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1.  Impaired Osteoblast Function in GPRC6A Null Mice 
Journal of Bone and Mineral Research  2009;25(5):1092-1102.
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.
PMCID: PMC3153369  PMID: 19874200
GPRC6A; G protein–coupled receptor (GPCR); osteoblast; bone mineral density; gene polymorphisms
2.  GPRC6A regulates prostate cancer progression 
The Prostate  2011;72(4):399-409.
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.
We examined if GPRC6A is expressed in human prostate cancer cell lines and used siRNA-mediated knockdown GPRC6A expression in prostate cancer cells to explore the function of GPRC6A in vitro. To assess the role GPRC6A in prostate cancer progression in vivo we intercrossed Gprc6a−/− mice onto the TRAMP mouse prostate cancer model.
GPRC6A transcripts were markedly increased in prostate cancer cell lines 22Rv1, PC-3 and LNCaP, compared to the normal prostate RWPE-1 cell line. In addition, a panel of GPRC6A ligands, including calcium, OC, and arginine, exhibited in prostate cancer cell lines a dose-dependent stimulation of ERK activity, cell proliferation, chemotaxis, and prostate specific antigen and Runx 2 gene expression. These responses were inhibited by siRNA-mediated knockdown of GPRC6A. Finally, transfer of Gprc6a deficiency onto a TRAMP mouse model of prostate cancer significantly retarded prostate cancer progression and improved survival of compound Gprc6a−/−/TRAMP mice.
GPRC6A is a novel molecular target for regulating prostate growth and cancer progression. Increments in GPRC6A may augment the ability of prostate cancer cells to proliferate in response to dietary and bone derived ligands.
PMCID: PMC3183291  PMID: 21681779
GPRC6A; GPCR; calcium; osteocalcin; siRNA; prostate cancer; cell proliferation; metastases
3.  Identification of a Novel Extracellular Cation-sensing G-protein-coupled Receptor* 
The Journal of biological chemistry  2005;280(48):40201-40209.
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.
PMCID: PMC1435382  PMID: 16199532
4.  Pharmacological characterization of mouse GPRC6A, an L-α-amino-acid receptor modulated by divalent cations 
British Journal of Pharmacology  2007;150(6):798-807.
Background and purpose:
GPRC6A is a novel member of family C of G protein-coupled receptors with so far unknown function. We have recently described both human and mouse GPRC6A as receptors for L-α-amino acids. To date, functional characterization of wild-type GPRC6A has been impaired by the lack of activity in quantitative functional assays. The aim of this study was thus to develop such an assay and extend the pharmacological characterization of GPRC6A.
Experimental approach:
We have engineered a novel cell-based inositol phosphate turnover assay for wild-type mouse GPRC6A based on transient co-expression with the promiscuous GαqG66D protein, known to increase receptor signalling sensitivity. This assay allowed for measurements of L-α-amino acid potencies. Furthermore, in combination with an assay measuring inward currents at Ca2+-activated chloride channels in Xenopus oocytes, the divalent cation-sensing ability of the receptor was examined.
Key results:
Using our novel assay, we demonstrate that the basic L-α-amino acids ornithine, lysine, and arginine are the most potent agonists at wild-type mouse GPRC6A. Using two different assay systems, we show that divalent cations do not activate the Gq signalling pathway of mouse GPRC6A per se but positively modulate the amino-acid response.
Conclusions and Implications:
This is the first reported assay for a wild-type GPRC6A successfully applied for quantitative pharmacological characterization of amino acid and divalent cation responses at mouse GPRC6A. The assay enables further search for GPRC6A ligands such as allosteric modulators, which may provide essential information about the physiological function of GPRC6A.
PMCID: PMC2013871  PMID: 17245368
GPRC6A; G protein-coupled receptor; promiscuous G proteins; L-α-amino acids; divalent cations; positive modulation
5.  Osteocalcin Promotes β-Cell Proliferation During Development and Adulthood Through Gprc6a 
Diabetes  2014;63(3):1021-1031.
Expanding β-cell mass through β-cell proliferation is considered a potential therapeutic approach to treat β-cell failure in diabetic patients. A necessary step toward achieving this goal is to identify signaling pathways that regulate β-cell proliferation in vivo. Here we show that osteocalcin, a bone-derived hormone, regulates β-cell replication in a cyclin D1–dependent manner by signaling through the Gprc6a receptor expressed in these cells. Accordingly, mice lacking Gprc6a in the β-cell lineage only are glucose intolerant due to an impaired ability to produce insulin. Remarkably, this regulation occurs during both the perinatal peak of β-cell proliferation and in adulthood. Hence, the loss of osteocalcin/Gprc6a signaling has a profound effect on β-cell mass accrual during late pancreas morphogenesis. This study extends the endocrine role of osteocalcin to the developmental period and establishes osteocalcin/Gprc6a signaling as a major regulator of β-cell endowment that can become a potential target for β-cell proliferative therapies.
