Urayama S, Kawakami A, Nakashima T, Tsuboi M, Yamasaki S, Hida A, Ichinose Y, Nakamura H, Ejima E, Aoyagi T, Nakamura T, Migita K, Kawabe Y, Eguchi K
The First Department of Internal Medicine, Nagasaki University School of Medicine, Japan.
J Lab Clin Med. 2000 Sep;136(3):181-93. doi: 10.1067/mlc.2000.108754.
Vitamin K2 is used for the treatment of osteoporosis, but the precise mode of action is still not clear. We investigated the effects of vitamin K2 on apoptosis of human osteoblasts. Human osteoblastic cell line MG63 cells and human primary osteoblast-like cells obtained from bone fragments in corrective surgery were used as human osteoblasts. Cells were cultured with or without various concentrations of vitamin K2 and tumor necrosis factor-alpha (TNF-alpha). We then determined the proliferative response, expression of Fas and Bcl-2-related proteins, and Fas-mediated apoptosis of these cells induced by anti-Fas immunoglobulin M (IgM). In addition, the effect of vitamin K2 in osteoblast apoptosis induced by Z-Leu-Leu-Leu-aldehyde (LLL-CHO), etoposide, or staurosporine was also examined. Human osteoblasts did not show spontaneous apoptosis in culture, even in the presence of vitamin K2 or TNF-alpha. Furthermore, proliferation of the cells was not influenced by vitamin K2 or TNF-alpha. Fas was functionally expressed on human osteoblasts, and the treatment with TNF-alpha significantly enhanced both Fas expression and Fas-mediated apoptosis of osteoblasts. The addition of vitamin K2 to the culture resulted in a dose-dependent inhibition of functional Fas expression on osteoblasts, in the presence or absence of TNF-alpha. Treatment of human osteoblasts with vitamin K2 clearly suppressed Bax expression of the cells, although the expression of Bcl-2 was not influenced by vitamin K2. Fas ligand (FasL) cDNA transformants were cytotoxic against osteoblasts, and the cytotoxicity was increased when osteoblasts were treated with TNF-alpha. The addition of vitamin K2 to osteoblasts significantly decreased the cytotoxic effects of FasL cDNA transformants. Furthermore, apoptosis of human osteoblasts induced by LLL-CHO, etoposide, or staurosporine was also clearly suppressed in vitamin K2-treated osteoblasts. Our results suggest that vitamin K2 inhibits apoptotic cell death of osteoblasts and maintains the number of osteoblasts. These actions may explain the therapeutic efficacy of vitamin K2 in osteoporosis.
维生素K2用于治疗骨质疏松症，但其确切作用方式尚不清楚。我们研究了维生素K2对人成骨细胞凋亡的影响。人成骨细胞系MG63细胞以及从矫正手术中的骨碎片获取的人原代成骨样细胞被用作人成骨细胞。细胞在有或无不同浓度的维生素K2和肿瘤坏死因子-α（TNF-α）的情况下进行培养。然后我们测定了这些细胞的增殖反应、Fas和Bcl-2相关蛋白的表达，以及抗Fas免疫球蛋白M（IgM）诱导的Fas介导的这些细胞的凋亡。此外，还检测了维生素K2对由Z-亮氨酰-亮氨酰-亮氨醛（LLL-CHO）、依托泊苷或星形孢菌素诱导的成骨细胞凋亡的影响。人成骨细胞在培养中未显示出自发性凋亡，即使存在维生素K2或TNF-α。此外，细胞的增殖不受维生素K2或TNF-α的影响。Fas在人成骨细胞上功能性表达，TNF-α处理显著增强了Fas表达和成骨细胞的Fas介导的凋亡。在有或无TNF-α的情况下，向培养物中添加维生素K2导致成骨细胞上功能性Fas表达呈剂量依赖性抑制。用维生素K2处理人成骨细胞明显抑制了细胞的Bax表达，尽管Bcl-2的表达不受维生素K2的影响。Fas配体（FasL）cDNA转化体对成骨细胞具有细胞毒性，当用TNF-α处理成骨细胞时细胞毒性增加。向成骨细胞中添加维生素K2显著降低了FasL cDNA转化体的细胞毒性作用。此外，在经维生素K2处理的成骨细胞中，由LLL-CHO、依托泊苷或星形孢菌素诱导的人成骨细胞凋亡也明显受到抑制。我们的结果表明，维生素K2抑制成骨细胞的凋亡性细胞死亡并维持成骨细胞数量。这些作用可能解释了维生素K2在骨质疏松症治疗中的疗效。
