Wu Xiaojun, Ahn Eun-Young, McKenna Margaret A, Yeo Hyeonju, McDonald Jay M
Department of Pathology, University of Alabama at Birmingham, 35294, USA.
J Biol Chem. 2005 Aug 19;280(33):29964-70. doi: 10.1074/jbc.M500710200. Epub 2005 Jun 17.
Promotion of osteoclast apoptosis is one therapeutic approach to osteoporosis. Calmodulin, the major intracellular Ca(2+) receptor, modulates both osteoclastogenesis and bone resorption. The calmodulin antagonist, trifluoperazine, rescues bone loss in ovariectomized mice (Zhang, L., Feng, X., and McDonald, J. M. (2003) Endocrinology 144, 4536-4543). We show here that a 3-h treatment of mouse osteoclasts with either of the calmodulin antagonists, tamoxifen or trifluoperazine, induces osteoclast apoptosis dose-dependently. Tamoxifen, 10 microm, and trifluoperazine, 10 microm, induce 7.3 +/- 1.8-fold and 5.3 +/- 0.9-fold increases in osteoclast apoptosis, respectively. In Jurkat cells, calmodulin binds to Fas, the death receptor, and this binding is regulated during Fas-mediated apoptosis (Ahn, E. Y., Lim, S. T., Cook, W. J., and McDonald, J. M. (2004) J. Biol. Chem. 279, 5661-5666). In osteoclasts, calmodulin also binds Fas. When osteoclasts are treated with 10 microm trifluoperazine, the binding between Fas and calmodulin is dramatically decreased at 15 min and gradually recovers by 60 min. A point mutation of the Fas death domain in the Lpr(-cg) mouse renders Fas inactive. Using glutathione S-transferase fusion proteins, the human Fas cytoplasmic domain is shown to bind calmodulin, whereas a point mutation (V254N) comparable with the Lpr(-cg) mutation in mice has markedly reduced calmodulin binding. Osteoclasts derived from Lpr(-cg) mice have diminished calmodulin/Fas binding and are more sensitive to calmodulin antagonist-induced apoptosis than those from wild-type mice. Both tamoxifen- and trifluoperazine-induced apoptosis are increased 1.6 +/- 0.2-fold in Lpr(-cg)-derived osteoclasts compared with osteoclasts derived from wild-type mice. In summary, calmodulin antagonists induce apoptosis in osteoclasts by a mechanism involving interference with calmodulin binding to Fas. The effects of calmodulin/Fas binding on calmodulin antagonist-induced apoptosis may open a new avenue for therapy for osteoporosis.
促进破骨细胞凋亡是治疗骨质疏松症的一种方法。钙调蛋白是主要的细胞内钙离子受体，可调节破骨细胞生成和骨吸收。钙调蛋白拮抗剂三氟拉嗪可挽救去卵巢小鼠的骨质流失（Zhang, L., Feng, X., and McDonald, J. M. (2003) Endocrinology 144, 4536 - 4543）。我们在此表明，用钙调蛋白拮抗剂他莫昔芬或三氟拉嗪对小鼠破骨细胞进行3小时处理，可剂量依赖性地诱导破骨细胞凋亡。10微摩尔的他莫昔芬和10微摩尔的三氟拉嗪分别使破骨细胞凋亡增加7.3±1.8倍和5.3±0.9倍。在Jurkat细胞中，钙调蛋白与死亡受体Fas结合，且这种结合在Fas介导的凋亡过程中受到调节（Ahn, E. Y., Lim, S. T., Cook, W. J., and McDonald, J. M. (2004) J. Biol. Chem. 279, 5661 - 5666）。在破骨细胞中，钙调蛋白也与Fas结合。当破骨细胞用10微摩尔三氟拉嗪处理时，Fas与钙调蛋白之间的结合在15分钟时显著降低，并在60分钟时逐渐恢复。Lpr(-cg)小鼠中Fas死亡结构域的点突变使Fas失活。使用谷胱甘肽S -转移酶融合蛋白，显示人Fas细胞质结构域与钙调蛋白结合，而与小鼠Lpr(-cg)突变相当的点突变（V254N）显著降低了钙调蛋白结合。源自Lpr(-cg)小鼠的破骨细胞的钙调蛋白/Fas结合减少，并且比源自野生型小鼠的破骨细胞对钙调蛋白拮抗剂诱导的凋亡更敏感。与源自野生型小鼠的破骨细胞相比，源自Lpr(-cg)小鼠的破骨细胞中，他莫昔芬和三氟拉嗪诱导的凋亡均增加了1.6±0.2倍。总之，钙调蛋白拮抗剂通过干扰钙调蛋白与Fas结合的机制诱导破骨细胞凋亡。钙调蛋白/Fas结合对钙调蛋白拮抗剂诱导凋亡的影响可能为骨质疏松症的治疗开辟一条新途径。