PMCID: PMC3931403  PMID: 24009262
6.  The GPCR, class C, group 6, subtype A (GPRC6A) receptor: from cloning to physiological function 
British Journal of Pharmacology  2014;171(5):1129-1141.
GPRC6A (GPCR, class C, group 6, subtype A) is a class C GPCR that has been cloned from human, mouse and rat. Several groups have shown that the receptor is activated by a range of basic and small aliphatic L-α-amino acids of which L-arginine, L-lysine and L-ornithine are the most potent compounds with EC50 values in the mid-micromolar range. In addition, several groups have shown that the receptor is either directly activated or positively modulated by divalent cations such as Ca2+ albeit in concentrations above 5 mM, which is above the physiological concentration in most tissues. More recently, the peptide osteocalcin and the steroid testosterone have also been suggested to be endogenous GPRC6A agonists. The receptor is widely expressed in all three species which, along with the omnipresence of the amino acids and divalent cation ligands, suggest that the receptor could be involved in a broad range of physiological functions. So far, this has mainly been addressed by analyses of genetically modified mice where the GPRC6A receptor has been ablated. Although there has been some discrepancies among results reported from different groups, there is increasing evidence that the receptor is involved in regulation of inflammation, metabolism and endocrine functions. GPRC6A could thus be an interesting target for new drugs in these therapeutic areas.
Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit
PMCID: PMC3952793  PMID: 24032653
amino acid sensing; divalent cation sensing; 7TM receptor; GPRC6A; knockout mouse
7.  Gprc5a deletion enhances the transformed phenotype in normal and malignant lung epithelial cells by eliciting persistent Stat3 signaling induced by autocrine Lif 
Cancer research  2010;70(21):8917-8926.
Signal transduction and activator of transcription 3 (Stat3) is activated by cytokines and growth factors in lung cancers and regulates expression of genes implicated in cell growth, survival, and transformation. Previously, we found that mice with a deletion of the G protein-coupled receptor, family C, group 5, member a (Gprc5a) gene develop lung tumors indicating that Gprc5a is a tumor suppressor. Herein, we show that epithelial cells from Gprc5a knockout mouse lung (Gprc5a−/− cells) survive better in vitro in medium deprived of exogenous growth factors and form more colonies in semi-solid medium than their counterparts from wildtype mice (Gprc5a+/+ cells). Stat3 Tyrosine 705 phosphorylation and expression of several Stat3-regulated anti-apoptotic genes were higher in Gprc5a−/− than in Gprc5a+/+ cells. Both cell types secreted Leukemia inhibitory factor (Lif), however, whereas Stat3 activation was persistent in Gprc5a−/− cells it was transient in Gprc5a+/+ cells. Lung adenocarcinoma cells isolated from Gprc5a−/− mice also exhibited autocrine Lif-mediated Stat3 activation. The level of Socs3, the endogenous Stat3 inhibitory protein, was higher in Gprc5a+/+ than in Gprc5a−/− cells and expression of the tumor suppressor stabilized Socs3. Inhibition of Stat3 signaling in Gprc5a−/− normal and cancer cells by the Jak2 inhibitor AG490 or by a dominant negative Stat3(Y705F) increased starvation-induced apoptosis and inhibited colony formation. These results demonstrate that persistent Stat3 activation is important for the survival and transformation of Gprc5a−/− lung cells and suggest that the tumor suppressive effects of Gprc5a are mediated, at least in part, by inhibition of Stat3 signaling via Socs3 stabilization.
PMCID: PMC2970717  PMID: 20959490
Stat3; Gprc5a; lung cancer; Lif; apoptosis
8.  A Flanking Gene Problem Leads to the Discovery of a Gprc5b Splice Variant Predominantly Expressed in C57Bl/6J Mouse Brain and in Maturing Neurons 
PLoS ONE  2010;5(4):e10351.
Gprc5b, a retinoic acid-inducible orphan G protein–coupled receptor (GPCR), is a member of the group C metabotropic glutamate receptor family proteins possibly involved in non-canonical Wnt signaling. Many GPCR transcripts are alternatively spliced, which diversifies this class of proteins in their cell- and tissue-specific signaling, regulatory and/or pharmacological properties. We previously generated p97FE65 isoform-specific knockout mice that showed learning/memory deficits. In this study, we further characterized the 97FE65 null mice using cDNA microarray and RT-PCR analyses.
