在鼠标破骨细胞前体Notch信号的刺激
Summary
Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.
Abstract
Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state.
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.
Introduction
哺乳动物Notch信号传导途径是同源的果蝇的相同的途径和由四个跨膜Notch受体（Notch1-4）和铁血（JAG1＆JAG2）和Delta样（DLL1，3-五膜结合配体，及4）家庭1。当在接收单元Notch受体被配体结合在一传送单元2 Notch信号被启动。在此反式激活，膜结合Notch配体上产生的膜结合Notch受体3,4的拉伸力。配体结合的拉伸力诱导的Notch受体由肿瘤坏死因子α促进受体的胞外切割位转换酶（TACE），随后通过含早老-γ分泌复合物（γ分泌）介导的细胞内切割事件的构象变化。 γ分泌释放缺口INTracellular域（NICD），该易位进入它形成具有CBF -1-苏（H）-Lag-1（CSL），主脑样（MAML）一个转录激活复合物的核和细胞类型特异性的因素来驱动表达的靶基因5。
的Notch信号传导的激活结果中，需要在体外 Notch途径激活的独特方法的机械元件。可溶性Notch配体可以结合Notch受体，但不能产生拉伸所需NICD释放力，而在同一时间竞争性地抑制细胞相关的Notch配体的结合。因此，除了可溶性Notch配体到培养基可以减轻正常Notch信号6,7。幸运的是，Notch配体可诱导NICD释放，如果他们被固定到合适的刚性基板5，8，9，10。上涂覆的配体培养基材播种细胞或施加涂覆配体珠的细胞可以既激活Notch信号，以及它们之间的选择主要取决于缺口刺激期望的定时。立即，临时Notch信号传导的激活，如将一个功能或分化测定的中点期间需要，Notch配体可以结合到琼脂糖珠，施加到培养的细胞，并在任何时间洗掉。为从培养期开始时更持续的Notch信号，组织培养板可涂之前细胞接种配体。
对于此协议的目的，方法被进行了使用小鼠的破骨细胞的前体，但在这里所描述的方法的方法和变体适用于各种各样的细胞类型6，11，12，13， </SUP> 14。破骨细胞是终末分化的造血行数的细胞，它们负责骨组织的再吸收，并且它们在骨损失15的多个障碍有关。因此， 在他们的单核/巨噬细胞系细胞前体破骨细胞和分子机制控制它们的功能是为了更好地理解破骨细胞和新骨再生疗法的发展至关重要分化的体外研究。而它现在普遍接受的是Notch信号起着破骨细胞的分化和功能的积极作用，在这两种Notch信号传导的刺激和破骨细胞的前体培养和分化的变化导致最初矛盾的发现16，17，18，19。在方法的差异进一步检查和利用遗传模型已经大大澄清Notch信号在破骨细胞的作用，但标准化的缺口刺激和培养方法的应用程序可以防止这类争议在其他类型的细胞20，21，22，23 Notch信号的未来研究。
有用于培养和分化小鼠破骨细胞前体的多种方法，并且，与用于刺激Notch信号改变方法，最好的方法将取决于实验问题。这里，我们的优选培养来自小鼠长骨冲洗骨髓细胞的粘附和非粘附的级分的方法将提交。这种方法有本质上不需要专门的设备并生产了适用于各种分化方法的细胞的优点。
Protocol
Representative Results
Discussion
该协议中的关键步骤
文化与破骨细胞前体体外分化为破骨细胞和骨再生，骨量保存治疗靶点识别的分子机制研究一个有益的平台。当鼠标培养破骨细胞的前体，最关键的因素是一个天真的状态前体的维护。作为巨噬细胞样细胞，破骨细胞前体填装到细菌成分和促炎细胞因子响应。在激活时，破骨细胞前体将不再有效地分化成破骨细胞。前体活化的主要原因是炎性细…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the University of Pennsylvania Center for Musculoskeletal Disorders (5 P30 AR050950-09), a grant from the AO Foundation (S-16-12A), the Philadelphia VA Medical Center Translational Musculoskeletal Research Center, and an intramural orthopaedic surgery departmental research development fund. JWA is supported by the University of Pennsylvania Postdoctoral Opportunities in Research and Teaching (PENN-PORT) fellowship funded by the National Institute of General Medical Sciences Institutional Research and Career Development Award (IRACDA; 5 K12 GM081259-08).
Materials
| Recombinant mouse M-CSF | Biolegend | 576402 | Available from multiple suppliers, test activity before experiments |
| Recombinant mouse RANKL | Shenandoah Biotechnology | 200-04 | Available from multiple suppliers, test activity before experiments |
| Recombinant human Jagged1-Fc | R&D Systems | 1277-JG-050 | Available from multiple suppliers, test activity before experiments |
| Protein G agarose beads | InvivoGen | gel-agg-2 | |
| Goat anti-human IgG Fc | Jackson ImmunoResearch | 109-001-008 | |
| Minimum Essential Medium powder | Sigma-Aldrich | M0894 | |
| Accutase cell dissociation reagent | ThermoFisher | A1110501 | Used to lift osteoclast precursors |
| Acid Phosphatase, Leukocyte (TRAP) Kit | Sigma-Aldrich | 387A-1KT | Used to stain differentiated osteoclasts |
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