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.     

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)     

Endothelin- and sarafotoxin-induced receptor-mediated calcium mobilization in a clonal murine osteoblast-like cell line, MC3T3-E1/B

Previous studies have demonstrated that, in osteoblast-like MC3T3-E1 cells, various endothelin peptides and their homologous sarafotoxins generate prostaglandin E₂ (PGE₂) release through an ET_A receptor subtype. In this study, biphasic Ca²⁺ signals elicited with endothelin (ET)-1, ET-2, ET-3, β-ET, S6a1, and S6b (ET/S6) were measured by microspectrofluorimetric methods in cell suspensions loaded with Fura-2 acetoxymethylester (Fura-2 AM). Phospholipase C (PLC)-dependent calcium activation mechanisms seem to be involved. We found evidence of Ca²⁺ release from thapsigargin-sensitive and non-thapsigargin-sensitive intracellular Ca²⁺ stores as well as Ca²⁺ transmembrane inflow through multiple voltage-independent and Ni²⁺-sensitive cation channels. Using an ET_A receptor antagonist, BQ-123, we showed that this receptor was coupled to Ca²⁺ mobilization. All agonists tested, except S6c (an ET_B-receptor-specific agonist) induced receptor desensitization. Our results demonstrate that the ET/S6-induced Ca²⁺ signaling pathway is mediated via an ET_A-receptor subtype in MC3T3-E1/B cells.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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。     

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.     

TNF-alpha and IL-1beta suppress N-cadherin expression in MC3T3-E1 cells.

Excessive production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) secondary to estrogen deficiency have been implicated as the cause of osteoporosis in postmenopausal woman. These cytokines appear to stimulate osteoclast precursor proliferation and activate mature osteoclast formation directly and possibly indirectly via osteoblasts. To investigate the other possible roles that these cytokines may play in stimulating the bone resorption process, we examined the effect of TNF-alpha and IL-1beta on cell-cell adhesion molecules, cadherins, in osteoblastic MC3T3-E1 cells. In this study, we investigated cadherin expression and the effect of TNF-alpha, IL-1beta, and parathyroid hormone (PTH) on the expression of cadherins in MC3T3-E1 cells. Confluent cultures of MC3T3-E1 cells were challenged with recombinant human TNF-alpha (1-100 U/ml), recombinant human IL-1beta (1-100 ng/ml) and human PTH(1-34) (1-100 ng/ml), respectively. The results show that MC3T3-E1 cells express functional cadherin molecules, N-cadherin and OB-cadherin. TNF-alpha (10-100 U/ml) and IL-1beta (10-100 ng/ml) suppressed N-cadherin without changing OB-cadherin expression, while PTH (1-100 ng/ml) had no effect on cadherin expression. These results raise the possibility that TNF-alpha and IL-1beta may compromise the cell-cell adhesion of osteoblasts which cover the bone surface. The ensuing compromised cell-cell adhesion of osteoblasts may in turn facilitate the direct adhesion of osteoclasts on the calcified bone matrix surface. These results implicate an indirect role for osteoblasts in the promotion of bone resorption by TNF-alpha and IL-1beta.     

Biological effects of low power laser irradiation on clonal osteoblastic cells (MC3T3-E1)

Cultured osteoblastic cells were studied to determine the effects of laser irradiation on their rates of proliferation, differentiation, and calcification. A continuous wave Helium-Neon laser with a wavelength of 632.8 nm was used for this study. Clonal osteoblastic cells (MC3T3-E1) were exposed to laser beam at various energy densities. The cell growth rate and DNA synthesis were increased by laser irradiation only in the growing phase of culture. During long-term culture, 45Ca accumulation was enhanced by laser irradiation at 1.0 J/cm2, with four sessions of irradiation resulting in a 46% increase over controls. In contrast, no significant increase in alkaline phosphatase activity was produced by laser irradiation. Electron microscopy revealed a tendency of enlargement of the Golgi apparatus in the laser-treated cells. These results suggest that laser irradiation photoactivates osteoblastic cells, accelerates osteoblastic cell growth and calcification in vitro, and therefore, may promote bone regeneration.     