Methodology/Principal Findings
We discovered a novel brain-specific C-terminal splice variant of Gprc5b, Gprc5b_v2, which was differentially expressed in p97FE65 wild type and null mouse brains. The null mice were generated in 129/Sv ES cells, and backcrossed to C57Bl/6J for ten generations. We found that expression of Gprc5b_v2 mRNA in the brains of p97FE65 null mice was dramatically down-regulated (more than 20 fold) compared to their wild type littermates. However, expression profiles of Gprc5b variants and SNP analysis surrounding the FE65 locus suggest that the down-regulation is unlikely due to the altered FE65 function, but rather is caused by gene retention from the 129/Sv ES cells. Consistently, in contrast to ubiquitously expressed Gprc5b_v1, Gprc5b_v2 was predominantly expressed in the brain tissues of C57Bl/6J mice. The alternative splicing of the 3′ terminal exon also altered the protein coding sequences, giving rise to the characteristic C-termini. Levels of Gprc5b_v2 mRNA were increased during neuronal maturation, paralleling the expression of synaptic proteins. Overexpression of both Gprc5b variants stimulated neurite-like outgrowth in a neuroblastoma cell line.
Our results suggest that Gprc5b-v2 may play a role during brain maturation and in matured brain, possibly through the regulation of neuronal morphology and protein-protein interaction. This study also highlights the fact that unexpected gene retention following repeated backcrosses can lead to important biological consequences.
PMCID: PMC2859937  PMID: 20436672
9.  EGFR phosphorylates and inhibits lung tumor suppressor GPRC5A in lung cancer 
Molecular Cancer  2014;13:233.
GPRC5A is a retinoic acid inducible gene that is preferentially expressed in lung tissue. Gprc5a– knockout mice develop spontaneous lung cancer, indicating Gprc5a is a lung tumor suppressor gene. GPRC5A expression is frequently suppressed in majority of non-small cell lung cancers (NSCLCs), however, elevated GPRC5A is still observed in a small portion of NSCLC cell lines and tumors, suggesting that the tumor suppressive function of GPRC5A is inhibited in these tumors by an unknown mechanism.
In this study, we examined EGF receptor (EGFR)-mediated interaction and tyrosine phosphorylation of GPRC5A by immunoprecipitation (IP)-Westernblot. Tyrosine phosphorylation of GPRC5A by EGFR was systematically identified by site-directed mutagenesis. Cell proliferation, migration, and anchorage-independent growth of NSCLC cell lines stably transfected with wild-type GPRC5A and mutants defective in tyrosine phosphorylation were assayed. Immunohistochemical (IHC) staining analysis with specific antibodies was performed to measure the total and phosphorylated GPRC5A in both normal lung and lung tumor tissues.
We found that EGFR interacted with GPRC5A and phosphorylated it in two conserved double-tyrosine motifs, Y317/Y320 and Y347/ Y350, at the C-terminal tail of GPRC5A. EGF induced phosphorylation of GPRC5A, which disrupted GPRC5A-mediated suppression on anchorage-independent growth of NSCLC cells. On contrary, GPRC5A-4 F, in which the four tyrosine residues have been replaced with phenylalanine, was resistant to EGF-induced phosphorylation and maintained tumor suppressive activities. Importantly, IHC analysis with anti-Y317/Y320-P sites showed that GPRC5A was non-phosphorylated in normal lung tissue whereas it was highly tyrosine-phosphorylated in NSCLC tissues.
GPRC5A can be inactivated by receptor tyrosine kinase via tyrosine phosphorylation. Thus, targeting EGFR can restore the tumor suppressive functions of GPRC5A in lung cancer.
PMCID: PMC4200229  PMID: 25311788
GPRC5A; EGFR; Tyrosine kinase; Lung cancer; Post-translation modification
10.  X-ray Crystal Structure of Bovine 3 Glu-Osteocalcin 
Biochemistry  2013;52(47):8387-8392.
The 3 Glu form of osteocalcin (3 Glu-OCN) is increased in serum during low vitamin K intake or oral anticoagulant use (warfarin). Previous reports using circular dichroism show it is less structured than 3 Gla Ca2+-osteocalcin and does not bind strongly to bone mineral. Recent studies have suggested a role for 3 Glu-OCN as a potential regulator of glucose metabolism. A G-protein-coupled receptor, GPRC6a, found in the pancreas and testes was identified as the putative osteocalcin receptor. The purpose of this study is to determine the high-resolution structure of bovine 3 Glu-OCN, using X-ray crystallography, to understand molecular interactions with mineral and the GPRC6a receptor. Diffraction quality crystals of thermally decarboxylated bovine osteocalcin were grown, and the crystal structure was determined to 1.88 Å resolution. The final refined structure contained residues 17–47 and, like 3 Gla Ca2+-OCN, consisted of three α-helices surrounding a hydrophobic core, a C23–C29 disulfide bond between two of the helices, and no bound Ca2+. Thus, the helical structure of 3 Glu-OCN is Ca2+-independent but similar to that of 3 Gla Ca2+-OCN. A reduced level of mineral binding could result from a lower number of Ca2+ coordinating ligands on 3 Glu-OCN. The structure suggests the GPRC6a receptor may respond to helical osteocalcin and will aid in providing molecular mechanistic insight into the role of 3 Glu-OCN in glucose homeostasis.
