Modulatory effects of luteolin on osteoblastic function and inflammatory mediators in osteoblastic MC3T3-E1 cells

The effects of luteolin on the function of osteoblastic MC3T3-E1 cells and the production of local factors in osteoblasts were investigated. Luteolin (1microM) caused a significant elevation of collagen content, alkaline phosphatase (ALP) activity, and osteocalcin secretion in the cells (P<0.05). The effect of luteolin in increasing collagen content and ALP activity was completely prevented by the presence of 10(-6)M cycloheximide and 10(-6)M tamoxifen, suggesting that luteolin's effect results from a newly synthesized protein component and might be partly involved in estrogen action. We then examined the effect of luteolin on the 3-morpholinosydnonimine (SIN-1)-induced production of oxidative stress markers [nitric oxide (NO) and prostaglan E(2) (PGE(2))] and cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6)] in osteoblasts. Luteolin (1 and 10microM) decreased the SIN-1-induced production of NO, PGE(2), TNF-alpha, and IL-6 in osteoblasts. These results suggest that inflammatory mediators can be regulated by luteolin stimulating osteoblastic function.     

Effect of IL‐1β, PGE2, and TGF‐β1 on the expression of OPG and RANKL in normal and osteoporotic primary human osteoblasts

The RANKL/RANK/OPG pathway is essential for bone remodeling regulation. Many hormones and cytokines are involved in regulating gene expression in most of the pathway components. Moreover, any deregulation of this pathway can alter bone metabolism, resulting in loss or gain of bone mass. Whether osteoblasts from osteoporotic and nonosteoporotic patients respond differently to cytokines is unknown. The aim of this study was to compare the effect of interleukin (IL)‐1β, proftaglandin E₂ (PGE₂), and transforming growth factor‐β1 (TGF‐β1) treatments on OPG and RANKL gene expression in normal (n = 11) and osteoporotic (n = 8) primary osteoblasts. OPG and RANKL mRNA levels of primary human osteoblastic (hOB) cell cultures were assessed by real‐time PCR. In all cultures, OPG mRNA increased significantly in response to IL‐1β treatment and decreased in response to TGF‐β1 whereas PGE₂ treatment had no effect. RANKL mRNA levels were significantly increased by all treatments. Differences in OPG and RANKL responses were observed between osteoporotic and nonosteoporotic hOB: in osteoporotic hOB, the OPG response to IL‐1β treatment was up to three times lower (P = 0.009), whereas that of RANKL response to TGF‐β1 was five times higher (P = 0.002) after 8 h of treatment, as compared with those in nonosteoporotic hOBs. In conclusion, osteoporotic hOB cells showed an anomalous response under cytokine stimulation, consistent with an enhanced osteoclastogenesis resulting in high levels of bone resorption. J. Cell. Biochem. 110: 304–310, 2010. © 2010 Wiley‐Liss, Inc.     

Stimulation of differentiation in sodium-dependent vitamin C transporter 2 overexpressing MC3T3-E1 osteoblasts

Sodium-dependent vitamin C transporter (SVCT) 2 facilitates reduced ascorbic acid (AA) transport in MC3T3-E1 osteoblasts. Our previous studies suggested that Zn-induced osteoblast differentiation and Ca2+-, PO4(3-)-stimulated osteopontin (OPN) expression might result from their up-regulation effect on SVCT2 expression and AA uptake. Here, we investigated the role of SVCT2 on osteoblast differentiation by using SVCT2-overexpressing cells. Two clones of SVCT2-introduced cells overexpressed SVCT2 mRNA by 2.8- and 3.1-fold those of control cells, which resulted in obvious increase of AA uptake by 2.1- and 2.4-fold in Vmax with no change in Km. Alkaline phosphatase activity, hydroxyproline content significantly increased in SVCT2-overexpressing cells, and the induction of OPN mRNA was through up-regulation of OPN promoter activity by SVCT2 overexpression. Moreover, SVCT2-overexpressing cells exhibited more ability to promote mineralization and increase calcium deposition under the stimulation of 5 mM beta-glycerophosphate. These findings indicate that SVCT2 stimulates osteoblast differentiation and mineralization.     

