ATP-mediated mineralization of MC3T3-E1 osteoblast cultures

While bone is hypomineralized in hypophosphatemia patients and in tissue-nonspecific alkaline phosphatase (Tnsalp)-deficient mice, the extensive mineralization that nevertheless occurs suggests involvement of other phosphatases in providing phosphate ions for mineral deposition. Although the source of phosphate liberated by these phosphatases is unknown, pyrophosphate, ATP, pyridoxal-5'-phosphate (PLP) and phoshoethanolamine (PEA) are likely candidates. In this study, we have induced mineralization of MC3T3-E1 osteoblast cultures using ATP, and have investigated potential phosphatases involved in this mineralization process. MC3T3-E1 osteoblasts were cultured for 12 days and treated either with beta-glycerophosphate (betaGP) or ATP. Matrix and mineral deposition was examined by biochemical, cytochemical, ultrastructural and X-ray microanalytical methods. ATP added at levels of 4-5 mM resulted in mineral deposition similar to that following conventional treatment with betaGP. Collagen levels were similarly normal in ATP-mineralized cultures and transmission electron microscopy and X-ray microanalysis confirmed hydroxyapatite mineral deposition along the collagen fibrils in the ECM. Phosphate release from 4 mM ATP into the medium was rapid and resulted in approximately twice the phosphate levels than after release from 10 mM betaGP. ATP treatment did not affect mineralization by altering the expression of mineral-regulating genes such as Enpp1, Ank, and Mgp, nor phosphatase genes indicating that ATP induces mineralization by serving as a phosphate source for mineral deposition. Levamisole, an inhibitor of TNSALP, completely blocked mineralization in betaGP-treated cultures, but had minor effects on ATP-mediated mineralization, indicating that other phosphatases such as plasma membrane Ca2+ transport ATPase 1 (PMCA1) and transglutaminase 2 (TG2) are contributing to ATP hydrolysis. To examine their involvement in ATP-mediated mineralization, the inhibitors cystamine (TG2 inhibitor) and ortho-vanadate (PMCA inhibitor) were added to the cultures - both inhibitors significantly reduced mineralization whereas suppression of the phosphate release by ortho-vanadate was minor comparing to other two inhibitors. The contribution of PMCA1 to mineralization may occur through pumping of calcium towards calcification sites and TG2 can likely act as an ATPase in the ECM. Unlike the GTPase activity of TG2, its ATPase function was resistant to calcium, demonstrating the potential for participation in ATP hydrolysis and mineral deposition within the ECM at elevated calcium concentrations.     

辐射对成骨细胞系MC3T3-E1细胞增殖和功能基因表达的影响

目的为了深入了解辐射对成骨细胞的影响,探讨成骨细胞系MC3T3-E1细胞受到辐射后的功能变化。方法将MC3T3-E1细胞体外培养,诱导成骨前体细胞和成骨细胞,经137Csγ射线照射后,用MTT法分析细胞的存活率,用实时定量PCR方法分析ALP、Run X2和M-CSF基因的mRNA表达。结果 MTT实验表明,随照射剂量增加,正常MC3T3-E1细胞生长率明显下降,而经过诱导分化的MC3T3-E1细胞生长率变化越来越不明显。实时定量PCR实验结果表明,经过137Csγ射线照射后,MC3T3-E1细胞的ALP,Run X2和M-CSF基因的mRNA表达出现明显的降低;经过诱导分化为成骨前体细胞的,ALP,Run X2和M-CSF基因的mRNA表达与相应的正常组相比没有明显的规律变化;经过诱导进一步分化成为成骨细胞的,ALP和Run X2表达下降,M-CSF表达呈现升高趋势。结论辐射抑制早期成骨细胞的增殖、发育和分化。随着成骨细胞的分化,辐射对成骨细胞的增殖和生长发育影响减小,但是对成骨细胞发挥调节破骨细胞功能的作用并没有减少。     

Bone-resorbing cytokines enhance release of macrophage colony-stimulating activity by the osteoblastic cell MC3T3-E1

Summary It has been observed that bone resorption in response to interleukin 1 (IL 1) or tumor necrosis factor (TNF) is accompanied by an increase in osteoclast number. Because the osteoclast is of hemopoietic lineage, recruitment could be regulated by colony-stimulating factors, one of which may be macrophage colony-stimulating factor (MCSF). In this study, we show that the constitutive release of M-CSF activity by the osteoblastic cell MC3T3-E1 is enhanced by the presence of recombinant IL 1α, recombinant TNFα, or by the concurrent presence of purified transforming growth factorβ (TGFβ) and epidermal growth factor (EGF). Increased release of CSF by the osteoblast in response to these agents may provide a signal for the growth and maturation of osteoclast precursors leading to subsequent bone resorption.     