PMCID: PMC4517604  PMID: 24138653
11.  Comparison of human chromosome 19q13 and syntenic region on mouse chromosome 7 reveals absence, in man, of 11.6Mb containing 4 mouse calcium sensing receptor related sequences: relevance to familial benign hypocalciuric hypercalcaemia type 3 
Familial benign hypocalciuric hypercalcaemia (FBHH) is a genetically heterogenous disorder that consists of three types designated, FBHH1, FBHH2 and FBHH3 whose chromosomal locations are 3q21.1, 19p and 19q13, respectively. FBHH1 is caused by mutations of a calcium sensing receptor (CaSR), but the abnormalities underlying FBHH2 and FBHH3 are unknown. FBHH3, also referred to as the Oklahoma variant (FBHHOk), has been mapped to a 12cM interval, flanked by D19S908 and D19S866. To refine the location of FBHH3, we pursued linkage studies using 24 polymorphic loci. Our results establish linkage between FBHH3 and 17 of these loci, and indicate that FBHH3 is located in a 4.1Mb region flanked centromerically by D19S112 and telomerically by rs245111, which in the syntenic region on mouse chromosome 7 contains 4 Casr related sequences (Gprc2a-rss). However, human homologues of these Gprc2a-rss were not found and a comparative analysis of the 22.0Mb human and 39.3Mb mouse syntenic regions showed evolutionary conservation of 2 segments that were inverted with loss from the human genome of 11.6Mb that contained the 4 Gprc2a-rss. Thus, FBHH3 cannot be due to Gprc2a-rss abnormalities. DNA sequence analysis of 12 other genes from the interval that were expressed in the parathyroids and/or kidneys did not detect any abnormalities, thereby indicating that these genes are unlikely to be the cause of FBHH3. The results of this study have refined the map location of FBHH3, which will facilitate the identification of another CaSR or a mediator of calcium homeostasis.
PMCID: PMC2842244  PMID: 19809483
calcium; linkage; rearrangement
12.  Comparison of human chromosome 19q13 and syntenic region on mouse chromosome 7 reveals absence, in man, of 11.6 Mb containing four mouse calcium-sensing receptor-related sequences: relevance to familial benign hypocalciuric hypercalcaemia type 3 
Familial benign hypocalciuric hypercalcaemia (FBHH) is a genetically heterogeneous disorder that consists of three designated types, FBHH1, FBHH2 and FBHH3, whose chromosomal locations are 3q21.1, 19p and 19q13, respectively. FBHH1 is caused by mutations of a calcium-sensing receptor (CaSR), but the abnormalities underlying FBHH2 and FBHH3 are unknown. FBHH3, also referred to as the Oklahoma variant (FBHHOk), has been mapped to a 12cM interval, flanked by D19S908 and D19S866. To refine the location of FBHH3, we pursued linkage studies using 24 polymorphic loci. Our results establish a linkage between FBHH3 and 17 of these loci, and indicate that FBHH3 is located in a 4.1 Mb region flanked centromerically by D19S112 and telomerically by rs245111, which in the syntenic region on mouse chromosome 7 contains four Casr-related sequences (Gprc2a-rss). However, human homologues of these Gprc2a-rss were not found and a comparative analysis of the 22.0 Mb human and 39.3 Mb mouse syntenic regions showed evolutionary conservation of two segments that were inverted with loss from the human genome of 11.6 Mb that contained the four Gprc2a-rss. Thus, FBHH3 cannot be attributed to Gprc2a-rss abnormalities. DNA sequence analysis of 12 other genes from the interval that were expressed in the parathyroids and/or kidneys did not detect any abnormalities, thereby indicating that these genes are unlikely to be the cause of FBHH3. The results of this study have refined the map location of FBHH3, which will facilitate the identification of another CaSR or a mediator of calcium homeostasis.
PMCID: PMC2842244  PMID: 19809483
calcium; linkage; rearrangement
13.  Comparative Functional Genomics Analysis of NNK Tobacco-Carcinogen Induced Lung Adenocarcinoma Development in Gprc5a-Knockout Mice 
PLoS ONE  2010;5(7):e11847.
