Action of prostaglandins on clonal osteoblastic MC3T3-E1 cells

Prostaglandins (PG's) are well known as an important local regulator of bone metabolism. In this study, we examined to characterize the effects of PGs on osteoblasts, using a clonal osteoblastic MC3T3-E1 cells. Among PG metabolites, PGE₂ is a main prostanoid released in bone tissues. MC3T3-E1 cells also produced predominantly PGE₂. PG E₂ at low doses (1–100 ng/ml) and PGE₁ increased activity of alkaline phosphatase (ALP), an marker enzyme of early differentiation of osteoblasts, with positive correlation of elevating intracellular cAMP content. The stimulatory effects are amplified by the addition of isobutylmethyl xanthine (IBMX) and mimicked those of forskolin, a direct activator of adenylate cyclase. those results suggest that PGE₂ at low doses and PGE₁ act predominantly on adenylate cyclase to stimulate the early differntiation of the cells. On the other hand, PGE₂ at high doses (500–2000 ng/ml) and PGF_2α stimulated DNA synthesis of the cells in a dose-related manner. In the same range of concentrations, PGE₂ and PGF_2α augmented the accumulation of inositol triphosphates. Further, the effect of these PGs on the DNA synthesis is negated by addition of H-7, a potent inhibitor of protein kinase C. These date suggest that PGE₂ at high doses and PGF_2α stimulate the proliferation of the cells via enhance of phosphatidyl inositol (PI) turnover system and following activation of protein kinase C. Since PGE₂ reveals diverse effects on the cells dependent on its concentration, it is difficult to clarify the mechanism of PGE₂ action. Thus, we chose PGF_2α to elucidate the stimulatory effect of PGs on the prolferation of the cells. At least 12h time-lag was present between PG F_2α-signal transduction and an increase in DNA synthesis, and α-amanitin and cyclohexamide counteracted the effects, suggesting that some proteins involved in DNA synthesis are produced by the addition of PGF_2α in the duration. Further, neutralizing anti IGF-I antibody blocked the stimulation of DNA synthesis by PGF_2α. However, PGF_2α didn't affect the endogenous production of IGF-I of the cells. On the other hand, PGF_2α greatly elevated level of IGF-I binding sites on the cells, and the increase appeared bout 3h earlier than did the stimulation of DNA synthesis, indicating increase in responsiveness of the cells to IGF-I. These results suggest that the proliferation of the cells is stimulated by synergistic action of PGF_2α and IGF-I produced endogenously.     

The effects of low dose X-irradiation on osteoblastic MC3T3-E1 cells in vitro

Background

Recently anti-CCP testing has become popular in the diagnosis of rheumatoid arthritis (RA). However, the inadequate reporting of the relevant diagnostic studies may overestimate and bias the results, directing scientists into making false decisions. The aim of the present study was to evaluate the reporting quality of studies used anti-CCP2 for the diagnosis of RA and to explore the impact of reporting quality on pooled estimates of diagnostic measures.

Methods

PubMed was searched for clinical studies investigated the diagnostic accuracy of anti-CCP. The studies were evaluated for their reporting quality according to STARD statement. The overall reporting quality and the differences between high and low quality studies were explored. The effect of reporting quality on pooled estimates of diagnostic accuracy was also examined.

Results

The overall reporting quality was relatively good but there are some essential methodological aspects of the studies that are seldom reported making the assessment of study validity difficult. Comparing the quality of reporting in high versus low quality articles, significant differences were seen in a relatively large number of methodological items. Overall, the STARD score (high/low) has no effect on the pooled sensitivities and specificities. However, the reporting of specific STARD items (e.g. reporting sufficiently the methods used in calculating the measures of diagnostic accuracy and reporting of demographic and clinical characteristics/features of the study population) has an effect on sensitivity and specificity.

Conclusions

The reporting quality of the diagnostic studies needs further improvement since the study quality may bias the estimates of diagnostic accuracy.     

Effect of interleukin 1 beta on osteoblastic clone MC3T3-E1 cells

Summary The effect of recombinant interleukin 1 Beta (IL-1(β)) was investigated on osteoblastic cell line MC3T3-E1 cloned from mouse calvaria. IL-1(β) stimulated cell proliferation which increased cell number and caused dose-related stimulation of DNA synthesis, with a maximal effect at a concentration of 12.5 U/ml; suppressed alkaline phosphatase activity and collagen synthesis maximally at 0.5 and 62.5 U/ml, respectively; and increased the amount of free [³H] hydroxyproline in the cultures, but the amount was quite low. Prostaglandin E₂ synthesis was also stimulated dose dependently by the presence of IL-1(β), with a maximal increase at 2.5 U/ml, at which concentration the prostaglandin E₂ level in the medium was 1.61±0.10 ng/ml. The increased prostaglandin E₂ synthesis did not affect either the IL-1(β)-mediated change in DNA or collagen synthesis or alkaline phosphatase activity. These results extend the possibility that IL-1(β) is to act as a regulator of bone formation.     

Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G-protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1>LPA4>LPA2>LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G-protein-coupled receptor LPA1.     

Properties of Suc-GPLGP-MCAase and dipeptidyl-aminopeptidase in mouse calvaria-derived osteoblastic cells (MC3T3-E1)

Summary In this study the properties of Suc-GPLGP-MCAase and pipeptidyl-aminopeptidase (DAP) in clone MC3T3-E1 cells which have osteoblastic ability were examined. The Suc-GPLGP-MCAase was the most active at pH 8.0 and its molecular weight was about 65,000 as judged by gel filtration method. The enzyme activity was increased by some metal ions such as Mn⁺², Ca⁺², and Mg⁺² but inhibited by Zn⁺² and Cu⁺². EDTA increased the enzyme activity to 23-fold. The enzyme activity was slightly inhibited by thiol inhibitors, N-ethylmaleimide, and p-chloromercuribenzoic acid by 16% and 27%, respectively. The DAP has an optimum pH at 7.5, and a molecular weight of about 100,000. The enzyme was completely inhibited by diisopropylfluorophosphate, but not by N-ethylmaleimide, iodoacetic acid, p-chloromercuribenzoic acid, phenylmethylsulfonyl fluoride, EDTA, and several metals. These results show that Suc-GPLGP-MCAase and DAP in MC3T3-E1 cells have different novel properties compared with these enzymes in the peripheral tissues.     

Enamel matrix derivative inhibits TNF-alpha-induced apoptosis in osteoblastic MC3T3-E1 cells.

OBJECTIVE: The purpose of this study was to examine the effect of enamel matrix derivative (EMD) on TNF-alpha-induced apoptosis in osteoblastic MC3T3-E1 cells. STUDY DESIGN: MC3T3-E1 cells were cultured at an initial density of 5000/cm 2 in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and allowed to adhere for 24 hours. Medium was then changed into DMEM with 0.5% FBS. After 16 hours, cells were treated with EMD (100 microg/mL) alone, tumor necrosis factor alpha (TNF-alpha) (20 ng/mL) alone, transforming growth factor beta 1 (TGF-beta1) (10 ng/mL) alone, TNF-alpha plus TGF-beta1, or TNF-alpha plus EMD. Cells cultured with DMEM and 0.5% FBS served as control. Following 24-hour incubation, apoptosis was assessed by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay, and quantified by cell death enzyme-linked immunosorbent assay (ELISA). RESULTS: Both TUNEL assay and cell death ELISA show that TNF-alpha induces apoptosis in MC3T3-E1 cells. TNF-alpha increases cell death by approximately 2-fold, which is attenuated by both EMD and TGF-beta1. CONCLUSION: Like TGF-beta1, EMD protects osteoblasts from inflammation-induced apoptosis.     

Expression of matrix metalloproteinases during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells.

The mouse calvarial osteoblast MC3T3-E1 cells released 92 kDa and 68 kDa of gelatinase activities into the conditioned media (CMs) from undifferentiated cells. When differentiation was induced by cultivating cells with ascorbate-2-phosphate (AscP), 68-kDa activity increased significantly in parallel with production of 60-kDa activity. These enzymes required Ca2+ and Zn2+ ions for their proteolytic activities. The 68-kDa activity was immunologically identified as latent matrix metalloproteinase 2 (MMP-2). The 92-kDa activity was deduced to be latent MMP-9 based on its molecular mass. The 60-kDa activity band was found to possess both gelatin and beta-casein hydrolyzing activities, indicating that this activity band might comprise the active form of MMP-2 and latent MMP-13. MC3T3-E1 cells were found to express MMP-2, MMP-13, and membrane type (MT)1-MMP genes by Northern blotting. MMP-2 was expressed constitutively. MMP-13 was up-regulated during the growth with AscP. MT1-MMP expression also was modulated by AscP; at the early stage of differentiation, its messenger RNA (mRNA) level increased and then decreased gradually to the control level. These changes in the profiles of MMPs observed here could be attributed to the maturation of collagenous extracellular matrix (ECM) induced by AscP.     

