Novel ipriflavone receptors coupled to calcium influx regulate osteoclast differentiation and function
Novel ipriflavone receptors coupled to calcium influx regulate osteoclast differentiation and function
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DOI：
10.1210/en.137.8.3544
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发表时间：
1996-08-01
期刊：
影响因子：
4.8
通讯作者：
Fujita, T
中科院分区：
文献类型：
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作者：
Miyauchi, A;Notoya, K;Fujita, T
依普利酮(7-异丙氧基异黄酮)是一种有效的抗吸收药物，用于治疗骨质疏松症。然而，其对破骨细胞及其前体细胞的作用机制尚不清楚，为了确定其机制是否涉及对破骨细胞或其前体细胞的直接作用，我们检测了依普列酮对破骨细胞及其前体细胞内游离钙离子([Ca+](I))的影响，并测定了H-3标记的依普列芬的特异性结合。高纯度的鸡破骨细胞前体细胞在3-6天内自发分化为多核破骨细胞，负载Fura-2，通过视频成像监测亚细胞内[Ca~(2+)](I)的分布。依普黄酮引起[Ca~(2+)](I)迅速升高，随后持续升高[EC(50)=5×10~(-7)M，263+/-74 nM(SE)(n=8)，升高10(-6)M，持续期]。在分化的鸡破骨细胞和分离的兔破骨细胞中的反应是相同的。胞外Ca~(2+)的内流可能是依普列酮引起[Ca~(2+)](I)变化的原因，因为这种反应可被0.5 mM LaCl3或含EGTA的无钙培养液取消。此外，加入依普列酮后，细胞膜附近可检测到较高的[Ca~(2+)](I)水平。由于尼卡地平(10(-7)M)和维拉帕米(10(-7)M)对依普列酮诱导的[Ca~(2+)](I)反应无影响，依普列酮主要通过二氢吡啶不敏感的Ca~(2+)通道诱导钙内流。[H-3]依普列酮结合研究表明，在前体细胞[解离常数(K-d)，7.60×10(-8)M，2.67×10(-6)M]和成熟破骨细胞(K-d，4.98×10(-8)M，3.70×10(-6)M)中均存在依普列酮结合位点(两类)。不同的禽类破骨细胞功能调节剂如雌二醇(10(-6)M)或维甲酸(10(-6)M)不能取代依普列酮的特异性结合，表明依普列酮受体不同于这些钙调节激素的受体。依普黄酮能显著抑制破骨细胞前体细胞的融合，从而剂量依赖性地抑制骨吸收和抗酒石酸酸性磷酸酶活性。在破骨细胞及其前体细胞中发现了与钙离子内流偶联的新型依普列酮受体。这些依普列芬受体可能为调节破骨细胞分化和功能提供了一种机制。
Ipriflavone (7-isopropoxyisoflavone) is an effective antiresorptive agent used to treat osteoporosis. However, the mechanism of its action on osteoclasts and their precursor cells is not well understood, To determine whether the mechanism involves direct effects on osteoclasts or their precursors, we examined the effects of ipriflavone on cytosolic free calcium ([Ca2+](i)) in osteoclasts and their precursors and measured specific binding of H-3-labeled ipriflavone. Highly purified chicken osteoclast precursors, which spontaneously differentiate into multinucleated osteoclasts in 3-6 days, were loaded with fura-2, and the subcellular [Ca2+](i) distribution was monitored by videoimaging. Ipriflavone induced a rapid increase in [Ca2+](i) followed by a sustained elevation [EC(50) = 5 X 10(-7) M, 263 +/- 74 nM (SE) (n = 8) above basal levels, by 10(-6) M ipriflavone, sustained phase]. The responses were the same in differentiated chicken osteoclasts and isolated rabbit osteoclasts. An influx of extracellular Ca2+ is likely to be responsible for the ipriflavone-induced change in [Ca2+](i) because the response was abolished by 0.5 mM LaCl3, or by Ca-free medium containing EGTA. Moreover, high [Ca2+](i) levels were detected adjacent to the cell membrane after ipriflavone addition. Ipriflavone induced Ca influx mainly through dihydropyridine-insensitive Ca2+ channels, because nicardipine (10(-7) M) and verapamil(10(-7) M) had no effects on ipriflavone-induced [Ca2+](i) responses. [H-3]Ipriflavone binding studies indicated the presence of specific ipriflavone binding sites (two classes), both in precursor cells [dissociation constant (K-d), 7.60 x 10(-8) M, 2.67 x 10(-6) M] and in mature osteoclasts (K-d, 4.98 x 10(-8) M, 3.70 x 10(-6) M). Specific ipriflavone binding was not displaced by various modulators of avian osteoclast function, such as estradiol (10(-6) M) or retinoic acid(10(-6) M), indicating that ipriflavone receptors differ from the receptors for these Ca-regulating hormones. The fusion of osteoclast precursor cells was significantly inhibited by ipriflavone, which led to dose-dependent inhibition of bone resorption and tartrate-resistant acid phosphatase activity. Novel specific ipriflavone receptors that are coupled to Ca2+ influx were demonstrated in osteoclasts and their precursor cells. These ipriflavone receptors may provide a mechanism to regulate osteoclast differentiation and function.