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

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

Triiodothyronine, a Regulator of Osteoblastic Differentiation: Depression of Histone H4, Attenuation of c-fos/c-jun, and Induction of Osteocalcin Expression

Thyroid hormones influence growth and differentiation of bone cells. In vivo and in vitro data indicate their importance for development and maintenance of the skeleton. Triiodothyronine (T3) inhibits proliferation and accelerates differentiation of osteoblasts. We studied the regulatory effect of T3 on markers of proliferation as well as on specific markers of the osteoblastic phenotype in cultured MC3T3-E1 cells at different time points. In parallel to the inhibitory effect on proliferation, T3 down-regulated histone H4 mRNA expression. Early genes (c-fos/c-jun) are highly expressed in proliferating cells and are down-regulated when the cells switch to differentiation. When MC3T3-E1 cells are cultured under serum-free conditions, basal c-fos/c-jun expressions are nearly undetectable. Under these conditions, c-fos/c-jun mRNAs can be stimulated by EGF, the effect of which is attenuated to about 46% by T3. In addition, T3 stimulated the expression at the mRNA and protein level of osteocalcin, a marker of mature osteoblasts and alkaline phosphatase activity. All these effects were more pronounced when cells were cultured for more than 6 days. These data indicate that T3 acts as a differentiation factor in osteoblasts by influencing the expression of cell cycle–regulated, of cell growth–regulated, and of phenotypic genes. Received: 10 May 1996 / Accepted: 5 June 1997     

续苓健骨汤含药血清对MC3T3-E1细胞分化及增殖的影响

目的探讨续苓健骨汤含药血清对MC3T3-E1成骨细胞分化及增殖的影响。方法制备续苓健骨汤含药血清,实验分为空白对照组、含药血清低剂量组、中剂量组和高剂量组。采用CCK-8法和流式细胞术检测续苓健骨汤含药血清对MC3T3-E1细胞增殖和细胞周期的影响;碱性磷酸酶(ALP)活性测定MC3T3-E1细胞的成骨分化能力;茜素红染色检测MC3T3-E1细胞的矿化能力;实时荧光定量PCR检测成骨分化基因Runx2、OC、Bmp2、Col1a1mRNA水平。结果与空白对照组比较,中、高剂量续苓健骨汤含药血清能促进MC3T3-E1细胞增殖、S期细胞比率和细胞增殖指数,并且呈现一定的剂量依赖性;同时中高剂量续苓健骨汤含药血清组能明显提高MC3T3-E1细胞ALP活性(P0.01)和钙化能力(P0.01),促进Runx2、OC、Bmp2、Col1a1 mRNA的表达(P0.05)。结论续苓健骨汤含药血清能促进成骨细胞MC3T3-E1的增殖,并通过上调骨形成相关基因Runx2、OC、BMP2、Col1a1的表达水平,提高MC3T3-E1细胞的成骨能力。     

健骨胶囊对成骨样细胞MC3T3-E1增殖及分化作用机制

目的 探讨国医大师刘柏龄“健骨胶囊”对MC3T3-E1成骨细胞分化及增殖的影响。方法 制备健骨胶囊水提物,采用CCK-8法和细胞迁移实验检测健骨胶囊提取物对MC3T3-E1细胞增殖和细胞迁移的影响;茜素红染色检测MC3T3-E1细胞的矿化能力;实时荧光定量PCR检测成骨分化基因Runx2、OCN、OPN、Col1a1、ALP、Bcl2、RASSF1A等mRNA表达水平;蛋白质印迹法Western blot检测Col1a1、Bcl2的蛋白表达水平。结果 通过实验结果比对得出,健骨胶囊提取物能促进MC3T3-E1细胞增殖、使细胞迁移率提高;同时健骨胶囊提取物组能明显提高MC3T3-E1细胞钙化能力(P＜0.01),促进Runx2、OCN、OPN、Col1a1、ALP、Bcl2的mRNA表达(P＜0.05),上调Col1a1、Bcl2蛋白量的表达。结论 健骨胶囊能促进成骨细胞MC3T3-E1的增殖及细胞迁移能力,并通过上调成骨基因的表达水平如Runx2、OCN、OPN、Col1a1、ALP、Bcl2等,提高MC3T3-E1细胞的成骨能力。     

Oxygen tension is an important mediator of the transformation of osteoblasts to osteocytes