Ghrelin stimulates proliferation and differentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells

Ghrelin is a 28-amino-acid peptide identified in the stomach as an endogenous ligand of the growth hormone secretagogue receptor (GHS-R) that strongly stimulates the release of growth hormone at the hypothalamus and pituitary level. Although GHS-Rs are expressed in a variety of peripheral tissues, little is known about its effect on bone independent of GH/IGF-1 axis. This study was undertaken to investigate whether ghrelin exerts a direct effect on osteoblasts. We identified mRNA and protein expression of GHS-R in primary osteoblasts as well as a number of osteoblastic cell lines, including MC3T3-E1, ROS 17/2.8, UMR-106, MG63, and SaOS2 cells. Treatment of ghrelin (10(-11) to 10(-7) M) to MC3T3-E1 cells showed dose-dependent stimulation of proliferation, which was abrogated by treatment with [d-Lys]-GHRP-6 (10(-3) M), a selective antagonist of the ghrelin receptor. Ghrelin activated ERK1/2 MAPK and pretreatment with MAPK kinase inhibitors, PD98059 attenuated the ghrelin-induced cell proliferation. Ghrelin also inhibited TNFalpha-induced apoptosis and suppressed caspase-3 activation that occurs in response to TNFalpha as well as during in vitro differentiation process. Moreover, ghrelin treatment enhanced in vitro osteoblast differentiation as evidenced by matrix mineralization, alkaline phosphatase activity, and osteoblast-specific gene expression. These results suggest that ghrelin promotes proliferation and differentiation and inhibits apoptosis of osteoblasts.     

Effects of triiodothyronine on morphology, growth behavior, and the actin cytoskeleton in mouse osteoblastic cells (MC3T3-E1)

We investigated the effects of thyroid hormone treatment on morphology, growth behavior, and cytoskeletal structures of long-term cultured MC3T3-El cells. Morphological investigations were carried out on native cells by phase contrast microscopy and on epon-embedded semithin sections. The area covered by the cell and matrix layers (tissue-like area), percent extracellular matrix, average height of tissue-like area, and length and height of single cells were measured histomorphometrically on the cross sections. F-actin was analyzed histochemically and quantitated after fluorochrome-labeled phalloidin staining using confocal microscopy and fluorometry. Significant differences between control and T₃-treated cells were found after confluency, but not in subconfluent cultures. Control cells continued to proliferate forming multilayers, and produced increasing amounts of extracellular matrix. In contrast, T₃-treated cells stopped to proliferate forming two cell layers at the maximum. These cells were flattened, distinctly enlarged, and polygonal in shape. Histochemical staining for F-actin revealed three different staining patterns, depending on the position of the cell within the multilayer of control cultures. Basal cells contained a large number of thick stress fibers in parallel arrangement. Intermediate cells exhibited only a few thick actin filament bundles located at the outermost periphery. The superficial cells were characterized by a large number of thin, parallel-oriented microfllament bundles extending across the entire cytoplasm. The actin pattern of T₃-treated cells resembled that of the basal cell layer of the control cells. The amount of F-actin increased with the prolonged T₃ treatment. We conclude from these data that the known specific cellular responses to T₃ treatment are accompanied by significant morphological alterations indicating pivotal effects of thyroid hormones on osteoblastic differentiation.     

Transforming growth factor-beta reduces the phenotypic expression of osteoblastic MC3T3-E1 cells in monolayer culture

Transforming growth factor beta (TGF-beta) regulates cell growth and differentiation. Since it is abundant in bone, we have studied the effect of the polypeptide upon the growth and phenotypic expression of murine osteoblastic cells in monolayer culture. Its actions were compared to those of epidermal growth factor (EGF), another hormonally active polypeptide known to alter bone cell function. Picogram amounts of TGF-beta were found to inhibit the growth and phenotype (alkaline phosphatase and cAMP response to parathyroid hormone) of the clonal nontransformed MC3T3-E1 osteoblastic cell line. EGF also inhibited phenotypic expression, although at higher (nanogram) concentrations, but stimulated cell growth. The low concentration of TGF-beta required to inhibit growth and phenotype of osteoblastic cells together with its abundance in bone suggest that TGF-beta may be an important regulator of bone cell function.     

Mechanisms of root canal sealers cytotoxicity on osteoblastic cell line MC3T3-E1.