A differentiation-inducing factor produced by the osteoblastic cell line MC3T3-E1 stimulates bone resorption by promoting osteoclast formation

We have reported that the differentiation-inducing factor (DIF) is present in conditioned medium of mouse osteoblast-like cell (MC3T3-E1) cultures. In the present study, the DIF from conditioned medium of MC3T3-E1 cells was partially purified and its biologic activity was examined. The DIF was purified by monitoring the induction of phagocytic activity of mouse myeloblastic leukemia cells (M1). The DIF induced differentiation of not only M1 cells but also mouse myelomonocytic cells (WEHI-3). Furthermore, the DIF increased the in vitro bone-resorbing activity and the osteoclast number in mouse calvaria. The increases were inhibited by the addition of either salmon calcitonin or indomethacin. When mouse bone marrow cells were cultured with the DIF for 8 days, formation of osteoclast-like multinucleated cells was stimulated dose dependently. The DIF from MC3T3-E1 cells appeared to be different from interleukin-1 (IL-1), tumor necrosis factor (TNF), and transforming growth factor beta (TGF-beta). These results suggest that the DIF partially purified from osteoblast-like cell cultures stimulates osteoclastic bone resorption by promoting differentiation and fusion of osteoclast progenitors to form multinucleated osteoclasts.     

Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures

Studies on various compounds of inorganic phosphate, as well as on organic phosphate added by post-translational phosphorylation of proteins, all demonstrate a central role for phosphate in biomineralization processes. Inorganic polyphosphates are chains of orthophosphates linked by phosphoanhydride bonds that can be up to hundreds of orthophosphates in length. The role of polyphosphates in mammalian systems, where they are ubiquitous in cells, tissues and bodily fluids, and are at particularly high levels in osteoblasts, is not well understood. In cell-free systems, polyphosphates inhibit hydroxyapatite nucleation, crystal formation and growth, and solubility. In animal studies, polyphosphate injections inhibit induced ectopic calcification. While recent work has proposed an integrated view of polyphosphate function in bone, little experimental data for bone are available. Here we demonstrate in osteoblast cultures producing an abundant collagenous matrix that normally show robust mineralization, that two polyphosphates (PolyP5 and PolyP65, polyphosphates of 5 and 65 phosphate residues in length) are potent mineralization inhibitors. Twelve-day MC3T3-E1 osteoblast cultures with added ascorbic acid (for collagen matrix assembly) and β-glycerophosphate (a source of phosphate for mineralization) were treated with either PolyP5 or PolyP65. Von Kossa staining and calcium quantification revealed that mineralization was inhibited in a dose-dependent manner by both polyphosphates, with complete mineralization inhibition at 10 μM. Cell proliferation and collagen assembly were unaffected by polyphosphate treatment, indicating that polyphosphate inhibition of mineralization results not from cell and matrix effects but from direct inhibition of mineralization. This was confirmed by showing that PolyP5 and PolyP65 bound to synthetic hydroxyapatite in a concentration-dependent manner. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) efficiently hydrolyzed the two PolyPs as measured by P_i release. Importantly, at the concentrations of polyphosphates used in this study which inhibited bone cell culture mineralization, the polyphosphates competitively saturated TNAP, thus potentially interfering with its ability to hydrolyze mineralization-inhibiting pyrophosphate (PP_i) and mineralizing-promoting β-glycerophosphate (in cell culture). In the biological setting, polyphosphates may regulate mineralization by shielding the essential inhibitory substrate pyrophosphate from TNAP degradation, and in the same process, delay the release of phosphate from this source. In conclusion, the inhibition of mineralization by polyphosphates is shown to occur via direct binding to apatitic mineral and by mixed inhibition of TNAP.     

