Effects of diastereoisomers of 1,25-dihydroxyvitamin D3-26,23-lactone on alkaline phosphatase and collagen synthesis in osteoblastic cells

The effects of the four diastereoisomers of 1,25-dihydroxyvitamin D3-26,23-lactone (1,25-(OH)2D3-26,23-lactone) on alkaline phosphatase (AP) activity and collagen and noncollagen protein synthesis were examined in cultures of the osteoblastic clone MC3T3-E1 cell line. The four lactone diastereoisomers had little effect on the protein and DNA content of the cells. The 23(S),25(S)- and 23(R),25(R)-1,25-(OH)2D3-26,23-lactones increased AP activity in a linear dose-dependent fashion. Maximal effects were observed at 100 and 1000 pg/ml, respectively. In contrast, the naturally occurring 23(S),25(R)-, 1,25-(OH)2D3-26,23-lactone and the 23(R),25(S)-1,25-(OH)2D3-26,23-lactone showed biphasic stimulatory effects on AP activity. At both 80 and 10,000 pg/ml, they stimulated maximum increases in alkaline phosphatase activity. At 80 pg/ml the 23(S),25(R)- and 23(R),25(S)-isomers stimulated an increase in collagen synthesis, while at 10,000 pg/ml these isomers and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) did not. Moreover, these two isomers (at 10,000 pg/ml) plus insulin or dexamethasone had an additive effect on AP activity, but not at 80 pg/ml. At 80 pg/ml but not at 10,000 pg/ml, the 23(S),25(R)-isomer had an additive effect on AP activity with the simultaneous addition of 25-hydroxyvitamin D3. Relative to 1,25-(OH)2D3, the binding affinities of 23(S),25(S)-, 23(R),25(R)-, 23(S),25(R)- and 23(R),25(S)-1,25-(OH)2D3-26,23-lactones were calculated to be 1/13.0, 1/131.8, 1/805.2, and 1/1083.3, respectively. No metabolites could be detected in the medium when [1-3H]23(S),25(R)-1,25-(OH)2D3-26,23-lactone (the naturally occurring diastereoisomer) was added to the cultures. However, the stimulative effects of 1,25-(OH)2D3 and the 23(S),25(R)-isomer at both concentrations were completely abolished by L-1-tosyl-amido-2-phenylethyl chloromethyl ketone. These results indicate that 1,25-(OH)2D3-lactone has a stimulative effect on osteoblastic cell functions in vitro. The naturally occurring 23(S),25(R)-1,25-(OH)2D3-lactone acts biphasically and may act on bone metabolism in vivo, possibly through a 1,25-(OH)2D3-receptor-mediated pathway.     

Effect of total glucosides of peony on the proliferation,differentiation,and mineralization of MC3T3-E1 osteoblastic cells

<正>Objective To observe the effect of total glucosides of peony(TGP) on the proliferation,differentiation,and mineralization of MC3 T3-E1 osteoblastic cells.Methods The effects of TGP on the proliferation and cell cycle of MC3 T3-E1 osteoblastic cells were determined by MTT     

Mouse osteoblastic cells (MC3T3-E1) at different stages of differentiation have opposite effects on osteoclastic cell formation

Using our new culture system for multinucleate cells (MNCs) that have many characteristics of osteoclasts, we examined the effects of factors produced by osteoblastic cells on osteoclastic cell formation. Conditioned medium (CM) from undifferentiated osteoblastic MC3T3-E1 cells during their growth phase inhibited MNC formation in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Diluted CM (1:81) from differentiated cells obtained after cultivation for more than 20 days stimulated MNC formation, but at lower dilutions inhibited their formation. Dialyzed CM (greater than 2000 mol wt) from the differentiated cells was more stimulatory than undialyzed CM and showed no inhibitory effect on MNC formation. The inhibitory effect was observed with filtered (less than 3000 mol wt) CMs and was specific for osteoblastic cell CM. Prostaglandin E2 (PGE2) was detected in the CM from undifferentiated or differentiated MC3T3-E1 cells at concentrations (317 +/- 66 and 1287 +/- 179 pg/ml, respectively) sufficient to inhibit MNC formation, and this inhibition was partially abolished with CM (at 3-fold dilution) in indomethacin-treated cells (PGE2, less than 20 pg/ml), suggesting PGE2-mediated inhibition of MNC formation and the presence of another factor(s) besides PGE2 that influenced MNC formation. In contrast to day 3 CM plus 1,25-(OH)2D3, day 60 CM plus 1,25-(OH)2D3 induced MNC formation even in the absence of GM-CSF, and this induction was inhibited by an antibody to GM-CSF. Secondary colony formation assays showed the presence of a GM-CSF-like factor in the day 60 CM. These findings indicate that osteoblastic cells are involved in the process of osteoclastic cell formation, with at least two soluble factors produced by osteoblasts, a GM-CSF-like factor, which is stimulatory, and PGE2, which is inhibitory. The effects of CMs also differed depending on the stage of osteoblast differentiation.     