Osteocytes are derived from osteoblasts, but reside in the mineralized bone matrix under hypoxic conditions. Osteocyte-like cells show higher expression of ORP150, which is induced by hypoxia, than osteoblast-like cells. Accordingly, we hypothesized that the oxygen tension may regulate the transformation of osteoblasts to osteocytes. MC3T3-E1 cells and calvariae from 4-day-old mice were cultured under normoxic (20% O₂) or hypoxic (5% O₂) conditions. To investigate osteoblastic differentiation and tranformation to osteocytes, alizarin red staining was done and the expression of various factors was assessed. Hypoxic culture promoted the increased synthesis of mineralized matrix by MC3T3-E1 cells. Alkaline phosphatase activity was initially increased during hypoxic culture, but decreased during osteogenesis. Osteocalcin production was also increased by hypoxic culture, but decreased after mineralization. Furthermore, expression of Dmp1, Mepe, Fgf23, and Cx43, which are osteocyte-specific or osteocyte-predominant proteins, by MC3T3-E1 cells was greater under hypoxic than under normoxic conditions. In mouse calvarial cultures, the number of cells in the bone matrix and cells expressing Dmp1 and Mepe were increased by hypoxia. In MC3T3-E1 cell cultures, ORP150 expression was only detected in the mineralized nodules under normoxic conditions, while its expression was diffuse under hypoxic conditions, suggesting that the nodules were hypoxic zones even in normoxic cultures. These findings suggest that a low oxygen tension promotes osteoblastic differentiation and subsequent transformation to osteocytes.     

The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)/carrier complex on bone repair

We investigated the effects of transplantation of osteoblastic cells with a bone morphogenetic protein (BMP)/carrier complex on bone repair by in vitro and in vivo experiments. Poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS) was used as a carrier for cell transplantation. In the in vitro experiments, three cell types, C3H10T1/2 cells, MC3T3-E1 cells, and primary osteoblastic cells, isolated from newborn rat calvariae (ROB cells), were cultured for 2 weeks on PGS alone or PGS containing BMP-2 (PGS/BMP). C3H10T1/2 cells cultured on PGS/BMP expressed several markers related to differentiation of both osteoblasts and chondrocytes, such as alkaline phosphatase (ALP) activity and mRNAs for osteocalcin and aggrecan, whereas the cells cultured on PGS alone expressed no such markers. MC3T3-E1 cells cultured on PGS/BMP exhibited a more ALP-positive cells than those cultured on PGS alone. PGS/BMP promoted ROB cell differentiation into both osteoblasts and chondrocytes. In the in vivo experiments, we transplanted ROB cells, which had been cultured on PGS alone or PGS/BMP in vitro for 2 weeks, into bone defects created in rat calvariae. Transplantation of ROB cells cultured on PGS alone generated little new bone. Transplantation of ROB cells cultured on PGS, which absorbed a low dose (10 ng) of rhBMP-2,; induced significantly higher bone mineral content than PGS/BMP alone, although application of a high dose (1 microg) of rhBMP-2 induced no difference in bone mineral content between transplantation of PGS/BMP with or without ROB cells. These results show that transplantation of osteoblastic cells after induction of osteoblast maturation in vitro by cultivation on PGS/BMP is a potent technique for cell therapy of bone repair.     

GLP-1RA艾塞那肽调控骨骼代谢的作用研究

目的 为了探讨胰高血糖素样肽-1 (glucagon-like peptide-1,GLP-1)受体激动剂对糖尿病患者的成骨细胞和破骨细胞的影响。方法 利用小鼠成骨细胞MC3T3-E1和小鼠单核巨噬细胞RAW264.7模拟成骨细胞和破骨细胞。在高糖培养基中分别加入不同浓度的艾塞那肽孵育72 h,利用RT-qPCR技术分别检测成骨细胞MC3T3-E1中GLP1-R、ALP、LRP-5和破骨细胞RAW 264.7中GLP1-R、Cathespin K的表达情况。结果 艾塞那肽诱导成骨细胞MC3T3-E1细胞中GLP-1R、ALP、LRP-5基因的表达显著升高,而明显抑制破骨细胞RAW 264.7中GLP1-R、Cathespin K的表达。结论 GLP-1受体激动剂艾塞那肽可能通过影响成骨细胞及破骨细胞的分化,从而对糖尿病引起的骨质疏松起到一定程度的改善。     

