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 μmol/L), while 13 genes down-regulated by NMDA (0.5 mmol/L) and up-regulated by MK801 (100 μmol/L). Pretreatment of osteoblasts with the specific PKC inhibitor Calphostin C (0.05 μmol/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.
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Introduction
L-Glutamate (Glu) is accepted as an excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS). Extracellular levels of glutamate are determined by the glutamate transporter1, 2. The diverse actions of L-glutamate in the CNS result from the existence of multiple glutamate receptors (GluR). These have been divided into two classes, metabotropic (mGluR) and ionotropic (iGluR), according to their molecular heterologies and differing intracellular signal transduction mechanisms. The iGluRs are further classified into N-methyl-D-aspartate (NMDA), DL-α-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA), and kainite(KA) receptors. NMDA receptors are glutamate-gated ion channels characterized by a very high Ca2+ conductance3.
In mammalian bone, NMDA receptors are expressed in osteoblasts and osteoclasts as revealed by RT-PCR, in situ hybridization, immunohistochemistry, and electrophy-siology4, 5. Bone cells and neurons possess similar, and in some cases identical glutamate signaling machinery and receptors5.
Bone loss is associated with a reduction in nerve endings that immunostain for glutamate. Glutamate-containing neuronal endings have been described in a dense and intimate network in bone tissue6. Chenu et al reported that bone loss was induced in ovarectomized (OVX) rats with a reduction in neuronal density and concluded there was a functional link between the nervous system and bone loss after ovariectomy7. Hinoi et al reported that administration of glutamate significantly prevented the decreased bone mineral density in both the femur and the tibia in ovariectomized mice in vivo8. All these findings suggest that the neurotransmitter glutamate may play a role in bone remodeling.
NMDA has been shown to promote the proliferation and differentiation of hippocampal neural progenitor cells (NPCs) in vitro by activating NMDA receptors9. NMDA is mitogenic for MC3T3-E1 osteoblastic cells and glutamate has been reported to promote the viability of primary human osteoblasts in vitro10. Blockade of NMDA receptors in rat primary osteoblasts inhibits expression of markers of bone formation in vitro4, 11. Previously, we demonstrated that activation of NMDA receptors promoted rat primary osteoblast differentiation and that one of the possible mechanism was ERK1/2 activation12.
In the central nervous system (CNS), activation of the Ca2+-permeable NMDA receptors results in an increase in Ca2+ influx. The Ca2+ signals then activate several Ca2+-dependent kinases13. Kinases which have been suggested to be important include protein kinases C and A (PKC and PKA), and PI3 kinase (PI3K). PKC is localized at excitatory synapses containing NMDA receptors and might be involved in NMDA-evoked ERK1/2 phosphorylation14. In hippocampal neurons NMDA played a neuroprotective role through the PKA signaling pathway15, and in striatal and cortical neurons, PI3K is critically involved in NMDA receptor activation16, 17, 18. Thus, the above signaling pathways may positively respond to signals from ionotropic types of glutamate receptors in the CNS.
Locally elevated extracellular Ca2+ levels have been suggested to play a role in regulating bone remodeling. Signaling pathways such as phospholipase C (PLC), PKC, mitogen-activated protein kinase (MAPK), and PI3K have been implicated in the modification of cellular function by Ca2+19, 20. Since these signaling pathways are involved in NMDA receptor regulation in the CNS and were also suggested to be important by our initial microarray analysis, we hypothesized that they are also involved in NMDA-induced bone remodeling. The aim of this study was to investigate the mechanism of the effects of NMDA and MK801 (the noncompetitive antagonist of NMDA receptors) on differentiation of rat primary osteoblasts.
Materials and methods
Primary cultures of osteoblasts
Osteoblasts were prepared from calvaria of 1-day-old Sprague-Dawley rats by a sequential enzymatic digestion method as described previously21. The bones were cut into chips and washed with calcium- and magnesium-free phosphate-buffered saline (PBS). Calvaria were incubated at 37 °C for 20 min with 0.25% trypsin and 1 h with 0.1% type II collagenase in PBS with gentle mixing. Incubation with type II collagenase was then repeated. Cells released from the bone chips were collected in α-modified minimum essential medium (α-MEM) containing 10% fetal bovine serum (FBS), 100 units/mL penicillin (Gibco) and 100 μg/mL streptomycin (Gibco), followed by centrifugation at 1000 revolutions per minute for 10 min. The resulting pellets were re-suspended in α-MEM containing 10% FBS. Cells were plated at appropriate density, and cultured at 37 °C under 5% CO2. Culture medium was changed every 2 d.
