Oscillatory fluid flow-induced shear stress decreases osteoclastogenesis through RANKL and OPG signaling

Physical activity creates deformation in bone that leads to localized pressure gradients that drive interstitial fluid flow. Due to the cyclic nature of the applied load, this flow is oscillatory by nature. Oscillatory fluid flow (OFF) may lead to positive bone remodeling through effects on both osteoblasts and osteoclasts but its effect on osteoclastogenesis is poorly understood. In this study, the effects of OFF on expression of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG), two important regulators of osteoclast differentiation, were investigated. In addition, its effect on osteoclast formation was quantified. ST-2 murine bone marrow stromal cells were plated on glass slides and cultured with 1,25-dihydroxyvitamin D(3) to express RANKL. Cells were exposed to various durations of OFF resulting in a peak shear stress of 1 Pa. Time course and dose-response studies were performed and real-time RT-PCR was used to quantify levels of RANKL, OPG mRNA. ST-2 cells exposed to OFF were also co-cultured with RAW 264.7 monocytes and osteoclast number quantified. Decrease in RANKL/OPG was maximal immediately after end of flow and there existed a significant increase in OPG and decrease in RANKL with increasing load duration of up to 2 h. OFF resulted in a decrease in osteoclast formation by ST-2 cells co-cultured with RAW 264.7 cells compared to co-culture of control (non-loaded) ST-2 cells with RAW 264.7 cells. These results suggest that indeed OFF is a potent regulator of bone remodeling, and that shift towards positive bone remodeling mediated by loading-induced fluid flow may occur via suppression of the formation of osteoclasts.     

通过骨基质表面培养板检测RANKL诱导破骨细胞骨侵蚀能力

目的探讨以骨基质表面培养板替代骨磨片鉴定破骨细胞骨侵蚀能力的应用方法。方法通过RANKL诱导破骨前体细胞RAW264. 7建立破骨细胞分化模型,运用TRAP染色检测破骨细胞分化程度,并以骨基质表面培养板替代骨磨片行骨陷窝试验检测破骨细胞骨侵蚀能力,以侵蚀面积反映骨侵蚀能力。结果不同浓度的RANKL因子可有效诱导破骨前体细胞RAW264. 7分化为成熟多核破骨细胞,呈浓度依赖性。成熟破骨细胞在骨基质表面培养板上形成不同面积的不规则侵蚀圆环,趋势与破骨细胞分化程度一致。结论使用骨基质表面培养板可有效反映破骨细胞形成及骨侵蚀能力,与TRAP染色结果一致,并且具有操作简便、结果直观、便于统计分析等优点。     

The generation of osteoclasts from RAW 264.7 precursors in defined,serum-free conditions

Osteoclasts are the unique cell type capable of resorbing bone. The discovery of the TNF-ligand family member, RANKL, has allowed more reliable study of these important cells. The mouse monocytic cell line, RAW 264.7, has been shown to readily differentiate into osteoclasts upon exposure to recombinant RANKL. Unlike primary osteoclast precursors, there is no requirement for the addition of macrophage colony stimulating factor (M-CSF). However, to date, their differentiation has always been studied in the context of added foetal calf serum (FCS). FCS is a complex and largely undefined mixture of growth factors and matrix proteins, and varies between batches. For this reason, osteoclastogenesis would ideally be studied in the context of a defined, serum-free medium. RAW 264.7 cells were cultured in serum-replete α-MEM or serum-deprived medium (SDM) shown previously to support the growth of human osteoclasts in a co-culture with normal osteoblasts. In SDM, in the presence of recombinant RANKL, RAW 264.7 cells readily differentiated into tartrate resistant acid phosphatase (TRAP) positive multinucleated osteoclast-like cells, a process that was enhanced with the addition of 1α,25-dihydroxyvitamin D₃ (1,25D). While the osteoclasts grown in SDM were smaller in size compared with those derived in serum-replete media, their resorptive capacity was significantly increased as indicated by a twofold increase in average resorption pit size. In conclusion, we describe a defined model for studying osteoclast differentiation and activity in the absence of serum, which will be ideal for studying the role of agonistic and antagonistic molecules in this process.     

