Transwell小室内建立小鼠成骨-破骨细胞共培养体系

目的 :Transwell小室内建立体外小鼠成骨-破骨细胞共培养体系,并检测体系对成骨及破骨细胞活性的影响。方法:体外培育小鼠成骨细胞MC3T3-E1和小鼠单核巨噬细胞RAW264.7,RANKL诱导小鼠单核巨噬细胞RAW264.7分化为成熟破骨细胞后,于Transwell小室内建立成骨-破骨细胞共培养体系。通过CCK-8实验、茜素红染色、TRAP染色检测细胞的成骨、破骨活性。采用PCR、Western Blot方法检测成骨细胞MC3T3-E1中OPG、ALP、RANKL、TGF-b1的基因表达以及RANKL的蛋白表达,检测破骨细胞RANK、NF-κB的基因表达和蛋白表达。结果 :小鼠成骨细胞MC3T3-E1和小鼠破骨细胞可在Transwell小室内建立共培养体系;共培养体系影响小鼠成骨细胞与破骨细胞的分化活性,镜下可见成骨细胞分化增多,破骨细胞分化稍减少。共培养体系中成骨细胞基因OPG(0.65±0.08)、ALP(0.16±0.01)较单独培养OPG(1.00±0.08)、ALP(1.01±0.16)表达下降,而TGF-b1(4.42±0.21)、RANKL(4.12±1.04)较单独培养组TGF-b1(1.00±0.10)、RANKL(1.00±0.09)表达上升;破骨细胞相关RANK(0.63±0.06)、NF-κB(0.64±0.08)基因表达较单独培养组的RANK(1.00±0.08)、NF-κB(1.00±0.09)下降,差异均有统计学意义。同时共培养组的OPG(0.43±0.05)、NF-κB(0.59±0.05)的蛋白表达较单独培养组的OPG(0.84±0.06)、NF-κb(1.13±0.03)减少;共培养组RANKL(0.54±0.03)的蛋白表达则较单独培养组的RANKL(0.31±0.03)增加,差异有统计学意义,均与基因表达变化趋势一致。结论:小鼠成骨细胞MC3T3-E1和小鼠破骨细胞可在Transwell小室内建立共培养体系,共培养体系中成骨细胞活性高于破骨细胞活性。     

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。     

模拟微重力条件下重组骨保护素融合蛋白(rhOPG-HSA)对破骨前体细胞Raw264.7抑制效应的研究

目的探讨在模拟微重力(Simulated microgravity,SMG)条件下重组骨保护素融合蛋白(rhOPG-HSA)对破骨前体细胞Raw264.7的抑制效应。方法在SMG条件下,分别培养破骨前体细胞Raw264.7与成骨细胞MC3T3-E1,利用ELISA法检测MC3T3-E1细胞培养液中骨保护素活性,用巨噬细胞集落刺激因子(M-CSF)以及可溶性破骨细胞分化因子(sRANKL)诱导Raw264.7分化为破骨细胞,通过抗酒石酸酸性磷酸酶(TRAP)染色法鉴定rhOPG-HSA抑制破骨细胞能力,利用半定量RT-PCR测定破骨细胞中TRAP的表达。结果 SMG条件下,骨保护素活性在72 h后显著降低(P<0.01);Raw264.7细胞经M-CSF及sRANKL诱导3、4、5 d后,与阴性对照组相比,TRAP阳性染色的破骨细胞用rhOPG-HSA处理后明显减少(P<0.05),RT-PCR测定结果表明破骨细胞TRAP的表达降低(P<0.05)。结论 SMG条件下,rhOPG-HSA能够抑制破骨前体细胞Raw264.7的分化。     