Improved understanding of lung cancer development and progression, including insights from studies of animal models, are needed to combat this fatal disease. Previously, we found that mice with a knockout (KO) of G-protein coupled receptor 5A (Gprc5a) develop lung tumors after a long latent period (12 to 24 months).
Methodology/Principal Findings
To determine whether a tobacco carcinogen will enhance tumorigenesis in this model, we administered 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) i.p. to 2-months old Gprc5a-KO mice and sacrificed groups (n = 5) of mice at 6, 9, 12, and 18 months later. Compared to control Gprc5a-KO mice, NNK-treated mice developed lung tumors at least 6 months earlier, exhibited 2- to 4-fold increased tumor incidence and multiplicity, and showed a dramatic increase in lesion size. A gene expression signature, NNK-ADC, of differentially expressed genes derived by transcriptome analysis of epithelial cell lines from normal lungs of Gprc5a-KO mice and from NNK-induced adenocarcinoma was highly similar to differential expression patterns observed between normal and tumorigenic human lung cells. The NNK-ADC expression signature also separated both mouse and human adenocarcinomas from adjacent normal lung tissues based on publicly available microarray datasets. A key feature of the signature, up-regulation of Ube2c, Mcm2, and Fen1, was validated in mouse normal lung and adenocarcinoma tissues and cells by immunohistochemistry and western blotting, respectively.
Our findings demonstrate that lung tumorigenesis in the Gprc5a-KO mouse model is augmented by NNK and that gene expression changes induced by tobacco carcinogen(s) may be conserved between mouse and human lung epithelial cells. Further experimentation to prove the reliability of the Gprc5a knockout mouse model for the study of tobacco-induced lung carcinogenesis is warranted.
PMCID: PMC2912294  PMID: 20686609
14.  G-Protein Coupled Receptor Family C, Group 5, Member A (GPRC5A) Expression Is Decreased in the Adjacent Field and Normal Bronchial Epithelia of Patients with Chronic Obstructive Pulmonary Disease and Non–Small-Cell Lung Cancer 
Understanding oncogenes and tumor suppressor genes expression patterns is essential for characterizing lung cancer pathogenesis. We have previously demonstrated that mGprc5a/hGPRC5A is a lung-specific tumor suppressor evidenced by inflammation-mediated tumorigenesis in Gprc5a-knockout mice. The implication of GPRC5A in human lung cancer pathogenesis, including that associated with inflammatory chronic obstructive pulmonary disease (COPD), a risk factor for the malignancy, remains elusive.
We sought to examine GPRC5A immunohistochemical expression in histologically normal bronchial epithelia (NBE) from lung disease-free never- and ever-smokers (n = 13 and n = 18, respectively), from COPD patients with (n = 26) and without cancer (n = 24) and in non-small cell lung cancers (NSCLCs) (n = 474). Quantitative assessment of GPRC5A transcript expression in airways (n = 6), adjacent NBEs (n = 29) and corresponding tumors (n = 6) from 6 NSCLC patients was also performed.
GPRC5A immunohistochemical expression was significantly lower in tumors compared to uninvolved NBE (p < 0.0001) and was positively associated with adenocarcinoma histology (p < 0.001). GPRC5A airway expression was highest in lung disease-free NBE, decreased and intermediate in NBE of cancer-free COPD patients (p = 0.004) and further attenuated and lowest in epithelia of COPD patients with adenocarcinoma and SCC (p < 0.0001). Furthermore, GPRC5A mRNA was significantly decreased in NSCLCs and corresponding NBE compared to uninvolved normal lung (p = 0.03).
Our findings highlight decreased GPRC5A expression in the field cancerization of NSCLC, including that associated with lung inflammation. Assessment of the use of GPRC5A expression as a risk factor for NSCLC development in COPD patients is warranted.
PMCID: PMC3622592  PMID: 23154545
Field cancerization; Chronic obstructive pulmonary disease; Non–small-cell lung cancer; g-protein coupled receptor family C; group 5; member A; gene expression
15.  Osteocalcin regulates murine and human fertility through a pancreas-bone-testis axis 
The Journal of Clinical Investigation  2013;123(6):2421-2433.
