A role of protein phosphatase 2C on osteoclast differentiation and activation

NF-κB配体受体激活剂（RANKL）是肿瘤坏死因子家族的一员，在破骨细胞的分化、激活和存活中发挥着关键作用。 RANKL与其在破骨细胞祖细胞或成熟破骨细胞上表达的受体RANK相互作用，然后诱导NF-κB和p38 MAPK等下游分子的激活。由于蛋白磷酸酶 2C (PP2C) 已被报道为 SAPK/p38 信号通路的负调节因子之一，因此我们研究了 PP2C 是否可能通过调节 RANKL/RANK 信号通路参与破骨细胞生成的可能性。本课题的研究结果可以得出以下结论： 1、人胚肾上皮293细胞转染全长RANK（RANKL的受体）表达质粒后，p38 MAPK磷酸化水平和NF-κB活性上调。我们发现PP2C的共表达抑制了p38 MAPK和NF-κB.2的激活；我们生成了293细胞，其稳定表达Myc表位标记的RANK，称为293-RANK细胞。当用 RANKL 刺激这些细胞时，观察到 p38 MAPK 磷酸化水平增加和 NF-κB 激活。 PP2C 的过表达抑制了这些 RANKL 诱导的 p38 磷酸化和 NF-κB 激活。3；RAW264 细胞是巨噬细胞/单核细胞细胞系，已知在 RANKL 刺激后分化为破骨细胞样多核细胞。在 RANKL 刺激后定期从 RAW264 细胞中制备总 mRNA，并进行定量实时 PCR 以分析 PP2C 表达。结果表明，RANKL 刺激后 PP2C mRNA 水平短暂增加。总之，我们的结果表明 PP2C 可能与破骨细胞分化的调节有关。

The receptor activator of NF-κB ligand (RANKL), which is a member of the tumor necrosis factor family, plays a key role in the differentiation, activation and survival of the osteoclasts. RANKL interacts with its receptor, RANK, which is expressed on osteoclast progenitors or mature osteoclasts, and then induce activation of downstream molecules such as NF-κB and p38 MAPK. Since protein phosphatase 2C (PP2C) has been reported as one of the negative regulators in SAPK/p38 signaling pathway, we examined the possibility if PP2C might be involved in osteoclastogenesis through the regulation of RANKL/RANK signaling pathway. The following conclusions can be drawn from the results of this research project.1;When human embryonic kidney epithelial 293 cells were transfected with a full-length RANK(a receptor of RANKL) expression plasmids, phosphorylation level of p38 MAPK and NF-κB activity were upregulated. We found that coexpression of PP2C suppressed activation of both p38 MAPK and NF-κB.2;We generated 293 cells, which stably express Myc epitope-tagged RANK, called 293-RANK cells. When these cells were stimulated with RANKL, increase in phosphorylation level in p38 MAPK and activation of NF-κB were observed. Overexpression of PP2C inhibited these RANKL-induced p38 phosphorylation and NF-κB activation.3;RAW264 cells, which are macrophage/monocyte cell line, are known to differentiate into osteoclast-like multinucleated cells following RANKL stimulation. Total mRNAs were prepared from RAW264 cells at regular intervals after RANKL stimulation and quantitative realtime PCR was performed in order to analyze PP2C expression. As a result, it was shown that PP2C mRNA levels increased transiently following RANKL stimulation.Taken together, our results suggest the possibility that PP2C might be concerned with the regulation of osteoclast differentiation.