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)     

Murine osteoblastlike cells and the osteogenic cell MC3T3-E1 release a macrophage colony-stimulating activity in culture

Summary The osteoclast may be of hematopoietic lineage and as such its development could be regulated by colony-stimulating factors. Since there is much interest as to whether osteoblasts influence bone resorption, we examined whether bone cells produce colony-stimulating activity. Both cells isolated from neonatal calvaria and the osteogenic cell MC3T3-E1 were found to constitutively release a colony-stimulating activity possessing characteristics of a macrophage colony-stimulating factor, as determined by basic biochemical purification and by identity of colonies induced in cultures of bone marrow cells. Release could be increased by the presence of the bone-resorbing agents lipopolysaccharide and 1,25 dihydroxyvitamin D₃. We conclude that the osteoblast may contribute to both the processes of osteoclast formation and of hematopoiesis through the secretion of colony-stimulating activity into the adjacent bone marrow.     

Inhibition ofin vitro mineralization by aluminum in a clonal osteoblastlike cell line,MC3T3-E1

Summary The direct effect of aluminum on mineralization was examined using an osteoblastlike cell line, MC3T3-E1. The mineralization process was quantitated by measuring⁴⁵Ca accumulation into the cell and matrix layer of MC3T3-E1 cells in culture. The accumulation of⁴⁵Ca into the cell and matrix layer increased dramatically after 13 days of culture without a parallel change in the DNA content of these cells. Because nodular clusters of cells appear around the same period in which a massive mineralization occurs, the marked increase in⁴⁵Ca accumulation after the 13th day of culture appears to represent deposition of⁴⁵Ca into the extracellular matrix. Thus, this culture system offers a useful model for making a quantitative estimation of osteoblast-mediated mineralizationin vitro. When aluminum was added to this system, the accumulation of⁴⁵Ca into the cell matrix layer was inhibited in a dose-dependent manner: 10⁻⁶ M aluminum reduced⁴⁵Ca accumulation to 40.8±2.7% of that in nontreated cells without affecting alkaline phosphatase activity or the DNA content of these cells. Because the concentration of aluminum used in this study is well within the range of serum aluminum levels seen in chronic dialysis patients, the direct effects of aluminum on osteoblast-mediated mineralization shown in the present study may underlie the development of so-called aluminum-induced “osteomalacia” in certain dialysis patients.     

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.     

Arachidonic acid stimulates cell growth in an osteoblastic cell line,MC3T3-E1, by noneicosanoid mechanism

Summary Arachidonic acid, added to α-minimum essential medium containing 10% fetal bovine serum at the final concentration of 10⁻⁴ M, significantly increased DNA content of an osteoblastic cell line, MC3T3-E1, along with an increase of DNA synthesis. No growth-stimulatory effect of arachidonic acid was observed under serum-free condition. α-Linolenic acid, which cannot be converted to arachidonic acid, also increased DNA content at 10⁻⁴ M. Additionally, the stimulatory effects of these fatty acids were not inhibited by simultaneous addition of 10⁻⁵ M of indomethacin. Indomethacin, when added to α-minimum essential medium with 10% fetal bovine serum, also significantly increased DNA content of MC3T3-E1 cells. These results suggest that arachidonic acid may potentiate the growth-stimulatory effect of serumderived growth factors probably via noneicosanoid mechanism. Rat osteogenic sarcoma cell line, UMR106, also showed an increase in DNA content with arachidonic acid treatment. Hence, it is suggested that arachidonic acid may stimulate proliferation of cells of osteoblastic lineage. It is also suggested that indomethacin, probably by blocking endogenous prostaglandin E₂ synthesis, stimulates cell growth in MC3T3-E1 cells.     

Modulation of thrombospondin gene expression during osteoblast differentiation in MC3T3-E1 cells.