Rat osteoblasts (1.5×105/cm2) were plated in 6-well plates in α-MEM containing 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL ascorbic acid (Sigma) and 5 mmol/L sodium β-glycerophosphate (Sigma) for measurement of differentiation markers. After 4 d of culture, cells were starved with serum-free α-MEM and 0.2% bovine serum albumin (BSA) for 12 h. Cultures were also exposed to fresh serum-free medium with 0.2% BSA with or without NMDA, MK801 (both from Tocris Cookson Ltd UK), or other inhibitors.
RNA, cDNA preparation, and quantitative real-time PCR
To ensure statistical significance of microarray analyses, cultures of osteoblasts were incubated in osteogenic medium with 0.2% BSA containing NMDA or MK801 for 48 h. Total RNA was isolated from primary rat calvarial cells with TRIzol reagent (Invitrogen). RNA (1 μg) was reverse transcribed to cDNA with the Invitrogen Superscript Kit. Corresponding probes were hybridized to Illumina GeneChip arrays and subjected to bioinformatics analyses. cDNA was amplified with the Takara SYBR Green RT-PCR kit using gene-specific primers (Table 1) in the Real-Time PCR Detection System (Roche). Quantification and normalization to GAPDH amplicons was performed. Statistical analyses were performed with Prism 5.0 (GraphPad Software, San Diego, CA).
As might be expected, many genes that have previously been demonstrated to be NMDA or MK801-regulated can be found in our lists of genes as commonly regulated in the treatment regimens (Table 2 or 3). This implies that the microarray results and analyses are reliable. To validate this further, we used quantitative real time RT-PCR to examine the effect of NMDA or MK801 on individual genes. We examined the following ten genes (Nov, Stc1, Anxa1, Tspan8, Dab2, Nfkb2, Mmp12, Fmo1, Colec12, and Fap) which were commonly up-regulated by NMDA and down-regulated by MK801 as shown in Table 2. We also examined thirteen genes (Cib1, Ddit3, Gaa, Herpud1, Ninj1, Trappc2, Rpo1-2, Slc20a1, Slc3a2, Trib3, Yars, Serpinb2, and Thop1) which were commonly down-regulated by NMDA and up-regulated by MK801 as shown in Table 3.
Gene ontology analysis
http://omicslab.genetics.ac.cn/GOEAST/php/illumina.php was applied to GO analysis. We chose only GO categories that had a P-value of <0.001 and a log odds-ratio of >1.5-fold.
Alkaline phosphatase synthesis
Rat calvarial osteoblasts were plated at a density of 1.5×105/well into 6-well culture plates. After treatments, cells were washed three times with ice-cold PBS, scraped into 0.5 mL of ice-cold 0.5% Triton X-100 solution and lysed by ultrasonication in an ice bath for 2 min. The supernatant was centrifuged at 14 000×g and 4 °C for 5 min, then stored frozen at -20 °C until measurement of alkaline phosphatase levels. For the determination of these levels, cell lysates were incubated in a 96-well plate with 100 μL of 0.1 mol/L NaHCO3-Na2CO3 buffer (pH 10.0) containing 0.5% Triton X-100, with p-nitrophenylphosphate as substrate for 30 min at 37 °C. The absorbance of p-nitrophenol liberated in the reactive solution was read at 405 nm. Diluted cell lysates were measured at 740 nm for total protein content using the BCA method (Bio-Rad Protein Assay kit, Bio-Rad Laboratories, Richmond, CA, USA). ALP activity in the cells was normalized for total protein content of the cell lysate.