OBJECTIVE: The cytotoxic mechanisms of root canal sealers (Sealapex, AH26, and N2 Universal) were studied in vitro with MC3T3-E1 osteoblastic cells. STUDY DESIGN: MC3T3-E1 cells were cotreated with root canal sealers and antioxidants, and concentrations of intracellular glutathione (GSH) and reactive oxygen species (ROS) were measured. DNA fragmentation was observed after treatment with the sealers. RESULTS: N-Acetylcysteine (NAC) prevented N2 Universal- and AH26-induced cytotoxicities. However, ascorbic acid and Trolox did not affect the cytotoxicity of the sealers. N2 Universal and AH26 significantly decreased the GSH pool within a 3-hour treatment period. Unlike GSH levels, the ROS levels were not altered by the sealers. Cytotoxicity of Sealapex was not affected by NAC, and there were no changes of GSH/glutathione disulfide levels in cells treated with Sealapex. CONCLUSION: Cytotoxicities of N2 Universal and AH26 are caused by an intracellular GSH depletion without a burst of ROS. Sealapex may cause cytotoxicity in a way different from N2 Universal and AH26.     

Microcarriers facilitate mineralization in MC3T3-E1 cells

Summary MC3T3-E1 cells showed mineral deposits after about 1 week of culture when incubated in the presence of microcarrier beads. These deposits appeared as white spots on the dish surface, and under light microscopy the cells showed multiple cell layers and mineralization around the microcarriers. The deposits stained positive with calcium-specific Von Kossa's method. Using conventional assay, alkaline phosphatase activity (ALP) and parathyroid hormone-stimulated intracellular cAMP production were lower in the microcarrier cultures than in the control, but using cytochemical methods, high alkaline phosphatase activity was found around the microcarriers. These results indicate that microcarriers facilitated the formation of multiple cell layers and provided a culture environment for mineralization.     

Response of osteoblastic clonal cell line (MC3T3-E1) to [Asu1,7]Eel calcitonin at a specific cell density or differentiation stage

Summary Clone MC3T3-E1 cells isolated from newborn mouse calvaria is an osteogenic cell line which retains an ability to differentiate into osteo-blastic cellin vitro. The effect of [Asu^1,7]eel calcitonin (ECT) on clonal MC3T3-E1 cells was investigated at different stages of differentiation. ECT caused an increase in alkaline phosphatase (ALP) activity. The stimulative effect was demonstrated to be dependent upon cell density or differentiation stage. At a cell density of 1.18×10⁵/cm² cells were incubated with ECT for 2 days. The treatment by ECT caused an increase in ALP activity. A specific response to ECT dependent on the cell density was observed in a narrow range of cell density. Moreover this range of cell density responsible to ECT was found to be a rapid differentiation stage of MC3T3-E1 cells. These results suggest that calcitonin stimulates differentiation of osteoblast. In addition to these results, cellular adenosine-3′, 5′-cyclic monophosphate (cAMP) level was raised by ECT treatment at a cell density of about 1.4×10⁵ cell/cm² and this response was also specific for cell density. At cell density lower or higher than this density no stimulative effect by ECT was observed. On the other hand, N⁶,O₂-dibutyryl adenosine-3′,5′-cyclic monophosphate (db-cAMP) and theophylline caused an increase in ALP activity in wide cell density range. These results indicate that an increase in ALP activity by ECT is mediated by intracellular cAMP and that the specific response to ECT dependent on the cell density is regulated in the process of cAMP formation and/or in the preceding process of cAMP formation.     

Cyclooxygenase inhibitors enhance cell growth in an osteoblastic cell line, MC3T3-E1

To elucidate the significance of endogenous prostaglandin E2 (PGE2) in osteoblastic cell function, we studied the effects of cyclooxygenase inhibitors on cell growth and alkaline phosphatase (ALP) activity in MC3T3-E1 cells. UMR-106 cells were also used as references in our experiments. MC3T3-E1 cells, cultured in alpha-minimal essential medium containing 10% fetal bovine serum, were shown to produce PGE2, which was markedly suppressed in the presence of indomethacin. Addition of indomethacin resulted in an increase in DNA content and [3H]thymidine incorporation. A similar growth stimulatory effect was observed when structurally different cyclooxygenase inhibitors, that is, acetyl salicylic acid (ASA), flurbiprofen, and piroxicam, were added. These cyclooxygenase inhibitors, however, differed in their effects on ALP activity. Indomethacin and ASA enhanced ALP activity, whereas flurbiprofen and piroxicam suppressed it. We then examined the effects of exogenous addition of PGE2. Although exogenous PGE2 at 6 x 10(-6) M slightly stimulated cell growth, it inhibited cell growth at 6 x 10(-8) M and 6 x 10(-7) M. ALP activity was reduced in a dose-dependent fashion by exogenous PGE2. These results suggest that PGE2 produced by MC3T3-E1 may be suppressing cell proliferation and that cyclooxygenase inhibitors, per se, may stimulate cell growth by inhibiting endogenous PGE2 production in MC3T3-E1 cells. UMR-106 cells also produced PGE2, although less than MC3T3-E1 cells. In UMR-106 cells, the cyclooxygenase inhibitors did not influence DNA content or ALP activity as distinctly as in MC3T3-E1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