重组人骨保护素与重组核因子κb活化因子受体蛋白对破骨前体细胞分化的影响

目的:对比重组人骨保护素(rhOPG-Fc)与重组核因子κb活化因子受体蛋白(rhRANK)对破骨前体细胞分化的影响.方法:采用成骨细胞与破骨前体细胞RAW264.7混合培养,在地塞米松、1,25 (OH) 2VitD3诱导下生成破骨细胞的方法.研究分3组:rhRANK组:10-5 g/L;rhOPG-Fc组:10-5 g/L;空白对照组.作用9d后观察破骨细胞数目和形态,抗酒石酸酸性磷酸酶(TRAP)染色阳性细胞个数,骨磨片吸收陷窝计数.结果:在空白对照组,小鼠成骨细胞与破骨前体细胞RAW264.7混合培养6d后,开始出现多核细胞,9d时可见大量成熟多核细胞,经TRAP染色证实为成熟破骨细胞,而rhRANK组及rhOPG-Fc组TRAP染色阳性多核细胞数较对照组均减少,特别是rhRANK组减少更明显.骨片吸收陷窝计数显示rhRANK组及rhOPG-Fc组较对照组也明显减少,而相对来说,rhRANK组较rhOPG-Fc组更少.结论:rhOPG-Fc与rhRANK均可以有效抑制破骨前体细胞分化成为成熟破骨细胞,且rhRANK较rhOPG-Fc抑制效果更明显.     

低分子量褐藻糖胶对破骨细胞凋亡的影响

目的探讨低分子量褐藻糖胶(LMWF)对小鼠单核细胞RAW264.7诱导成熟破骨细胞凋亡的影响。方法通过100ng/m L RANKL诱导RAW264.7细胞株分化为破骨细胞,经TRAP特异性染色和骨吸收陷窝对诱导后的细胞进行鉴定。鉴定成功后,用100 ng/m L RANKL诱导RAW264.7细胞株5 d后,使用含有LMWF的培养基继续培养3 d,通过对TRAP阳性细胞计数和分析骨吸收面积来观察低分子量褐藻糖胶对破骨细胞的抑制和骨吸收功能情况;采用流式细胞术检测LMWF对破骨细胞凋亡的影响,capsase-3活性测试试剂盒检测LMWF对capsase-3活性进行测定;RT-PCR检测LMWF对成熟破骨细胞BAX与BCL-2基因表达的影响。结果单纯采用100 ng/m L的RANKL可成功诱导成熟的、有功能的破骨细胞。LMWF可以明显抑制RANKL诱导成熟破骨细胞的形成以及成熟破骨细胞的骨吸收功能;流式细胞术显示LMWF可增加成熟破骨细胞的早期凋亡率;并且能升高capsase-3的活性;PCR显示LMWF可明显下调破骨细胞凋亡相关的BCL-2和上调BAX基因mRNA表达,降低BCL-2/BAX的比值。结论低分子量褐藻糖胶可抑制破骨细胞的活性与骨吸收能力,促进破骨细胞凋亡,其主要机制是通过下调BCL-2和上调BAX mRNA基因表达实现的。     

c-myc is required for osteoclast differentiation.

The role of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL)-a tumor necrosis factor (TNF)-related cytokine-in osteoclast formation has been established clearly. However, the downstream signaling pathways activated by this cytokine remain largely unknown. To identify genes that play a role in osteoclastogenesis, we used RAW 264.7 mouse monocytes as a model system for the differentiation of multinucleated osteoclasts from mononucleated precursors. RAW 264.7 cells were induced with RANKL to form multinucleated giant osteoclast-like cells (OCLs) that expressed a number of osteoclast-specific markers and were able to form resorption pits on both calcium phosphate films and bone slices. This system was used to identify genes that are regulated by RANKL and may play a role in osteoclast differentiation. The proto-oncogene c-myc was strongly up-regulated in RANKL-induced OCLs but was absent in undifferentiated cells. Expression of Myc partners Max and Mad, on the other hand, was constant during OCL differentiation. We expressed a dominant negative Myc in RAW 264.7 cells and were able to block RANKL-induced OCL formation. Northern Blot analysis revealed a delay and a significant reduction in the level of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K. We conclude that c-myc is a downstream target of RANKL and its expression is required for RANKL-induced osteoclastogenesis.     