Intensity-related differences in collagen post-translational modification in MC3T3-E1 osteoblasts after exposure to low- and high-intensity pulsed ultrasound

Low-intensity pulsed ultrasound (LIPUS) has distinct effects on biologic mineralization at intensities of <100 mW/cm2. Intensity-dependent differences in the pattern of accelerated mineralization may be due to different alterations in regulation of collagenous matrix formation. However, little is known about the influence of LIPUS on collagen metabolism in the context of mineralization processes. Therefore, we attempted to evaluate differential effects of two intensities of pulsed ultrasound (30 vs. 120 mW/cm2) on collagen post-translational modification and mineralization in osteoblastic MC3T3-E1 cells. Murine osteoblastic MC3T3-E1 cells were exposed to pulsed ultrasound (1.5-MHz, 200-ms burst sine wave at 1.0-kHz frequency, either 30 or 120 mW/cm2 SATA, for 20 min/day from Day 14 to Day 35 postconfluence). Expression patterns of lysyl oxidase (LO), procollagen-lysine, 2-oxyglutarate, 5-dioxigenase 1 (PLOD1, LH1), and 2 (PLOD2, LH2) was examined using quantitative PCR. Quantitative analysis of reducible immature cross-links (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible mature cross-links (pyridinoline and deoxypyridinoline) as well as analysis of the maturation of immature to mature cross-links were performed. Exposure to 30 mW/cm2 LIPUS upregulated LH2 mRNA expression and enzyme activity compared to controls. It was associated with increased relative amounts of telopeptidyl hydroxylysine (Hyl)-derived cross-links beginning on Day 14, upregulated LO mRNA expression, increased total reducible and nonreducible cross-links, and increased ratios of newly formed nonreducible to reducible cross-links. Similarities in the pattern of cross-link formation and calcium deposition in matrices between 30 mW/cm2 LIPUS-treated MC3T3-E1 cultures and bone suggest that 30 mW/cm2 LIPUS may promote the maturation of collagenous matrix as a scaffold for calcification. In contrast, exposure to 120 mW/cm2 ultrasound increased calcium accumulation compared to control at Day 35, but increases were delayed until Day 25. No differences in the extent and pattern of cross-links were observed compared to controls. These results suggest that the promotion of mineralization induced by 120 mW/cm2 may be attributed to other factors involved in mineralization process rather than cross-link pattern. Our results demonstrated the existence of differential effects of lower versus higher intensities of ultrasound on mineralization processes in vitro.     

二甲双胍调控PI3K/Akt/mTOR通路减轻糖皮质激素诱导的成骨细胞凋亡

目的探讨二甲双胍(Met)调控磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)信号通路对糖皮质激素地塞米松(Dex)诱导的成骨细胞凋亡的影响。方法将体外培养的小鼠胚胎成骨细胞前体细胞MC3T3-E1分为对照组(正常培养)、Dex组(以Dex处理)、Met组(以Dex和Met共同处理)、Met+IGF-1组(以Dex、Met和PI3K/Akt/m TOR通路活化剂IGF-1共同处理)、Met+NVP-BEZ235组(以Dex、Met和PI3K/Akt/m TOR通路抑制剂NVP-BEZ235共同处理),采用免疫印迹法(WB)检测MC3T3-E1细胞中PI3K、Akt、磷酸化(p)-Akt、m TOR和p-mTOR蛋白表达水平,通过噻唑蓝(MTT)法检测MC3T3-E1细胞存活率、流式细胞术检测MC3T3-E1细胞凋亡率、实时荧光定量PCR检测MC3T3-E1细胞中Bcl-2和Bax mRNA表达水平、Caspase-3活性测定试剂盒检测MC3T3-E1细胞Caspase-3活性、JC-1探针检测MC3T3-E1细胞线粒体膜电位变化。结果与对照组比较,Dex组细胞中PI3K、p-Akt、p-mTOR蛋白表达水平和细胞存活率、Bcl-2 mRNA表达水平以及线粒体膜电位均明显降低,而细胞凋亡率、Bax mRNA表达水平和Caspase-3活性均明显升高(P0.05);与Dex组比较,Met组细胞中PI3K、p-Akt、p-mTOR蛋白表达水平和细胞存活率、Bcl-2 mRNA表达水平以及线粒体膜电位均明显升高,而细胞凋亡率、Bax mRNA表达水平、Caspase-3活性明显降低(P0.05);给予IGF-1作用后Met对MC3T3-E1细胞的作用效果明显增强,而给予NVP-BEZ235作用后Met对MC3T3-E1细胞的作用效果明显减弱(P0.05)。结论 Met可通过激活PI3K/Akt/m TOR通路抑制线粒体凋亡途径,减轻糖皮质激素Dex诱导的成骨细胞凋亡。     

