Functional analysis of apoptosis-related proteins in osteoclasts

骨骼系统的动态平衡是由骨重建过程维持的，骨重建过程依赖于成骨细胞的骨形成和破骨细胞的骨吸收之间的微妙平衡。破骨细胞是主要负责骨吸收的多核巨细胞，在完成任务后迅速发生凋亡。我们已报道促凋亡的Bcl2家族分子Bim在破骨细胞的凋亡中起重要作用，并且在破骨细胞的骨吸收活性中起着关键作用。然而，抗凋亡分子Bcl2、Bclxl对破骨细胞存活和骨吸收活性的影响尚未完全阐明。因此，我们将Bcl2家族分子视为有机分子复合体，建立了bcl2-/-、bcl2-/-bim+/-小鼠和bclx条件性基因敲除小鼠，并分析了这些分子对破骨细胞存活和骨吸收活性的影响。我们在体外实验中证明了抗凋亡分子Bcl2和Bclxl促进了破骨细胞的存活。体外实验和体内实验表明，BclxL抑制破骨细胞的骨吸收活性，而Bcl2虽然是抗凋亡的Bcl2亚家族，但对破骨细胞的功能有一定的促进作用。Bcl2-/-小鼠的健康状况不佳和过早死亡，阻碍了对bcl2基因缺失在骨骼组织中作用的体内分析。因此，我们产生了bcl2-/-bim+/-小鼠，在这些小鼠中，一个bim等位基因被敲除，代表它们在成年后正常生长。通过对bcl2-/-bim+/-小鼠的分析，我们发现bcl2对于维持成年小鼠正常的骨内环境平衡是必不可少的。因此，除了对细胞的生死进行调控外，Bcl2家族蛋白在细胞的功能上也起着关键的作用。

Homeostasis of the skeletal system is maintained by a bone remodeling process, which is dependent on a delicate balance between bone formation by osteoblasts and bone resorption by osteoclasts. Osteoclasts are multinucleated giant cells primarily responsible for bone resorption, which rapidly underwent apoptosis after finishing their tasks. We have previously reported that proapoptotic Bcl-2 family molecule Bim played an essential role for apoptosis of osteoclasts, and moreover played a pivotal role on bone resorption activity of osteoclasts. However, the roles of anti-apoptotic molecule Bcl-2, Bcl-xL on survival and bone resorption activity of osteoclasts have not been fully elucidated. We therefore regarded Bcl-2 family molecules as the organic molecular complex, and generated bcl-2-/-, bcl-2-/- bim+/- mice, and bcl-x conditional knockout mice and analyzed the roles of the molecules on survival and bone resorption activity of osteoclasts. We demonstrated that anti-apoptotic molecule Bcl-2 and Bcl-xL promoted the survival of osteoclasts in ex vivo experiments. Remarkably, Bcl-xL suppressed the bone resorption activity of osteoclasts ; in contrast, Bcl-2 reasonably promoted the function of osteoclasts in ex vivo and in vivo experiments though they are the same anti-apoptotic Bcl-2 subfamily. The in vivo analysis of the role of bcl-2 deficiency in the skeletal tissue is hampered by the ill health and early death of bcl-2-/-mice. We therefore generated bcl-2-/- bim+/-mice in which a single bim allele was knocked out and represented that they grew normally into adults. We revealed that Bcl-2 was essential for maintaining normal bone homeostasis in the adult mice by analyzing bcl-2-/- bim+/-mice. Thus, Bcl-2 family proteins play pivotal roles on the function of respective cells in addition to regulation of their life and death.