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Modulation of cellular differentiation by N-methyl-D-aspartate receptors in osteoblasts¹

Laboratory of Molecular Pharmacology, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa 920-0934, Japan

²Correspondence: Laboratory of Molecular Pharmacology, Kanazawa University Graduate School of Natural Science and Technology, 13-1 Takara-machi, Kanazawa, Ishikawa 920-0934, Japan. E-mail, yyoneda{at}anet.ne.jp

SPECIFIC AIM

In bone, constitutive expression is seen with certain subtypes of ionotropic (iGluR) and metabotropic (mGluR) glutamate (Glu) receptors in human and rodent osteoblasts and osteoclasts. Imbalance between bone-forming osteoblast and bone-resorbing osteoclast would lead to pathogenesis as well as etiology of certain metabolic bone diseases including osteoporosis, Paget’s disease, and osteopetrosis. Recent studies give rise to the possibility that L-Glu may be one of the endogenous paracrine and/or autocrine factors used for intercellular communications in bone cells. Although N-methyl-D-aspartate (NMDA) receptors are supposed to modulate bone resorption by osteoclasts, a possible physiological role of heteromeric NMDA receptor channels expressed in osteoblasts is not fully clarified to date. This study aimed to elucidate possible functional roles of NMDA receptors expressed in primary cultures of rat osteoblasts.

PRINCIPAL FINDINGS

1. Expression of NMDA receptor subunits
Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed constitutive expression of mRNA for NR1 and NR2D subunits, but not for NR2A, NR2B, and NR2C subunits, in rat calvarial osteoblasts cultured for 7 to 28 days in vitro (DIV). Of NR subunits tested, immunoreactivities were detected at cellular surroundings, but not in the nuclei, with both NR1 and NR2D subunits in osteoblasts cultured for 7 DIV. No immunoreactive cells were found with NR2A, NR2B, and NR2C under these experimental conditions.

2. Exposure to MK-801
Osteoblasts were cultured for different days from 3 to 28 DIV in the presence of the NMDA channel antagonist dizocilpine (MK-801) at a concentration range of 10 to 100 µM for determinations of alkaline phosphatase activity and Ca²⁺ accumulation. Osteoblastic cells were at a proliferative phase between 1 and 6 DIV with concomitant formation of several confluent cell layers; subsequently cells were more refractive and round than the background cell layers by 10 to 11 DIV, followed by formation of some nodules and clusters with matrix. The matrix started to mineralize and calcify within 15 DIV. Alkaline phosphatase activity and Ca²⁺ accumulation increased in proportion to the duration of culture from 3 to 14 DIV, with relatively high levels for up to 28 DIV. Sustained exposure to MK-801 significantly prevented these increases in osteoblasts cultured for 7 to 28 DIV in a concentration-dependent manner, whereas no significant changes were seen at an early phase of up to 5 DIV even in the persistent presence of MK-801 at different concentrations. MK-801 failed to significantly affect the number of living cells irrespective of the duration of exposure from 3 to 28 DIV. In osteoblasts cultured for 28 DIV in the presence of 100 µM MK-801, expression of osteocalcin was markedly inhibited compared with those cells cultured in the absence of MK-801 (Fig. 1 ). In control osteoblasts cultured for 28 DIV, some nodules and clusters were formed with matrix whereas MK-801 at 100 µM reduced formation of these nodules.

View larger version (84K): [in this window] [in a new window]   Figure 1. Osteocalcin expression in osteoblasts cultured with MK-801. Osteoblasts were cultured in -MEM in either the presence or absence of MK-801 at 100 µM for 28 DIV, followed by fixation with 4% paraformaldehyde for subsequent immunocytochemical analysis using the anti-osteocalcin antibody. Typical micrographs are shown with similar results in 3 independent determinations.

3. Exposure to other NMDA receptor antagonists
NMDA domain antagonist and glycine domain antagonist were invariably effective in significantly inhibiting accumulation of Ca²⁺ in osteoblasts cultured for 28 DIV in a concentration-dependent manner, in addition to MK-801.

4. Timing of MK-801 exposure
Accumulation of Ca²⁺ was similarly inhibited after sustained exposure to MK-801 for 25 days from DIV 3 to 28 and for 28 days from DIV 0 to 28; less potent inhibition was seen with Ca²⁺ accumulation in osteoblasts exposed to MK-801 for 21 days from DIV 7 to 28. MK-801 did not significantly affect Ca²⁺ accumulation in osteoblasts at 100 µM when exposed after 11 DIV up to 21 DIV for consecutive periods of 7 to 17 days until the day of harvest.

5. Expression of CBFA1
Gel retardation electrophoresis revealed that MK-801 significantly inhibited DNA binding activity of core binding factor 1 (CBFA1) in osteoblasts when exposed for 7 to 28 DIV without significantly altering it in cells exposed for 3 to 5 DIV. Similarly, expression of CBFA1 protein was almost completely abolished in osteoblasts cultured in the presence of MK-801 for 11 to 28 DIV (Fig. 2 a, b), with a significant reduction of >60% in cells exposed for 7 DIV (Fig. 2a ). In osteoblasts cultured in the presence of MK-801 for 3 to 5 DIV, MK-801 failed to significantly affect expression of CBFA1 protein. MK-801 was also effective in inhibiting expression of mRNA for CBFA1 when exposed to osteoblasts for 7 to 21 DIV (Fig. 2c ).

View larger version (45K): [in this window] [in a new window]   Figure 2. CBFA1 expression in osteoblasts cultured with MK-801. Osteoblasts were cultured in -MEM in either the presence or absence of MK-801 at 100 µM for 3 to 11 DIV followed by preparation of nuclear extracts at each stage and subsequent determination of expression of CBFA1 on immunoblot assays using the anti-CBFA1 antibody. Typical blots are shown in the left panels where each lane corresponds to a sample from one well of culture plates; densitometric percentages are shown in the right panel where each value represents the mean ± SE from 4 different experiments. **P<0.01, significantly different from each control value obtained in the absence of MK-801.