The osteoblast-derived hormone osteocalcin promotes testosterone biosynthesis in the mouse testis by binding to GPRC6A in Leydig cells. Interestingly, Osteocalcin-deficient mice exhibit increased levels of luteinizing hormone (LH), a pituitary hormone that regulates sex steroid synthesis in the testes. These observations raise the question of whether LH regulates osteocalcin’s reproductive effects. Additionally, there is growing evidence that osteocalcin levels are a reliable marker of insulin secretion and sensitivity and circulating levels of testosterone in humans, but the endocrine function of osteocalcin is unclear. Using mouse models, we found that osteocalcin and LH act in 2 parallel pathways and that osteocalcin-stimulated testosterone synthesis is positively regulated by bone resorption and insulin signaling in osteoblasts. To determine the importance of osteocalcin in humans, we analyzed a cohort of patients with primary testicular failure and identified 2 individuals harboring the same heterozygous missense variant in one of the transmembrane domains of GPRC6A, which prevented the receptor from localizing to the cell membrane. This study uncovers the existence of a second endocrine axis that is necessary for optimal male fertility in the mouse and suggests that osteocalcin modulates reproductive function in humans.
PMCID: PMC3668813  PMID: 23728177
16.  Osteocalcin Induces Release of Glucagon-Like Peptide-1 and Thereby Stimulates Insulin Secretion in Mice 
PLoS ONE  2013;8(2):e57375.
The uncarboxylated form (ucOC), but not the γ-carboxylated form (GlaOC), of the bone-derived protein osteocalcin stimulates insulin secretion and regulates energy metabolism in insulin target tissues. Glucagon-like peptide–1 (GLP-1) is an insulin secretagogue that is released from the gut in response to food intake. We have now found that Gprc6a, a putative ucOC receptor, is expressed in epithelial cells of the mouse small intestine as well as in STC-1 enteroendocrine cells. Secretion of GLP-1 by STC-1 cells was stimulated by ucOC but not by GlaOC. The serum GLP-1 concentration in mice was increased by intraperitoneal or oral administration of ucOC, whereas GlaOC was effective in this regard only after oral application. Serum insulin levels were also increased by ucOC, and this effect was potentiated by an inhibitor of dipeptidyl peptidase IV and blocked by a GLP-1 receptor antagonist. Intravenous injection of ucOC in mice increased the serum GLP-1 concentration, and also increased the serum level of insulin. Our results suggest that ucOC acts via Gprc6a to induce GLP-1 release from the gut, and that the stimulatory effect of ucOC on insulin secretion is largely mediated by GLP-1.
PMCID: PMC3577726  PMID: 23437377
17.  G protein-coupled receptor, family C, group 5 (GPRC5B) downregulation in spinal cord neurons is involved in neuropathic pain 
Korean Journal of Anesthesiology  2014;66(3):230-236.
G protein-coupled receptor, family C, group 5 (GPRC5B), a retinoic acid-inducible orphan G-protein-coupled receptor (GPCR), is a member of the group C metabotropic glutamate receptor family proteins presumably related in non-canonical Wnt signaling. In this study, we investigated altered GPRC5B expression in the dorsal horn of the spinal cord after spinal nerve injury and its involvement in the development of neuropathic pain.
After induction of anesthesia by intraperitoneal injection of pentobarbital (35 mg /kg), the left L5 spinal nerve at the level of 2 mm distal to the L5 DRG was tightly ligated with silk and cut just distal to the ligature. Seven days after nerve injury, animals were perfused with 4% paraformaldehyde, and the spinal cords were extracted and post-fixed at 4℃ overnight. To identify the expression of GPRC5B and analyze the involvement of GPRC5B in neuropathic pain, immunofluorescence was performed using several markers for neurons and glial cells in spinal cord tissue.
After L5 spinal nerve ligation (SNL), the expression of GPRC5B was decreased in the ipsilateral part, as compared to the contralateral part, of the spinal dorsal horn. SNL induced the downregulation of GPRC5B in NeuN-positive neurons in the spinal dorsal horn. However, CNPase-positive oligodendrocytes, OX42-positive microglia, and GFAP-positive astrocytes were not immunolabeled with GPRC5B antibody in the spinal dorsal horn.
These results imply that L5 SNL-induced GPRC5B downregulation may affect microglial activation in the spinal dorsal horn and be involved in neuropathic pain.
PMCID: PMC3983420  PMID: 24729846
GPCR5B; Microglial activation; Neuroglial cell; Neuropathic pain; Spinal nerve injury
18.  The emerging roles of GPRC5A in diseases 
Oncoscience  2014;1(12):765-776.
The ‘Retinoic Acid-Inducible G-protein-coupled receptors’ or RAIG are a group comprising the four orphan receptors GPRC5A, GPRC5B, GPRC5C and GPRC5D. As the name implies, their expression is induced by retinoic acid but beyond that very little is known about their function. In recent years, one member, GPRC5A, has been receiving increasing attention as it was shown to play important roles in human cancers. As a matter of fact, dysregulation of GPRC5A has been associated with several cancers including lung cancer, breast cancer, colorectal cancer, and pancreatic cancer. Here we review the current state of knowledge about the heterogeneity and evolution of GPRC5A, its regulation, its molecular functions, and its involvement in human disease.