Apocynin stimulates osteoblast differentiation and inhibits bone-resorbing mediators in MC3T3-E1 cells

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH-oxidase. In the present study, the effect of apocynin on the function of osteoblastic MC3T3-E1 cells was studied. Apocynin caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and mineralization in the cells (P < 0.05). Antimycin A (AMA), which inhibits complex III of the electron transport system, has been used as a reactive oxygen species (ROS) generator in biological systems. We exposed cultured osteoblastic MC3T3-E1 cells to AMA with or without pretreatment with apocynin. Apocynin significantly (P < 0.05) increased cell survival, calcium deposition, and osteoprotegerin release and decreased the production of ROS and osteoclast differentiation inducing factors such as TNF-α, IL-6, and receptor activator of nuclear factor-kB ligand (RANKL) in the presence of AMA. These results demonstrate that apocynin can protect osteoblasts from mitochondrial dysfunction-induced toxicity and may have positive effects on skeletal structure.     

Effect of Azithromycin on Mineralized Nodule Formation in MC3T3-E1 Cells

Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.     

Sodium fluoride induces apoptosis and alters bcl-2 family protein expression in MC3T3-E1 osteoblastic cells

Chronic excessive fluoride intake is known to be toxic and can lead to fluorosis and bone pathologies. However, the cellular mechanisms underlying NaF-induced cytotoxicity in osteoblasts are not well understood. The objectives of this study were to determine the effects of fluoride treatment on MC3T3-E1 osteoblastic cell viability, cell cycle analysis, apoptosis and the expression levels of bcl-2 family members: bcl-2 and bax. MC3T3-E1 cells were treated with 10⁻⁵; 5 × 10⁻⁵; 10⁻⁴; 5 × 10⁻⁴ and 10⁻³ M NaF for up to 48 h. NaF was found to reduce cell viability in a temporal and concentration dependent manner and promote apoptosis even at low concentrations (10⁻⁵ M). This increased apoptosis was due to alterations in the expression of both pro-apoptotic bax and anti-apoptotic bcl-2. The net result was a decrease in the bcl-2/bax ratio which was found at both the mRNA and protein levels. Furthermore, we also noted that NaF-induced S-phase arrest during the cell cycle of MC3T3-E1 cells. These data suggest that fluoride-induced osteoblast apoptosis is mediated by direct effects of fluoride on the expression of bcl-2 family members.     

Fluid shear stress induces Runx‐2 expression via upregulation of PIEZO1 in MC3T3‐E1 cells

Mechanically induced biological responses in bone cells involve a complex biophysical process. Although various mechanosensors have been identified, the precise mechanotransduction pathway remains poorly understood. PIEZO1 is a newly discovered mechanically activated ion channel in bone cells. This study aimed to explore the involvement of PIEZO1 in mechanical loading (fluid shear stress)‐induced signaling cascades that control osteogenesis. The results showed that fluid shear stress increased PIEZO1 expression in MC3T3‐E1 cells. The fluid shear stress elicited the key osteoblastic gene Runx‐2 expression; however, PIEZO1 silencing using small interference RNA blocked these effects. The AKT/GSK‐3β/β‐catenin pathway was activated in this process. PIEZO1 silencing impaired mechanically induced activation of the AKT/GSK‐3β/β‐catenin pathway. Therefore, the results demonstrated that MC3T3‐E1 osteoblasts required PIEZO1 to adapt to the external mechanical fluid shear stress, thereby inducing osteoblastic Runx‐2 gene expression, partly through the AKT/GSK‐3β/β‐catenin pathway.     

