Sayumi Fujimori

Nrf2 negatively regulates osteoblast differentiation via interfering with Runx2-dependent transcriptional activation.

Nrf2 (nuclear factor E2 p45-related factor 2) is believed to be a transcription factor essential for the regulation of many detoxifying and antioxidative genes in different tissues. In the present study, we investigated the role of Nrf2 in the regulation of osteoblastic differentiation. nrf2 mRNA expression was significantly up-regulated in femur isolated from ovariectomized mice, whereas in situ hybridization analysis revealed that up-regulation of nrf2 mRNA was mainly found in osteoblasts attached on cancellous bone in femur of ovariectomized mice. Expression of Nrf2 protein was also seen in osteoblasts in neonatal mouse tibia and calvaria. In osteoblastic MC3T3-E1 cells stably transfected with nrf2 expression vector, significant inhibition was seen in the maturation-dependent increase in alkaline phosphatase activity as well as the mineralized matrix formation. Stable overexpression of nrf2 significantly impaired Runx2 (runt-related transcription factor 2)-dependent stimulation of osteocalcin promoter activity and recruitment of Runx2 on osteocalcin promoter without affecting the expression of runx2 mRNA. Coimmunoprecipitation and mammalian two-hybrid assay revealed a physical interaction between Runx2 and Nrf2, whereas cellular distribution of endogenous Runx2 was not apparently changed by nrf2 overexpression in MC3T3-E1 cells. Alternatively, Nrf2 bound to antioxidant-responsive element-like-2 sequence of osteocalcin promoter. The inhibition by nrf2 on runx2-dependent osteocalcin promoter activity was partially prevented by the introduction of reporter of deletion mutant for ARE-like-2 sequence of osteocalcin promoter. These data suggest that Nrf2 may negatively regulate cellular differentiation through inhibition of the Runx2-dependent transcriptional activity in osteoblasts.

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

N‐Methyl‐d‐aspartate (NMDA) receptors for the central neurotransmitter l‐glutamate (Glu) have been shown to be present in both osteoblasts and osteoclasts. Sustained exposure to the NMDA channel antagonist dizocilpine (MK‐801) significantly prevented increases in both alkaline phosphatase activity and Ca2+ accumulation in a concentration‐dependent manner in osteoblasts cultured for 7–28 days in vitro (DIV), without significantly affecting cell survivability. Osteocalcin expression was markedly reduced in the presence of MK‐801 in osteoblasts cultured for 28 DIV. Both an NMDA domain antagonist and a glycine domain antagonist similarly prevented Ca2+ accumulation in osteoblasts exposed for 28 consecutive DIV. MK‐801 was effective in significantly inhibiting Ca2+ accumulation determined at 28 DIV in osteoblasts exposed before 7 DIV but was ineffective in cells exposed after 11–21 DIV. Sustained exposure to MK‐801 significantly inhibited DNA binding activity and expression of core binding factor α‐1 (CBFA1) in osteoblasts exposed after 7 DIV up to 28 DIV, but not in those exposed before 7 DIV. These results suggest that heteromeric NMDA receptor channels may be functionally expressed to regulate mechanisms underlying cellular differentiation rather than proliferation and/or maturation through modulation of expression of CBFA1 in cultured rat calvarial osteoblasts.

Facilitation of glutamate release by ionotropic glutamate receptors in osteoblasts

Constitutive expression of mRNA was seen for the vesicular glutamate transporter brain-specific Na(+)-dependent inorganic phosphate cotransporter (BNPI), but not differentiation-associated Na(+)-dependent inorganic phosphate cotransporter, in rat calvarial osteoblasts cultured for 7 and 21 days in vitro (DIV). Three different agonists for ionotropic glutamate receptors (iGluR) at 1mM, as well as 50mM KCl, significantly increased the release of endogenous L-glutamate from osteoblasts cultured for 7DIV when determined 5 min after the addition by using a high performance liquid chromatograph. The inhibitor of desensitization of DL-alpha-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA) receptors cyclothiazide significantly potentiated and prolonged the release of endogenous L-glutamate evoked by AMPA in a dose-dependent manner. The release evoked by AMPA was significantly prevented by the addition of an AMPA receptor antagonist as well as by the removal of Ca(2+) ions. These results suggest that endogenous L-glutamate could be released from intracellular vesicular constituents associated with BNPI through activation of particular iGluR subtypes expressed in cultured rat calvarial osteoblasts.

