Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior.
Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior.
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RANKL刺激的小鼠RAW 264.7细胞向破骨细胞分化的时间和行为。
DOI：
10.3390/biology10020117
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发表时间：
2021-02-04
期刊：
影响因子：
4.2
通讯作者：
Zito F
中科院分区：
文献类型：
--
作者：
Lampiasi N;Russo R;Kireev I;Strelkova O;Zhironkina O;Zito F
多核细胞的形成是成熟破骨细胞生成的关键。要做到这一点，单核破骨前细胞会迁移到其他破骨前细胞附近，从而融合在一起。这一精细调控的过程依赖于相邻细胞之间的“感觉”和“识别”。在这项研究中，我们重点研究了破骨细胞前融合的时间和行为，并强调了破骨细胞分化4天期间形态和细胞骨架组织的变化。首次描述了有趣的细胞延伸，推测其功能是作为迁移中介。关键蛋白的亚细胞定位与破骨细胞成熟时间相关。特别是，首次在两个有丝分裂原激活的蛋白激酶和一个主要转录因子之间显示出与核相互排斥的关系。破骨细胞融合过程中涉及的一些基因表达的不同趋势与破骨细胞分化的时机有关。尽管还需要进一步的研究，但我们相信这项研究将有助于理解调控破骨细胞形成的初始过程的机制，包括迁移和融合，这反过来对于许多与骨相关的疾病的治疗具有重要意义，如骨质疏松、骨化症和类风湿性关节炎。多核细胞的发育对破骨细胞(OCs)的成熟和功能至关重要。我们的目标是扩展关于破骨细胞发生的知识，重点是OC前融合的时机和行为。RAW 264.7细胞是一种小鼠单核-巨噬细胞系，为破骨细胞发生机制的体外研究提供了一种有价值和广泛使用的工具。用核因子κ-B受体激活剂配体(RANKL)处理细胞1~4天，用透射电子显微镜、免疫荧光和定量聚合酶链式反应检测细胞形态、细胞骨架结构、蛋白质分布和OC特异性基因表达。核因子κ-B受体激活剂刺激后两天开始出现多核细胞，随后几天细胞数量和大小增加，并伴有形态和细胞骨架结构的改变。在用小麦胚凝集素(WGA)-异硫氰酸荧光素(FITC)标记的细胞内，在三天内观察到有趣的细胞延伸。RANK、TRAF6、p-p38、pERK1/2和NFATc1的胞膜、胞浆和胞核分布与OCS成熟时间有关。调控破骨细胞发生的转录因子(NFATc1、c-fos、relA、MITF)、RANKL信号转导相关分子(TRAF6)、细胞骨架调节(RhoA)、融合(DC-STAMP)、迁移(MMP9)和OC特异性酶(TRAP、CTSK)的基因表达与OC分化时间呈现不同趋势。我们的发现为RAW 264.7细胞在RANKL刺激过程中发生的形态和分子变化提供了一个综合的观点，这对于更好地理解OCS的成熟过程是重要的。
The formation of multinucleated cells is critical for mature osteoclast generation. For this to happen, mononuclear pre-osteoclasts migrate in proximity to other pre-osteoclasts to fuse together. This finely regulated process is dependent on the “feeling” and “recognition” between neighboring cells. In this study, we focused on pre-osteoclast fusion timing and behavior, and highlighted changes in morphological and cytoskeletal organization during 4 days of osteoclast differentiation. For the first time, interesting cellular extensions have been described, with the presumed function to serve as migration mediators. The sub-cellular localization of key proteins correlated with the osteoclast maturation timing. In particular, for the first time, a relationship of mutual exclusion from the nuclei has been shown between two mitogen activated protein kinases and a master transcription factor. The different trends in the expression of some genes involved in the osteoclast fusion process were related to osteoclast differentiation timing. Although further investigation is needed, we are confident that this study will contribute to the understanding of the mechanisms regulating the initial processes of osteoclastogenesis, including migration and fusion, which in turn are of fundamental importance for the management of many bone-related diseases, such as osteoporosis, osteopetrosis and rheumatoid arthritis. The development of multi-nucleated cells is critical for osteoclasts (OCs) maturation and function. Our objective was to extend knowledge on osteoclastogenesis, focusing on pre-OC fusion timing and behavior. RAW 264.7 cells, which is a murine monocyte-macrophage cell line, provide a valuable and widely used tool for in vitro studies on osteoclastogenesis mechanisms. Cells were treated with the receptor activator of nuclear factor κ-B ligand (RANKL) for 1–4 days and effects on cell morphology, cytoskeletal organization, protein distribution, and OC-specific gene expression examined by TEM, immunofluorescence, and qPCR. Multinucleated cells began to appear at two days of Receptor Activator of Nuclear factor κ-B Ligand (RANKL) stimulation, increasing in number and size in the following days, associated with morphological and cytoskeletal organization changes. Interesting cellular extensions were observed in three days within cells labeled with wheat germ agglutinin (WGA)-Fluorescein isothiocyanate (FITC). The membrane, cytoplasmic, or nuclear distribution of RANK, TRAF6, p-p38, pERK1/2, and NFATc1, respectively, was related to OCs maturation timing. The gene expression for transcription factors regulating osteoclastogenesis (NFATc1, c-fos, RelA, MITF), molecules involved in RANKL-signaling transduction (TRAF6), cytoskeleton regulation (RhoA), fusion (DC-STAMP), migration (MMP9), and OC-specific enzymes (TRAP, CtsK), showed different trends related to OC differentiation timing. Our findings provide an integrated view on the morphological and molecular changes occurring during RANKL stimulation of RAW 264.7 cells, which are important to better understand the OCs’ maturation processes.
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