The levels of expression of two related extracellular matrix protein genes, thrombospondins 1 and 2 (TSP1 and TSP2), were analyzed in the mouse osteogenic cell line, MC3T3-E1. To monitor differentiation, we also measured two potential markers of the osteoblastic phenotype, alkaline phosphatase (ALP) activity, and alpha 1(I) collagen mRNA levels. TSP1 mRNA levels increased 10- to 15-fold during the first nine days of osteoblastic conversion, and then dropped to a level still significantly above baseline values. This increase in TSP1 mRNA closely paralleled that observed in ALP activity. In contrast, TSP2 mRNA levels were unchanged throughout the 21-day time course. These findings suggest that TSP1 is a marker for osteoblast differentiation and could play a role in the cellular changes that accompany acquisition of the osteoblastic phenotype in MC3T3-E1 cells.     

Laser irradiation promotes the proliferation of mouse pre-osteoblast cell line MC3T3-E1 through hedgehog signaling pathway

Low-level laser could promote osteoblast proliferation, and it has been applied in clinical practice to promote wound healing and tissue regeneration. However, the mechanism related to laser irradiation remains unclear. This study aimed to investigate the effects of low-level laser irradiation on the cell proliferation and the expressions of hedgehog signaling molecules Indian hedgehog (Ihh), Ptch, and Gli in vitro. In our present study, the MTT method was used to evaluate the effect on cell proliferation of laser irradiation on MC3T3-E1 cells. And cell cycle was examined by flow cytometry. Gene and protein expressions of hedgehog signaling molecules, including Ihh, Ptch, Smoothened (Smo), and Gli, were examined by qRT-PCR and western blot analysis. The results showed that laser irradiation at dosage of 3.75 J/cm² enhances the proliferation of MC3T3-E1 cells compared with control groups (p = 0.00). Moreover, laser irradiation (3.75 J/cm²) increased the cell amount at S phase (p = 0.00). In addition, the expressions of Ihh, Ptch, Smo, and Gli were significantly increased compared to the control during laser irradiation (3.75 J/cm²)-induced MC3T3-E1 osteoblast proliferation. After adding the hedgehog signaling inhibitor CY (cyclopamine), cell proliferation and Ihh, Ptch, Smo, and Gli expressions were inhibited (p = 0.00), and the cell amount at S phase was reduced compared with combination groups (p = 0.00). These results indicated that laser irradiation promotes proliferation of MC3T3-E1 cells through hedgehog signaling pathway. Our findings provide insights into the mechanistic link between laser irradiation-induced osteogenesis and hedgehog signaling pathway.     

Relationship between collagen synthesis and expression of the osteoblast phenotype in MC3T3-E1 cells.

The MC3T3-E1 mouse calvaria-derived cell line has been used to study the role of collagen synthesis in osteoblast differentiation. MC3T3-E1 cells, like several previously characterized osteoblast culture systems, expressed osteoblast markers and formed a mineralized extracellular matrix only after exposure to ascorbic acid. Mineralization was stimulated further by beta-glycerol phosphate. Ultrastructural observations indicated that the extracellular matrix produced by ascorbic acid-treated cells was highly organized and contained well-banded collagen fibrils. Expression of osteoblast markers followed a clear temporal sequence. The earliest effects of ascorbic acid were to stimulate type I procollagen mRNA and collagen synthesis (24 h after ascorbate addition), followed by induction of alkaline phosphatase (48-72 h) and osteocalcin (96-144 h) mRNAs. Procollagen mRNA, which was expressed constitutively in the absence of ascorbate, increased only twofold after vitamin C addition. In contrast, alkaline phosphatase and osteocalcin mRNAs were undetectable in untreated cultures. Actions of ascorbic acid on osteoblast marker gene expression are mediated by increases in collagen synthesis and/or accumulation because (1) parallel dose-response relationships were obtained for ascorbic acid stimulation of collagen accumulation and alkaline phosphatase activity, and (2) the specific collagen synthesis inhibitors, 3,4-dehydroproline and cis-4-hydroxyproline, reversibly blocked ascorbic acid-dependent collagen synthesis and osteoblast marker gene expression.     