Demonstration of expression of mRNA for particular AMPA and kainate receptor subunits in immature and mature cultured rat calvarial osteoblasts

Reverse transcription polymerase chain reaction revealed expression of mRNA for particular subunits of ionotropic glutamate receptors (iGluR) in primary cultures of rat calvarial osteoblastic cells under immature to mature states. These included GluR3, KA1 and KA2 subunits, in addition to NR1 and NR2D subunits. These results suggest that glutamate may play an unidentified role in mechanisms associated with cellular development through particular subunits of iGluR in rat calvarial osteoblasts.

A molecular mechanism of pyruvate protection against cytotoxicity of reactive oxygen species in osteoblasts.

We demonstrated previously that exogenous pyruvate has a protective action against cell death by hydrogen peroxide in cultured osteoblasts through a mechanism associated with its antioxidative property. In the present study, we have evaluated possible participation of monocarboxylate transporters (MCTs) responsible for the bidirectional membrane transport of pyruvate in the cytoprotective property in osteoblasts. Expression of the MCT2 isoform was found in cultured rat calvarial osteoblasts and in osteoblasts located on mouse tibia at both mRNA and protein levels. The accumulation of [14C]pyruvate occurred in a temperature- and pH-dependent manner in osteoblasts cultured for 7 days with high sensitivity to a specific MCT inhibitor, whereas pyruvate was released into extracellular spaces from cultured osteoblasts in a fashion sensitive to the MCT inhibitor. Transient overexpression of the MCT2 isoform led to reduced vulnerability to the cytotoxicity of hydrogen peroxide with an increased activity of [14C]pyruvate accumulation in murine osteoblastic MC3T3-E1 cells. Ovariectomy significantly decreased the content of pyruvate in femoral bone marrows in mice in vivo, whereas daily i.p. administration of pyruvate at 0.25 g/kg significantly prevented alterations of several histomorphometric parameters as well as cancellous bone loss in femurs by ovariectomy on 28 days after the operation. These results suggest that MCTs may be functionally expressed by osteoblasts to play a pivotal role in mechanisms related to the cytoprotective property of pyruvate.

Modulation of cellular proliferation and differentiation through GABAB receptors expressed by undifferentiated neural progenitor cells isolated from fetal mouse brain

In this study, we have attempted to evaluate the possible role of metabotropic GABAB receptors (GABABR) expressed by neural progenitor cells prepared from neocortex of embryonic Std‐ddY mice. Immunocytochemical analysis confirmed the validity of isolation procedures of neural progenitors, while round spheres were formed with clustered cells during culture with epidermal growth factor (EGF) for 10 days. A reverse transcription polymerase chain reaction analysis revealed constitutive expression of GABAAR, GABABR, and GABACR subtypes in undifferentiated progenitors and neurospheres formed within 10 days. Exposure to GABA led to concentration‐dependent increases in the total area and proliferation activity of neurospheres at 10–300 µM, while the GABABR agonist baclofen at 100 µM significantly increased the size of neurospheres expressing both GABABR1 and GABABR2 subunits in a manner sensitive to a GABABR antagonist. By contrast, a significant decrease was seen in the total areas of neurospheres prepared from mice deficient of the GABABR1 subunit. In neurospheres of GABABR1‐null mice, a significant increase was induced in the number of cells immunoreactive for a glial marker protein, with a concomitant decrease in that of a neuronal marker protein, upon spontaneous differentiation after the removal of EGF. These results suggest that GABABR may be functionally expressed by neural progenitor cells to preferentially promote the commitment toward a neuronal lineage after the activation of cellular proliferation toward self‐replication in the developing mouse brain. J. Cell. Physiol. 216: 507–519, 2008.

