Kuniaki Suzuki

Long-term effects of prostaglandin E2 on the mineralization of a clonal osteoblastic cell line (MC3T3-E1)

Prostaglandin (PG) E2 is thought to be a mediator of the effect of mechanical stress on bone formation, but its effects on osteoblasts have not yet been fully described. Here, the effects of the continuous application of PGE2 and indomethacin, an inhibitor of prostaglandin G/H synthase (cyclo-oxygenase), on the proliferation, differentiation and mineralization of a clonal osteoblastic cell line, MC3T3-E1, were investigated. The cells were cultured in media with either a high (1 microg/ml) or a low (1 ng/ml) concentration of PGE2, with indomethacin (1 microg/ml) and, as a control, with neither agent. The effects of PGE2 and indomethacin were assessed quantitatively. Indomethacin and a high concentration of PGE2 increased the total protein compared to the control and low-PGE2 cultures. 7 days after confluence, alkaline phosphatase (ALP) activity within the cells and extracellular matrices increased. This increase was highest with indomethacin and lowest with a high concentration of PGE2. ALP activity also increased in the medium, but only 21 days after confluence; the effects of the agents were similar to those on the cells and matrices. The accumulation of calcium, inorganic phosphate and hydroxyproline was highest with indomethacin. PGE2 production was at its maximum when the cells were at confluence and was inhibited by indomethacin. Specific [3H]PGE2 binding to the microsomal fraction of the cell was also measured to examine the expression of the PGE2 receptor. The amount of [3H]PGE2 binding per mg of protein was highest at confluence, then decreased and again increased in the mineralizing stage. These results suggest that indomethacin increases ALP activity and the accumulation of mineralized tissue in MC3T3-E1 cells, presumably by inhibiting the production of PGE2. PGE2 could signal the suppression of mineralization as early as confluence.

Histamine stimulates production of osteoclast differentiation factor/receptor activator of nuclear factor-κB ligand by osteoblasts

Histamine H(1),H(2), and H(3) receptors are expressed by osteoblastic MC3T3-E1 (E1) cells derived from mouse calvaria. Expression of the osteoclast differentiation factor (ODF)/receptor activator of nuclear factor-kappaB ligand (RANKL) transcript was induced in E1 cells and bone marrow stromal cells (ST2). Histamine markedly increased the steady-state level of ODF/RANKL mRNA in a dose-dependent manner. The effect of histamine on expression of ODF/RANKL mRNA by E1 cells was transient, with a peak at 6h. Western blot analysis revealed that histamine increased production of ODF/RANKL protein by E1 cells at 12h. In cocultures of E1 cells and mouse bone marrow cells, histamine stimulated osteoclastogenesis in the presence of 1,25-dihydroxyvitamin D(3) and this effect was blocked by preincubation with neutralizing antibody against ODF/RANKL. These results suggest that histamine regulates osteoclastogenesis, at least in part, through induction of ODF/RANKL expression by osteoblasts and bone marrow stromal cells.

Effect of mineral trioxide aggregate on rat clonal dental pulp cells: expression of cyclooxygenase-2 mRNA and inflammation-related protein via nuclear factor kappa B signaling system.

INTRODUCTION Recently, mineral trioxide aggregate (MTA) has been routinely used for endodontic treatment. It is well-known that MTA induced secondary dentin formation in pulp cavity when it was applied to dentin, whereas its cytotoxicities were unclear. The purpose of this study was to evaluate the effect of MTA on rat clonal dental pulp cells, RPC-C2A. METHODS This study was conducted to observe the response of RPC-C2A cells on MTA with reverse-transcriptase polymerase chain reaction, Western blot analysis, and enzyme immunoassay. Data were compared by analysis of variance. Statistical significance was established at P <.01. RESULTS MTA significantly caused an up-regulation of cyclooxygenase-2 (COX-2) and inducible form of nitric oxide synthase (iNOS) mRNA expression. Furthermore, MTA caused inhibitory kappa B (IkappaB) phosphorylation and translocation of nuclear factor-kappa B (NF-kappaB) subunits to nucleus. Curucumin, an inhibitor of NF-kappaB activation, suppressed MTA-induced COX-2 and iNOS mRNA expressions. In addition, MTA increased the production of prostaglandin E(2) in comparison with the controls. CONCLUSIONS MTA induces inflammation via NF-kappaB signaling system.