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

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

震荡流体剪切力通过ERK5信号通路促进成骨细胞增殖

目的探讨震荡流体剪切力(oscillatory shear stress,OSS)通过ERK5信号通路在诱导成骨细胞增殖中发挥的作用。方法对成骨MC3T3-E1细胞进行不同的处理,分为正常组、OSS组、XMD8-92组和OSS+XMD8-92组。采用MTT实验分别测定4组细胞的增殖活性并绘制生长曲线;蛋白免疫印迹法分别检测P-ERK5、ERK5和Cyclin D1等蛋白水平变化。结果 OSS可显著增加成骨MC3T3-E1细胞增殖活性,但此效应可被ERK5高选择性抑制剂XMD8-92阻断。OSS可显著上调Cyclin D1的表达,而XMD8-92可显著下调OSS诱导的Cyclin D1的表达。结论 OSS通过激活ERK5信号通路促进成骨细胞增殖,Cyclin D1是ERK5信号通路下游的重要靶点基因。     

Stimulation of osteoblastic cell differentiation by Notch.

Notch is a transmembrane protein that plays a critical role in the determination of cellular differentiation pathways. Although its importance in the development of mesenchymal tissues has been suggested, its role in skeletal tissues has not been well investigated. Northern blot experiments showed the expression of Notch1 in MC3T3-E1 osteoblastic cells at early differentiation stages. When a Notch1 cytoplasmic domain (Notch-IC [NIC]) delivered by an adenovirus vector was expressed in osteoblastic MC3T3-E1 cells, a significant increase in calcified nodule formation was observed in long-term cultures. Activation of endogenous Notch in MC3T3-E1 by coculturing them with Delta-like-1 (Dll1)-expressing myeloma cells also resulted in a stimulation of calcified nodule formation. Not only affecting nodule formation, Notch activation also had effects on osteoblastic differentiation of multipotent mesenchymal cells. Osteoblastic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein 2 (BMP-2) was significantly stimulated, whereas adipogenic differentiation was suppressed strongly, resulting in a dominant differentiation of osteoblastic cells. NIC expression in primary human bone marrow mesenchymal stem cells (hMSCs) also induced both spontaneous and stimulated osteoblastic cell differentiation. These observations suggest that osteoblastic cell differentiation is regulated positively by Notch and that Notch could be a unique and interesting target molecule for the treatment of osteoporosis.     

ERK5信号通路介导流体剪切力对MC3T3-E1成骨细胞MMPs、TIMPs表达的影响

目的观察流体剪切力(fluid shear stress,FSS)作用下,MC3T3-E1成骨细胞中细胞基质金属蛋白酶(matrix metalloproteinases,MMPs)和基质金属蛋白酶抑制剂(tissue inhibitors of metalloproteinases,TIMPs)的表达情况,并探讨ERK5信号通路在其中的作用。方法对MC3T3-E1成骨细胞进行不同的处理,分为正常组、XMD8-92组、FSS组和FSS+XMD8-92组,对FSS组施加12 dyn/cm~2流体剪切力,采用蛋白免疫印迹法分别检测P-ERK5、ERK5、MMPs和TIMPs蛋白水平的变化。结果生理强度(12 dyn/cm~2)的流体剪切力作用于MC3T3-E1成骨细胞45 min后能显著上调MMPs的表达,下调TIMPs的表达,但此效应可被ERK5高选择性抑制剂XMD8-92阻断。结论 ERK5信号通路调控流体剪切力对成骨细胞MMPs、TIMPs蛋白的表达。     

Fidgetin-like 1 gene inhibited by basic fibroblast growth factor regulates the proliferation and differentiation of osteoblasts.