Calyculin A stimulates the expression of TNF-alpha mRNA via phosphorylation of Akt in mouse osteoblastic MC3T3-E1 cells

Intracellular phosphatase activity has been recognized to play a central role in signal transduction. In the present study, we investigated the effects of calyculin A, an inhibitor of protein phosphatases, on the expression of TNF-alpha mRNA and the possible signaling pathways in mouse osteoblastic MC3T3-E1 cells. The result of semiquantitative RT-PCR showed that calyculin A increased the expression of TNF-alpha mRNA in MC3T3-E1 cells. Pre-treatment of LY294002 and Wortmannin, inhibitors of PI3K, inhibited the calyculin A-stimulated TNF-alpha mRNA expression. Western blot result disclosed that calyculin A increased the phosphorylation status of Akt at Ser473. However, U0126 and SB203580, specific inhibitor of MEK1/2 and p38MAPK, respectively, had no effect on calyculin A-stimulated expression of TNF-alpha mRNA. BAY11-7085 and CAPE, inhibitors of NF-kappaB activity, did not alter the calyculin A-stimulated TNF-alpha mRNA expression. Indirect immunofluorescent study confirmed that NF-kappaB was not translocated to the nucleus by calyculin A treatment. Our present results suggest that inhibition of phosphatase activity by calyculin A stimulate the phosphorylation of Akt at Ser473 by PI3K/Akt signaling pathway, resulting in the expression TNF-alpha mRNA.     

左归丸含药血清通过p38 MAPK信号通路诱导MC3T3-E1成骨细胞的分化

目的 探讨p38MAPK信号通路对左归丸含药血清干预MC3I3-E1成骨细胞分化的影响.方法 制备大鼠含药血清,选用p38特异阻滞剂SB203580,实验分为空白对照组、SB203580组、左归丸组、左归丸加SB203580组、倍美力组、倍美力加SB203580组.孵育48 h后,采用PNPP法检测碱性磷酸酶(ALP)活性,采用Westem印迹法分析ALP蛋白表达水平;孵育7d,采用改良钙钴染色法检测ALP活性;孵育14d,采用茜素红染色法测定矿化沉积情况.结果 与空白对照组比较,左归丸组显著促进MC3T3-E1成骨细胞分化,明显上调ALP蛋白表达水平(P＜0.01);与左归丸组比较,左归丸加SB203580组显著抑制左归丸含药血清对MC3T3-E1成骨细胞的分化的促进作用,明显下调ALP蛋白表达水平(P＜0.01).结论 左归丸含药血清可能部分通过p38MAPK信号通路干预MC3T3-E1成骨细胞分化.     

Interleukin (IL)-4 and IL-13 inhibit the differentiation of murine osteoblastic MC3T3-E1 cells