骨碎补-续断药对对成骨/破骨代谢的双向调控作用及其对Hif1ɑ基因的影响

目的 分析骨碎补-续断药对对成骨/破骨代谢的调控作用并初步探讨其机制。方法 分别制备空白、低剂量、中剂量及高剂量的骨碎补-续断含药血清。采用CCK-8法、ALP染色法和茜素红染色法,分别观察含药血清对MC3T3-E1细胞增殖、成骨和矿化能力的影响;采用CCK-8法和TRAP染色法观察含药血清对RAW264.7细胞增殖和破骨分化能力的影响。采用系统药理学的方法分析药物的可能作用靶点,并结合RT-PCR和Western-blot的方法验证。结果 中剂量和高剂量骨碎补-续断含药血清可促进MC3T3-E1细胞的增殖,抑制RAW264.7细胞的增殖,中剂量组和高剂量组间没有明显差异;同时,中、高剂量含药血清可促进MC3T3-E1细胞的ALP活性和钙化能力,抑制RAW264.7细胞的TRAP活性,中剂量和高剂量间没有显著性差异。骨碎补-续断药对的可能作用靶点涉及HIF1ɑ,RT-PCR和Western-blot结果证实中剂量含药血清可提高MC3T3-1细胞Hif1ɑ基因的mRNA和蛋白水平及RAW264.7细胞的HIF1ɑ蛋白水平。结论 骨碎补-续断药对具有促进成骨代谢,抑制破骨代谢的作用,HIF...     

破骨细胞参与时IGF-1对成骨细胞的促同化作用

目的探讨IGF-1对成骨细胞(osteoblast,OB)的促同化作用是否需要破骨细胞(Osteoclast,OC)的协同。方法体外培养MC3T3小鼠成骨细胞及RAW264.7小鼠单核巨噬细胞,RAW264.7细胞经核因子κB受体活化因子配基(Receptor activator of nuclear factor kappa-B ligand,RANKL)诱导分化为破骨细胞。以50 ng/ml重组人胰岛素样生长因子-1(rhIGF-1)分别干预成骨细胞及分化成熟的破骨细胞,Wstern blotting验证IGF-1受体的活化。以0、10、50、100 ng/ml的rhIGF-1分别干预成骨细胞、破骨细胞及共培养的成、破骨细胞。12 h后终止培养,收集破骨细胞经IGF-1干预后的条件培养基(OC conditioned medium after IGF-1 treatment,OCCM)对另一组新接种的成骨细胞干预12 h。ELISA检测3组成骨细胞培基中骨钙素(bone Gla protein,BGP)、RANKL的含量,Real-time PCR检测成骨细胞中Bgp基因的表达。结果 RANKL诱导培养8 d后RAW264.7细胞形成TRAP阳性,成熟的多核破骨细胞;Wstern blotting检测表明rhIGF-1可有效得使成、破骨细胞中的IGF-1受体发生磷酸化;ELISA与Real-time PCR测定结果显示,IGF-1直接干预OB时,Bgp基因表达水平和培养基中BGP及RANKL蛋白的含量均与无干预的对照组无明显区别;而OCCM干预及共培养的OB组,当IGF-1初始干预浓度为10 ng/ml时BGP、RANKL含量及Bgp基因的表达水平显著提高,共培养组与OCCM培养组间无明显区别。结论 IGF-1对OB的促同化作用依赖于OC的参与,两种细胞间的联系可能是通过可溶性的细胞因子来完成。     

负载丹酚酸B的破骨前体细胞膜纳米颗粒对成骨细胞和破骨细胞分化的影响

目的 通过制备破骨前体细胞膜纳米颗粒（nanoparticles，NPs）负载丹酚酸B（salvianolic acid，SalB），构建载药纳米颗粒SalB-NPs，观察其对破骨细胞及成骨细胞分化的影响。方法 采用超声裂解、挤膜的方法制备NPs，并将NPs与SalB共孵育后，使用200 nm聚碳酸酯膜挤出，获得SalB-NPs。在诱导小鼠原代破骨细胞分化和成骨前体细胞（MC3T3-E1）成骨分化的过程中，按照处理方式不同，分为对照组、SalB组、SalB-NPs组。采用透射电镜、纳米粒度及ZETA电位仪和Western Blot对材料进行表征，采用噻唑蓝检测试剂盒检测材料对RAW 264.7和MC3T3-E1细胞活力的影响，采用高效液相色谱法检测SalB的释放率和装载率。采用抗酒石酸酸性磷酸酶（TRAP）染色评价破骨细胞分化能力，通过碱性磷酸酶（ALP）染色和茜素红染色评估成骨分化能力，采用Real time-PCR检测破骨细胞分化及成骨分化相关基因表达水平。结果 制备的纳米颗粒直径在200 nm左右，同时表达RANK蛋白。TRAP染色显示SalB-NPs显著抑制破骨细胞的形成，下调破骨分化相关基因水平，与对照组和SalB组相比，差异有统计学意义（P＜0.05）。使用成骨诱导培养基诱导MC3T3-E1细胞成骨分化，14 d ALP染色和21 d茜素红染色均显示SalB-NPs组ALP活性和钙盐沉积量较SalB组明显增加，差异有统计学意义（P＜0.05）。结论 SalB-NPs体外发挥促进成骨分化、抑制破骨细胞形成双重功效，作为骨质疏松的治疗药物开发，有很好的应用前景，未来仍需在骨质疏松模型动物进一步验证其治疗效果。     