Western blotting
Cells treated with 0.5 mmol/L NMDA and wortmannin (100 nmol/L) (Cell Signaling, USA) for the indicated times were lysed and the protein concentrations were determined by Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, CA). For Western blotting, 50 mg of total cell lysates was subjected to SDS-polyacrylamide gel electrophoresis. The protein was transferred to polyvinylidene difluoride membranes using transfer buffer (50 mmol/L Tris, 190 mmol/L glycin, and 10% methanol) at 120 V for 2 h. The membranes were incubated with blocking buffer (50 mmol/L Tris, 200 mmol/L NaCl, 0.2% Tween 20, and 3% bovine serum albumin) overnight at 4 °C. After washing three times with washing buffer (blocking buffer without 3% bovine serum albumin) for 10 min each, the blot was incubated with primary antibody (PI3K, phosphorylated PI3K) for 12 h, followed by horseradish peroxidaselabeled secondary antibody for 1 h. The membranes were washed again, and detection was performed using an ECL kit (ECL Plus Western Blotting Detection System, GE Healthcare UK Limited, Little Chalfont, UK) and exposed to film.
Statistical analysis
Each experiment was performed with at least three independent samples. The results are expressed as the mean±SD, unless otherwise indicated. Statistical significance of the observed differences was analyzed by one-way ANOVA where appropriate. A P value <0.05 was considered statistically significant.
Results
cDNA microarray and gene ontology analysis
Genes whose expression was changed in opposite directions by NMDA and MK801 treatment were defined as being commonly up-regulated or down-regulated genes. According to the ratio values, 353 genes were up-regulated by NMDA, 106 genes down-regulated by MK801 and hence 83 genes were the commonly up-regulated genes. There were 50 genes down-regulated by NMDA, 297 genes up-regulated by MK801 and 27 were the commonly down-regulated genes (Figure 1). We then examined the significant GO categories and genes by http://omicslab.genetics.ac.cn/GOEAST/php/illumina.php. A P-value of <0.001 and a log odds-ratio >1.5-fold were selected as the significant criteria. This narrowed the numbers of genes to 10 in the group of common genes that were up-regulated by NMDA and down-regulated by MK801 (Table 2), and 13 in the group of common genes down-regulated by NMDA and up-regulated by MK801 (Figure 2, Table 3).
Effects of inhibiting PKC, PKA, and PI3K on indexes of osteoblast differentiation
From the results of the microarray analysis as well as some previously published work in other cell types, we noted several genes (Anxa1, Mmp12, Stc1, Trib3, NF-κB, Ddit3) involved in PKC, PKA, and PI3K signaling pathways (see discussion). To determine the involvement of PKC in NMDA-mediated osteoblast differentiation, serum-starved cells were pretreated with Calphostin C (0.05 μmol/L), a specific inhibitor of PKC, for 90 min followed by coincubation with 0.5 mmol/L NMDA for 48 h. Cell morphology was assessed using a commercial ALP staining kit. Basal levels of ALP activity were unaffected by treatment of osteoblastic cells with Calphostin C alone. However, this treatment did lead to an observable reduction in alkaline phosphatase levels (Figure 3).
To determine whether activation of PKA is involved in NMDA-induced osteoblast differentiation, cells were pretreated with the PKA inhibitor H-89 (20 nmol/L). After incubation in serum-free medium for 12 h, cells were pretreated with 20 nmol/L H-89 for 90 min, followed by coincubation with 0.5 mmol/L NMDA for 48 h. Inhibition of PKA by H-89 treatment led to a decrease in NMDA-stimulated ALP activity levels (Figure 3). The ability of this inhibitor to curtail the effects of NMDA on osteoblast differentiation suggests that PKA activation is involved in NMDA-induced osteoblast differentiation.
We further explored signal transduction components related to NMDA-induced osteoblast differentiation by examining the involvement of PI3K using the PI3K inhibitor wortmannin. Cells were pretreated with wortmannin (100 nmol/L) for 90 min, followed by coincubation with 0.5 mmol/L NMDA for 48 h. This treatment protocol also led to a decrease in NMDA-stimulated ALP activity levels (Figure 3). The ability of these three inhibitors to curtail the effects of NMDA on markers of osteoblast differentiation suggests that activation of PKC, PKA, and PI3K is involved in the phenomenon of NMDA-induced osteoblast differentiation.
To assess whether PI3K were activated by NMDA, we assessed phosphorylation of PI3K using phospho-antibodies against phosphorylated peptides derived from PI3K. We found that 0.5 mmol/L NMDA induced a rapid increase in PI3K phosphorylation with maximal levels at 15 min. Prolonged NMDA stimulation up to 30 min, however, resulted in a decrease of phosphorylated PI3K levels toward baseline (Figure 4A).
