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

核因子受体激活剂κB配体(RANKL)是肿瘤坏死因子家族的成员，在破骨细胞的分化、激活和存活中起着关键作用。RANKL与其受体RANK相互作用，RANK表达在破骨细胞前体细胞或成熟破骨细胞上，然后诱导下游分子如NF-κB和p38MAPK的激活。鉴于蛋白磷酸酶2C(PP2C)已被报道为SAPK/p38信号通路中的负调控因子之一，我们探讨了PP2C是否可能通过调节RANKL/RANK信号通路而参与破骨细胞的发生。本研究结果可得出以下结论：1.将RANK受体全长RANK表达载体导入人胚胎肾上皮细胞293细胞后，p38MAPK的磷酸化水平和NF-κB活性均上调。我们发现PP2C的共表达抑制了p38MAPK和NF-κB的激活；我们产生了稳定表达Myc表位标记的RANK的293细胞，称为293-RANK细胞。经RANKL刺激后，p38MAPK的磷酸化水平增加，并激活了NF-κB。PP2C的过表达抑制了RANKL诱导的p38磷酸化和核因子-κB的激活。3.RAW264细胞是巨噬细胞/单核细胞系，在RANKL刺激后可分化为破骨细胞样多核细胞。RANKL刺激后，每隔一定时间从RAW264细胞中提取总mRNAs，实时定量聚合酶链式反应分析PP2C的表达。结果表明，在RANKL刺激后，PP2C的mRNA水平一过性升高，提示PP2C可能与破骨细胞分化的调控有关。

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.