Udagawa N, Takahashi N, Jimi E, Matsuzaki K, Tsurukai T, Itoh K, Nakagawa N, Yasuda H, Goto M, Tsuda E, Higashio K, Gillespie M T, Martin T J, Suda T
Department of Biochemistry, School of Dentistry, Showa University, Tokyo, Japan.
Bone. 1999 Nov;25(5):517-23. doi: 10.1016/s8756-3282(99)00210-0.
We previously reported that osteoblasts/stromal cells are essentially involved in the activation as well as differentiation of osteoclasts through a mechanism involving cell-to-cell contact between osteoblasts/stromal cells and osteoclast precursors/osteoclasts. Osteoclast differentiation factor (ODF, also called RANKL/OPGL/TRANCE) and macrophage colony-stimulating factor (M-CSF, also called CSF-1) are two essential factors produced by osteoblasts/stromal cells for osteoclastogenesis. In other words, osteoblasts/stromal cells were not necessary to generate osteoclasts from spleen cells in the presence of both ODF/RANKL and M-CSF. In the present study, we examined the precise roles of ODF/RANKL and M-CSF in the activation of osteoclasts induced by calvarial osteoblasts. Osteoclasts were formed in mouse bone marrow cultures on collagen gel-coated dishes in response to a soluble form of ODF/RANKL (sODF/sRANKL) and M-CSF, and recovered by collagenase digestion. When recovered osteoclasts were further cultured on plastic dishes, most of the osteoclasts spontaneously died within 24 h. Osteoclasts cultured for 24 h on dentine slices could not form resorption pits. Addition of sODF/sRANKL to the recovered osteoclasts markedly enhanced their survival and pit-forming activity. M-CSF similarly stimulated the survival of osteoclasts, but did not induce their pit-forming activity. When primary mouse osteoblasts were added to the recovered osteoclasts, resorption pits were formed on dentine slices. Bone-resorbing factors such as 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, or prostaglandin E2 enhanced pit-forming activity of osteoclasts only in the presence of osteoblasts. M-CSF-deficient osteoblasts prepared from op/op mice similarly enhanced pit-forming activity of osteoclasts. The pit-forming activity of osteoclasts induced by sODF/sRANKL or osteoblasts was completely inhibited by simultaneous addition of osteoprotegerin/osteoclastogenesis inhibitory factor, a decoy receptor of ODF/RANKL. Primary osteoblasts constitutively expressed ODF/RANKL mRNA, and its level was upregulated by treatment with 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, and prostaglandin E2. These results, obtained by using an assay system that unequivocally assesses osteoclast activation, suggest that ODF/RANKL but not M-CSF mediates osteoblast-induced pit-forming activity of osteoclasts, and that bone-resorbing factors stimulate osteoclast activation through upregulation of ODF/RANKL by osteoblasts/stromal cells.
我们之前报道过，成骨细胞/基质细胞通过一种涉及成骨细胞/基质细胞与破骨细胞前体/破骨细胞之间细胞间接触的机制，在破骨细胞的激活以及分化过程中发挥着重要作用。破骨细胞分化因子（ODF，也称为RANKL/OPGL/TRANCE）和巨噬细胞集落刺激因子（M-CSF，也称为CSF-1）是成骨细胞/基质细胞产生的两种破骨细胞生成必需因子。也就是说，在同时存在ODF/RANKL和M-CSF的情况下，成骨细胞/基质细胞并非从小鼠脾细胞生成破骨细胞所必需的。在本研究中，我们检测了ODF/RANKL和M-CSF在颅骨成骨细胞诱导的破骨细胞激活中的具体作用。在胶原凝胶包被的培养皿中，小鼠骨髓培养物在可溶性形式的ODF/RANKL（sODF/sRANKL）和M-CSF作用下形成破骨细胞，然后通过胶原酶消化回收。当回收的破骨细胞在塑料培养皿上进一步培养时，大多数破骨细胞在24小时内自发死亡。在牙本质切片上培养24小时的破骨细胞不能形成吸收陷窝。向回收的破骨细胞中添加sODF/sRANKL可显著提高其存活率和陷窝形成活性。M-CSF同样刺激破骨细胞的存活，但不诱导其陷窝形成活性。当将原代小鼠成骨细胞添加到回收的破骨细胞中时，牙本质切片上形成了吸收陷窝。骨吸收因子如1α,25-二羟基维生素D3、甲状旁腺激素或前列腺素E2仅在有成骨细胞存在时增强破骨细胞的陷窝形成活性。从op/op小鼠制备的M-CSF缺陷型成骨细胞同样增强了破骨细胞的陷窝形成活性。sODF/sRANKL或成骨细胞诱导的破骨细胞的陷窝形成活性在同时添加骨保护素/破骨细胞生成抑制因子（ODF/RANKL的诱饵受体）时被完全抑制。原代成骨细胞组成性表达ODF/RANKL mRNA，其水平在受到1α,25-二羟基维生素D3、甲状旁腺激素和前列腺素E2处理后上调。这些通过明确评估破骨细胞激活的检测系统获得的结果表明，介导成骨细胞诱导的破骨细胞陷窝形成活性的是ODF/RANKL而非M-CSF，并且骨吸收因子通过成骨细胞/基质细胞上调ODF/RANKL来刺激破骨细胞激活。
