Abstract
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. © 2010 American Society for Bone and Mineral Research.
Highlights
IntroductionGPRC6A is a recently identified member of family C of G protein–coupled receptors (GPCRs) that senses extracellular cations, osteocalcin, and amino acids.[1,2,3,4] Transcripts for GPRC6A are expressed in many tissues and organs, including lung, liver, spleen, heart, kidney, blood vessels, skeletal muscle, testis, brain, and bone.[1,2,3,4,5] Consistent with its broad expression, ablation of GPRC6A is associated with multiple abnormalities, including glucose intolerance, hepatic steatosis, abnormal steroid biogenesis, and osteopenia, suggesting that GPRC6A may directly or indirectly regulate anabolic responses in multiple organs.[6]Bone has been proposed to be a special tissue compartment where the combination of calcium, osteocalcin, and amino acids might constitute important extracellular signals regulating bone formation.[4]
Bone has been proposed to be a special tissue compartment where the combination of calcium, osteocalcin, and amino acids might constitute important extracellular signals regulating bone formation.[4]. While osteoblast-mediated bone formation is coupled to osteoclast-mediated bone resorption through the production by osteoblastic stromal cells of osteoprotegerin (OPG) and receptor activator of NF-kB ligand (RANKL),(7) there is emerging evidence for reverse coupling by factors released from osteoclast-mediated degradation of mineralized bone matrix, such as amino acids and calcium, that act on osteoblasts to fill in the resorptive cavity.[8] several mouse models with a primary increase in osteoclast-mediated bone resorption, including OPG and calcitonin null mice,(9,10) have a secondary increase in osteoblast-mediated bone formation
1096 Journal of Bone and Mineral Research these cells, we evaluated the response to calcium, calcimimetic, and arginine of cultured of bone marrow stromal cell cultures (BMSCs) and calavarial osteoblasts obtained from wild-type and GPRC6AÀ/À mice (Fig. 3)
Summary
GPRC6A is a recently identified member of family C of G protein–coupled receptors (GPCRs) that senses extracellular cations, osteocalcin, and amino acids.[1,2,3,4] Transcripts for GPRC6A are expressed in many tissues and organs, including lung, liver, spleen, heart, kidney, blood vessels, skeletal muscle, testis, brain, and bone.[1,2,3,4,5] Consistent with its broad expression, ablation of GPRC6A is associated with multiple abnormalities, including glucose intolerance, hepatic steatosis, abnormal steroid biogenesis, and osteopenia, suggesting that GPRC6A may directly or indirectly regulate anabolic responses in multiple organs.[6]Bone has been proposed to be a special tissue compartment where the combination of calcium, osteocalcin, and amino acids might constitute important extracellular signals regulating bone formation.[4]. Since dietary protein-derived chemical signals may be derived from their metabolism into free amino acids,(13) circulating levels of amino acids and calcium may modulate signaling pathways in bone. Both osteoblasts and osteoclasts respond to extracellular calcium in vitro through a putative extracellular amino acid and calcium-sensing GPCR.[11,14,15]. Preliminary characterization of the skeleton of GPRC6AÀ/À mice indicates that loss of this receptor is associated with decreased bone mineral density (BMD) and impaired mineralization of bone,(6) but the presence of other abnormalities confounded the ability to ascertain the direct and indirect effects of GPRC6A on skeletal function. To determine if GPRC6A is a potential candidate for the purported extracellular calciumsensing receptor in osteoblasts, we performed a more detailed assessment of the skeletal phenotype of GPRC6AÀ/À mice, examined the function of primary osteoblasts and bone marrow stromal cells derived from these mutant mice ex vivo, and evaluated whether polymorphisms in GPRC6A are associated with skeletal abnormalities in humans by a gene association analysis
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