To determine whether activation of PI3K is involved in NMDA-induced osteoblast differentiation, cells were pretreated with the PI3K inhibitor wortmannin. After incubation in serum-free medium for 12 h, cells were pretreated with 100 nmol/L wortmannin for 90 min, followed by coincubation with 0.5 mmol/L NMDA for 15 min. Inhibition of PI3K by wortmannin treatment led to a decrease in NMDA-stimulated phosphorylated PI3K levels (Figure 4B). The ability of this inhibitor to curtail the effects of NMDA on osteoblast differentiation suggests that PI3K activation is involved in NMDA-induced osteoblast differentiation (Figure 5).
Discussion
Our data suggest that NMDA promotes osteoblast differentiation via PKA, PKC and PI3K signaling pathways. These findings also demonstrate that NMDA directly acts on and affects osteoblasts and that the NMDA receptors expressed in primary rat calvaria osteoblasts are functional.
Within the large family of iGluRs, NMDARs constitute a subfamily identifiable by a specific molecular composition and unique pharmacological and functional properties22, 23. The most commonly used agonist at the glutamate recognition site of NMDA receptors is NMDA itself. However, it is not a substrate that promotes glutamate uptake24. Activation of NMDA receptors has been shown to be important in normal expression of bone matrix proteins4, 12. We have previously demonstrated that NMDA increased osteoblast ALP activity and osteocalcin (OC) in a dose-dependent manner, while the NMDA receptor antagonist MK801 reduced these effects12. We have also observed that NMDA increases ALP activity and OC expression in a time-dependent manner and that the peak of this increase reached 48 h after treatment (data not shown). Together, these observations suggest that NMDA regulates osteoblast differentiation via NMDA receptors.
In the present study, we employed cDNA microarray analysis, which was expected to be more sensitive than cDNA subtraction analysis, for the detection of specific genes related to the stimulation of NMDA receptors in rat osteoblasts. Annexin 1 (Anxa1) is one member of a family of phospholipid and calcium-binding proteins of which 20 are known at present25. It translocates from the cytoplasm to the outer cell surface via a Ca2+-dependent mechanism26, 27, 28. It is a substrate for protein kinase C and protein tyrosine kinases and has multiple phosphorylation sites as well as calcium and phospholipid binding properties29. Anxa1 was also shown to be activated as a consequence of sequential PI3K-PKC activation30, 31. Matrix metalloproteinases (MMPs) are a family of secreted or transmembrane zinc-dependent endopeptidases. The activation of MMP3, MMP12 and MMP13 was correlated with activation of the PI3K/Akt signaling cascade in microglial cells32. In epithelial cells and fibroblasts, MMP12 and MMP13 could be up-regulated in a PI3K, PKCδ and ERK1/2-dependent manner33. The stanniocalcins, comprising Stc1 and Stc2, are secreted homodimeric glycoprotein hormones with little homology to other proteins34. Stc1 was originally identified as a hormone secreted by the corpuscles of the stannius inteleost fish35 and in fish appears to function primarily in the prevention of hypercalcaemia mediated via the ability of Stc1 to reduce calcium uptake by gills and inhibit intestinal calcium transport in the gut36, 37, 38. In human endothelial cells Stc1 mRNA expression was up-regulated primarily through PKC, ERK and Ca2+ signaling pathways39. Trib3 (tribbles 3) is a mammalian homologue of Drosophila tribbles40, 41, 42. Expression of Trib3 was found to be PI3K-dependent in prostate cancer cells43. Furthermore, it has been suggested that the tribbles protein family is involved in regulation of the MAP kinase pathway44. Nuclear factor-κB (NF-κB) is a ubiquitous heterodimeric transcription factor that regulates inflammation and cell survival and differentiation45, 46. Many different kinases, including PKA, PKC, GSK-3β, PI3K, AKT, p38, NIK and even IKK, have been shown to induce phosphorylation of NF-κB directly or indirectly45, 46, 47. Regulation of NF-κB has also previously been suggested to depend on the PLCβ/PKC pathway48, 49. The PI3K/Akt signaling pathway seems able to positively regulate NF-κB activity50, 51. Studies have demonstrated that different stress response pathways mediating Ddit3 (DNA-damage inducible transcript 3) expression are regulated by protein kinases52, 53. Ddit3 is reported to be induced during serum starvation, glutamine deprivation, growth to confluency and upon exposure to tunicamycin, which promotes accumulation of proteins in the ER by preventing glycosylation54. Ddit3 protein was increased in pancreatic β-cells grown in high glucose that were exposed to U0126 to block ERK1/2 activity55. Our data showed Anxa1, MMP12, Stc1 and NF-κB were all up-regulated by NMDA in osteoblasts, while Trib3 and Ddit3 were down-regulated. In addition, it suggests that ERK, PKA, PKC and PI3K signaling pathways may be involved with changes of these genes' expression levels in NMDA-treated osteoblasts.
