Supperession of Osteoclast Differentiation by Protein Phosphatase 2C

在前期的研究中我们建立了稳定表达RANKL受体RANK的293-RANK细胞系，发现在293-RANK细胞中过表达PP2C β或PP2CE可抑制RANKL诱导的p38和NF-κ B的活化。在此基础上，进一步研究了PP2C β和PP2C ε在破骨细胞生成中可能的生物学作用。从本研究项目的结果中可以得出以下结论. PP2C β或PP2C ε的过表达抑制了RANKL诱导的JNK和p38的活化，但不抑制ERK的活化。RANKL刺激的位于p38和TAKI上游的MKK6的活化也受到PP2CP或PP2C ε过表达的抑制，TAKI是MKK6的上游激酶。这表明PP2C β或PP2C ε可以通过灭活TAM来抑制RANKL/RANK信号通路。RAW264细胞通过RANKL处理形成破骨细胞样细胞，用编码PP2C β siRNA的质粒瞬时转染。RANKL处理后，与空载体转染细胞相比，PP2C β siRNA质粒转染细胞的TRAP活性和多核细胞数量增加。提示PP2C β的抑制可促进RAW264细胞向破骨细胞样细胞的分化。由于我们产生的PP2C β敲除小鼠（-/-）是致命的，我们使用PP2C β杂合（+/-）小鼠来检查杂合（+/-）小鼠和野生型（+/+）同窝小鼠在骨髓基质细胞的骨细胞分化方面是否存在任何差异。结果，在用M-CSF和RANKL处理72小时后，杂合（+/-）小鼠细胞中的TRAP活性高于其野生型（+/+）同窝小鼠。提示PP2C β可能对M-CSF和RANKL介导的破骨细胞生成具有抑制作用，为PP2C β抑制破骨细胞生成提供了有力证据。

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.