PMCID: PMC4303886  PMID: 25621293
GPRC5A; RAI3; tumor suppressor; oncogene; dual-behavior; cancer
19.  Regulation of male fertility by the bone-derived hormone osteocalcin 
Molecular and cellular endocrinology  2013;382(1):10.1016/j.mce.2013.10.008.
Traditionally, bone has been viewed as a relatively static tissue only fulfilling mechanical and scaffolding function. In the past decade however, this classical view of the bone has considerably evolved towards a more complex picture. It is now clear that the skeleton is not only a recipient for hormonal input but it is also an endocrine organ itself. Through the secretion of an osteoblast-derived molecule, osteocalcin, the skeleton regulates glucose homeostasis and male reproductive functions. When undercarboxylated, osteocalcin acts following its binding to a G-coupled receptor, GPRC6A, on pancreatic β cells to increase insulin secretion, on muscle and white adipose tissue to promote glucose homeostasis and on Leydig cells of the testis to favor testosterone biosynthesis. More recently, it was also shown that osteocalcin acts via a pancreas-bone-testis axis that regulates, independently of and in parallel to the hypothalamus-pituitary-testis axis, male reproductive functions by promoting testosterone biosynthesis. Lastly, in trying to expand the biological relevance of osteocalcin from mouse to human, it was shown that GPRC6A is a potential new susceptibility locus for primary testicular failure in humans. Altogether, these results shed new light on the importance of the endocrine role of the skeleton and also provide credence to the search for additional endocrine functions of this organ.
PMCID: PMC3850748  PMID: 24145129
20.  Novel Bone Endocrine Networks Integrating Mineral and Energy Metabolism 
Current osteoporosis reports  2013;11(4):391-399.
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.
PMCID: PMC4011556  PMID: 24193547
Bone; osteoblast; osteocyte; extracellular matrix; mineralization; fibroblastic growth factors; alpha-Klotho; fibroblastic growth factor receptor; hypophosphatemia; vitamin D; Cyp27b1; Cyp24; PTH; G-protein coupled receptors; GPRC6A; L-arginine; testosterone; osteocalcin; insulin resistance; insulin secretion; metabolic syndrome; hypophosphatemia
21.  Enhancement of lung tumorigenesis in a Gprc5a Knockout mouse by chronic extrinsic airway inflammation 
Molecular Cancer  2012;11:4.
Although cigarette smoking is the principal cause of lung carcinogenesis, chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, has been identified as an independent risk factor for lung cancer. Bacterial colonization, particularly with non-typeable Haemophilus influenzae (NTHi), has been implicated as a cause of airway inflammation in COPD besides cigarette smoke. Accordingly, we hypothesized that lung cancer promotion may occur in a chronic inflammatory environment in the absence of concurrent carcinogen exposure.
Herein, we investigated the effects of bacterial-induced COPD-like inflammation and tobacco carcinogen-enhanced tumorigenesis/inflammation in the retinoic acid inducible G protein coupled receptor knock out mouse model (Gprc5a-/- mouse) characterized by late-onset, low multiplicity tumor formation. Three-month-old Gprc5a-/- mice received 4 intraperitoneal injections of the tobacco-specific carcinogen, NNK, followed by weekly exposure to aerosolized NTHi lysate for 6 months. The numbers of inflammatory cells in the lungs and levels of several inflammatory mediators were increased in Gprc5a-/- mice treated with NTHi alone, and even more so in mice pretreated with NNK followed by NTHi. The incidence of spontaneous lung lesions in the Gprc5a-/- mice was low, but NTHi exposure led to enhanced development of hyperplastic lesions. Gprc5a-/- mice exposed to NNK alone developed multiple lung tumors, while NTHi exposure increased the number of hyperplastic foci 6-fold and the tumor multiplicity 2-fold. This was associated with increased microvessel density and HIF-1α expression.
We conclude that chronic extrinsic lung inflammation induced by bacteria alone or in combination with NNK enhances lung tumorigenesis in Gprc5a-/- mice.
PMCID: PMC3281775  PMID: 22239913
lung cancer; inflammation; COPD; Gpcr5a; NTHi
22.  MicroRNA-31 negatively regulates peripherally derived regulatory T-cell generation by repressing retinoic acid-inducible protein 3 
Nature Communications  2015;6:7639.
