Expression of Functional Metabotropic Glutamate Receptors in Primary Cultured Rat Osteoblasts

Gu, Yuchun; Publicover, Stephen J.

doi:10.1074/jbc.m004520200

Cited by 84 publications

(73 citation statements)

References 55 publications

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“…To our knowledge, this paper deals with the first direct demonstration of the functional expression of AMPAR by cultured rodent chondral cells. Although several previous studies have already demonstrated the functional expression of different subtypes of iGluRs 20,21) and mGluRs in bone, 22,23) no direct evidence is available for a role of AMPAR in mechanisms relevant to the exocytotic release of endogenous Glu from cartilage in the literature to date. Therefore, the further potentiation by the AMPAR desensitization blocker CTZ is highly suggestive of the possible involvement of AMPAR expressed by cultured chondrocytes in mechanisms underlying the release of endogenous Glu through exocytosis from cartilage.…”

Section: Discussionmentioning

confidence: 96%

Release of Endogenous Glutamate by AMPA Receptors Expressed in Cultured Rat Costal Chondrocytes

Wang

Hinoi

Takemori

et al. 2005

Biological & Pharmaceutical Bulletin

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L-Glutamate (Glu) is one of the most abundant free amino acids with a major excitatory neurotransmitter role in the vertebrate central nervous system, while recent trends are toward a role in neuronal differentiation, migration and survival in the developing brain.2-4) The actions of extracellular Glu are mediated by membranous receptors, which can be divided into two major groups. 4) One is ionotropic Glu-gated ion channels (iGluRs) that are further classified into DL-aamino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA), kainite (KA) and N-methyl-D-aspartate (NMDA) subtypes according to sequential similarities as well as responsiveness to different agonists and antagonists, 5,6) whereas the other is G-protein-coupled metabotropic receptors (mGluRs) that are a member of the class 3 G protein-coupled receptor family. 7,8) In addition to GluRs, vesicular Glu transporters (VGLUTs) are essential for signal output through the condensation of Glu into vesicular constituents for subsequent exocytotic release. Within the central nervous system (CNS), both VGLUT-1 9) and VGLUT-2 10) isoforms are supposed to suffice for the definition of an excitatory neuronal phenotype, while VGLUT3 is expressed in a number of cells shown to release Glu through exocytosis including dopaminergic, GABAergic and serotonergic neurons as well as astrocytes. 11)Recently, evidence that glutamatergic signaling is also functional in non-neuronal tissues, such as bone, pancreas and skin, is accumulating in the literature.12,13) Glu may act as a more widespread "cytokine" rather than a "neurotransmitter" to influence a variety of cellular activities in different tissue types. 12,13) Recent studies have raised the possibility that Glu may be one of the endogenous paracrine (autocrine) factors used for intercellular communications in bone. 14,15) The addition of an NMDA receptor antagonist inhibits cell differentiation in cultured osteoclasts, 16) for example, while Glu induces elevation of intracellular free Ca 2ϩ in a manner sensitive to antagonism by the NMDA receptor antagonist dizocilpine in osteoblasts. 17) We have also recently demonstrated the exacerbation of osteoblastic differentiation by different NMDA receptor antagonists.18) In addition to NMDA receptors, osteoblasts constitutively express mRNA for non-NMDA receptors such as GluR3 subunit of AMPA receptors (AMPAR) as well as KA1 and KA2 subunits of KA receptors, 19) while AMPAR modulate the exocytotic release of Glu from cultured osteoblasts. 20) By contrast, no much attention has been paid to the possible functional expression by cartilage of particular glutamatergic signaling molecules required for the neurotransmission in the brain to date. Therefore, we have attempted to mimic our previous studies on cultured rat osteoblasts for the release of endogenous Glu using cultured rat chondrocytes. In the present study, functional expression was for the first time shown with a particular subtype of iGluRs in cartilage. MATERIALS AND METHODSMaterials Leica CM 3050s cryostat and a fluorescen...

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Section: Discussionmentioning

confidence: 96%

Release of Endogenous Glutamate by AMPA Receptors Expressed in Cultured Rat Costal Chondrocytes

Wang

Hinoi

Takemori

et al. 2005

Biological & Pharmaceutical Bulletin

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“…38 We have also demonstrated marked suppression of the cellular differentiation by sustained exposure to different NMDA receptor antagonists in a manner dependent on Runx2, which is the master regulator of osteoblast differentiation, in cultured rat calvarial osteoblasts. 39 Functional expression is also shown with mGluR1 isoform in rat femoral osteoblasts 40 and with both mGluR4 and mGluR8 isoforms in rat calvarial osteoblasts, 41 respectively. In addition to the aforementioned analyses on different molecules required for glutamatergic signal input in osteoblasts, on the other hand, the possible functional expression of GluRs has also been evaluated in osteoclasts in the literature.…”