The RANKL/RANK/OPG pathway

Understanding of osteoclast formation and activation has advanced considerably since the discovery of the RANKL/RANK/OPG system in the mid 1990s. Osteoblasts and stromal stem cells express receptor activator of NF-jB ligand (RANKL), which binds to its receptor, RANK, on the surface of osteoclasts and their precursors. This regulates the differentiation of precursors into multinucleated osteoclasts and osteoclast activation and survival both normally and in most pathologic conditions associated with increased bone resorption. Osteoprotegerin (OPG) is secreted by osteoblasts and osteogenic stromal stem cells and protects the skeleton from excessive bone resorption by binding to RANKL and preventing it from interacting with RANK. The RANKL/OPG ratio in bone marrow is thus an important determinant of bone mass in normal and disease states. RANKL/RANK signaling also regulates lymph node formation and mammary gland lactational hyperplasia in mice, and OPG protects large arteries of mice from medial calcification. This article reviews the roles of the RANKL/RANK/OPG system in bone and other tissues.     

Osteoprotegerin and its Ligand: A New Paradigm for Regulation of Osteoclastogenesis and Bone Resorption

In just 3 years, striking new advances have been made in understanding the molecular mechanisms that govern the crosstalk between osteoblasts/stromal cells and hematopoietic osteoclast precursor cells that leads to osteoclastogenesis. Led first by the discovery of osteoprotegerin (OPG), a naturally occurring protein with potent osteoclastogenesis inhibitory activity, rapid progress was made to the isolation of RANKL, a transmembrane ligand expressed on osteoblasts/stromal cells that binds to RANK, a transmembrane receptor on hematopoietic osteoclast precursor cells. The interaction of RANK and RANKL initiates a signaling and gene expression cascade that results in differentiation and maturation of osteoclast precursor cells to active osteoclasts capable of resorbing bone. OPG acts as a decoy receptor, binding to RANKL and blocking its interaction with RANK, inhibiting osteoclast development. Many of the calciotropic hormones and cytokines, including 1,25(OH)₂D₃, PTH, PGE₂ and IL-11, appear to act through a dual capacity to inhibit production of OPG and stimulate production of RANKL. Estrogen, on the other hand, appears to inhibit production of RANKL and RANKL-stimulated osteoclastogenesis. Recently, the results of the first clinical trial with OPG supported its potential as a therapeutic agent for diseases such as osteoporosis. The new understanding provided by the RANK/RANKL/OPG paradigm for both differentiation of osteoclasts and their activation has had tremendous impact on the field and opened new avenues for development of possible treatments of diseases characterized by excessive bone resorption.     

Isolation of human osteoclasts formed in vitro: hormonal effects on the bone-resorbing activity of human osteoclasts

Osteoclasts are multinucleated cells that carry out bone resorption. Analysis of the direct effect of hormones on the bone-resorbing activity of human osteoclasts has been limited by difficulties in isolating these cells from the human skeleton. In this study, human osteoclasts formed from cultures of peripheral blood mononuclear precursors (PBMCs) on a Type-I collagen gel were isolated by collagenase treatment for investigating their resorptive activity. PBMCs were cultured in the presence of M-CSF, soluble RANKL, dexamethasone, and 1,25(OH)2D3. The isolated multinucleated cells expressed the osteoclast markers, TRAP, VNR, cathepsin K, calcitonin receptors and were capable of extensive lacunar resorption. Calcitonin inhibited the motility and resorptive activity of osteoclasts. RANKL significantly stimulated osteoclast resorption, but 1,25(OH)2D3, PTH, and OPG did not. These findings indicate that calcitonin and RANKL act directly on human osteoclasts to inhibit and stimulate osteoclast bone-resorbing activity, respectively, and that PTH, 1,25(OH)2D3, and OPG are more likely to influence osteoclast activity indirectly. This technique of human osteoclast isolation should permit the effects of cellular and hormonal/humoral factors on the bone-resorbing activity of mature human osteoclasts to be assessed independently of any effect such factors have on osteoclast formation. It should also make it possible to examine directly the resorptive activity and other characteristics of osteoclasts in specific bone disorders such as Paget's disease.     