柚皮苷对高糖作用下MC3T3-E1细胞活力和Akt通路相关因子表达的影响

目的探讨柚皮苷（NG）对高糖环境下MC3T3-E1细胞活力的影响及可能的分子机制。 方法体外培养小鼠MC3T3-E1细胞，实验分5组：对照组（正常无血清培养基）、高糖组（含25 mmol/L葡萄糖）、0.1 μmol/L +高糖组（0.1 μmol/L NG + 25 mmol/L葡萄糖）、1 μmol/L +高糖组（1 μmol/L NG+25 mmol/L葡萄糖）、10 μmol/L +高糖组（10 μmol/L NG+25 mmol/L葡萄糖）。药物干预后，采用CCK-8法检测细胞活力；实时荧光定量PCR（qPCR）法检测细胞成骨特异性转录因子（Runx2）、胰岛素样生长因子-1（IGF-1）、蛋白激酶（Akt1）mRNA的表达；蛋白质印迹法（Western blot）检测细胞碱性磷酸酶（ALP）、Akt1、IGF-1蛋白的表达。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。 结果CCK8检测结果显示，与对照组比较，高糖组细胞OD值（12 h：0.90±0.01比0.80±0.01，24 h：1.00±0.05比0.84±0.01，48 h：1.09±0.03比0.90±0.01）均降低，差异有统计学意义（P < 0.01）；与高糖组比较，0.1 μmol/L+高糖组细胞OD值（24 h：0.84±0.01比0.93±0.05，48 h：0.90±0.01比0.99±0.01）、1 μmol/L +高糖组和10 μmol/L+高糖组OD值（12 h：0.80±0.01比0.92±0.01、1.01±0.32，24 h：0.84±0.01比1.01±0.04、1.16±0.03，48 h：0.90±0.01比1.12±0.02、1.20±0.02）均升高，差异有统计学意义（P < 0.05）。与对照组比较，高糖组细胞Runx2、IGF-1、Akt1的mRNA的表达水平（24 h：1.00比0.34±0.02、1.00比0.34±0.01、1.00比0.15±0.02）、（48 h：1.00比0.72±0.03、1.00比1.09±0.07、1.00比0.38±0.04）降低，差异有统计学意义（P < 0.01）。与高糖组比较，1 μmol/L +高糖组和10 μmol/L+高糖组细胞Runx2、IGF-1、Akt1的mRNA表达水平（24 h：0.34±0.02比0.62±0.09、0.64±0.05，0.34±0.01比0.77±0.03、1.02±0.07，0.15±0.02比0.24±0.08、0.4±0.09）、（48 h：0.72±0.03比1.27±0.02、1.37±0.02，1.09±0.07比2.44±0.19、2.73±0.04，0.38±0.04比0.86±0.06、1.43±0.03）均升高，差异有统计学意义（P < 0.05）。与对照组比较，高糖组细胞ALP、Akt1、IGF-1蛋白表达水平（48 h：1.00比0.72±0.02、1.00比0.89±0.03、1.00比0.09±0.01）均降低，差异有统计学意义（P < 0.05）；与高糖组比较，0.1 μmol/L+高糖组、1 μmol/L+高糖组和10 μmol/L+高糖组ALP、Akt1、IGF-1蛋白表达水平（48 h：0.72±0.02比1.92±0.02、2.30±0.30、3.09±0.10，0.89±0.03比1.50 ± 0.03、1.43±0.04、1.40±0.13，0.09±0.01比1.75±0.01、2.30±0.31、2.07±0.07）均升高，差异有统计学意义（P < 0.05）。 结论NG逆转高糖诱导的MC3T3-E1细胞活力减退；同时改善高糖的抑制作用，促进MC3T3-E1细胞IGF-1、AKt-1、Runx2 mRNA和IGF-1、AKt-1、ALP蛋白的表达。     

小鼠前成骨细胞MC3T3-E1在自组装多肽水凝胶支架中的成骨分化

目的研究MC3T3-E1细胞在自组装多肽水凝胶支架上的生长和成骨分化.方法在多肽水凝胶支架RADA16上接种MC3T3-E1细胞,荧光染色观察细胞形态和存活情况;组织化学染色检测MC3T3-E1细胞碱性磷酸酶活性以及细胞外钙质沉积;RT-PCR分析成骨特异性基因的表达.结果 MC3T3-E1细胞在水凝胶支架RADA16上粘附铺展良好,呈纺锤样形态.诱导培养后支架上的细胞有较高水平的碱性磷酸酶表达和矿化基质沉积.此外,骨分化特异性基因骨桥蛋白和骨涎蛋白也有表达,且表达量随培养时间的延长而增多.结论 在自组装水凝胶内MC3T3-E1细胞可向成骨方向分化,并能在凝胶内产生矿化的细胞外基质.     