Cisplatin sensitivity of oral squamous carcinoma cells is regulated by Na + ,K + -ATPase activity rather than copper-transporting P-type ATPases, ATP7A and ATP7B

PurposeCisplatin (CDDP) is one of the major chemotherapeutic drugs, but tumor cells’ acquired resistance to CDDP limits its therapeutic potentials. One of the main reasons of resistance is reduced drug accumulation. The mechanism by which tumor cells accumulate reduced CDDP is not well elucidated yet. The aim of this study was to investigate what regulates intracellular CDDP accumulation.MethodsSix different types of oral squamous carcinoma cells were used in this study. Assessment of CDDP sensitivity was determined by measuring the ATP level of the cells. Intracellular CDDP and copper (Cu) accumulation were measured and CDDP efflux study was conducted. Assessment of Na+,K+-ATPase α and β subunits, ATP7A and ATP7B was done by western blotting. Specific activities of Na+,K+-ATPase and copper-transporting P-type ATPase (Cu2+-ATPase) were detected and a role of Na+,K+-ATPase inhibitor in intracellular CDDP accumulation was examined.ResultsAmong the cells HSC-3 and BHY cells were found most CDDP-sensitive and CDDP-resistant, respectively. The most CDDP-sensitive HSC-3 cells exhibited an increased intracellular cisplatin accumulation, high Na+,K+-ATPase activity and over-expressed Na+,K+-ATPase α and β subunits, ATP7A and ATP7B, compared to the most CDDP-resistant BHY cells, but there were no such differences between the two in the CDDP efflux level or Cu2+-ATPase activity. Moreover, pretreatment with Na+,K+-ATPase inhibitor markedly reduced intracellular cisplatin accumulation.ConclusionsNa+,K+-ATPase activity is responsible for regulating intracellular CDDP accumulation in oral squamous carcinoma cells rather than Cu2+-ATPase.

Effects of bisphosphonates on osteoclastogenesis in RAW264.7 cells.

Bisphosphonates are used as therapeutic agents for the management of osteoporosis and other bone diseases. However, the precise effects and mechanisms of bisphosphonates on osteoclastogenesis are unclear, as previous studies have reported contradictory findings and no studies have circumstantially assessed the effects of bisphosphonates on osteoclastogenesis. Therefore, the aim of this study was to determine the effects of bisphosphonates on osteoclastogenesis in RAW264.7 (RAW) cells. To examine the direct effects of bisphosphonates on osteoclast differentiation via receptor activator of nuclear factor-κB (RANK) ligand (RANKL), RAW cells were cultured with bisphosphonates. Addition of bisphosphonates to RAW cells led to a significant decrease in the number of osteoclasts and large osteoclasts (≥ 8 nuclei) in a bisphosphonate concentration-dependent and time-dependent manner. The cytotoxicity of non-nitrogen-containing bisphosphonates was specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells. Resorption activity was significantly diminished by treatment with bisphosphonates, thus confirming that bisphosphonates impair the absorptive activity of osteoclasts. We also investigated the effects of bisphosphonates on the mRNA expression of genes associated with osteoclastogenesis, osteoclast-specific markers and apoptosis-related genes using quantitative real-time PCR. The results suggest that bisphosphonates suppress osteoclast differentiation and infusion, and induce osteoclast apoptosis. With regard to osteoclast apoptosis induced by bisphosphonates, we further investigated the detection of DNA fragmentation and Caspase-Glo 3/7 assay. DNA fragmentation was confirmed after treatment with bisphosphonates, while caspase-3/7 activity increased significantly when compared with controls. In conclusion, bisphosphonates directly inhibited RANKL-stimulated osteoclast differentiation and fusion in RAW cells. It was confirmed that bisphosphonates impair osteoclast resorption activity and induce apoptosis. The effects of non-nitrogen-containing bisphosphonates were also specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells.