Production of IL-7 is increased in ovariectomized mice,but not RANKL mRNA expression by osteoblasts/stromal cells in bone,and IL-7 enhances generation of osteoclast precursors in vitro

Osteoclastogenic cytokines produced by T and B lineage cells and interleukin (IL)-7-induced expansion of the pool size of osteoclast precursors have been suggested to play an important role in acceleration of osteoclastogenesis induced by estrogen deficiency. However, the contribution of increased RANKL produced by osteoblasts/stromal cells to increase osteoclastogenesis in a mouse model of estrogen-deficient osteoporosis and in vitro effects of IL-7 on osteoclast precursor generation remain controversial. Thus, we investigated the effect of ovariectomy (OVX) of mice on production of RANKL, osteoprotegerin (OPG), and IL-7 in bone and the effect of IL-7 on osteoclast precursor generation in vitro. OVX did not significantly stimulate mRNA expressions of RANKL and OPG in whole femurs. Because the epiphysis, but not the femoral shaft (diaphysis) or bone marrow, is the main site of osteoclastogenesis, it is important to specifically analyze mRNA expression by osteoblasts/stromal cells at these parts of the femur. Therefore, we isolated RNA from bone marrow cell-free epiphysis, diaphysis, and flushed-out bone marrow and examined mRNA expression. The results showed no significant changes of RANKL and OPG mRNA expression in any part of the femur. In addition, OVX did not significantly affect RANKL and OPG mRNA expression by the adherent stromal cells isolated from flushed-out bone marrow cells but did stimulate RANKL mRNA expression by B220⁺ cells in the nonadherent cell fraction. On the other hand, OVX increased IL-7 mRNA expression in the femur as well as IL-7 concentrations in bone fluid. In cultures of unfractionated bone cells isolated by vigorous agitation of minced whole long bones to release the cells tightly attached to the bone surfaces, but not in cocultures of clonal osteoblasts/stromal cells and flushed-out bone marrow cells, IL-7 stimulated generations of osteoclasts as well as osteoclast precursors. These data suggest that increased RANKL production by osteoblasts/stromal cells is unlikely to play a central role in acceleration of osteoclastogenesis in estrogen deficiency of mice and that IL-7 stimulates osteoclast precursor generation, presumably through an action of IL-7 on the cells attached to bone rather than on cells contained in the bone marrow cell population.     

Osteoblasts of calvaria induce higher numbers of osteoclasts than osteoblasts from long bone

Several studies have demonstrated the existence of functional differences between osteoclasts harbored in different bones. The mechanisms involved in the occurrence of such a heterogeneity are not yet understood. Since cells of the osteoblast lineage play a critical role in osteoclastogenesis, osteoclast heterogeneity may be due to osteoblasts that differ at the different bone sites. In the present study we evaluated possible differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. Osteoblasts were isolated from calvaria and long bone of mice and co-cultured with osteoclast precursors obtained from bone marrow of both types of bone, spleen and peripheral blood. Irrespective of the source of the precursors, a significantly higher number of TRACP-positive multinucleated cells were formed with calvaria osteoblasts. The expression of osteoclastogenesis related genes was analyzed by qPCR. OPG was significantly higher expressed by long bone osteoblasts. The RANKL/OPG ratio and TNF-α gene expression were significantly higher in calvaria osteoblast cultures. OPG added to the culture system inhibited osteoclastogenesis in both groups. Blocking TNF-α had no effect on osteoclastogenesis. Calvaria and long bone osteoblasts were pre-stimulated with VitD3 for 5 days. Subsequently, osteoclast precursors were added to these cultures. After a co-culture of 6 days, it was shown that VitD3 pre-stimulation of long bone osteoblasts strongly improved their capacity to induce osteoclast formation. This coincided with an increased ratio of RANKL/OPG. Taken together, the data demonstrated differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. This appeared to be due to differences in the expression of RANKL and OPG. VitD3 pre-stimulation improved the ability of long bone osteoblasts to induce osteoclast formation. Our findings demonstrate bone-site specific differences in osteoblast-mediated formation of osteoclasts. The data may suggest that the heterogeneity of osteoclasts is partially due to the way the osteoblasts induce their formation.     

Fibroblastic stromal cells express receptor activator of NF-kappa B ligand and support osteoclast differentiation.