Cell death by pyruvate deficiency in proliferative cultured calvarial osteoblasts

Cell survival was significantly decreased in primary cultured rat calvarial osteoblasts in vitro at Day 0, 1, and 3 by replacement of the standard culture medium (alpha-modified minimum essential medium; alpha-MEM) with Dulbeccos modified eagles medium (DMEM). Decreased cell survival was also observed following medium replacement in cultures of murine calvaria-derived osteoblastic cell line MC3T3-E1. Staining with Hoechst33342 revealed apoptotic cells with fragmented or condensed nuclei, while a fraction of the cell culture was stained with propidum iodide, indicating necrosis. Marked increases in DNA binding of both activator protein-1 and nuclear factor-kappaB were found in nuclear extracts of cells following medium replacement. The addition of either pyruvate or cysteine at each concentration found in alpha-MEM almost entirely prevented cell death associated with medium replacement at Day 3. These results suggest that pyruvate and cysteine may be essential factors for cell growth and survival in osteoblast cultures at the proliferative phase.

Functional GABAB receptors expressed in cultured calvarial osteoblasts

In immature and mature primary cultured rat calvarial osteoblasts, both mRNA and corresponding proteins were constitutively expressed for 2 splice variants of GABA(B) receptor (GABA(B)R) subunits but not for any known GABA(A) and GABA(C) receptor subunits. The agonist for GABA(B)R baclofen significantly inhibited cAMP formation induced by forskolin in a manner sensitive to the antagonist 2-hydroxysaclofen. Similar expression was seen with mRNA for GABA(B)R-1a and -1b splice variants in the murine calvarial osteoblast cell line MC3TC-E1 cells cultured for 7-21 days in vitro (DIV). In these MC3T3-E1 cells, baclofen not only inhibited the activity of alkaline phosphatase, but also exacerbated Ca2+ accumulation, throughout the culture period up to 28 DIV. These results suggest that GABA may play an unidentified role in mechanisms associated with cellular proliferation, differentiation, and/or development through functional GABA(B)R constitutively expressed in cultured osteoblasts.

Constitutive expression of heterologous N-methyl-D-aspartate receptor subunits in rat adrenal medulla

An RT‐PCR analysis revealed constitutive expression of mRNA for N‐methyl‐D‐aspartate (NMDA) receptor (NR)‐1, NR‐2C, and NR‐2D subunits in rat adrenal and pituitary glands, in addition to brain and retina. Constitutive expression of mRNA was detected for the NR‐2A subunit in pituitary but not adrenal gland. Although on Western blotting assays adrenal medulla exhibited expression of NR‐1 subunit protein without expression of NR‐2C and NR‐2D subunit proteins, an immunohistochemical investigation clearly showed selective localization of proteins for NR‐1, NR‐2C, and NR‐2D subunits in adrenal medulla but not in adrenal cortex. Prior treatment with different glycosidases invariably resulted in a marked increase in immunoreactivity to the anti‐NR‐1 antibody in both hippocampus and adrenal medulla. An intraperitoneal injection of a blocker of NMDA receptor channel, but not NMDA itself, led to marked potentiation of DNA binding activity of the transcription factor activator protein‐1 in adrenal nuclear extracts 2 hr after administration. These results suggest that heteromeric NMDA receptor channels may be constitutively and functionally expressed with glycosylation of NR‐1 subunit under the influence of tonic stimulation by circulating agonists such as L‐glutamate in rat adrenal medulla.

Blockade by N-methyl-D-aspartate of elevation of activator protein-1 binding after stress in rat adrenal gland.

Cold immobilization stress induced a marked elevation of expression of activator protein‐1 (AP1) complex in rat hypothalamus, pituitary, adrenal, and gastric mucosa, but not in other discrete brain structures examined, when determined immediately after stress for 3 hr. Adrenal AP1 binding linearly increased with the duration of stress up to 6 hr, whereas the increase was seen in both adrenal cortex and medulla of rats stressed for 3 hr. In adrenals, the elevation exhibited decline profiles different from those of expression of cAMP response element binding protein. Western blotting revealed that stress for 3 hr induced significant increases in expression of the components of AP1 complex, c‐Fos, c‐Jun, and Jun‐B proteins, in adrenals, without markedly affecting expression of Fos‐B, Fra‐2, and Jun‐D proteins. The prior systemic administration of N‐methyl‐D‐aspartate (NMDA) led to significant prevention of the elevation after stress for 3 hr in adrenals, whereas the NMDA antagonist dizocilpine alone induced a marked increase in adrenal AP1 binding, without altering the elevation by stress. These results suggest that stress may modulate de novo protein synthesis at the level of gene transcription by AP1 complex through a molecular mechanism associated with NMDA receptor channels in rat adrenal glands.