| Budget Amount *help |
¥16,630,000 (Direct Cost: ¥15,400,000、Indirect Cost: ¥1,230,000)
Fiscal Year 2007: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000)
Fiscal Year 2006: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2005: ¥7,200,000 (Direct Cost: ¥7,200,000)
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| Research Abstract |
Receptor activator of NF-kappa B ligand (RANKL) is a transmembrane glycoprotein, which plays an essential role in the development of osteoclasts. Extracellular portion of RANKL is cleaved proteolytically to produce soluble form of RANKL, the process called ectodomain shedding, but the molecular mechanism of RANKL shedding and its physiological and pathological role have not been clarified yet. To identify molecules involved in the regulation of RANKL shedding, we developed a novel screening system, in which expression plasmids encoding secreted placental alkaline phosphatase fused with mouse C-terminally truncated RANKL were co-transfected with candidate genes of RANKL sheddases. We have screened 100,000 clones. One of the positive clones, splice variant of CAPRI (Ca^<2+>-promoted Ras inactivator), which had been identified to be a member of RAS-GAP (GTPase-activating protein), was found to have a positive effect to RANKL shedding. CAPRI is a molecule which suppresses the activity of R
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as in Ca2+ dependent manner, but its variant which we identified (ΔCAPRI) lacks GAP related domain, then it will be a dominant negative form of CAPRI. Expression vector for active form of Ras stimulates shedding of RANKL in our assay system. We also found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 had strong RANKL shedding activity. In western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in reduction of the lower and the higher molecular weight products, respectively. Suppression of MMP14 in primary osetoblasts resulted in an increase in membrane-bound RANKL, and promoted osteoclastogenesis in cocultures with bone marrow cells. In addition, osteoclasts from MMP14-deficient mice efficiently induced osteoclastogenesis, which is consistent with the increased osteoclastogenesis in vivo. These results suggest that RANKL shedding is a process of post-transcriptional regulation of RANKL, which downregulates local osteoclastogenesis, and MMP14 plays a critical role. Less
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