Osteoclast formation in bone is supported by osteoblasts expressing receptor activator of NF-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) expression. Numerous osteotropic factors regulate expression levels of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts, thereby affecting osteoclast differentiation. However, not only in RANKL widely expressed in soft tissues, but osteoclasts have been noted in extraskeletal lesions. We found that cultured skin fibroblastic cells express RANKL, M-CSF, and OPG messenger (mRNA). Stimulation by 1 alpha,25 dihydroxyvitamin D3 [1,25(OH)2D3] plus dexamethasone (Dex) augmented RANKL and diminished OPG mRNA expression in fibroblastic cells and caused the formation of numerous osteoclasts in cocultures of skin fibroblastic cells with hemopoietic cells or monocytes. The osteoclasts thus formed expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin (CT) receptors and formed resorption pits in cortical bone. Osteoclast formation also was stimulated (in the presence of Dex) by prostaglandin E2 (PGE2), interleukin-11 (IL-11), IL-1, tumor necrosis factor-alpha (TNF-alpha), and parathyroid hormone-related protein (PTHrP), factors which also stimulate osteoclast formation supported by osteoblasts. In addition, granulocyte-macrophage-CSF (GM-CSF), transforming growth factor-beta (TGF-beta), and OPG inhibited osteoclast formation in skin fibroblastic cell-hemopoietic cell cocultures; CT reduced only osteoclast nuclearity. Fibroblastic stromal cells from other tissues (lung, respiratory diaphragm, spleen, and tumor) also supported osteoclast formation. Thus, RANKL-positive fibroblastic cells in extraskeletal tissues can support osteoclastogenesis if osteolytic factors and osteoclast precursors are present. Such mesenchymally derived cells may play a role in pathological osteolysis and may be involved in osteoclast formation in extraskeletal tissues.     

E-cadherin is important for cell differentiation during osteoclastogenesis

E-cadherin, a protein responsible for intercellular adhesion between epithelial cells, is also expressed in the monocyte/macrophage lineage. In this study we have explored the involvement of E-cadherin during receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclast differentiation. Osteoclastogenesis involves a period of precursor expansion followed by multiple fusion events to generate a multinuclear osteoclast that is capable of bone resorption. We asked whether E-cadherin participated in early precursor interactions and recognition or was a component of the osteoclast fusion machinery. Here, we show that endogenous E-cadherin expression is the highest during early stages of osteoclast differentiation, with surface expression visible on small precursor cells (fewer than four nuclei per cell) in both RAW 264.7 cells and primary macrophages. Blocking E-cadherin function with neutralizing antibodies prior to the onset of fusion delayed the expression of TRAP, Cathepsin K, DC-STAMP and NFATc1 and significantly diminished multinucleated osteoclast formation. Conversely, E-cadherin-GFP overexpressing macrophages displayed earlier NFATc1 nuclear translocation along with faster formation of multinucleated osteoclasts compared to control macrophages. Through live imaging we identified that disrupting E-cadherin function prolonged the proliferative phase of the precursor population while concomitantly decreasing the proportion of migrating precursors. The lamellipodium and polarized membrane extensions appeared to be the principal sites of fusion, indicating precursor migration was a critical factor contributing to osteoclast fusion. These findings demonstrate that E-cadherin-mediated cell–cell contacts can modulate osteoclast-specific gene expression and prompt differentiating osteoclast precursors toward migratory and fusion activities.     

Involvement of receptor activator of NFkappaB ligand and tumor necrosis factor-alpha in bone destruction in rheumatoid arthritis