In the CNS, activation of NMDA receptors increases calcium influx, which is associated with spatial long term memory in the Morris water maze56 through the PKA signaling pathway56, 57, 58. Repetitive stimulation of glutamatergic NMDA receptors result in PKA and PKC activation in vivo59. The high Ca2+ permeability of NMDA receptor channels in bone is a characteristic similar to that of receptors expressed in the CNS5, 60. Ca2+ flux could trigger a number of second messenger responses that could ultimately be responsible for the anabolic effects of NMDA stimulation in bone. The MAPK, PKC, PKA and PI3K signaling pathways can all be directly activated downstream of calcium influx61. The activation of protein kinase C (PKC) and/or protein kinase A (PKA) plays an essential role in osteoblast differentiation62. In our previous study, we have observed activation of the ERK1/2 signaling pathway when osteoblasts were treated with NMDA12. With this background information, we further pursued the results of our microarray analysis information and specifically studied the PKC, PKA and PI3K signaling pathways to see if these signaling pathways are involved in NMDA-mediated osteoblast activation, independent of ERK1/2. We found that cells staining for ALP were significantly decreased by treatment with the PKA inhibitor H-89 or the PKC inhibitor Calphostin C. However, the ALP staining cells recovered after treatment with NMDA, which suggests a mechanism for NMDA-induced osteoblast differentiation that is, at least partly, dependent on PKA and PKC signaling. NMDA increased PI3K phosphorylation which is kinetically correlated to concomitant ERK1/2 phosphorylation in rat striatal neurons63. It was reported that intracellular Ca2+- regulated pathways signal through PI3K in osteoblasts subjected to mechanical strain64. The ability of the chemical inhibitor of PI3K, wortmannin, to significantly reduce NMDA-induced expression of the osteoblast differentiation marker ALP suggests that PI3K is involved in NMDA-mediated osteoblastic cell differentiation.
In summary, we have demonstrated that activation of NMDA receptors promotes osteoblast differentiation via PKA, PKC and PI3K signaling mechanisms which are independent of ERK1/2 signaling. The screening for specific genes carried out in this work allowed the discovery of signaling pathways which are associated with these genes. These findings yield new avenues by which to look for therapeutic targets in osteoporosis and provide useful direction for further investigation of the mechanisms involved.
Author contribution
Dr Jie-li LI designed the project, performed the research and wrote the paper; Dr Lin ZHAO worked on the data for revision; Dr Bin CUI helped on detailed research work; Dr Lian-fu DENG and Dr Guang NING contributed overview advice; Dr Jian-min LIU provided fundings on all the work and designed the project.
References
Nakanishi N, Shneider NA, Axel R . A family of glutamate receptor genes: evidence for the formation of heteromultimeric receptors with distinct channel properties. Neuron 1990; 5: 569–81.
Yoneda Y, Kuramoto N, Kitayama T, Hinoi E . Consolidation of transient ionotropic glutamate signals through nuclear transcription factors in the brain. Prog Neurobiol 2001; 63: 697–719.
Wisden W, Seeburg PH . Mammalian ionotropic glutamate receptors. Curr Opin Neurobiol 1993; 3: 291–8.
Hinoi E, Fujimori S, Yoneda Y . Modulation of cellular differentiation by N-methyl-D-aspartate receptors in osteoblasts. FASEB J 2003; 17: 1532–4.
Gu Y, Genever PG, Skerry TM, Publicover SJ . The NMDA type glutamate receptors expressed by primary rat osteoblasts have the same electrophysiological characteristics as neuronal receptors. Calcif Tissue Int 2002; 70: 194–203.