Glabridin protects osteoblastic MC3T3-E1 cells against antimycin A induced cytotoxicity

Mitochondrial dysfunction, particularly respiratory chain disruption, is often responsible for aging-related bone diseases. In this study, the protective effects of glabridin, an isoflavan isolated from licorice root, against pharmacological inhibition of the respiratory chain were studied using osteoblastic MC3T3-E1 cells treated with antimycin A, which inhibits complex III of the electron transport system. Glabridin restored mitochondrial membrane potential dissipation, ATP loss, inactivation of complex IV, intracellular calcium elevation, and cytochrome c release that was induced by antimycin A treatment. This compound also prevented cell death. These results imply that glabridin protects osteoblasts from antimycin A-induced cell death via improved mitochondrial function. Glabridin scavenged ROS and mitochondrial superoxide anions generated by antimycin A. In addition, glabridin prevented antimycin A-induced nitrotyrosine increase and thioredoxin reductase inactivation, suggesting that glabridin may be useful for protecting mitochondria against a burst of oxidative stress. Since phosphoinositide 3-kinase (PI3K) and cAMP-response element-binding protein (CREB) signaling is known to be pro-survival, we determined whether PI3K and CREB activation is associated with the cytoprotective effects of glabridin in the MC3T3-E1 cells. Glabridin restored antimycin A-induced inactivation of PI3K and CREB, suggesting that PI3K and CREB-dependent pathways may be involved in glabridin-induced cytoprotective responses. Our study demonstrates that glabridin reduces mitochondrial dysfunction induced during aging, and could significantly prevent osteoblast damage in osteoporotic patients.     

Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells

Covalent intermolecular cross-linking of collagen is initiated by the action of lysyl oxidase (LOX) on the telopeptidyl lysine and hydroxylysine residues. Recently, several LOX isoforms, i.e., LOX-like proteins 1-4 (LOXL1-4), have been identified but their specific tissue distribution and functions are still largely unknown. In this study, mRNA expression of LOX and LOXL1-4 in MC3T3-E1 osteoblastic cells was screened by RT-PCR and quantitatively analyzed by real-time PCR during cell differentiation and matrix mineralization. The results demonstrated that LOX and all LOXLs, except LOXL2, were expressed in this cell line and that the expression pattern during cell differentiation and matrix mineralization was distinct from one another. This indicates that the expression of LOX and its isoforms is highly regulated during osteoblast differentiation, suggesting their distinct roles in collagen matrix stabilization and subsequent mineralization.     

MC3T3-E1前成骨细胞培养时间对矿化结节表达的影响

目的:观察MC3T3-E1前成骨细胞不同培养时间点矿化结节的形态,探讨一个既节省实验时间与经费,又便于观察矿化结节形态差异的实验方法。方法:将MC3T3-E1前成骨细胞按培养时间分为四组(14、21、28、35天组),各组实验结束时行茜素红染色,光学显微镜下观察矿化结节的形态变化。结果:各组均见红色的矿化结节形成,随培养时间延长,染色面积增大,密度增高,14天时结节轮廓清晰,结节间距较大,21天时结节面积增大,28天时结节边界超出视野,35天时视野内大片深染,结节轮廓不清。结论:在本实验周期内,MC3T3-E1前成骨细胞培养14至21天通过茜素红染色可以较清晰地观察矿化结节,其中培养14天时即可观察到结节大小、数量及形态,考虑到实验时间及经费的因素,我们认为MC3T3-E1前成骨细胞培养14天后行茜素红染色是观察不同因素对其矿化产生影响的适宜时间点。     

Chemotactic responses of osteoblastic MC3T3-E1 cells toward zinc chloride

Although zinc (Zn) is known to participate in bone formation, its exact role in the remodeling of this tissue has not been fully clarified. The present study was designed to investigate whether Zn has a role at the resorptive sites in vitro. We investigated the migration of osteoblastic MC3T3-E1 cells in response to Zn using a Boyden chamber assay. Exposure of MC3T3-E1 cells to Zn stimulated the migration of MC3T3-E1 cells. Checkerboard analysis revealed that the migration of MC3T3-E1 cells toward Zn was a directional (chemotaxis) rather than a random (chemokinesis) motion. Pretreatment of MC3T3-E1 cells with pertussis toxin completely blocked the chemotactic response of cells to Zn, indicating that it is mediated by G-protein-coupled receptors. Because the bone is one of the major Zn storage sites, we suggest that Zn released from bone-resorptive sites plays an important role in the recruitment of osteoblasts and bone renewal.     