氧化应激活化RAW264.7巨噬细胞对MC3T3-E1成骨细胞迁移、增殖及成骨基因表达影响的实验研究

目的探索氧化应激活化RAW264.7巨噬细胞对MC3T3-E1成骨细胞迁移、增殖及成骨基因表达的影响。方法取MC3T3-E1细胞系及RAW264.7细胞系,分别培养传至第7代进行实验。利用不同浓度(0、25、50、100μmol/L)H_2O_2刺激RAW264.7细胞,MTS检测培养1、3、6 h后细胞增殖率,采用超氧化物歧化酶(superoxide dismutase,SOD)检测试剂盒检测培养1 h时细胞内SOD含量,筛选H2O2引起RAW264.7细胞氧化应激的最佳浓度和作用时间,并对应收集RAW264.7细胞(加或不加H_2O_2处理)24 h上清液。取第7代MC3T3-E1细胞,分别以100μL无血清DMEM培养基(A组)、未氧化应激RAW264.7细胞上清液(B组)、氧化应激RAW264.7细胞上清液(C组)培养,采用MTS法检测细胞增殖情况,行划痕实验检测细胞迁移能力;另取细胞分为4组,空白对照组,以完全培养基培养;阳性对照组,以含50μg/m L维生素C及10 nmol/L的β甘油磷酸钠的完全培养基进行成骨诱导培养;正常对照组,以含50μg/m L维生素C及10 nmol/L的β甘油磷酸钠的完全培养基及未氧化应激RAW264.7细胞上清液培养;实验组,以含50μg/m L维生素C及10 nmol/L的β甘油磷酸钠的完全培养基及氧化应激RAW264.7细胞上清液培养。培养3、7、14 d,RT-PCR检测细胞内成骨相关基因ALP、Runx2、骨桥蛋白(osteopontin,OPN)、Ⅰ型胶原蛋白(collagen typeⅠ,COL-Ⅰ)、骨钙素(osteocalcin,OC)、骨唾液酸蛋白(bone sialoprotein,BSP)表达水平。结果 MTS和SOD检测结果显示:25μmol/L H_2O_2刺激1 h为构建RAW264.7细胞氧化应激模型最佳浓度和作用时间。细胞生长检测显示:1、2、3 d B、C组细胞增殖率显著高于A组(P0.05),C组低于B组,其中2、3 d时组间比较差异有统计学意义(P0.05)。划痕实验显示12 h时B、C组MC3T3-E1细胞迁移显著快于A组,C组快于B组,细胞迁移距离比较差异有统计学意义(P0.05);24 h时B、C组划痕已被细胞爬满。除3 d外,实验组各时间点ALP、Runx2、OC、BSP m RNA相对表达量均较阳性对照组明显降低,OPN、COL-Ⅰm RNA相对表达量较空白对照组降低,差异有统计学意义(P0.05)。结论 H_2O_2构建RAW264.7细胞氧化应激模型的最佳浓度为25μmol/L、作用时间为1 h。氧化应激活化RAW264.7巨噬细胞上清液能促进MC3T3-E1成骨细胞迁移、抑制其增殖及成骨相关基因的表达。     