The FIGNL1 gene was proven to be a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). In this in vitro study, the AAA proteins inhibited osteoblast proliferation and stimulated osteoblast differentiation. We showed that FIGNL1 may play some regulatory role in osteoblastogenesis. INTRODUCTION: The fidgetin-like 1 (FIGNL1) gene encodes a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). Although the FIGNL1 protein localizes to both the nucleus and cytoplasm, the function of FIGNL1 remains unknown. In a previous study, we identified several genes that mediate the anabolic effects of basic fibroblast growth factor (bFGF) on bone by using microarray data. FIGNL1 was one of the genes that downregulated >2-fold in MC3T3-E1 cells after treatment with bFGF. Therefore, this study was aimed to identify and confirm the function of FIGNL1 on osteoblastogenesis. MATERIALS AND METHODS: We examined the effect of the FIGNL1 gene on proliferation, differentiation, and apoptosis in mouse osteoblast cells (MC3T3-E1 and mouse primary calvarial cells) using flow cytometry, RT-PCR, cell proliferation assay, and cell death assay. MC3T3-E1 cells and mouse calvarial cells were transfected with small interfering RNA (siRNA) directed against the FIGNL1 or nontargeting control siRNA and examined by cell proliferation and cell death assays. Also, FIGNL1 was fused to enhance green fluorescent protein (EGFP), and the EGFP-fused protein was transiently expressed in MC3T3-E1 cells. RESULTS: Reduced expression of FIGNL1 by bFGF and TGF-beta1 treatment was verified by RT-PCR analysis. Overexpression of FIGNL1 reduced the proliferation of MC3T3-E1 and calvarial cells, more than the mock transfected control cells did. In contrast, siFIGNL1 transfection significantly increased the proliferation of osteoblasts, whereas overexpression of FIGNL1 did not seem to alter apoptosis in osteoblasts. Meanwhile, overexpression of FIGNL1 enhanced the mRNA expression of alkaline phosphatase (ALP) and osteocalcin (OCN) in osteoblasts. In contrast, siFIGNL1 decreased the expression of ALP and OCN. A pEGFP-FIGNL1 transfected into MCT3-E1 cells had an initially ubiquitous distribution and rapidly translocated to the nucleus 1 h after bFGF treatment. CONCLUSIONS: From these results, we proposed that FIGNL1, a subfamily member of the AAA family of proteins, might play some regulatory role in osteoblast proliferation and differentiation. Further analyses of FIGNL1 will be needed to better delineate the mechanisms contributing to the inhibition of proliferation and stimulation of osteoblast differentiation.     

Differential expression of neuroleukin in osseous tissues and its involvement in mineralization during osteoblast differentiation.

Osteoblast differentiation is a multistep process that involves critical spatial and temporal regulation of cellular processes marked by the presence of a large number of differentially expressed molecules. To identify key functional molecules, we used differential messenger RNA (mRNA) display and compared RNA populations isolated from the defined transition phases (proliferation, matrix formation, and mineralization) of the MC3T3-E1 osteoblast-like cell line. Using this approach, a complementary DNA (cDNA) fragment was isolated and identified as neuroleukin (NLK), a multifunctional cytokine also known as autocrine motility factor (AMF), phosphoglucose isomerase (PGI; phosphohexose isomerase [PHI]), and maturation factor (MF). Northern analysis showed NLK temporal expression during MC3T3-E1 cell differentiation with a 3.5-fold increase during matrix formation and mineralization. Immunocytochemical studies revealed the presence of NLK in MC3T3-E1 cells as well as in the surrounding matrix, consistent with a secreted molecule. In contrast, the NLK receptor protein was detected primarily on the cell membrane. In subsequent studies, a high level of NLK expression was identified in osteoblasts and superficial articular chondrocytes in bone of 1-, 4-, and 8-month-old normal mice, as well as in fibroblasts, proliferating chondrocytes, and osteoblasts within a fracture callus. However, NLK was not evident in hypertrophic chondrocytes or osteocytes. In addition, treatment of MC3T3 cells with 6-phosphogluconic acid (6PGA; a NLK inhibitor) resulted in diminishing alkaline phosphatase (ALP) activity and mineralization in MC3T3-E1 cells, especially during the matrix formation stage of differentiating cells. Taken together, these data show specific expression of NLK in discrete populations of bone and cartilage cells and suggest a possible role for this secreted protein in bone development and regeneration.