Serre CM, Farlay D, Delmas PD, Chenu C . Evidence for a dense and intimate innervation of the bone tissue, including glutamate-containing fibers. Bone 1999; 25: 623–9.
Burt-Pichat B, Lafage-Proust MH, Duboeuf F, Laroche N, Itzstein C, Vico L, et al. Dramatic decrease of innervation density in bone after ovariectomy. Endocrinology 2005; 146: 503–10.
Hinoi E, Takarada T, Uno K, Inoue M, Murafuji Y, Yoneda Y . Glutamate suppresses osteoclastogenesis through the cystine/glutamate antiporter. Am J Pathol 2007; 170: 1277–90.
Joo JY, Kim BW, Lee JS, Park JY, Kim S, Yun YJ, et al. Activation of NMDA receptors increases proliferation and differentiation of hippocampal neural progenitor cells. J Cell Sci 2007; 120: 1358–70.
Fatokun AA, Stone TW, Smith RA . Hydrogen peroxide-induced oxidative stress in MC3T3-E1 cells: the effects of glutamate and protection by purines. Bone 2006; 39: 542–51.
Peet NM, Grabowski PS, Laketic-Ljubojevic I, Skerry TM . The glutamate receptor antagonist MK801 modulates bone resorption in vitro by a mechanism predominantly involving osteoclast differentiation. FASEB J 1999; 13: 2179–85.
Li JL, Cui B, Qi L, Li XY, Deng LF, Ning G, et al. NMDA enhances stretching-induced differentiation of osteoblasts through the ERK1/2 signaling pathway. Bone 2008; 43: 469–75.
Mabuchi T, Kitagawa K, Kuwabara K, Takasawa K, Ohtsuki T, Xia Z, et al. Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo. J Neurosci 2001; 21: 9204–13.
Wang JQ, Fibuch EF, Mao L . Regulation of mitogen-activated protein kinases by glutamate receptors. J Neurochem 2007; 100: 1–11.
Valera E, Sánchez-Martín FJ, Ferrer-Montiel AV, Messeguer A, Merino JM . NMDA-induced neuroprotection in hippocampal neurons is mediated through the protein kinase A and CREB (cAMP-response element-binding protein) pathway. Neurochem Int 2008; 53: 148–54.
Perkinton MS, Ip JK, Wood GL, Crossthwaite AJ, Williams RJ . Phosphatidylinositol 3-kinase is a central mediator of NMDA receptor signalling to MAP kinase (Erk1/2), Akt/PKB and CREB in striatal neurones. J Neurochem 2002; 80: 239–54.
Fuller G, Veitch K, Ho LK, Cruise L, Morris BJ . Activation of p44/p42 MAP kinase in striatal neurons via kainate receptors and PI3 kinase. Brain Res Mol Brain Res 2001; 89: 126–32.
Lafon-Cazal M, Perez V, Bockaert J, Marin P . Akt mediates the anti-apoptotic effect of NMDA but not that induced by potassium depolarization in cultured cerebellar granule cells. Eur J Neurosci 2002; 16: 575–83.
Brown EM, MacLeod RJ . Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 2001; 81: 239–97.
Huang Z, Cheng SL, Slatopolsky E . Sustained activation of the extracellular signal-regulated kinase pathway is required for extracellular calcium stimulation of human osteoblast proliferation. J Biol Chem 2001; 276: 21351–8.
Danciu TE, Adam RM, Naruse K, Freeman MR, Hauschka PV . Calcium regulates the PI3K-Akt pathway in stretched osteoblasts. FEBS Lett 2003; 536: 193–7.
Dingledine R, Borges K, Bowie D, Traynelis SF . The glutamate receptor ion channels. Pharmacol Rev 1999; 51: 7–61.
Cull-Candy SG, Leszkiewicz DN . Role of distinct NMDA receptor subtypes at central synapses. Sci STKE 2004: re16.
Kew JN, Kemp JA . Ionotropic and metabotropic glutamate receptor structure and pharmacology. Psychopharmacology (Berl) 2005; 179: 4–29.
Moss SE . Ion channels. Annexins taken to task. Nature 1995; 378: 446–7.