Effect of N-acetylcysteine on Rat Dental Pulp Cells Cultured on Mineral Trioxide Aggregate

INTRODUCTION The purpose of this study was to evaluate the cytotoxicity of mineral trioxide aggregate (MTA) and its potential detoxification by an antioxidant amino acid, N-acetylcysteine (NAC). METHODS Rat dental pulp cells extracted from rat maxillary incisors were directly cultured on MTA with or without NAC in culture medium. The number of cells and their spreading behavior were both assessed 24 hours after seeding. The intracellular levels of reactive oxygen species (ROS) and glutathione (GSH) were also assessed after 24 hours of culture. RESULTS The number of cells attached to MTA was 60% greater when NAC was added to the culture medium. In addition, the area and perimeter of the cells were found to be 2-fold greater in the culture containing NAC. Cells cultured on MTA alone showed large ROS concentrations, which disappeared when the medium was supplemented with NAC. The intracellular GSH level, however, increased 3.5-fold with NAC addition. CONCLUSIONS This study demonstrated that the presence of NAC in environments can substantially improve attachment and spreading behaviors of dental pulp cells on MTA. This biological effect was associated with an improvement in the cellular redox system by NAC and warrants further exploration of NAC for determining its therapeutic value in improving the biocompatibility of MTA.

A phosphotyrosine-containing quenched fluorogenic peptide as a novel substrate for protein tyrosine phosphatases.

Mca-Gly-Asp-Ala-Glu-Tyr(PO(3)H(2))-Ala- Ala-Lys(DNP)-Arg-NH(2), where Mca is (7-methoxycoumarin-4-yl)acetyl and DNP is 2,4-dinitrophenyl, was synthesized as a fluorogenic substrate for protein tyrosine phosphatases (PTPs). In the peptide, the fluorescent Mca group is quenched efficiently by the DNP group. Although the fluorescence intensity of the substrate was practically unchanged upon PTP-catalysed dephosphorylation, it increased approx. 120-fold upon subsequent treatment with chymotrypsin. Analysis by HPLC showed that chymotrypsin cleaved only the dephosphorylated substrate at the Tyr-Ala bond. Thus with the aid of chymotrypsin, dephosphorylation of the substrate can be measured fluorometrically. A strictly linear correlation was observed between PTP concentration and dephosphorylation rate. The fluorogenic substrate was dephosphorylated by some PTPs much more rapidly than the corresponding (32)P-labelled substrate used for comparison, whereas alkaline phosphatase dephosphorylated the two substrates at similar rates. The fluorogenic substrate is therefore more specific for PTPs than the radiolabelled substrate. The assay with the fluorogenic substrate could be applied to the estimation of kinetc parameters and measurement of PTP activity in crude-enzyme preparations. The lower detection limit of our assay (1 microM substrate in 200 microliter of reaction mixture) was estimated to be 0.2-0.4 pmol, whereas it was estimated to be about 1 pmol in the assay that used (32)P-labelled peptide (specific radioactivity of approx. 1000 c.p.m. /pmol). Our assay is simple, specific, highly sensitive and non-radioisotopic, and hence would contribute greatly to the development of PTP biology.

Inhibition of Na+,K+-ATPase by cisplatin and its recovery by 2-mercaptoethanol in human squamous cell carcinoma cells.

Na+,K+-ATPase (EC 3.6.1.37) is assumed to be involved in the transport of cisplatin [cis-diamminedichloroplatinum(II)] into cells and to act as a modulator of 5-fluorouracil (5-FU) in combination therapy of cisplatin and 5-FU. Whereas inhibition of Na+,K+-ATPase activity by cisplatin is expected to have effects on both anti-cancer therapy and nephrotoxicity, the inhibition mechanism remains to be elucidated. We studied the inhibition of Na+,K+-ATPase activity by cisplatin using an enzyme partially purified from Ca9-22 cells derived from a human squamous cell carcinoma of the gingiva. Cisplatin inhibited the Na+,K+-dependent ATP hydrolysis activity, and this inhibition depended on both the concentration of cisplatin and the preincubation time with cisplatin. The time-dependent inhibition was thought to be caused by a slow change of cisplatin from the inactive to the active form. We further tested the effect of cisplatin on the partial reactions of the enzyme, Na+-dependent ATP hydrolysis and K+-dependent pnitrophenylphosphate hydrolysis activities to determine which step in the reaction sequence of Na+,K+-ATPase was inhibited. Cisplatin inhibited both activities depending on its concentration and the preincubation time, whereas the Na+-dependent ATP hydrolysis activity was inhibited even at lower concentrations. Formation of a phosphointermediate of Na+,K+-ATPase was also inhibited by cisplatin depending on the concentration and preincubation time. Cisplatin (500 microM) and 8-fold higher concentration of 2-mercaptoethanol (2-ME; 4 mM) prevented inactivation of the enzyme by cisplatin, and the Na+,K+-ATPase activity inhibited by pretreatment with cisplatin was also recovered almost completely by 2-ME. These results suggest that the active form of cisplatin inhibits the Na+,K+-ATPase activity by inhibiting the formation of a phosphointermediate of the enzyme and that the inhibition by cisplatin is arrested by an addition of thiol group.

