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Multiple signaling pathways involved in stimulation of osteoblast differentiation by N-methyl-D-aspartate receptors activation in vitro.

ABSTRACT: AIM: Glutamate receptors are expressed in osteoblastic cells. The present study was undertaken to investigate the mechanisms underlying the stimulation of osteoblast differentiation by N-methyl-D-aspartate (NMDA) receptor activation in vitro. METHODS: Primary culture of osteoblasts was prepared from SD rats. Microarray was used to detect the changes of gene expression. The effect of NMDA receptor agonist or antagonist on individual gene was examined using RT-PCR. The activity of alkaloid phosphotase (ALP) was assessed using a commercial ALP staining kit. RESULTS: Microarray analyses revealed that 10 genes were up-regulated by NMDA (0.5 mmol/L) and down-regulated by MK801 (100 ?mol/L), while 13 genes down-regulated by NMDA (0.5 mmol/L) and up-regulated by MK801 (100 ?mol/L). Pretreatment of osteoblasts with the specific PKC inhibitor Calphostin C (0.05 ?mol/L), the PKA inhibitor H-89 (20 nmol/L), or the PI3K inhibitor wortmannin (100 nmol/L) blocked the ALP activity increase caused by NMDA (0.5 mmol/L). Furthermore, NMDA (0.5 mmol/L) rapidly increased PI3K phosphorylation, which could be blocked by pretreatment of wortmannin (100 nmol/L). CONCLUSION: The results suggest that activation of NMDA receptors stimulates osteoblasts differentiation through PKA, PKC, and PI3K signaling pathways, which is a new role for glutamate in regulating bone remodeling.

SUBMITTER: Li JL

PROVIDER: S-EPMC4003127 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Multiple signaling pathways involved in stimulation of osteoblast differentiation by N-methyl-D-aspartate receptors activation in vitro.

Li Jie-li JL Zhao Lin L Cui Bin B Deng Lian-fu LF Ning Guang G Liu Jian-min JM

Acta pharmacologica Sinica 20110620 7

<h4>Aim</h4>Glutamate receptors are expressed in osteoblastic cells. The present study was undertaken to investigate the mechanisms underlying the stimulation of osteoblast differentiation by N-methyl-D-aspartate (NMDA) receptor activation in vitro.<h4>Methods</h4>Primary culture of osteoblasts was prepared from SD rats. Microarray was used to detect the changes of gene expression. The effect of NMDA receptor agonist or antagonist on individual gene was examined using RT-PCR. The activity of alk ...[more]

PMID: 21685927

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Project description:The N-methyl-D-aspartate receptor (NMDAR) provides a pathway for glutamate-mediated inter-cellular communication, best known for its role in the brain but with multiple examples of functionality in non-neuronal cells. Data previously published by others and us provided ex vivo evidence that NMDARs regulate platelet and red blood cell (RBC) production. Here, we summarize what is known about these hematopoietic roles of the NMDAR. Types of NMDAR subunits expressed in megakaryocytes (platelet precursors) and erythroid cells are more commonly found in the developing rather than adult brain, suggesting trophic functions. Nevertheless, similar to their neuronal counterparts, hematopoietic NMDARs function as ion channels, and are permeable to calcium ions (Ca2+). Inhibitors that block open NMDAR (memantine and MK-801) interfere with megakaryocytic maturation and proplatelet formation in primary culture. The effect on proplatelet formation appears to involve Ca2+ influx-dependent regulation of the cytoskeletal remodeling. In contrast to normal megakaryocytes, NMDAR effects in leukemic Meg-01 cells are diverted away from differentiation to increase proliferation. NMDAR hypofunction triggers differentiation of Meg-01 cells with the bias toward erythropoiesis. The underlying mechanism involves changes in the intracellular Ca2+ homeostasis, cell stress pathways, and hematopoietic transcription factors that upon NMDAR inhibition shift from the predominance of megakaryocytic toward erythroid regulators. This ability of NMDAR to balance both megakaryocytic and erythroid cell fates suggests receptor involvement at the level of a bipotential megakaryocyte-erythroid progenitor. In human erythroid precursors and circulating RBCs, NMDAR regulates intracellular Ca2+ homeostasis. NMDAR activity supports survival of early proerythroblasts, and in mature RBCs NMDARs impact cellular hydration state, hemoglobin oxygen affinity, and nitric oxide synthase activity. Overexcitation of NMDAR in mature RBCs leads to Ca2+ overload, K+ loss, RBC dehydration, and oxidative stress, which may contribute to the pathogenesis of sickle cell disease. In summary, there is growing evidence that glutamate-NMDAR signaling regulates megakaryocytic and erythroid cells at different stages of maturation, with some intriguing differences emerging in NMDAR expression and function between normal and diseased cells. NMDAR signaling may provide new therapeutic opportunities in hematological disease, but in vivo applicability needs to be confirmed.

Dataset Information

Multiple signaling pathways involved in stimulation of osteoblast differentiation by N-methyl-D-aspartate receptors activation in vitro.

Publications

Multiple signaling pathways involved in stimulation of osteoblast differentiation by N-methyl-D-aspartate receptors activation in vitro.

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