Philip JG, Flower RJ, Buckingham JC . Glucocorticoids modulate the cellular disposition of lipocortin 1 in the rat brain in vivo and in vitro. Neuroreport 1997; 8: 1871–6.
Taylor AD, Cowell AM, Flower J, Buckingham JC . Lipocortin 1 mediates an early inhibitory action of glucocorticoids on the secretion of ACTH by the rat anterior pituitary gland in vitro. Neuroendocrinology 1993; 58: 430–9.
Taylor AD, Christian HC, Morris JF, Flower RJ, Buckingham JC . An antisense oligodeoxynucleotide to lipocortin 1 reverses the inhibitory actions of dexamethasone on the release of adrenocorticotropin from rat pituitary tissue in vitro. Endocrinology 1997; 138: 2909–18.
Alldridge LC, Harris HJ, Plevin R, Hannon R, Bryant CE . The annexin protein lipocortin 1 regulates the MAPK/ERK pathway. J Biol Chem 1999; 274: 37620–8.
Solito E, Mulla A, Morris JF, Christian HC, Flower RJ, Buckingham JC . Dexamethasone induces rapid serine-phosphorylation and membrane translocation of annexin 1 in a human folliculostellate cell line via a novel nongenomic mechanism involving the glucocorticoid receptor, protein kinase C, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Endocrinology 2003; 144: 1164–74.
John C, Cover P, Solito E, Morris J, Christian H, Flower R, et al. Annexin 1-dependent actions of glucocorticoids in the anterior pituitary gland: roles of the N-terminal domain and protein kinase C. Endocrinology 2002; 143: 3060–70.
Ito S, Kimura K, Haneda M, Ishida Y, Sawada M, Isobe K . Induction of matrix metalloproteinases (MMP3, MMP12, and MMP13) expression in the microglia by amyloid-beta stimulation via the PI3K/Akt pathway. Exp Gerontol 2007; 42: 532–7.
Shukla A, Barrett TF, Nakayama KI, Nakayama K, Mossman BT, Lounsbury KM . Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCdelta-dependent pathway in murine lung. Faseb J 2006; 20: 997–9.
Wagner GF, Dimattia GE . The stanniocalcin family of proteins. J Exp Zool A Comp Exp Biol 2006; 305: 769–80.
Wagner GF, Hampong M, Park CM, Copp DH . Purification, characterization, and bioassay of teleocalcin, a glycoprotein from salmon corpuscles of Stannius. Gen Comp Endocrinol 1986; 63: 481–91.
Lafeber FP, Flik G, Wendelaar Bonga SE, Perry SF . Hypocalcin from Stannius corpuscles inhibits gill calcium uptake in trout. Am J Physiol 1988; 254: R891–6.
Lu M, Wagner GF, Renfro JL . Stanniocalcin stimulates phosphate reabsorption by flounder renal proximal tubule in primary culture. Am J Physiol 1994; 267: R1356–62.
Sundell K, Björnsson BT, Itoh H, Kawauchi H . Chum salmon (Oncorhynchus keta) stanniocalcin inhibits in vitro intestinal calcium uptake in Atlantic cod (Gadus morhua). J Comp Physiol [B] 1992; 162: 489–95.
Holmes DI, Zachary IC . Vascular endothelial growth factor regulates stanniocalcin-1 expression via neuropilin-1-dependent regulation of KDR and synergism with fibroblast growth factor-2. Cell Signal 2008; 20: 569–79.
Grosshans J, Wieschaus E . A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila. Cell 2000; 101: 523–31.
Mata J, Curado S, Ephrussi A, Rørth P . Tribbles coordinates mitosis and morphogenesis in Drosophila by regulating string/CDC25 proteolysis. Cell 2000; 101: 511–22.
Seher TC, Leptin M . Tribbles, a cell-cycle brake that coordinates proliferation and morphogenesis during Drosophila gastrulation. Curr Biol 2000; 10: 623–9.
Schwarzer R, Dames S, Tondera D, Klippel A, Kaufmann J . TRB3 is a PI 3-kinase dependent indicator for nutrient starvation. Cell Signal 2006; 18: 899–909.
Kiss-Toth E, Bagstaff SM, Sung HY, Jozsa V, Dempsey C, Caunt JC, et al. Human tribbles, a protein family controlling mitogen-activated protein kinase cascades. J Biol Chem 2004; 279: 42703–8.