Interleukin-4 (IL-4) inhibits the spontaneous and stimulated bone resorption resulting from the inhibition of osteoclast formation, as well as osteoclastic activity. Since IL-13 shares some biological properties with IL-4, it was recently reported that IL-13 inhibits bone resorption. The present study was designed to determine the effects of murine IL-4 (IL-4) and murine IL-13 (IL-13) on the murine osteoblastic cell line MC3T3-E1. IL-4 and IL-13 stimulated 3H-thymidine incorporation in the MC3T3-E1 cells and its proliferation in dose dependent manners. A spontaneous increase in alkaline phosphatase (ALP) activity in the cells after plating was inhibited by IL-4 or IL-13, and both cytokines blunted an increase in ALP activity by human parathyroid hormone (PTH) (1-34). PTH-stimulated cyclic AMP (cAMP) production was inhibited by pretreatment with IL-4 and IL-13 for 48 hr in dose dependent manners. Pretreatment with IL-4 and IL-13 for 48 hr caused a decrease in PTH-induced cAMP production at any stimulatory concentration. However, the effective dose (ED50) was unchanged by the pretreatment with these cytokines. Pretreatment with IL-4 and IL-13 did not modulate cAMP generation by forskolin. In contrast, cAMP generation by PGE2 is greater in the cells treated with the cytokines compared to those without the cytokines. These results indicate that IL-4 and IL-13 act on MC3T3-E1 cells in the same manner, stimulating cell proliferation, but inhibiting cell differentiation. The inhibition of osteoblast differentiation by IL-4 and IL-13 may be associated with a decrease in PTH actions on osteoblasts.     

Effects of 1,25-dihydroxyvitamin D3 and cortisol on bovine and human parathyroid cells

Incubation of bovine parathyroid cells with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) decreased both preproparathyroid mRNA levels and parathyroid hormone (PTH) secretion. There was a fall to 56.6 +/- 13.7% (mean +/- S.E.M.) and 65.1 +/- 9.3% in mRNA levels and PTH secretion respectively at 1 nmol 1,25-(OH)2D3/l, and 41.1 +/- 13.6% and 42.0 +/- 12.1% at 10 nmol 1,25-(OH)2D3/l after 24 h. After 48 h in 0.1 nmol 1,25-(OH)2D3/l, mRNA levels had fallen to 35.3 +/- 12.6% and PTH secretion to 32.1 +/- 5.0%. In human adenomatous cells, however, incubation with 1,25-(OH)2D3 (10 nmol/l) had no effect on either mRNA levels or PTH secretion even after 48 h. This lack of sensitivity of adenomatous cells to 1,25-(OH)2D3 was not due to an absence of receptors (3847 +/- 39 receptors/ng cytosolic protein in adenomatous cells compared with 4068 +/- 371 in bovine cells) or receptors being of low affinity. Cortisol (1 mumol/l) caused a reduction in the number of receptors for 1,25-(OH)2D3 in bovine parathyroid cells of approximately 20% within 24 h of incubation, but no change in affinity. This decrease was accompanied by abolition of the response to 1,25-(OH)2D3 and was reversible, in that withdrawal of cortisol for the final 24 h of incubation was sufficient for the response to return, the number of receptors having returned to control values. These results suggest that only a small percentage of receptors for 1,25-(OH)2D3 in bovine parathyroid cells may be functional at any one time.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of lovastatin on proteasome activities in highly purified rabbit 20 S proteasome preparations and mouse MC3T3-E1 osteoblastic cells