JNK抑制剂SP600125对骨质疏松症保护作用的实验研究

目的 探讨JNK抑制剂SP600125对骨质疏松症模型成骨细胞和破骨细胞分化及功能的影响。方法 使用CCK8试验检测RAW264.7细胞、BMMs细胞和成骨细胞的增殖活性;使用相关染色试验探究JNK抑制剂SP600125的成骨化作用和对破骨细胞分化及功能的影响;使用RT-PCR分别检测BMMs细胞中破骨细胞及成骨细胞特异性基因mRNA表达水平;使用蛋白印迹试验检测RAW264.7细胞中JNK通路和NF-κB通路的活化水平;使用DCFH-DA法检测RAW264.7细胞中ROS水平。结果 CCK8试验结果显示,当SP600125浓度≤20 μmmol/L,对RAW264.7细胞、BMMs细胞和成骨细胞的增殖活性无显著影响;SP600125不影响成骨细胞钙结节的形成并抑制破骨细胞分化和功能;SP600125抑制的破骨细胞特异性基因的表达但不改变成骨细胞特异性基因的表达;SP600125抑制RANKL诱导的RAW264.7细胞中JNK通路和NF-κB通路的激活及ROS水平的升高。结论 JNK抑制剂SP600125能够抑制破骨细胞的分化及骨吸收功能,但对成骨细胞的分化无显著影响。     

柚皮苷对共培养体系中成骨细胞活性及破骨细胞分化的影响

目的研究在成骨细胞-破骨细胞共培养体系中,柚皮苷对成骨细胞活性和破骨细胞分化的影响。方法将成骨细胞(MC3T3-E1细胞株)和破骨细胞(RAW264.7细胞株)以2∶1的比例分别培养至Transwell小室的上室和下室。根据培养基是否含有柚皮苷分为对照组和柚皮苷(2ng/ml组、20 ng/ml组、200 ng/ml组),培养7 d后对下室破骨细胞进行TRAP染色和骨吸收陷窝鉴定;荧光定量PCR分析成骨细胞骨保护素(osteoprotegerin,OPG)、核因子κB受体活化因子配体(receptor activator for nuclear factor-κB ligand,RANKL)基因和破骨细胞分化基因活化T细胞核因子-1(NFATc-1)、活化T细胞核因子-2(NFATc-2)和核因子κB受体激活蛋白(receptor activator of NF-κB,RANK)的相对表达量。结果与对照组相比,柚皮苷可以促进成骨细胞OPG、RANKL的表达量且可提高OPG/RANKL的比值,差异有统计学意义(P0.05);20 ng/ml柚皮苷TRAP(+)细胞数(5.82±3.37)明显少于对照组(20.56±7.69),差异有统计学意义(P0.05);柚皮苷可抑制破骨细胞NFATc-1、NFATc-2的表达,促进RANK的表达,差异有统计学意义(P0.05)。结论柚皮苷可促进共培养条件体系中成骨细胞OPG和RANKL的表达以及抑制破骨细胞的分化,与上调OPG/RANKL的比值有关。     

黄瓜籽总皂苷对MC3T3-E1细胞分化及SPARC、OPG/RANKL表达的影响

目的观察黄瓜籽总皂苷提取物(cucumber seed saponins,CSS)对小鼠成骨细胞MC3T3-E1增殖、分化和矿化的影响,以及与骨质疏松相关的SPARC、OPG/RANKL/RANK信号通路的作用。方法通过MTT实验、碱性磷酸酶(alkaline phosphatase,ALP)活性检测、茜素红染色,考察不同浓度CSS对MC3T3-E1细胞增殖、分化及矿化的影响;采用RT-PCR方法检测SPARC、OPG/RANKL mRNA表达水平; Western blot检测SPARC、OPG/RANKL的蛋白表达量。结果与阳性对照组相比,CSS能明显促进MC3T3-E1细胞增殖(P0.05),CSS高、中剂量组能明显提高MC3T3-E1细胞ALP活性及钙化结节数量(P0.05);与空白组相比,CSS不同剂量组均明显上调SPARC、OPG/RANKL mRNA及蛋白表达水平(P0.05)。结论黄瓜籽总皂苷能够促进成骨细胞MC3T3-E1的增殖、分化及矿化能力,并通过上调SPARC、OPG/RANKL的表达水平提高MC3T3-E1细胞的成骨能力。     

