An inducible, ligand-independent receptor activator of NF-κB gene to control osteoclast differentiation from monocytic precursors.
An inducible, ligand-independent receptor activator of NF-κB gene to control osteoclast differentiation from monocytic precursors.
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NF-κB基因的诱导，独立于配体的受体活化剂，可控制与单核细胞前体分化的破骨细胞分化。
DOI：
10.1371/journal.pone.0084465
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Giachelli CM
中科院分区：
文献类型：
--
作者：
Rementer CW;Wu M;Buranaphatthana W;Yang HY;Scatena M;Giachelli CM
破骨细胞是骨吸收细胞，对牙齿和骨骼的正常形成和维持至关重要。破骨细胞缺乏可导致异位骨化（HO），这种病理对关节、瓣膜和血管的机械功能尤其有害。另一方面，破骨细胞过度活跃是骨质疏松症的主要原因。一种可靠的方法来控制破骨细胞的产生将有助于作为潜在的自体细胞治疗HO，以及抗骨质疏松药物的高通量药物筛选。在本报告中，我们描述了细胞工程方法的发展，以控制单核前体细胞分化为破骨细胞。核因子κB受体激活因子（RANK）的寡聚化是破骨细胞从单核细胞/巨噬细胞前体分化的必要条件。我们设计了一种小鼠单核细胞系RAW264.7来表达一种融合蛋白，该融合蛋白包括细胞内RANK信号域和fk506衍生的二聚化域，该二聚化域与小分子化学诱导物（CID）结合。用CID处理表达该融合蛋白的病毒感染细胞，观察抗酒石酸酸性磷酸酶活性的剂量依赖性诱导，以及多核破骨细胞的形成。此外，NF-κB信号以cid依赖的方式上调，表明细胞内有效的RANK信号传导。功能性cid诱导的破骨细胞在二维和三维体外吸收实验中都具有强大的矿物质吸收活性。此外，cid诱导的破骨细胞与天然rankl诱导的破骨细胞具有相同的寿命。最重要和最关键的是，这些工程细胞分化成对强效破骨细胞抑制剂骨保护素具有抗性的破骨细胞。综上所述，这些研究首次描述了一种诱导控制单核细胞前体分化为破骨细胞的方法，这可能对未来开发工程化自体细胞疗法以及高通量药物测试系统有用，以治疗不依赖于骨保护素的破骨细胞过度活性疾病。
Osteoclasts are bone-resorbing cells that are critical for the normal formation and maintenance of teeth and skeleton. Osteoclast deficiency can contribute to heterotopic ossification (HO), a pathology that is particularly detrimental to the mechanical functions of joints, valves and blood vessels. On the other hand, osteoclast over-activity is a major cause of osteoporosis. A reliable method for controlled generation of osteoclasts would be useful as a potential autologous cell therapy for HO, as well as high-throughput drug screening for anti-osteoporotic drugs. In this report, we describe the development of a cell engineering approach to control monocytic precursor cell differentiation to osteoclasts. Oligomerization of receptor activator of nuclear factor κB (RANK) is known to be essential for osteoclast differentiation from monocyte/macrophage precursors. We engineered a murine monocytic cell line, RAW264.7 to express a fusion protein comprising the intracellular RANK signaling domain and FK506-derived dimerization domains that bind to a small molecule chemical inducer of dimerization (CID). Virally infected cells expressing this fusion protein were treated with CID and dose-dependent induction of tartrate-resistant acid phosphatase activity, as well as multinucleated osteoclast formation were observed. Furthermore, NF-κB signaling was upregulated in a CID-dependent fashion, demonstrating effective RANK intracellular signaling. Functionally CID-induced osteoclasts had robust mineral resorptive activity in both two-dimensional and three-dimensional in vitro resorption assays. In addition, the CID-induced osteoclasts have the same life span as native RANKL-induced osteoclasts. Most importantly and crucially, the engineered cells differentiated into osteoclasts that were resistant to the potent osteoclast inhibitor, osteoprotegerin. Taken together, these studies are the first to describe a method for inducible control of monocytic precursor differentiation to osteoclasts that may be useful for future development of an engineered autologous cell therapy as well as high-throughput drug testing systems to treat diseases of osteoclast over-activity that are independent of osteoprotegerin.
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