Peripherally derived regulatory T (pTreg) cell generation requires T-cell receptor (TCR) signalling and the cytokines TGF-β1 and IL-2. Here we show that TCR signalling induces the microRNA miR-31, which negatively regulates pTreg-cell generation. miR-31 conditional deletion results in enhanced induction of pTreg cells, and decreased severity of experimental autoimmune encephalomyelitis (EAE). Unexpectedly, we identify Gprc5a as a direct target of miR-31. Gprc5a is known as retinoic acid-inducible protein 3, and its deficiency leads to impaired pTreg-cell induction and increased EAE severity. By generating miR-31 and Gprc5a double knockout mice, we show that miR-31 promotes the development of EAE through inhibiting Gprc5a. Thus, our data identify miR-31 and its target Gprc5a as critical regulators for pTreg-cell generation, suggesting a previously unrecognized epigenetic mechanism for dysfunctional Treg cells in autoimmune diseases.
Peripherally derived regulatory T cells (pTreg) exhibit immunosuppressive capacity. Here, the authors show that microRNA-31 acting through inhibiting its direct target Gprc5a negatively regulates pTreg generation and promotes the development of experimental autoimmune encephalomyelitis.
PMCID: PMC4510656  PMID: 26165721
23.  A Gprc5a Tumor Suppressor Loss of Expression Signature Is Conserved, Prevalent, and Associated with Survival in Human Lung Adenocarcinomas12 
Neoplasia (New York, N.Y.)  2010;12(6):499-505.
Increasing the understanding of the impact of changes in oncogenes and tumor suppressor genes is essential for improving the management of lung cancer. Recently, we identified a new mouse lung-specific tumor suppressor—the G protein-coupled receptor 5A (Gprc5a). Microarray analysis of the transcriptomes of lung epithelial cells cultured from normal tracheas of Gprc5a knockout and wild-type mice defined a loss-of-Gprc5a gene signature, which revealed many aberrations in cancer-associated pathways. To assess the relevance of this mouse tumor suppressor to human lung cancer, the loss-of-Gprc5a signature was cross species compared with and integrated with publicly available gene expression data of human normal lung tissue and non-small cell lung cancers. The loss-of-Gprc5a signature was prevalent in human lung adenocarcinomas compared with squamous cell carcinomas or normal lung. Furthermore, it identified subsets of lung adenocarcinomas with poor outcome. These results demonstrate that gene expression patterns of Gprc5a loss in nontumorigenic mouse lung epithelial cells are evolutionarily conserved and important in human lung adenocarcinomas.
PMCID: PMC2887090  PMID: 20563252
24.  Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors 
Nature Communications  2012;3:1329-.
Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1β during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca2+ pathway. The resulting increase in the intracellular calcium concentration triggers inflammasome assembly and Caspase-1 activation. We identified necrotic cells as one source for excess extracellular calcium triggering this activation. In vivo, increased calcium concentrations can amplify the inflammatory response in the mouse model of carrageenan-induced footpad swelling, and this effect was inhibited in GPRC6A−/− mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation.
Levels of extracellular calcium can increase at sites of infection and inflammation; however, the physiological significance of this has been unclear. This work shows that extracellular calcium acts as a danger signal, triggering the NLRP3 inflammasome via two G protein-coupled receptors.
PMCID: PMC3535422  PMID: 23271661
25.  High Milk Consumption Does Not Affect Prostate Tumor Progression in Two Mouse Models of Benign and Neoplastic Lesions 
PLoS ONE  2015;10(5):e0125423.
Epidemiological studies that have investigated whether dairy (mainly milk) diets are associated with prostate cancer risk have led to controversial conclusions. In addition, no existing study clearly evaluated the effects of dairy/milk diets on prostate tumor progression, which is clinically highly relevant in view of the millions of men presenting with prostate pathologies worldwide, including benign prostate hyperplasia (BPH) or high-grade prostatic intraepithelial neoplasia (HGPIN). We report here a unique interventional animal study to address this issue. We used two mouse models of fully penetrant genetically-induced prostate tumorigenesis that were investigated at the stages of benign hyperplasia (probasin-Prl mice, Pb-Prl) or pre-cancerous PIN lesions (KIMAP mice). Mice were fed high milk diets (skim or whole) for 15 to 27 weeks of time depending on the kinetics of prostate tumor development in each model. Prostate tumor progression was assessed by tissue histopathology examination, epithelial proliferation, stromal inflammation and fibrosis, tumor invasiveness potency and expression of various tumor markers relevant for each model (c-Fes, Gprc6a, activated Stat5 and p63). Our results show that high milk consumption (either skim or whole) did not promote progression of existing prostate tumors when assessed at early stages of tumorigenesis (hyperplasia and neoplasia). For some parameters, and depending on milk type, milk regimen could even exhibit slight protective effects towards prostate tumor progression by decreasing the expression of tumor-related markers like Ki-67 and Gprc6a. In conclusion, our study suggests that regular milk consumption should not be considered detrimental for patients presenting with early-stage prostate tumors.
PMCID: PMC4418739  PMID: 25938513

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