二甲基乙二酰基甘氨酸对小鼠胚胎成骨细胞前体细胞增殖和分化的影响

目的 :探讨低氧模拟剂二甲基乙二酰基甘氨酸(dimethyloxalylglycine,DMOG)对小鼠胚胎成骨细胞前体细胞(MC3T3-E1)的增殖、成骨分化及血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响。方法:将MC3T3-E1细胞接种到培养板24h后,实验组培养基中分别加入50μM(50μM组)和200μM(200μM组)的DMOG,对照组加入完全培养液。分别于培养1、3、5d时采用MTT法检测MC3T3-E1细胞增殖情况,5、10d行碱性磷酸酶(alkaline phosphatase,ALP)染色及ALP活性检测成骨细胞分化,21d用茜素红染色检测MC3T3-E1细胞钙结节的形成并进行定量分析,采用ELISA法检测MC3T3-E1培养3d情况时上清液中的VEGF蛋白含量,并用实时荧光定量PCR法检测MC3T3-E1细胞VEGF mRNA的相对表达量。结果:培养1d时对照组、50μM组和200μM组的MC3T3-E1的光密度(optical density,OD)值分别为0.041±0.009、0.074±0.019、0.086±0.044,3d时分别为0.123±0.027、0.148±0.020、0.224±0.061,5d时分别为0.297±0.044、0.325±0.084、0.354±0.038,1d、3d时50μM组和200μM组与同时间点对照组比较均有显著性差异(P0.05),3d时50μM组与200μM组有显著性差异(P0.05)。培养5d和10d时对照组ALP染色颜色较深,50μM组颜色中等,200μM组颜色较浅;5d时对照组ALP活性为1.943±0.072,50μM组为1.632±0.051,200μM组为1.319±0.065;10d时对照组ALP活性为3.734±0.067,50μM组为3.381±0.070,200μM组为2.831±0.086。三组间同时间点比较均有统计学差异(P0.05),同组10d时与5d时比较均有统计学差异(P0.05)。茜素红染色200μM组可见少量红色结节,50μM组可见中等量红色结节,对照组可见大量红色结节;对照组茜素红含量(μg/ml)为56.178±7.940,50μM组为41.922±2.438,200μM组为31.929±1.922,三组间比较均有统计学差异(P0.05)。培养3d时对照组细胞培养上清液中VEGF蛋白含量(ng/孔)为9.063±0.603,50μM组为12.123±0.870,200μM组为15.540±0.581,三组间比较均有统计学差异(P0.05);50μM组VEGF mRNA表达量为对照组的1.792±0.067,200μM组为对照组的3.963±0.092,三组间比较均有统计学差异(P0.05)。结论 :低氧模拟剂DMOG可促进MC3T3-E1细胞增殖和VEGF表达,抑制其成骨分化。     

Effect of microfibrillar collagen on growth and differentiation of osteoblast-like MC3T3-E1 cells

Effect of microfibrillar collagen on the proliferation and differentiation of osteoblastic cells was studied using MC3T3-E1 (E1) cells. In order to achieve direct contact of microfibrillar collagen with E1 cells, they were embedded in denatured collagen gel, and DNA content, [³H] thymidine incorporation, alkaline phosphatase activity, and⁴⁵Ca accumulation were examined after long-term culture. Microfibrillar collagen embedded with E1 cells increased DNA content and stimulated DNA synthesis of E1 cells in a dose-dependent manner. In vitro mineralization induced by E1 cells was also stimulated by microfibrillar collagen in a dose-dependent manner: 1mg/ml of microfibrillar collagen stimulated⁴⁵Ca accumulation by about 3 fold, and 10 mg/ml by 5 fold. Alkaline phosphatase activity was not affected by the presence of microfibrillar collagen. Because the interaction of specific RGD tripeptide recognition site on collagen fiber with cell surface adhesion receptors is proposed to affect the proliferation and differentiation of various cells, it is suggested that the interaction of osteoblastic cells with collagen fibers plays an important role in the regulation of proliferation and the expression of osteoblastic phenotype in these cells.     

Mitogenic Action of Adenosine on Osteoblast-Like Cells, MC3T3-E1

The purpose of this study was to investigate the mechanisms by which adenosine stimulates proliferation of osteoblast-like cells, MC3T3-E1. Adenosine by itself induces the stimulation of cell proliferation and accentuates the mitogenecity of PDGFs (AA and BB homodimers) for the cells. 8-Cyclopentyl-1,3-dimethylxanthine (CPX), a nonselective adenosine receptor antagonist, partially inhibited adenosine-induced DNA synthesis in a competitive manner, suggesting that the mitogenic action of adenosine is, at least in part, mediated by xanthine-sensitive receptors. In pertussis-toxin (PTX)-pretreated cells, adenosine- but not PDGF-BB-stimulated DNA synthesis was partially inhibited, and CPX did not exert a further inhibitory effect, suggesting an involvement of PTX-sensitive G-protein downstream of CPX-sensitive receptor. When adenosine uptake was prevented with dipyridamole, the stimulation of proliferation by adenosine was not decreased at all, indicating that the CPX-insensitive part of adenosine action is not associated with the uptake of adenosine and subsequent incorporation into the nucleotide pool. Adenosine did not influence the basal level or the PDGF-BB-induced increase in [Ca²⁺]i. Since it is known that the cAMP pathway acts in inhibiting osteoblast proliferation, the mitogenic action of adenosine would be dependent on neither the cAMP pathway nor the phospholipase C/Ca²⁺ pathway. It has been concluded that adenosine exerts a mitogenic effect via two pathways at least, one mediated by xanthine-sensitive receptor and PTX-sensitive G-protein and the other through an unknown xanthine- and PTX-insensitive process. Received: 21 May 1995 / Accepted: 28 June 1997