2006 Fiscal Year Final Research Report Summary
The analysis of the mechanism of chondrocyte differentiation and apoptosis
| Project/Area Number |
17591550
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Orthopaedic surgery
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| Research Institution | The University of Tokyo |
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Principal Investigator |
AKIYAMA Toru The University of Tokyo, Faculty of Medicine, Research Associate, 医学部附属病院, 助手 (40376471)
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| Co-Investigator(Kenkyū-buntansha) |
CHUNG Ungil The University of Tokyo, Graduate school of Medicine, Assistant Professor, 大学院医学系研究科, 助教授 (30345053)
TANAKA Sakae The University of Tokyo, Faculty of Medicine, Lecturer, 医学部附属病院, 講師 (50282661)
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| Project Period (FY) |
2005 – 2006
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| Keywords | apoptosis / enchondral ossification / Bcl-2 family molecule / inorganic phosphate |
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| Research Abstract |
During endochondral ossification, chondrocytes undergo hypertrophic differentiation and die by apoptosis. The level of inorganic phosphate(Pi) elevates at the site of cartilage mineralization, and previous studies have proposed that Pi entry into hypertrophic chondrocytes may act as an apoptogen. The Bcl-2 family proteins, which consist of pro- and anti-apoptotic members, are major regulators of mitochondria-initiated apoptosis. To investigate their role in determining the cell fate of chondrocytes, we used chondrogenic ATDC5 cells that differentiate into hypertrophic state in the presence of insulin. When differentiated ATDC5 cells were treated with Pi, they mineralized the surrounding matrix and underwent rapid apoptosis as evidenced by nuclear condensation, Caspase-3 ＆ 7 activation, and Lamin proteolysis. With this system, we analyzed the role of pro- and anti-apoptotic Bcl-2 members in chondrocyte apoptosis. Among 15 pro-apoptotic Bcl-2 members, 7 molecules increased their expressi
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on levels during the course of hypertrophic differentiation, and two in response to Pi stimulation. Of these, gene silencing of a proapoptotic BH3-only molecule bnip3 by RNA interference significantly suppressed Pi-induced apoptosis. Conversely, among anti-apoptotic members examined, overexpression of bcl-xL suppressed, and its knockdown promoted apoptosis. The susceptibility to apoptosis by bcl-xL knockdown was partially restored by simultaneous silencing of bnip3. Pi treatment markedly upregulated the protein level of Bnip3 without affecting Bcl-xL levels. Bnip3 was associated with Bcl-xL and attenuated its anti-apoptotic effect in chondrocytes. Immunohistological examination of murine growth plates revealed that Bcl-xL expressed uniformly in the growth plate chondrocytes, whereas Bnip3 expression was exclusively localized in the hypertrophic chondrocytes. Furthermore, Bnip3 expression was decreased in hypertrophic chondrocytes in mice fed with a low phosphate diet, and the abnormalities in the growth plate were almost completely rescued. In summary, increase in Pi levels in hypertrophic zone causes upregulation of Bnip3 in chondrocytes, which binds to Bcl-xL and consequently impairs its anti-apoptotic effect, and finally causes apoptosis of the cells. Less
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