| Project/Area Number |
16390528
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Morphological basic dentistry
|
|---|
| Research Institution | KYUSHU UNIVERSITY |
|---|
Principal Investigator |
KUKITA Toshio Kyushu University, Faculty of Dental Science, Professor, 歯学研究院, 教授 (70150464)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KUKITA Akiko Saga University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (30153266)
NAGATA Kengo Kyushu University, Faculty of Dental Science, Research Associate, 歯学研究院, 助手 (90189134)
|
|---|
| Project Period (FY) |
2004 – 2006
|
| Project Status |
Completed (Fiscal Year 2006)
|
| Budget Amount *help |
¥14,300,000 (Direct Cost: ¥14,300,000)
Fiscal Year 2006: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2005: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 2004: ¥7,700,000 (Direct Cost: ¥7,700,000)
|
|---|
| Keywords | osteoclast / differentiation / DC-STAMP / fusion / membrane protein with 7 transmembrane domains / 膜表面抗原 / モノクローナル抗体 / 遺伝子改変動物 / 活性化 / Kat1抗原 / siRNA / 骨吸収 |
|---|
| Research Abstract |
We have firstly developed the cell line to research the fusion mechanism of mononuclear pre-osteoclasts. RAW-D clone, a subclone of murine macrophage-like RAW264 cells, is the most suitable clone highly sensitive to RANKL to differentiate into osteoclasts. As the 2nd step, we have searched gene specifically expressed in RAW-D cells after stimulation with RANKL by use of RNA subtraction techniques. For the 3rd step, we have evaluated the function of obtained genes. We have cloned genome of DC-STAMP from appropriate BAC clone and constructed the targeting vector followed by introducing the vector into ES cells. However, we could not obtain stable ES cells targeted DC-STAMP gene. We have modified the design of the targeting vector and introduced it into ES cells. Now we are trying the efforts to obtain stable ES clone. On the other hand, we have evaluated the function of DC-STAMP in osteoclastogenesis by use of several in vitro cell culture systems. We have transfected RAW-D cells with fu
… More
ll length cDNA for DC-STAMP and ΔT7 DC-STAMP ligated into the expression vector pCIneo. Both of these cDNA construct significantly augmented osteoclastogenesis induced by RANKL. As DC-STAMP and ΔT7 DC-STAMP significantly stimulated formation of osteoclasts with numerous nuclei, it was estimated that DC-STAMP has pivotal roles in the fusion among pre-osteoclasts. By the way, we have ligated DC-STAMP and AT7 DC-STAMP into pEGFPN1 to prepare GFP-fusion protein at C-terminus of these molecules. When we transfected these constructs into RAW-D cells, to our surprise, a significant induction of TRAP-positive cells (also induced TRAP-positive multinucleated cell) was observed. Even in the absence of RANKL. These data suggest a presence of regulatory domain at the C-terminal portion of DC-STAMP. Loss-of-function studies by use of specific siRNA clearly showed a marked inhibition of osteoclastogenesis involving multinucleation process. It is now elucidated that DC-STAMP is a key regulatory molecule in osteoclastogenesis especially in fusion process of pre-osteoclasts. Our findings would open a new aspect of therapeutic way to regulate inflammatory bone destruction. Less
|