Amino acid derivative-mediated detoxification and functionalization of dual cure dental restorative material for dental pulp cell mineralization.

Current dental restorative materials are only used to fill the defect of hard tissues, such as dentin and enamel, because of their cytotoxicity. Therefore, exposed dental pulp tissues in deep cavities must be first covered by a pulp capping material like calcium hydroxide to form a layer of mineralized tissue. However, this tissue mineralization is based on pathological reaction and triggers long-lasting inflammation, often causing clinical problems. This study tested the ability of N-acetyl cysteine (NAC), amino acid derivative, to reduce cytotoxicity and induce mineralized tissue conductivity in resin-modified glass ionomer (RMGI), a widely used dental restorative material having dual cure mechanism. Rat dental pulp cells were cultured on untreated or NAC-supplemented RMGI. NAC supplementation substantially increased the percentage of viable cells from 46.7 to 73.3% after 24-h incubation. Cell attachment, spreading, proliferative activity, and odontoblast-related gene and protein expressions increased significantly on NAC-supplemented RMGI. The mineralization capability of cells, which was nearly suppressed on untreated RMGI, was induced on NAC-supplemented RMGI. These improved behaviors and functions of dental pulp cells on NAC-supplemented RMGI were associated with a considerable reduction in the production of intracellular reactive oxygen species and with the increased level of intracellular glutathione reserves. These results demonstrated that NAC could detoxify and functionalize RMGIs via two different mechanisms involving in situ material detoxification and antioxidant cell protection. We believe that this study provides a new approach for developing dental restorative materials that enables mineralized tissue regeneration.

Effect of the release from mechanical stress on osteoclastogenesis in RAW264.7 cells

The effects of mechanical stress release on osteoclastogenesis may be as important as those of mechanical stress application. However, the direct effects of mechanical stress on the behavior of osteoclasts has not been thoroughly investigated and there is limited information on the results of the release from mechanical stress. In this study, the effects of mechanical stress application and its release on osteoclast differentiation were examined. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts derived from RAW264.7 cells were measured and the expression of osteoclast differentiation genes, which was altered in response to the release from mechanical stress according to the Flexercell tension system was evaluated by real-time PCR. Osteoclast differentiation and fusion were suppressed by mechanical stress application and were rapidly induced after mechanical stress release. The mRNA expression of the osteoclast specific genes, TRAP, matrix metalloproteinase-9 (MMP-9), cathepsin-K (cath-k), calcitonin receptor (CTR), ATPase H+ transporting vacuolar proton pump member I (ATP6i), chloride channel-7 (ClC7) and dendritic cell-specific transmembrane protein (DC-STAMP) was decreased with mechanical stress application, and increased up to 48 h after the release from it. These alterations in gene mRNA expression were associated with the number of osteoclasts and large osteoclasts. Inducible nitric oxide synthetase (iNOS) mRNA was increased with mechanical stress and decreased after its release. Nitric oxide (NO) production was increased with mechanical stress. Nuclear factor of activated T cells cytoplasmic (NFATc) family mRNAs were not altered with mechanical stress, but were up-regulated up to 48 h after the release from it. These findings indicate that the suppression of osteoclast differentiation and fusion induced by mechanical stress is the result of NO increase via iNOS, and that the promotion of osteoclast differentiation and fusion after the release from mechanical stress is related to the NFATc family genes, whose expression remained constant during mechanical stress but was up-regulated after the release from mechanical stress.