A number of clinical studies suggest that the use of the lipid-lowering agents collectively referred to as statins (hydroxymethyl glutaryl coenzyme A [HMG-CoA] reductase inhibitors) is associated with increased bone density, reduced fracture risk, and net bone anabolism. Statins (< or =5 micromol/L) stimulate rodent bone formation, but the mechanistic basis remains unclear. Since statins and the proteasome inhibitor lactacystin are structurally similar, and high doses (> or =40 micromol/L) of statins can inhibit the chymotryptic activity of the proteasome, it has been hypothesized that statins exert their anabolic effects on bone, in part, by inhibiting the proteasome, the major eukaryotic intracellular regulatory protease. This hypothesis conflicts with reports that statins stimulate proteasome activity and that proteasome-catalyzed degradation of specific substrates is required for cell proliferation, differentiation, and survival. Our chief objective was to determine the effects of statins (< or =10 micromol/L) on the chymotryptic activity of the proteasome in the 20 S proteasome and intact murine MC3T3-E1 cells cultured to low density (preosteoblasts) or high density (differentiated osteoblasts). Lovastatin (0.001 micromol/L to 5.0 micromol/L) stimulated the chymotryptic activity of the highly purified 20 S proteasome. Preosteoblasts and differentiated osteoblasts treated with 1, 5, or 10 micromol/L lovastatin for 1 hour exhibited morphologic abnormalities that were ameliorated by preincubation and treatment with 20 micromol/L mevalonate. The chymotryptic activity of the preosteoblast proteasome increased after 2 days of 1.0 micromol/L or 5.0 micromol/L lovastatin treatment. In addition, the DNA and protein contents of 1.0 micromol/L or 5.0 micromol/L lovastatin-treated preosteoblast cultures were lower those that observed in vehicle-, 0.01 micromol/L lovastatin-, or 0.10 micromol/L lovastatin-treated cultures. The chymotryptic activity of the proteasome was much lower in differentiated osteoblasts than in preosteoblasts. Two days of treatment with 1 micromol/L lovastatin modestly stimulated the chymotryptic activity of the proteasome in differentiated osteoblasts, but had no effects on total protein or DNA, compared to cultures treated with vehicle or lower doses of lovastatin. Thus, the data support the hypothesis that statins stimulate proteasome activities in highly purified proteasome preparations and preosteoblastic cells. Treating preosteoblastic or differentiated MC3T3-E1 cells with lovastatin concentrations > or = 1 micromol/L resulted in abnormal morphology and reduced the DNA and protein levels in preosteoblastic cultures, confirming the adverse effects of statins previously reported for other cells. In conclusion, the hypothesis that lovastatin exerts its anabolic effects on bone by inhibiting the proteasome activity of the osteoblast was refuted, and the effects of lovastatin on MC3T3-E1 cells were found to be highly dose- and development-dependent.     

Phenotype-associated changes in the effects of 1,25-dihydroxyvitamin D3 on alkaline phosphatase and bone GLA-protein of rat osteoblastic cells

The effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on alkaline phosphatase (AP) activity and the synthesis of gamma-carboxy-glutamic acid containing protein (BGP) were compared in phenotypically distinct cloned cell lines derived from the osteoblast-like rat osteogenic sarcoma line ROS 17/2-8. 1,25(OH)2D3 stimulated AP activity and BGP synthesis in phenotypes which exhibited relatively low basal AP activity and high basal BGP levels. In contrast 1,25(OH)2D3 inhibited AP activity in phenotypes that exhibited high basal AP activity. The latter cells had undetectable BGP levels and the synthesis of this protein failed to respond to the 1,25(OH)2D3 stimulus. In the cells that responded to 1,25(OH)2D3 with an increase in AP activity the effect of the hormone on AP could be blocked by actinomycin-D. However in the cells that responded to 1,25(OH)2D3 with inhibition of AP the effect of the hormone on AP was not influenced by actinomycin-D. The directly opposite effects of 1,25(OH)2D3 on the AP activity of the respective clones did not change qualitatively at different stages of culture and could not be accounted by differences in the 1,25(OH)2D3 receptor status nor by different effects of the hormone on cell proliferation. These data raise the possibility that the response of AP to 1,25(OH)2D3 in osteoblastic cells depends on their state of phenotypic differentiation. The stimulatory effect of the hormone in low AP-producing cells might be related to differentiation promoting properties of 1,25(OH)2D3. The inhibitory effect of 1,25(OH)2D3 on AP, unlike the stimulatory effect of the hormone does not appear to be mediated by the classical mechanism of 1,25(OH)2D3 action on the genome and might be associated with dedifferentiated osteoblastic cells.     