Matrix mineralization in MC3T3-E1 cell cultures initiated by β-glycerophosphate pulse

MC3T3-E1 cells, grown in the presence of serum and ascorbate, express alkaline phosphatase and produce an extensive collagenous extracellular matrix that can be mineralized by the addition of β-glycerophosphate (β-GP). In the present work, we study the influence of concentration and duration of β-GP treatment on the mineralization pattern in 4-week-old cell cultures. Amount and structure of mineral deposition were monitored by von Kossa staining, light, and electron microscopy, as well as small-angle X-ray scattering (SAXS) of unstained specimens. SAXS measures the total surface of the mineral phase and is therefore preferentially sensitive to very small crystals (typically <50 nm). It was used to determine the ratio (M) of small crystals to collagen matrix. A variety of mineralization patterns was observed to occur simultaneously, some associated with collagen within nodules or in deeper layers of the cultures and some independent of it. At a β-GP concentration of 10 mmol, mineralization was initiated after about 24 h and continued to increase, irrespective of whether the high level of β-GP was maintained or reduced to 2 mmol. With shorter pulses (<24 h), no significant mineralization was observed in the week following β-GP pulse. With continuous treatment at 5 mmol β-GP, the first signs of mineralization were detected 14 days after the beginning of treatment in the 4-week-old cultures, but no mineralization at all occurred at lower β-GP concentrations. When cells were grown without ascorbic acid for 4 weeks, only two cell layers without collagen matrix were found. In these cultures, no mineralization detectable by SAXS could be induced with β-GP. These data indicate that, in viable cells, high doses of β-GP are essential for the nucleation of mineral crystals, but not for the progression of mineralization once crystals had been nucleated. In contrast, when 4-week-old cell cultures were devitalized, M was found to increase immediately, even at 2 mmol β-GP. These results suggest that, in MC3T3-E1 cell cultures, cell viability is essential for prevention of spontaneous mineralization of the extracellular matrix.     

Fluid shear-induced ATP secretion mediates prostaglandin release in MC3T3-E1 osteoblasts.

ATP is rapidly released from osteoblasts in response to mechanical load. We examined the mechanisms involved in this release and established that shear-induced ATP release was mediated through vesicular fusion and was dependent on Ca2+ entry into the cell through L-type voltage-sensitive Ca2+ channels. Degradation of secreted ATP by apyrase prevented shear-induced PGE2 release. INTRODUCTION: Fluid shear induces a rapid rise in intracellular calcium ([Ca2+]i) in osteoblasts that mediates many of the cellular responses associated with mechanotransduction in bone. A potential mechanism for this increase in [Ca2+]i is the activation of purinergic (P2) receptors resulting from shear-induced extracellular release of ATP. This study was designed to determine the effects of fluid shear on ATP release and the possible mechanisms associated with this release. MATERIALS AND METHODS: MC3T3-E1 preosteoblasts were plated on type I collagen, allowed to proliferate to 90% confluency, and subjected to 12 dynes/cm2 laminar fluid flow using a parallel plate flow chamber. ATP release into the flow media was measured using a luciferin/luciferase assay. Inhibitors of channels, gap junctional intercellular communication (GJIC), and vesicular formation were added before shear and maintained in the flow medium for the duration of the experiment. RESULTS AND CONCLUSIONS: Fluid shear produced a transient increase in ATP release compared with static MC3T3-E1 cells (59.8 +/- 15.7 versus 6.2 +/- 1.8 nM, respectively), peaking within 1 minute of onset. Inhibition of calcium entry through the L-type voltage-sensitive Ca2+ channel (L-VSCC) with nifedipine or verapamil significantly attenuated shear-induced ATP release. Channel inhibition had no effect on basal ATP release in static cells. Ca(2+)-dependent ATP release in response to shear seemed to result from vesicular release and not through gap hemichannels. Vesicle disruption with N-ethylmaleimide, brefeldin A, or monensin prevented increases in flow-induced ATP release, whereas inhibition of gap hemichannels with either 18alpha-glycyrrhetinic acid or 18beta-glycyrrhetinic acid did not. Degradation of extracellular ATP with apyrase prevented shear-induced increases in prostaglandin E2 (PGE2) release. These data suggest a time line of mechanotransduction wherein fluid shear activates L-VSCCs to promote Ca2+ entry that, in turn, stimulates vesicular ATP release. Furthermore, these data suggest that P2 receptor activation by secreted ATP mediates flow-induced prostaglandin release.     

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.