Section: Discussionmentioning

confidence: 99%

Glutamate Suppresses Osteoclastogenesis through the Cystine/Glutamate Antiporter

Hinoi

Takarada

Uno

et al. 2007

The American Journal of Pathology

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Previous studies have demonstrated functional expression of different glutamate receptor subtypes (GluRs) in both osteoblasts and osteoclasts. In the present study, we investigated the possible functional expression by osteoclasts of different glutamatergic signaling machineries including GluRs. In disagreement with the aforementioned prevailing view, no mRNA expression was found for all GluRs examined in primary cultured mouse osteoclasts differentiated from bone marrow precursors. Constitutive expression of mRNA was seen with glutamate transporters, such as excitatory amino acid transporters and cystine/glutamate antiporter, in primary osteoclasts. Glutamate significantly inhibited osteoclastogenesis at a concentration over 500 mol/L in both primary osteoclasts and preosteoclastic RAW264.7 cells without affecting the cell viability in a manner sensitive to the antiporter inhibitor. In RAW264.7 cells stably overexpressing the cystine/glutamate antiporter, the inhibition by glutamate was more conspicuous than in cells transfected with empty vector alone. The systemic administration of glutamate significantly prevented the decreased bone mineral density in both femur and tibia in addition to increased osteoclastic indices in ovariectomized mice in vivo. These results suggest that glutamate may play a pivotal role in mechanisms associated with osteoclastogenesis through the cystine/glutamate antiporter functionally expressed by osteoclasts devoid of any GluRs cloned to date. (Am J

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“…Non-CaR osteoblast mechanisms have been suggested (35), for instance, certain metabotropic glutamate receptors that are present in bone (40) and can be activated both by Ca 2ϩ o and by Gd 3ϩ (41). Nevertheless, the Ca 2ϩ o -and Gd 3ϩ -sensitive subtype metabotropic glutamate receptor 1␣ is not expressed in osteoblasts (42). In addition, the concentrations of Ca 2ϩ o and Gd 3ϩ required to evoke metabotropic glutamate receptor 1␣ responses are considerably higher than those used here (41).…”

Section: Osteoblast Gene Expression In Frc Cellsmentioning

confidence: 99%

Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones

Dvorák

Siddiqua

Ward

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

393

262

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We investigated the direct effects of changes in free ionized extracellular calcium concentrations ([Ca 2؉ ]o) on osteoblast function and the involvement of the calcium-sensing receptor (CaR) in mediating these responses. CaR mRNA and protein were detected in osteoblast models, freshly isolated fetal rat calvarial cells and murine clonal osteoblastic 2T3 cells, and in freshly frozen, undecalcified preparations of human mandible and rat femur. In fetal rat calvarial cells, elevating [Ca 2؉ ]o and treatment with gadolinium, a nonpermeant CaR agonist, resulted in phosphorylation of the extracellular signal-regulated kinases 1 and 2, Akt, and glycogensynthase kinase 3␤, consistent with signals of cell survival and proliferation. In agreement, cell number was increased under these conditions. Expression of the osteoblast differentiation markers core binding factor ␣1, osteocalcin, osteopontin, and collagen I mRNAs was increased by high [ (2, 3) and alter the levels of expression of some differentiation markers (4, 5). During mineralization, decreases in [Ca 2ϩ ] o are also likely to occur (6), but the effect of lowering [Ca 2ϩ ] o in bone cells has not been extensively addressed.The mechanism of [Ca 2ϩ ] o -sensing by osteoblasts is unclear. The parathyroid extracellular calcium-sensing receptor (CaR) is a key player in the maintenance of a constant systemic [Ca 2ϩ ] o , predominantly through regulation of parathyroid hormone (PTH) secretion and urinary calcium excretion (7,8). CaR is also present in osteoblasts (9 and references therein), where a functional role is currently debated. Recently two studies have shown that CaRdeficient mice exhibit an essentially normal skeletal phenotype when the hyperparathyroidism resulting from the lack of the parathyroid CaR is prevented (10, 11). Thus, it remains unclear whether the osteoblast CaR is a true regulator of bone function or whether its expression is vestigial (12).In this study, we investigated the effects of both decreasing and increasing [Ca 2ϩ ] o on osteoblast proliferation and intracellular signaling events, the expression of several osteoblast differentiation markers [core binding factor ␣1 (Cbfa1, also termed Runx2 and Osf2), osteocalcin (OC), osteopontin (OP), and type I collagen (collaI)], the activity of alkaline phosphatase (AlP), and mineralized nodule formation in the absence of systemic calciotropic factors, namely PTH and vitamin D. We further investigated the role played by the CaR in these events using an alternative, nonpermeant CaR agonist, gadolinium (Gd 3ϩ ) and a CaR inhibitor, NPS 89636 (a ''calcilytic''). We used well characterized osteoblast models, freshly isolated fetal rat calvarial cells (FRC) (13) and the clonal murine osteoblast cell line, 2T3 cells (14). The expression of CaR in freshly frozen sections of rat and human bone was also determined. Materials and MethodsAnimals. Sprague-Dawley rats (Charles River Breeding Laboratories) were killed by cervical dislocation and used in accordance to the U.K. Animals Scientific Procedures...

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Expression of Functional Metabotropic Glutamate Receptors in Primary Cultured Rat Osteoblasts

Cited by 84 publications

References 55 publications

Release of Endogenous Glutamate by AMPA Receptors Expressed in Cultured Rat Costal Chondrocytes

Release of Endogenous Glutamate by AMPA Receptors Expressed in Cultured Rat Costal Chondrocytes

Glutamate Suppresses Osteoclastogenesis through the Cystine/Glutamate Antiporter

Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones

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