Osteoclastogenesis is Influenced by Modulation of Gap Junctional Communication with Antiarrhythmic Peptides

Osteoclasts are formed by the fusion of mononuclear precursor cells of the monocyte–macrophage lineage. Among several putative mechanisms, gap-junctional intercellular communication (GJC) has been proposed to have a role in osteoclast fusion and bone resorption. We examined the role of GJC in osteoclastogenesis and in vitro bone resorption with mouse bone marrow hematopoietic stem cells and RAW 264.7 cells. Blocking of gap junctions with 18-α-glycyrrhetinic acid (18GA) led to inhibition of osteoclastogenesis and in vitro bone resorption. Similarly, the GJC inhibitor GAP27 inhibited osteoclast formation. GJC modulation with the antiarrhythmic peptides (AAPs) led to increased amounts of multinuclear RAW 264.7 osteoclasts as well as increased number of nuclei per multinuclear cell. In the culture of bone marrow hematopoietic stem cells in the presence of bone marrow stromal cells AAP reduced the number of osteoclasts, and coculture of MC3T3-E1 preosteoblasts with RAW 264.7 macrophages prevented the action of AAPs to promote osteoclastogenesis. The present data indicate that AAPs modulate the fusion of the pure culture of cells of the monocyte–macrophage lineage. However, the fusion is influenced by GJC in cells of the osteoblast lineage.     

Prostate-specific antigen induces apoptosis of osteoclast precursors: potential role in osteoblastic bone metastases of prostate cancer

BACKGROUND: Prostate cancer is frequently associated with bone metastases with marked osteoblastic changes and low osteoclastic activity but its mechanism is not well understood. We previously reported that prostate-specific antigen (PSA) stimulated the proliferation and the activation of osteoblasts. In this study, we investigated the effect of PSA on osteoclastogenesis. METHODS: Two human prostate cancer cell lines and PSA were directly injected into human adult bone (HAB) implanted into NOD/SCID mice, followed by morphological analysis. RAW 264.7 cells, murine osteoclast precursor, were treated with PSA. RESULTS: PSA-producing LNCaP and PSA caused a significant decrease of osteoclast precursors and osteoclasts in HAB accompanied by osteoblast proliferation and new bone formation, while PSA-nonproducing PC3 showed increasing osteoclasts with osteolysis. PSA induced apoptosis of RAW 264.7 cells in vitro. PSA-induced apoptosis was dependent of enzymatic activity of PSA and was specific to immature tartrate-resistant acid phosphatase-negative mononuclear RAW 264.7 cells. CONCLUSIONS: PSA plays a crucial role for osteoblastic bone metastasis by promoting both osteoblasts proliferation and apoptosis of osteoclast precursors.     

Platelet-derived growth factor BB secreted from osteoclasts acts as an osteoblastogenesis inhibitory factor.

Osteoclasts and osteoblasts are responsible for strict bone maintenance with a balance between bone formation and resorption by interacting with each other. Recently, it has been revealed that osteoblasts/stromal cells regulate differentiation of osteoclasts/hematopoietic cells by two factors, the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) on the plasma membrane, and secreted osteoprotegerin (OPG). However, no factors have yet been reported by which osteoclasts/hematopoietic cells regulate osteoblasts/stromal cells. To elucidate the possibility of signal transduction from osteoclasts to osteoblasts, we studied the conditioned medium of mouse osteoclast-like myeloma cell line RAW264.7 treated with RANKL. We found that this medium contains a factor that inhibits differentiation of mouse osteoblast precursor-like cell line MC3T3-E1 to osteoblasts induced by bone morphogenetic protein 4 (BMP-4) and named this factor osteoblastogenesis inhibitory factor (OBIF). OBIF was purified by successive three-step chromatography by heparin affinity, anion exchange, and reversed-phase columns. Osteoblastogenesis inhibitory activity made one peak in each chromatography step, showing the factor is a single entity. Active fractions were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and bands of proteins were excised, digested by trypsin, and analyzed by liquid chromatography equipped with tandem mass spectrometry (LC/MS/MS). Consequently, we have identified this factor to be platelet-derived growth factor BB (PDGF BB) homodimer. Furthermore, this identification of PDGF BB as OBIF was confirmed by neutralization of the inhibitory activity of the medium with anti-PDGF antibody. These results show, for the first time, that osteoclasts regulate osteoblasts directly and suggest that PDGF BB is a key factor in bone remodeling.     