Silverman N, Maniatis T . NF-kappaB signaling pathways in mammalian and insect innate immunity. Genes Dev 2001; 15: 2321–42.
Ghosh S, Karin M . Missing pieces in the NF-kappaB puzzle. Cell 2002; 109 Suppl: S81–96.
Sun SC, Xiao G . Deregulation of NF-kappaB and its upstream kinases in cancer. Cancer Metastasis Rev 2003; 22: 405–22.
Gebken J, Lüders B, Notbohm H, Klein HH, Brinckmann J, Müller PK, et al. Hypergravity stimulates collagen synthesis in human osteoblast-like cells: evidence for the involvement of p44/42 MAP-kinases (ERK 1/2). J Biochem 1999; 126: 676–82.
Shahrestanifar M, Fan X, Manning DR . Lysophosphatidic acid activates NF-kappaB in fibroblasts. A requirement for multiple inputs. J Biol Chem 1999; 274: 3828–33.
Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB . NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 1999; 401: 82–5.
Romashkova JA, Makarov SS . NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling. Nature 1999; 401: 86–90.
Carrier F, Zhan Q, Alamo I, Hanaoka F, Fornace AJ Jr. Evidence for distinct kinase-mediated pathways in gadd gene responses. Biochem Pharmacol 1998; 55: 853–61.
Papathanasiou MA, Kerr NC, Robbins JH, McBride OW, Alamo I Jr, Barrett SF, et al. Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C. Mol Cell Biol 1991; 11: 1009–16.
Kaufman RJ . Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev 1999; 13: 1211–33.
Lawrence M, Shao C, Duan L, McGlynn K, Cobb MH . The protein kinases ERK1/2 and their roles in pancreatic beta cells. Acta Physiol (Oxf) 2008; 192: 11–7.
Abel T, Nguyen PV, Barad M, Deuel TA, Kandel ER, Bourtchouladze R . Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampus-based long-term memory. Cell 1997; 88: 615–26.
Huang YY, Li XC, Kandel ER . cAMP contributes to mossy fiber LTP by initiating both a covalently mediated early phase and macromolecular synthesis-dependent late phase. Cell 1994; 79: 69–79.
Frey U, Huang YY, Kandel ER . Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons. Science 1993; 260: 1661–4.
Peng HY, Cheng YW, Lee SD, Ho YC, Chou D, Chen GD, et al. Glutamate-mediated spinal reflex potentiation involves ERK1/2 phosphorylation in anesthetized rats. Neuropharmacology 2008; 54: 686–98.
Mayer ML, Westbrook GL . Permeation and block of N-methyl-D-aspartic acid receptor channels by divalent cations in mouse cultured central neurones. J Physiol 1987; 394: 501–27.
Bray JG, Mynlieff M . Involvement of protein kinase C and protein-kinase A in the enhancement of L-type calcium current by GABA(B) receptor activation in neonatal hippocampus. Neuroscience 2011; 179: 62–72.
Carpio L, Gladu J, Goltzman D, Rabbani SA . Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways. Am J Physiol Endocrinol Metab 2001; 281: E489–99.
Mao LM, Tang QS, Wang JQ . Regulation of extracellular signal-regulated kinase phosphorylation in cultured rat striatal neurons. Brain Res Bull 2009; 78: 328–34.
Hinoi E, Fujimori S, Nakamura Y, Yoneda Y . Group III metabotropic glutamate receptors in rat cultured calvarial osteoblasts. Biochem Biophys Res Commun 2001; 281: 341–6.
Acknowledgements
This work is supported by the National Natural Science Foundation of China (No 30570881) and is supported partially by grants from the Division of Endocrinology and Metabolic Diseases, E-Institute of Shanghai Universities (E03007) and Shanghai Education Commission (No Y0204). We thank Genminix Informatics Ltd Co for assistance with the microarray analysis.
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Li, Jl., Zhao, L., Cui, B. et al. Multiple signaling pathways involved in stimulation of osteoblast differentiation by N-methyl-D-aspartate receptors activation in vitro. Acta Pharmacol Sin 32, 895–903 (2011). https://doi.org/10.1038/aps.2011.38
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DOI: https://doi.org/10.1038/aps.2011.38
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