Bone loss represents a major unsolved problem in rheumatoid arthritis (RA). The skeletal complications of RA consist of focal bone erosions and periarticular osteoporosis at sites of active inflammation, and generalized bone loss with reduced bone mass. New evidence indicates that osteoclasts are key mediators of all forms of bone loss in RA. TNF-alpha is one of the most potent osteoclastogenic cytokines produced in inflammation and is pivotal in the pathogenesis of RA. Production of tumor necrosis factor-alpha (TNF-alpha) and other proinflammatory cytokines in RA is largely CD4(+) T-cell dependent and mostly a result of interferon-gamma (IFN-gamma) secretion. Synovial T cells contribute to synovitis by secreting IFN-gamma and interleukin (IL)-17 as well as directly interacting with macrophages and fibroblasts through cell-to-cell contact mechanisms. Activated synovial T cells express both membrane-bound and soluble forms of receptor activator of NF-kappaB ligand (RANKL). In rheumatoid synovium, fibroblasts also provide an abundant source of RANKL. Furthermore, TNF-alpha and IL-1 target stromal-osteoblastic cells to increase IL-6, IL-11, and parathyroid hormone-related protein (PTHrP) production as well as expression of RANKL. In the presence of permissive levels of RANKL, TNF-alpha acts directly to stimulate osteoclast differentiation of macrophages and myeloid progenitor cells. In addition, TNF-alpha induces IL-1 release by synovial fibroblasts and macrophages, and IL-1, together with RANKL, is a major survival and activation signal for nascent osteoclasts. Consequently, TNF-alpha and IL-1, acting in concert with RANKL, can powerfully promote osteoclast recruitment, activation, and osteolysis in RA. The most convincing support for this hypothesis has come from in vivo studies of animal models. Protection of bone in the presence of continued inflammation in arthritic rats treated with osteoprotegerin (OPG) supports the concept that osteoclasts mediate bone loss, providing further evidence that OPG protects bone integrity by downregulating osteoclastogenesis and promoting osteoclast apoptosis. Modulation of the RANKL/OPG equilibrium in arthritis may provide additional skeletal benefits, such as chondroprotection. The nexus between T-cell activation, TNF-alpha overproduction, and the RANKL/OPG/RANK ligand-receptor system points to a unifying paradigm for the entire spectrum of skeletal pathology in RA. Strategies that address osteoclastic bone resorption will represent an important new facet of therapy for RA.     

高糖及TNF-α对RAW264.7细胞向破骨细胞分化的影响

【摘要】 目的 观察高糖及TNF-α的培养条件对RAW264.7细胞向破骨细胞诱导分化的影响。方法 在正常、高糖(30 mmol/L)及TNF-α(10 μmol/L)条件下培养RAW264.7细胞后,加入浓度为100 ng/mL的细胞核转录因子κB受体激活物的配体(receptor activator of NF-κB ligand, RANKL)为诱导剂,诱导RAW264.7向破骨细胞分化;诱导9天后,抗酒石酸酸性磷酸酶(TRAP)染色,比较各组TRAP+细胞数,RT-PCR及Western Blot检测各组破骨细胞标志基因CTR和MMP-9的表达。结果 不同的培养条件下RANKL均能诱导RAW264.7分化为成熟的破骨细胞,其中TNF-α环境中RAW264.7形成的TRAP+阳性细胞数、CTR和MMP-9的表达最高,而在高糖环境下最低。结论 TNF-α可以促进RAW264.7向破骨细胞分化,而高糖对这个过程可能是抑制作用,这一现象符合Ⅰ型和Ⅱ糖尿病患者骨质破坏的表现;高糖及TNF-α的培养条件下RANKL对RAW264.7的作用可模拟糖尿病足病变微环境中OC的诱导分化的过程。     

破骨细胞分化调节机制的研究进展

破骨细胞起源于骨髓的单核髓性造血干细胞, 是一种具有骨吸收功能的多核巨细胞, 其在骨代谢方面起着关键性的作用, 因而机体对于破骨细胞的调控非常严格。破骨细胞动员和分化成熟过程是一个复杂而又精细的多级调控过程, 在相关调控机制中, OPG/RANKL/RANK系统起着分化调节枢纽的作用, 是调节破骨细胞分化和功能的关键信号途径。最近研究发现破骨细胞和免疫细胞在骨代谢领域相互联系紧密, 这也为骨疾病的治疗提供了新的治疗靶点。另外破骨细胞凋亡在骨代谢中的作用越来越受重视, 但其相关机制还不是很明确, 仍需要深入研究。     