吡格列酮对正常MC3T3-E1成骨细胞增殖、凋亡以及功能蛋白表达的影响

目的探讨不同浓度吡格列酮(PIO)对正常糖浓度培养下MC3T3-E1小鼠早期成骨细胞增殖、凋亡及功能蛋白分泌表达的影响。方法体外培养MC3T3-E1细胞,分为正常对照组、不同浓度PIO干预组(5μmol/L、10μmol/L、20μmol/L、40μmol/L),分别干预24、48 h。CCK-8检测细胞增殖,流式细胞仪检测细胞凋亡率,RIA法和ELISA法分别检测相关功能蛋白骨钙素(OCN)、碱性磷酸酶(ALP),RT-PCR检测过氧化物酶体增殖物激活受体γ(PPARγ)、成骨因子runt相关基因2(Runx2)及骨形成蛋白-2(BMP-2)mRNA表达水平。结果 (1)与对照组比较,MC3T3-E1细胞增殖能力在5μmol/L PIO干预组增加显著(P<0.05)、在PIO大于5μmol/L～10μmol/L干预组则降低(P<0.05)。干预时间由24 h延长至48 h,各组细胞增殖能力呈不同程度增加,但在20、40μmol/L PIO干预的两组增加无统计学差别。(2)对照组及各PIO浓度干预干预24 h,细胞凋亡率分别为:1.97%、0.43%、13.0%、48.30%、81.00%;(3)随着PIO浓度从0～40μmol/L范围内的增加,MC3T3-E1细胞的PPARγmRNA表达水平呈增加趋势(P<0.05);Runx2 mRNA表达量在5μmol/L PIO浓度组时较对照组增加,在剂量较高时(PIO≥10μmol/L)随浓度升高表达下降。(4)细胞功能蛋白ALP、OCN的分泌及BMP-2的表达在5μmol/L PIO浓度组时最高,PIO≥10μmol/L时,上述蛋白量逐渐下降(P<0.05);随干预时间从24～48 h延长,各PIO浓度干预组ALP以及BMP-2量增加(P<0.05),而OCN无显著改变(P>0.05)。结论 PIO对正常糖浓度培养的MC3T3-E1细胞具有双向作用:低浓度促进细胞增殖,高浓度则促进凋亡。PPARγ适当激活可促使成骨细胞通过Runx2增加功能蛋白的合成;过度激活,则表现出细胞毒性。     

Characteristics of steroid hormone receptors in cultured MC3T3-E1 osteoblastic cells and effect of steroid hormones on cell proliferation

Summary We examined the binding characteristics of three kinds of steroid hormones—estrogen, androgen, and glucocorticoid—in cultured MC3T3-E1 mouse osteoblastic cells by whole-cell binding assay. The binding studies revealed the presence of a single class of high-affinity binding sites for [³H]17-estradiol, [³H]mibolerone (a synthetic androgen), and [³H]triamcinolone acetonide (a synthetic glucocorticoid). The numbers of binding sites for these steroid hormones were found to be 4534±819, 14312±1884, and 24898±655 sites/cell; and the Kd values were 8.57±0.62 x 10^–10M, 1.12±0.19 x 10^–9 M, and 6.08±1.24 x ^–10M, respectively. We also examined the effects of steroid hormones on the proliferation of MC3T3-E1 cells. 17-estradiol significantly stimulated the proliferation of the cells (130–150% of control). Dihydrotestosterone also significantly stimulated the proliferation of the cells (115% of control); the effect was, however, much less potent than that of 17-estradiol, although the number of binding sites was approximately three times more than that of 17 estradiol. Triamcinolone acetonide and dexamethasone had no effect on cell proliferation. These results suggest that estrogen and androgen act directly on osteoblastic cells through a receptor-mediated mechanism, and that androgen is much less potent than estrogen in stimulating the proliferation of MC3T3-E1 osteoblastic cells.