2 or RGS4, the RhoA inhibitor C3 toxin, the dominant negative Gαq-(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.

GPRC6A Null Mice Exhibit Osteopenia, Feminization and Metabolic Syndrome

Background GPRC6A 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 Findings In 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.

GPRC6A mediates responses to osteocalcin in β-cells in vitro and pancreas in vivo.

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.

GPRC6A Mediates the Non-genomic Effects of Steroids

The identity of the putative G-protein coupled receptor (GPCR) that mediates the non-genomic effects of androgens is unknown. We present in vitro and in vivo evidence that the orphan GPRC6A receptor, a widely expressed calcium and amino acid sensing GPCR, transduces the non-genomic effects of testosterone and other steroids. Overexpression of GPRC6A imparts the ability of extracellular testosterone to illicit a rapid, non-genomic signaling response in HEK-293 cells lacking the androgen receptor. Conversely, testosterone-stimulated rapid signaling and phosphorylation of ERK is attenuated in bone marrow stromal cells derived from GPRC6A−/− mice and in 22Rv1 prostate cancer cells after siRNA-mediated knockdown of GPRC6A. Compared with wild-type controls, GPRC6A−/− null mice exhibit significantly less ERK activation and Egr-1 expression in both bone marrow and testis in response to pharmacological doses of testosterone in vivo. In addition, testosterone administration results in suppression of luteinizing hormone in wild-type male mice, but paradoxically stimulates serum luteinizing hormone levels in GPRC6A−/− null mice. These results suggest that GPRC6A is functionally important in regulating non-genomic effects of androgens in multiple tissues.

Multiligand specificity and wide tissue expression of GPRC6A reveals new endocrine networks.

Emerging evidence supports the hypothesis that the skeleton is an endocrine organ that regulates energy metabolism through the release of the osteoblast-derived hormone, osteocalcin (Ocn). This bone-pancreas endocrine network is controversial because important gaps remain to be filled in our knowledge of the physiological effects of Ocn in multiple organs and the complex alterations in other hormonal networks induced by Ocn administration. A key step toward understanding the integrative regulation of energy metabolism by bone is the identification of GPCR family C group 6 member A (GPRC6A) as the Ocn receptor. GPRC6A is an amino acid-sensing G protein-coupled receptor highly expressed in β-cells and is activated by recombinant Ocn in vitro and in vivo but that is widely expressed in tissues other than the pancreas and is capable of sensing multiple structurally unrelated ligands, including l-amino acids, cations, and anabolic steroids in addition to Ocn. The broad expression and multiligand specificity of GPRC6A is identifying both systemic and paracrine regulation of seemingly disparate biological processes, ranging from energy metabolism, sexual reproduction, hypothalamic-pituitary function, bone formation, and prostate cancer. Consistent with the existence of more complex endocrine networks, ablation of GPRC6A in Gprc6a(-/-) mice results in complex metabolic abnormalities, including obesity, glucose intolerance, hepatic steatosis, insulin resistance, hyperphosphatemia, osteopenia, plus several hormonal abnormalities, including decreased circulating testosterone, IGF-I, and insulin and increased estradiol, LH, GH, and leptin. Recombinant Ocn also regulates testosterone production by the testes and male fertility through a GPRC6A-dependent mechanism, and testosterone regulation of LH secretion is abnormal in Gprc6a(-/-) mice. Thus, GPRC6A, as the biologically relevant receptor for Ocn, defines not only a molecular mechanism for linking bone metabolism with metabolic regulation of β-cells and sexual reproduction but also as a receptor shared by testosterone and dietary factors, and it is also involved in multiple endocrine networks integrating the functions of pancreas, muscle, liver, fat, testes, bone, and the hypothalamic-pituitary axis with alterations in both environmental and endogenous ligands.

GPRC6A regulates prostate cancer progression

GPRC6A is a nutrient sensing GPCR that is activated in vitro by a variety of ligands, including amino acids, calcium, zinc, osteocalcin (OC), and testosterone. The association between nutritional factors and risk of prostate cancer, the finding of increased expression of OC in prostate cancer cells, and the association between GPRC6A and risk of prostate cancer in Japanese men implicates a role of GPRC6A in prostate cancer.

Antiandrogen gold nanoparticles dual-target and overcome treatment resistance in hormone-insensitive prostate cancer cells.

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.

Impaired osteoblast function in GPRC6A null mice

GPRC6A is a widely expressed orphan G protein–coupled receptor that senses extracellular amino acids, osteocalcin, and divalent cations in vitro. GPRC6A null (GPRC6A−/−) mice exhibit multiple metabolic abnormalities including osteopenia. To investigate whether the osseous abnormalities are a direct function of GPRC6A in osteoblasts, we examined the function of primary osteoblasts and bone marrow stromal cell cultures (BMSCs) in GPRC6A−/− mice. We confirmed that GPRC6A−/− mice exhibited a decrease in bone mineral density (BMD) associated with reduced expression of osteocalcin, ALP, osteoprotegerin, and Runx2‐II transcripts in bone. Osteoblasts and BMSCs derived from GPRC6A−/− mice exhibited an attenuated response to extracellular calcium‐stimulated extracellular signal‐related kinase (ERK) activation, diminished alkaline phosphatase (ALP) expression, and impaired mineralization ex vivo. In addition, siRNA‐mediated knockdown of GPRC6A in MC3T3 osteoblasts also resulted in a reduction in extracellular calcium‐stimulated ERK activity. To explore the potential relevance of GPRC6A function in humans, we looked for an association between GPRC6A gene polymorphisms and BMD in a sample of 1000 unrelated American Caucasians. We found that GPRC6A gene polymorphisms were significantly associated with human spine BMD. These data indicate that GRPC6A directly participates in the regulation of osteoblast‐mediated bone mineralization and may mediate the anabolic effects of extracellular amino acids, osteocalcin, and divalent cations in bone.

Novel bone endocrine networks integrating mineral and energy metabolism.

The skeleton is an endocrine organ that regulates energy metabolism through the release of the osteoblast-derived hormone, osteocalcin (Ocn), and phosphate and vitamin D homeostasis through the secretion by osteoblasts and osteocytes of the novel hormone, FGF23 Ocn activates a widely expressed G-protein coupled receptor, GPRC6A, to regulate insulin secretion by pancreatic β−cells, testosterone secretion by testicular Leydig cells, fatty acid metabolism in the liver, and insulin sensitivity of muscle and fat, as well as other functions. FGF23 targets a limited number of tissues, including kidney, parathyroid gland, choroid plexus, and pituitary gland that co-express FGF receptors and α-Klotho complexes. Ectodomain shedding and secretion of a soluble form of Klotho also is purported to act as an anti-ageing hormone. Further elucidation of these novel endocrine networks is likely to lead to new appreciation of the cooperation between various organ systems to regulate phosphate, vitamin D, and energy metabolism.