破骨细胞血系起源的活细胞成像观察

目的:采用活细胞成像技术,观察血系单核细胞诱导形成破骨细胞的全过程,旨在进一步阐明破骨细胞血系起源的发生及其细胞动力学。方法:取成年SPF级纯种雄性SD大鼠1只,体重280 g,腹主动脉采血8 ml,经密度梯度离心分离单个核细胞,在RANKL与M-CSF诱导下,分为倒置相差显微镜观察组、抗酒石酸酸性磷酸酶染色组、噬骨试验扫描电镜观察组、活细胞成像组4组进行培养。倒置相差显微镜观察组从培养开始,在数字显微成像系统下,每天观察记录1次;抗酒石酸酸性磷酸酶染色组培养21 d作酶活性染色鉴定;噬骨试验扫描电镜观察组培养21 d取出骨磨片作扫描电镜观察;活细胞成像组采用多点位缩时电影法对整个培养过程进行长达35 d的连续观察记录。结果:诱导培养2周后,倒置相差显微镜观察可见大量多核细胞形成,外形呈圆形、梭形、扇形、椭圆形及不规则突起状;抗酒石酸酸性磷酸酶染色绝大部分多核细胞与单核细胞均呈阳性反应;骨磨片扫描电镜观察可见较多骨吸收陷窝、坑洼及沟道,还有位于陷窝及沟道内正在行使骨吸收功能的破骨细胞;活细胞成像观察到起源于周围血的多核破骨细胞是由单核细胞、单核细胞与多核细胞及多核细胞之间相互融合而成,其细胞间的融合均发生在贴壁状态,显微缩时电影观察显示破骨细胞形态表现复杂多变。结论:大鼠周围血单核细胞在RANKL和M-CSF诱导下,可以向破骨细胞分化,形成具有骨吸收功能的多核破骨细胞。破骨细胞的形成是发生在贴壁状态下多种形式的细胞融合过程,破骨细胞的粘附特性对其存活及功能发挥至关重要。破骨细胞具有吞噬功能,其形态结构动态多变。破骨细胞不仅是一种多核细胞,还可能包括单核破骨细胞。破骨细胞通过融合形成多核巨细胞的特性,可能是其适应功能需求与骨吸收效率的一种特殊生物学行为。实验结果进一步证实了破骨细胞的血系起源学说,并为深入阐明破骨细胞的细胞动力学与细胞生物学特性提供了新的实验研究依据。     

The small molecule harmine regulates NFATc1 and Id2 expression in osteoclast progenitor cells

Small molecule compounds that potently affect osteoclastogenesis could be useful as chemical probes for elucidating the mechanisms of various biological phenomena and as effective therapeutic strategies against bone resorption. An osteoclast progenitor cell-based high-throughput screening system was designed to target activation of NFAT, which is a key event for osteoclastogenesis. Orphan ligand library screening using this system identified the β-carboline derivative harmine, which is a highly potent inhibitor of dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A), to be an NFAT regulator in osteoclasts. RAW264.7 cells highly expressed DYRK1A protein, and in vitro phosphorylation assay demonstrated that harmine directly inhibited the DYRK1A-mediated phosphorylation (in-activation) of NFATc1. Harmine promoted the dephosphorylation (activation) of NFATc1 in RAW264.7 cells within 24h, and it significantly increased the expression of NFATc1 in RAW264.7 cells and mouse primary bone marrow macrophages (BMMs) both in the presence and absence of RANKL stimulation. Although harmine promoted NFATc1 expression and stimulated target genes for osteoclastogenesis, cell-cell fusion and the formation of TRAP-positive multinucleated osteoclasts from RAW264.7 cells and BMMs was significantly inhibited by harmine treatment. Meanwhile, harmine remarkably promoted the expression of inhibitor of DNA binding/differentiation-2 (Id2), which is a negative regulator for osteoclastogenesis, in RAW264.7 cells and BMMs. An Id2-null-mutant showed slightly increased osteoclast formation from BMMs, and the harmine-mediated inhibition of osteoclast formation was abolished in the BMMs of Id2-null-mutant mice. These results suggest that harmine is a potent activator of NFATc1 that interferes with the function of DYRK1A in osteoclast precursors and also up-regulates Id2 protein, which may dominantly inhibit expression pathways associated with cell-cell fusion, thereby leading to the disruption of the fusion events mediating osteoclastogenesis. The small molecule harmine is therefore expected to provide an experimental tool for investigating signaling cascades in osteoclastogenesis, especially those centered on DYRK1A-mediated NFATc1 and Id2 regulation.