THSD4基因在小鼠间充质干细胞及MC3T3-E1细胞成骨分化中的作用

目的 探讨THSD4基因对小鼠间充质干细胞和MC3T3-E1细胞成骨分化的影响。方法 提取绝经后骨质疏松症患者的骨髓间充质干细胞进行基因测序分析,与骨关节炎患者的骨髓间充质干细胞进行比较,分析基因表达差异。通过提取不同分化阶段的小鼠骨髓间充质干细胞（M-BMSC）及MC3T3-E1细胞的mRNA来检测THSD4 基因以及成骨分化的标志性基因（ALP、Runx2、Osx）的表达水平。通过构建慢病毒表达载体来实现对M-BMSC及MC3T3-E1细胞中THSD4的敲减及过表达,并观察其对M-BMSC及MC3T3-E1细胞成骨分化能力的影响。结果 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且通过KEGG以及GO富集分析发现THSD4基因可能与PI3K-AKT信号通路及Wnt信号通路相关。随着成骨诱导分化时间的延长,THSD4 mRNA和成骨分化标志性基因（ALP、Runx2、Osx）mRNA在MC3T3-E1以及M-BMSC中表达量均逐渐增加。过表达THSD4可以增强MC3T3-E1细胞和M-BMSC的成骨分化能力,而敲减THSD4则减弱了MC3T3-E1细胞和M-BMSC的成骨分化能力。结论 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且THSD4基因可以增强MC3T3-E1细胞以及M-BMSC的成骨分化能力。     

BMP9 Reduces Bone Loss in Ovariectomized Mice by Dual Regulation of Bone Remodeling

Bone remodeling is dynamic and is tightly regulated through bone resorption dominated by osteoclasts and bone formation dominated by osteoblasts. Imbalances in this process can cause various pathological conditions, such as osteoporosis. Bone morphogenetic protein 9 (BMP9), a biomolecule produced and secreted by the liver, has many pharmacological effects, including anti-liver fibrosis, antitumor, anti-heart failure, and antidiabetic activities. However, the effects of BMP9 on the regulation of osteoblast and osteoclast functions and the underlying molecular mechanism(s) have not yet been investigated. In this study, BMP9 increased the expression of osteoblastogenic gene markers, such as ALP, Cola1, OCN, RUNX2, and OSX, and ALP activity in MC3T3-E1 cells by upregulating LGR6 and activating the Wnt/β-catenin pathway. BMP9 also suppressed receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation of bone marrow macrophages (BMMs) by inhibiting the Akt-NF-κB-NFATc1 pathway. More importantly, in an ovariectomy (OVX) mouse model, BMP9 attenuated bone loss and improved bone biomechanical properties in vivo by increasing bone-forming activity and suppressing bone resorption activity. Accordingly, our current work highlights the dual regulatory effects that BMP9 exerts on bone remodeling by promoting bone anabolic activity and inhibiting osteoclast differentiation in OVX mice. © 2020 American Society for Bone and Mineral Research.     

Effects of geranylgeranoic acid in bone: induction of osteoblast differentiation and inhibition of osteoclast formation.

Retinoids are known to be of special importance for normal bone growth and development. Recently, we reported that retinoids not only induced osteoblast differentiation, but also inhibited osteoclast formation in vitro. In this study, we examined the osteogenic effects of geranylgeranoic acid (GGA), a chemically synthesized acyclic retinoid, in bone in vitro and in vivo. GGA not only suppressed proliferation of osteoblastic MC3T3-E1 cells, but also up-regulated differentiation markers of osteoblasts such as alkaline phosphatase (ALP) activity and expression of osteopontin (OP) messenger RNA (mRNA). In contrast, GGA inhibited osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] in cocultures of mouse bone marrow cells and primary osteoblasts. Treatment of stromal ST2 cells with GGA restored the 1alpha,25(OH)2D3- or prostaglandin E2 (PGE2)-induced suppression of osteoprotegerin (OPG) mRNA expression. GGA inhibited osteoclast formation induced by macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of nuclear factor kappaB ligand (sRANKL) in the culture of bone marrow macrophages. Thus, it is likely that GGA inhibits osteoclast formation by affecting both osteoblasts and osteoclast progenitors in the coculture system. Furthermore, in vivo, GGA increased bone mineral density (BMD) of total as well as distal femur in a P6 strain of senescence-accelerated mice (SAMP6). These results indicate that GGA increases bone mass by maintaining a positive balance of bone turnover by inducing osteoblast differentiation and suppressing osteoclast formation.     