Inhibition of interleukin-1 production by 1,25-dihydroxyvitamin D3

The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], inhibits the proliferation of T lymphocytes and production of growth-promoting factors (including interleukin-2) (IL2) in CTLL2 murine cells. In this study, we investigated the role of monocytes in this hormone-mediated inhibitory effect, by testing the effects of 1,25-(OH)2D3 on the ability of the mitogenic lectin phytohemagglutinin (PHA) to induce T cell activation in either a monocyte-dependent or phorbol myristate acetate (PMA)-driven (monocyte-independent) system. The results indicate that proliferation of T cells and production of growth-promoting factors are inhibited by 1,25-(OH)2D3 only in the monocyte-dependent system. Preincubation of monocytes with 1,25-(OH)2D3 for various periods of time and subsequent removal of the hormone resulted in inhibition of the PHA-driven proliferation of T cells. Preincubation for 2 h resulted in 20% inhibition, while preincubation for 36 h reduced proliferation to 50% of the control value [no 1,25-(OH)2D3 exposure]. These data suggested that monocytes are important participants in 1,25-(OH)2D3-mediated events. Therefore, we tested the effects of the hormone on the production of IL1, a monocyte-derived product thought to be involved in the induction of IL2 release and the subsequent development of the T cell proliferative response. 1,25-(OH)2D3 inhibited the production of both extracellular and cell-associated immunoreactive IL1 alpha and IL1 beta. Indomethacin, a prostaglandin synthetase inhibitor, did not alter the inhibitory properties of 1,25-(OH)2D3, suggesting that prostaglandins are not responsible for the inhibitory phenomenon. We conclude that part of the ability of 1,25-(OH)2D3 to inhibit T cell proliferation may be due to direct effects on monocytes by down-regulating IL-1 production. However, it is unlikely that the immunoregulatory properties of 1,25-(OH)2D3 on T cells are mediated solely through monocytes, and it is possible that the hormone also exerts its influence directly on T cells.     

Normal matrix mineralization induced by strontium ranelate in MC3T3-E1 osteogenic cells

There is growing evidence that strontium ranelate (SR; S12911-2, PROTELOS; Institut de Recherches Internationales Servier, Courbevoie, France), a compound containing 2 atoms of stable strontium (Sr), influences bone cells and bone metabolism in vitro and in vivo. We previously reported that SR increases bone mass in rats and mice by stimulating bone formation and inhibiting bone resorption. We also showed that short-term treatment with SR enhances osteoblastic cell recruitment and function in short-term rat calvaria cultures. Because Sr incorporates into the bone matrix, it was of interest to determine whether SR may affect matrix mineralization in long-term culture. To this goal, osteogenic mouse calvaria-derived MC3T3-E1 osteoblastic cells were cultured for up to 14 days in the presence of ascorbic acid and phosphate to induce matrix formation and mineralization. Matrix formation was determined by incorporation of tritiated proline during collagen synthesis. Matrix mineralization was quantified by measuring the number and surface of mineralized nodules using a digital image analyzer. In this model, 1,25(OH)2 vitamin D (1 nmol/L) used as internal control, increased alkaline phosphatase (ALP) activity, an early osteoblast marker, on days 4, 10, and 14 of culture. Treatment with SR (1 mmol/L Sr(2+)) increased ALP activity at days 4 and 14 of culture. SR also increased collagen synthesis at days 4 and 10 of culture. In contrast, 1,25(OH)2 vitamin D (1 nmol/L) inhibited collagen synthesis at 4 to 14 days of culture. Long-term treatment with SR (0.1 to 1 mmol/L Sr(2+)) dose dependently increased Sr concentration into the calcified nodules, but did not alter matrix mineralization in long-term culture, as shown by the ratio of the surface of mineralized nodules to the number of mineralized nodules on day 14 of culture. These results show that long-term treatment with SR increases collagenous matrix formation by MC3T3-E1 osteoblasts without inducing deleterious effect on matrix mineralization.     

1,25-Dihydroxyvitamin D3 up-regulates the 1,25-dihydroxyvitamin D3 receptor in vivo.

The level of mRNA encoding the 1,25-dihydroxyvitamin D3 receptor in the intestine of vitamin D-deficient rats given 1,25-dihydroxyvitamin D3 was determined by Northern blot analysis using a 32P-labeled cDNA probe to the 1,25-dihydroxyvitamin D3 receptor. mRNA levels increased 10-fold above deficiency levels at 6 and 12 hr after an intravenous dose of 1,25-dihydroxyvitamin D3, returning to predosing levels at 24 hr. Total receptor protein level determined by an immunoradiometric assay was increased 2-fold at 12 hr. No change in unoccupied receptor levels determined by ligand-binding assay was observed during this period. These results suggest that 1,25-dihydroxyvitamin D3 increases receptor mRNA and total receptor level to maintain constant levels of unoccupied receptor.     

