Nakamura Megumi, Kondo Hiroshi, Shimada Yukiko, Waheed Abdul A, Ohno-Iwashita Yoshiko
Department of Protein Biochemistry, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
Exp Cell Res. 2003 Nov 1;290(2):381-90. doi: 10.1016/s0014-4827(03)00343-4.
Recently it has been shown that cholesterol plays indispensable roles in the function of cholesterol-rich microdomains (rafts), such as in ligand-mediated signal transduction. Using a perfringolysin O derivative (BCtheta) that binds selectively to cholesterol in rafts without causing membrane damage (Proc. Natl. Acad. Sci. USA 98 (2001) 4926), we have investigated the effect of in vitro replicative aging of human diploid fibroblasts, TIG-1, on the distribution of plasma membrane cholesterol. The amount of BCtheta-labeled membrane cholesterol decreased during replicative aging of TIG-1 cells, whereas total cholesterol increased somewhat. The relationship was confirmed by double staining with BCtheta and senescence-associated-beta-galactosidase, a biomarker of senescent cells. Cell fractionation experiments revealed decreases in both cholesterol in rafts and a raft marker, flotillin, during replicative aging. In addition, hydroxyurea-induced prematurely senescent cells also showed a lower level of BCtheta-labeled cholesterol than untreated cells, despite maintaining the total amount of cholesterol. When TIG-1 cells were cultured in cholesterol-deficient medium, BCtheta labeling was first diminished and then premature senescence was induced. Taken together with the reduced signaling capacity of aged cells, these results suggest that plasma membrane cholesterol in raft microdomains is more sensitive to senescence than total cholesterol and is a primary target in aging.
最近研究表明，胆固醇在富含胆固醇的微结构域（脂筏）的功能中发挥着不可或缺的作用，比如在配体介导的信号转导过程中。我们使用一种产气荚膜梭菌溶素O衍生物（BCtheta），它能选择性地结合脂筏中的胆固醇而不引起膜损伤（《美国国家科学院院刊》98 (2001) 4926），研究了人二倍体成纤维细胞TIG-1体外复制性衰老对质膜胆固醇分布的影响。在TIG-1细胞的复制性衰老过程中，BCtheta标记的膜胆固醇量减少，而总胆固醇略有增加。用BCtheta和衰老相关β-半乳糖苷酶（一种衰老细胞的生物标志物）进行双重染色证实了这种关系。细胞分级分离实验表明，在复制性衰老过程中，脂筏中的胆固醇和脂筏标志物flotillin均减少。此外，羟基脲诱导的早衰细胞尽管总胆固醇量保持不变，但BCtheta标记的胆固醇水平也低于未处理的细胞。当TIG-1细胞在胆固醇缺乏的培养基中培养时，BCtheta标记首先减少，然后诱导早衰。结合衰老细胞信号传导能力的降低，这些结果表明，脂筏微结构域中的质膜胆固醇比总胆固醇对衰老更敏感，是衰老过程中的主要靶点。
