Supperession of Osteoclast Differentiation by Protein Phosphatase 2C

在之前的研究项目中，我们建立了293-RANK细胞系，稳定表达RANKL受体RANK，发现293-RANK细胞中过表达PP2Cβ或PP2CE可抑制RANKL诱导的p38和NF-κB的活化。基于这些结果，我们进一步研究了PP2Cβ和PP2Cε在破骨细胞发生中的生物学作用。从这个研究项目的结果可以得出以下结论：过表达PP2Cβ或PP2Cε可抑制RANKL诱导的JNK和p38的激活，但对ERK无抑制作用。RANKL刺激了位于p38上游的MKK6的活化，而MKK6的上游激酶TAKI也被PP2CP或PP2Cε的过表达抑制。这表明PP2Cβ或PP2Cε可以通过tam2的失活抑制RANKL/RANK信号通路。RAW264细胞经RANKL处理后形成破骨细胞样细胞，用编码PP2Cβ siRNA的质粒瞬时转染。在RANKL处理下，转染PP2Cβ siRNA质粒的细胞TRAP活性和多核细胞数量明显高于转染空载体的细胞。提示抑制PP2Cβ.3可促进RAW264细胞向破骨细胞样细胞的分化。由于我们培育的PP2Cβ敲除小鼠（-/-）是致命的，我们使用PP2Cβ杂合（+/-）小鼠来检测杂合（+/-）小鼠和野生型（+/+）幼崽在骨髓基质细胞的破骨细胞分化方面是否有任何差异。结果，在M-CSF和RANKL作用72小时后，杂合子（+/-）小鼠细胞中的TRAP活性高于野生型（+/+）小鼠。提示PP2Cβ可能在体内对M-CSF和RANKL介导的破骨细胞形成有抑制作用。这些结果提供了强有力的证据，证明PP2Cβ对破骨细胞的发生具有抑制作用。

In the former research project we established the cell line called 293-RANK cells stably expressing RANK, the receptor of RANKL, and showed that overexpressed PP2Cβ or PP2CE in 293-RANK cells suppressed the activation of p38 and NF-κB induced by RANKL. Based on those results, further investigation was performed to elucidate possible biological roles of PP2Cβ and PP2Cε in osteoclastogenesis. The following conclusions can be drawn from the results of this research project.1. Overexpression of PP2Cβ or PP2Cε suppressed activation of JNK as well p38 but not ERK, induced by RANKL. RANKL stimulated activation of MKK6 located upstream of p38 and TAKI, which is an upstream kinase of MKK6, was also suppressed by overexpression of PP2CP or PP2Cε. This indicates that PP2Cβ or PP2Cε can suppress RANKL/RANK signaling pathway via inactivation of TAM.2. RAW264 cells, which form osteoclast-like cells by treatment with RANKL, were transiently transfected with plasmids encoding PP2Cβ siRNA. With RANKL treatment, TRAP activity and number of multinucleated cells were increased in transfected cells with PP2Cβ siRNA plasmids compared to those in transfected cells with empty vectors. It is suggested that differentiation of RAW264 cells into osteoclast-like cells is promoted by inhibition of PP2Cβ.3. Because PP2Cβ knockout mice(-/-), which we generated, were lethal, we used PP2Cβ heterozygous(+/-) mice to examine if there is any difference between heterozygous(+/-) mice and wild type(+/+) littermates in osteoclastic differentiation from their bone marrow stromal cells. As a result, after treatment for 72hr with M-CSF and RANKL, TRAP activity in cells from heterozygous(+/-) mice was higher than those from their wild type(+/+) littermates. This suggests that PP2Cβ might have suppressive effect on M-CSF and RANKL mediated osteoclaast formation in vivo.These results provide strong evidence that PP2Cβ has a suppressive effect on osteoclastogenesis.