The effects of various cell growth factors and cyclosporin A, an immunosuppressive agent, on cloned osteoblastic cell line, MC3T3-E1 cells

Cloned MC3T3-E1 cells which have retained several osteoblast-like characteristics were derived from newborn mouse calvaria. In order to elucidate the function of osteoblasts, the effects of 1,25(OH)2D3, interleukin (IL)-1 beta, IL-3, interferon(INF)-gamma and epidermal growth factor(EGF) on the activity of alkaline phosphatase(Al-P'ase), DNA synthesis and the production of prostaglandin E2(PGE2) in MC3T3-E1 cells were studied. The influence of cyclosporin A(CSA), a potent immunosuppressive agent, was also studied. The following results were obtained: 1. 1,25(OH)2D3 increased the incorporation of [45Ca]Cl2 into matrix and accelerated the calcification of MC3T3-E1 cells. 2. Al-P'ase activity and the incorporation of [3H]-thymidine into MC3T3-E1 cells were increased by 1,25(OH)2D3 but decreased by IL-1 beta, INF-gamma, IL-3 and EGF. 3. IL-1 beta increased and INF-gamma decreased PG-E2 production by MC3T3-E1 cells. 4. CSA decreased either Al-P'ase activity or incorporation of [3H]-thymidine, and increased PG-E2 production in MC3T3-E1 cells. CSA which was simultaneously incubated with these various cell growth factors, showed a similar effect to that of CSA alone. These results suggest that cytokines produced from immune cells, could affect osteoblasts besides that of calcium regulating hormones like parathyroid hormone and 1,25(OH)2D3, implying a probability for the participation of immunocompetent cells in the regulation of bone metabolism.     

Immunocytochemical localization of the 1,25-dihydroxyvitamin D3 receptor in target cells

We have used a monoclonal antibody (9A7) against the purified avian 1,25-dihydroxyvitamin D3 receptor to develop an immunocytochemical technique for visualization of the protein in fixed tissues and cultured cells. In Bouin's-fixed, chick intestine, 1,25-dihydroxyvitamin D3 receptor-like immunoreactivity was localized mainly in nuclei of epithelial cells and was more abundant in the crypt than in the villar cells. Receptor staining was low or undetectable in liver hepatocytes but was present in nuclei of cells lining the hepatic sinusoids. In rat brain, receptor-like immunoreactivity was abundant and widely distributed, but did not always coincide with the presence of vitamin D-dependent calcium-binding protein; 1,25-dihydroxyvitamin D3 receptor was absent from cerebellar Purkinje cells that contained abundant calcium-binding protein. In disaggregated rat bone cells, receptor immunoreactivity was present in mononuclear cells including osteoblasts and fibroblasts but was absent from osteoclasts. Two separate clones of osteoblast-like, rat osteosarcoma cells, shown in previous studies to be either receptor positive (17/2.8) or negative (24.1), demonstrated nuclear immunoreactivity in exact concordance with receptor levels as determined by ligand binding. The phenomenon of hormone-induced up-regulation of receptor was visualized in receptor-positive 3T6 fibroblasts by demonstration of markedly enhanced nuclear reactivity in cells treated with 10(-7) M 1,25-dihydroxyvitamin D3 for 48 h. Our studies demonstrate the feasibility of the immunocytochemical approach to visualize the 1,25-dihydroxyvitamin D3 receptor in target tissues and show that it is predominantly a nuclear protein in the relatively unoccupied and fully activated states. Moreover, the vitamin D-dependent calcium binding is not a universal marker for 1,25-dihydroxyvitamin D3 action. Rather, our observations suggest that the expression of the 1,25-dihydroxyvitamin D3 receptor may be connected with the state of cellular differentiation.     

红霉素对聚甲基丙烯酸甲酯磨损颗粒刺激下MC3T3-E1细胞成骨作用的影响

目的 探讨红霉素对聚甲基丙烯酸甲酯(PMMA)磨损颗粒刺激下MC3T3-E1细胞成骨作用的影响及其潜在机制.方法 对MC3T3-E1细胞予以不同浓度(0.0μg/ml、0.2μg/ml、1.0μg/ml、5.0μg/ml和10.0μg/ml)的红霉素进行预处理,后加入PMMA磨损颗粒进行干预刺激,72 h后对MC3T3...