Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-κB ligand:: Modulation of the expression by osteotropic factors and cytokines
Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-κB ligand:: Modulation of the expression by osteotropic factors and cytokines
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DOI：
10.1006/bbrc.2000.3379
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发表时间：
2000-09-07
影响因子：
3.1
通讯作者：
Sakai, H
中科院分区：
文献类型：
--
作者：
Nakashima, T;Kobayashi, Y;Sakai, H
多种体液因子调节破骨细胞生成。成骨细胞/基质谱系细胞上表达的 NF-κ B 配体 (RANKL) 受体激活剂通过与其受体 RANK（在后者细胞群上表达）结合，在将基本分化信号转导至破骨细胞谱系细胞方面发挥着关键作用；然而，检测RANKL蛋白表达的困难阻碍了我们研究体液因子对RANKL表达的调节。为了测定RANKL的蛋白表达，我们建立了一种新的方法，称为配体-受体沉淀（LRP）蛋白质印迹分析，该方法可以利用RANKL和RANK/骨保护素（OPG）之间的特异性结合特性来特异性浓缩靶蛋白。普通蛋白质印迹法未检测到核后上清液中的 RANKL 蛋白表达，但 LRP 蛋白质印迹分析清楚地表明，RANKL 在小鼠成骨细胞/基质细胞上作为膜结合蛋白产生，并被金属蛋白酶裂解为可溶形式。刺激破骨细胞生成的细胞因子，例如 IL-1β、IL-6、IL-11、IL-17 和 TNF-α，会增加成骨细胞/基质细胞中 RANKL 的表达，同时减少 OPG 的表达。相反，抑制破骨细胞生成的细胞因子，例如 IL-13、INF-γ 和 TGF-β1 抑制 RANKL 和/或增强 OPG 的表达；表达。证明了膜结合 RANKL 和可溶性 RANKL 之间的功能差异，这表明膜结合 RANKL 在小鼠骨髓细胞培养物发育的破骨细胞生成中比可溶性 RANKL 更有效。本研究表明了 LRP Western blot 分析的有用性，该分析表明体液因子对破骨细胞生成的调节部分是通过调节 RANKL 和 OPG 的表达来实现的；在成骨细胞/基质谱系细胞中。 (C) 2000 年学术出版社。
A variety of humoral factors modulate the osteoclastogenesis. Receptor activator of NF-kappa B ligand (RANKL) expressed on osteoblast/stromal lineage cells plays a pivotal role to transduce an essential differentiation signal to osteoclast lineage cells through binding to its receptor, RANK, expressed on the latter cell population; however, the difficulty to detect RANKL protein expression hampers us in investigating the regulation of RANKL expression by humoral facotrs. To determine protein expression of RANKL, we have established a new method, named as a ligand-receptor precipitation (LRP) Western blot analysis, which can specifically concentrate the target protein by the use of specific binding characteristic between RANKL and RANK/osteoprotegrin (OPG). RANKL protein expression in the postnuclear supernatant was not detected by common Western blotting but LRP Western blot analysis clearly showed that RANKL is produced as a membrane-bound protein on murine osteoblasts/stromal cells, and cleaved into a soluble form by metalloprotease. Cytokines stimulating the osteoclastogenesis, such as IL-1 beta, IL-6, IL-11, IL-17, and TNF-alpha, increased the expression of RANKL with decrease of OPG expression in osteoblasts/stromal cells. In contrast, cytokines inhibiting the osteoclastogenesis, such as IL-13, INF-gamma, and TGF-beta 1 suppressed the expression of RANKL, and/or augmented OPG; expression. Functional difference between membrane-bound and soluble RANKL was demonstrated, which showed that membrane-bound RANKL works more efficiently than soluble RANKL in the osteoclastogenesis developed from murine bone marrow cell culture. The present study indicates the usefulness of LRP Western blot analysis, which shows that the modulation of osteoclastogenesis by humoral factors is achieved, in part, by regulation of the expression of RANKL and OPG; in osteoblast/stromal lineage cells. (C) 2000 Academic Press.