Effects of secreted frizzled-related protein 3 on osteoblasts in vitro.

To examine if sFRP3s act as decoy receptors for Wnt, we examined the effects of recombinant sFRP3 on mouse osteoblast proliferation and differentiation. We found that sFRP3 unexpectedly increased osteoblast differentiation, suggesting it may act through other mechanisms besides acting as a decoy receptor for Wnt's. INTRODUCTION: Secreted frizzled-related proteins (sFRPs) are a truncated form of frizzled receptor, missing both the transmembrane and cytosolic domains. Because previous studies have shown that sFRPs bind and act as decoy receptors for Wnt proteins that promote osteoblast differentiation, we postulated that sFRP3 acts as an inhibitor of osteoblast differentiation. MATERIALS AND METHODS: We examined the effects of mouse recombinant sFRP3 and/or Wnt-3A on cell proliferation and differentiation using MC3T3-E1 mouse osteoblasts and primary cultures of mouse bone marrow stromal cells. We evaluated the effects of sFRP3 on beta-catenin levels using Western immunoblot analyses. RESULTS: We found that sFRP3 suppressed osteoblast cell number in a dose-dependent manner that was the result of a decrease in proliferation and not because of an increase in apoptosis. Surprisingly, sFRP3 increased osteoblast differentiation, which could not be explained based on sFRP3 acting as a decoy receptor for stimulatory Wnt's. Furthermore, sFRP3 did not inhibit Wnt3A-induced increase in alkaline phosphatase (ALP) activity. Wnt3A, but not sFRP3 treatment, increased cellular beta-catenin levels, and sFRP3 failed to block Wnt3A-induced increase in cellular beta-catenin levels. Treatment with endostatin, an agent known to degrade beta-catenin, did not inhibit sFRP3-induced increase in ALP activity. sFRP1, like sFRP3, inhibited proliferation and stimulated ALP activity in MC3T3-E1 mouse osteoblasts. CONCLUSIONS: Based on our findings, we conclude that sFRP3 decreased osteoblast proliferation and unexpectedly increased parameters of osteoblast differentiation. Based on our findings, we propose that sFRP3 may stimulate differentiation through a beta-catenin-independent pathway in addition to its previously known function as a decoy receptor for Wnt's.     

Insulin-like growth factor I production is essential for anabolic effects of thyroid hormone in osteoblasts.

Thyroid hormone (T3) and insulin-like growth factor I (IGF-I) are critical regulators of skeletal function. T3 increases IGF-I production in bone. To assess the potential role of IGF-I as a mediator of T3 actions, we characterized phenotypic markers of osteoblast activity in two osteoblast models, normal mouse osteoblasts and MC3T3-E1 cells, exposed to T3 alone or under conditions that interfere with IGF-I actions. T3 significantly increased osteoblast 3H-proline incorporation, alkaline phosphatase (ALP), and osteocalcin. Both alphaIR3, a neutralizing monoclonal antibody to the IGF-I receptor, and JB1, an IGF-I analogue antagonist, attenuated the stimulatory effects of T3. T3 effects also were decreased in cells transfected with antisense oligonucleotide (AS-ODN) to the IGF-I receptor gene. Both IGF-I and T3 had mitogenic effects that were inhibited by the antagonists. IGF-I by itself did not stimulate 3H-proline incorporation, ALP, and osteocalcin in the models used, revealing that although IGF-I is essential for the anabolic effects of T3, it acts in concert with other factors to elicit these phenotypic responses.     

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.