Bordetella bronchiseptica dermonecrotizing toxin stimulates protein synthesis in an osteoblastic clone, MC3T3-E1 cells

Abstract The effects of Bordetella bronchiseptica dermonecrotizing toxin on protein synthesis in an osteoblastic clone, MC3T3-E1 cells, were investigated. The rate of protein synthesis in the serum-starved cells was increased by the toxin after a latent period of about 4 h, and reached 2.5 times that of the control 24 h after addition of toxin. The toxin raised the level of protein synthesis even in actively proliferating cells. The stimulatory effect of the toxin on protein synthesis occurred earlier than other toxic events so far reported, such as the stimulation of DNA synthesis and the inhibition of osteoblastic differentiation, and was apparently dependent on the toxin concentrations over the range 0.05 ng ml⁻¹ to 6.0 ng ml⁻¹. Therefore, the stimulatory effect of the toxin on protein synthesis could be useful in determining the mode of action of the toxin.     

Osteoprotegerin (OPG) and RANKL expression and distribution in developing human craniomandibular joint

During embryogenesis the bone tissue of craniomandibular joint (CMJ) is formed through two pathways: intramembranous ossification and endochondral ossification. The development process is under the control of regulatory factors.The osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kappaB ligand are key regulators of osteoclastogenesis. The aim of this study is the localization of OPG and RANKL mRNA and protein in the foetal CMJ by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: OPG and RANKL mRNA and protein were co-localized in the same cell types; OPG and RANKL were specially immunolocated in osteogenic cells; immunolabeling was often seen in the nucleus and cytoplasm of otherwise negative hypertrophic chondrocytes; IHC and ISH labeling decreased from proliferative to hypertrophic chondrocytes; early osteocytes showed dual protein expression and some of the mature osteocytes were ISH-negative; periosteal osteoclasts and chondroclasts were mostly stained by IHC and variably labeled by ISH; the new bone matrix and trabecular borders showed intense immunolabeling. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism in the CMJ development and their extracellular presence in the new bone matrix and trabecular borders suggests a local regulatory role.     

Pancreatic polypeptide is secreted from and controls differentiation through its specific receptors in osteoblastic MC3T3-E1 cells

Although the neuropeptide Y (NPY) family has been demonstrated to control bone metabolism, the role of pancreatic polypeptide (PP), which has structural homology with NPY and peptide YY (PYY) to share the NPY family receptors, in peripheral bone tissues has remained unknown. In the present study, we studied the regulatory roles of PP and its Y receptors using MC3T3-E1 cells, a murine transformed osteoblastic cell line, as a model for osteoblastic differentiation. We found that (1) PP mRNA was detected and increased during cell-contact-induced differentiation in MC3T3-E1 cells; (2) the immunoreactivity of PP was detected by radioimmunoassay and increased in culture medium during differentiation; (3) all the types of NPY family receptor mRNAs (Y1, Y2, Y4, Y5, and y6) were found to increase during differentiation; (4) PP stimulated differentiation in MC3T3-E1 cells in terms of ALP mRNA and BMP-2 mRNA. These findings suggested that MC3T3-E1 cells produce and secrete PP, which may in turn stimulate the differentiation of MC3T3-E1 through its specific receptors in an autocrine manner.     

The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation

While the role of p75^NTR signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75^NTR on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75^NTR-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75^NTR in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75^NTR, as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75^NTR-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75^NTR signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation.     

不同温度下成骨细胞增殖及其OPG/RANKL mRNA表达的影响

目的:探讨不同温度下对小鼠成骨细胞MC3T3-E1的增殖以及OPG/RANKL表达水平的影响。方法:1.以小鼠成骨细胞MC3T3-E1为体外实验模型,MTT法检检测细胞的增殖情况。2.RT-PCR方法检测MC3T3-E1OPG/RANKL mRNA的表达水平。结果:设定对照组为37℃,高于对照组(38℃-39℃-40℃-41℃-42℃)分别作用于MC3T3-E1细胞1小时/天,连续1周,可刺激细胞增殖,OD值显著增加(P<0.05)。同时可增加OPG mRNA表达,降低RANKL mRNA表达,呈温度梯度依赖性。结论:热刺激促进MC3T3-E1细胞增殖,同时通过调节OPG/RANKL mRNA的表达,直接促进骨形成,抑制骨吸收。