| Budget Amount *help |
¥3,850,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥450,000)
Fiscal Year 2007: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
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.
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