CONCLUSIONS AND SIGNIFICANCE

In this study, the essential importance lies in the exacerbation of osteoblastic differentiation by different NMDA receptor antagonists. Sustained exposure to MK-801 invariably led to marked inhibition of expression of mRNA and the corresponding protein for the master regulator of bone differentiation CBFA1 in addition to its DNA binding activity in primary cultured rat calvarial osteoblasts when determined after the stage of cellular differentiation. The data cited in this study give support to the proposal that NMDA receptors could predominantly modulate cell differentiation rather than proliferation through a mechanism associated with expression of CBFA1 during cellular maturation in osteoblasts. To our knowledge, this paper deals with the first direct demonstration of modulation of expression of CBFA1 absolutely required for cell differentiation in the nucleus through heteromeric NMDA receptor channels expressed at the cell surface in cultured rat calvarial osteoblasts. Several reports have already demonstrated the functional expression of heteromeric NMDA receptor channels in bone cells, although the possible functional significance is not fully clarified in osteoblasts compared with that in osteoclasts.

CBFA1 is a transcription factor essential for the growth of osteoblasts as a master regulator at the differentiation stage. Disruption of CBFA1 prevents skeltogenesis, and heterozygous mutations lead to cleidocranial dysplasia, an autosomal dominant disorder. CBFA1 is also shown to regulate expression of several genes such as collagenase-3, type (I) collagen, osteoprotegerin, bone sialoprotein, osteopontin, and alkaline phosphatase in addition to osteocalcin. The present findings that MK-801 prevented alkaline phosphatase activities and osteocalcin expression, therefore, argue in favor of the idea that the anti-osteogenetic property of MK-801 is due to inhibition of expression of particular target genes essential for differentiation and maturation of osteoblasts through reduced CBFA1 DNA binding. Reduced expression of both CBFA1 protein and mRNA could account for the reduction of CBFA1 DNA binding after sustained exposure to MK-801. The mechanism underlying reduced expression by MK-801 of CBFA1 protein and mRNA, however, remains to be elucidated. It is highly conceivable that MK-801 may prevent NMDA signaling at the cell surface to induce suppression of expression of mRNA for CBFA1 at the level of gene transcription in the nucleus. The possibility that MK-801 may promote degradation of CBFA1 protein at the level of post-translation is not ruled out. In fact, degradation of CBFA1 protein is accelerated by a variety of agents with the ability to elevate cAMP level, such as parathyroid hormone, through a ubiquitin-proteasome pathway. Since immunoblotting analysis was conducted using nuclear extracts for the anti-CBFA1 antibody in this study, the idea that MK-801 may prevent nuclear translocation of CBFA1 protein required for binding to cis-element and subsequent transcriptional regulation of the aforementioned osteoblast-specific genes is not excluded so far. At any rate, NMDA receptor channels could participate at least in part in molecular mechanisms underlying regulation of cellular differentiation by CBFA1 through modulation of its gene expression in osteoblasts.

Activation of NMDA receptors is shown to result in marked expression of the nuclear transcription factor activator protein-1 (AP1) complex composed of Jun and Fos family members, particularly central structures in vivo and in vitro. Accumulating evidence that members of AP1 complex participate in the regulation of cell proliferation, differentiation, and maturation in bone is available in the literature. Transgenic mice overexpressing c-Fos protein in bone develop osteosarcoma, whereas double transgenic mice with both c-Fos and c-Jun proteins develop osteosarcomas at a higher frequency than single transgenic mice with c-Fos protein alone, with mice overexpressing c-Jun being normal. Overexpression of DeltaFosB in transgenic mice leads to increases in bone formation throughout the skeleton and bone mass during development, leading to osteosclerosis. Transgenic mice overexpressing Fra-1 also develop a progressive increase in bone mass leading to osteosclerosis. These findings give rise to the idea that blockade of NMDA receptors may lead to inhibition of osteoblastogenesis. All GluR expressed in cultured osteoblasts would be under tonic stimulation by Glu present in culture medium. Common negative effects of antagonists for different domains within NMDA receptor channels on various osteoblastic activities favor the idea that these antagonists would prevent the cellular growth of osteoblasts through inhibition of the opening process of NMDA receptor channels under sustained stimulation by Glu at each concentration used. Sympathetic and sensory nerve fibers innervate into bone, whereas glutamatergic innervation is distributed in bone. The endogenous agonist Glu could be supplied by these glutamatergic nerve fivers as well as by osteoblasts themselves, in addition to by blood circulation, to osteoblasts. The similarity between osteoblasts and neurons could be proposed by the data cited above.

It thus appears that NMDA receptor channels could be expressed in osteoblasts to play a role crucial for osteogenesis in bone. Bone cells could express all molecular machineries required for Glu signaling in the central nervous system (CNS). These include Glu transporters, iGluR, mGluR, and vesicular Glu transporter. Accordingly, Glu seems to play dual roles in mechanisms underlying maintenance of homeostasis as an excitatory neurotransmitter in the CNS and an autocrine and/or paracrine mediator in bone.

View larger version (23K): [in this window] [in a new window]   Figure 3. Schematic representation for possible significance of NMDA receptors expressed in osteoblasts. The endogenous agonist Glu could play a role as an autocrine signal mediator through activation of NMDA receptors after exocytotic release, which leads to expression of CBFA1 responsible for modulation of cellular commitment, differentiation, and/or maturation in osteoblasts.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0820fje; doi: 10.1096/fj.02-0820fje

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