Sasaki Takahisa
Department of Oral Histology, School of Dentistry, Showa University, Tokyo 142-8555, Japan.
Microsc Res Tech. 2003 Aug 15;61(6):483-95. doi: 10.1002/jemt.10370.
The differentiation and functions of osteoclasts (OC) are regulated by osteoblast-derived factors such as receptor activator of NFKB ligand (RANKL) that stimulates OC formation, and a novel secreted member of the TNF receptor superfamily, osteoprotegerin (OPG), that negatively regulates osteoclastogenesis. In examination of the preosteoclast (pOC) culture, pOCs formed without any additives expressed tartrate-resistant acid phosphatase (TRAP), but showed little resorptive activity. pOC treated with RANKL became TRAP-positive OC, which expressed intense vacuolar-type H(+)-ATPase and exhibited prominent resorptive activity. Such effects of RANKL on pOC were completely inhibited by addition of OPG. OPG inhibited ruffled border formation in mature OC and reduced their resorptive activity, and also induced apoptosis of some OC. Although OPG administration significantly reduced trabecular bone loss in the femurs of ovariectomized (OVX) mice, the number of TRAP-positive OC in OPG-administered OVX mice was not significantly decreased. Rather, OPG administration caused the disappearance of ruffled borders and decreased H(+)-ATPase expression in most OC. OPG deficiency causes severe osteoporosis. We also examined RANKL localization and OC induction in periodontal ligament (PDL) during experimental movement of incisors in OPG-deficient mice. Compared to wild-type OPG (+/+) littermates, after force application, TRAP-positive OC were markedly increased in the PDL and alveolar bone was severely destroyed in OPG-deficient mice. In both wild-type and OPG-deficient mice, RANKL expression in osteoblasts and fibroblasts became stronger by force application. These in vitro and in vivo studies suggest that RANKL and OPG are important regulators of not only the terminal differentiation of OC but also their resorptive function. To determine resorptive functions of OC, we further examined the effects of specific inhibitors of H(+)-ATPase, bafilomycin A1, and lysosomal cysteine proteinases (cathepsins), E-64, on the ultrastructure, expression of these enzymes and resorptive functions of cultured OC. In bafilomycin A1-treated cultures, OC lacked ruffled borders, and H(+)-ATPase expression and resorptive activity were significantly diminished. E-64 treatment did not affect the ultrastructure and the expression of enzyme molecules in OC, but significantly reduced resorption lacuna formation, by inhibition of cathepsin activity. Lastly, we examined the expression of H(+)-ATPase, cathepsin K, and matrix metalloproteinase-9 in odontoclasts (OdC) during physiological root resorption in human deciduous teeth, and found that there were no differences in the expression of these molecules between OC and OdC. RANKL was also detected in stromal cells located on resorbing dentine surfaces. This suggests that there is a common mechanism in cellular resorption of mineralized tissues such as bone and teeth.
破骨细胞（OC）的分化和功能受成骨细胞衍生因子的调节，如刺激OC形成的核因子κB受体激活剂配体（RANKL），以及肿瘤坏死因子受体超家族的一个新的分泌成员骨保护素（OPG），它对破骨细胞生成起负调节作用。在对前破骨细胞（pOC）培养的研究中，未添加任何添加剂而形成的pOC表达抗酒石酸酸性磷酸酶（TRAP），但几乎没有吸收活性。用RANKL处理的pOC变成TRAP阳性的OC，其表达强烈的液泡型H(+) - ATP酶并表现出显著的吸收活性。RANKL对pOC的这种作用可通过添加OPG而完全被抑制。OPG抑制成熟OC中皱褶缘的形成并降低其吸收活性，还诱导一些OC凋亡。虽然给予OPG显著减少了去卵巢（OVX）小鼠股骨中的小梁骨丢失，但给予OPG的OVX小鼠中TRAP阳性OC的数量并未显著减少。相反，给予OPG导致大多数OC中皱褶缘消失并降低H(+) - ATP酶表达。OPG缺乏会导致严重的骨质疏松症。我们还研究了在OPG缺陷小鼠切牙实验性移动过程中牙周膜（PDL）中RANKL的定位和OC诱导情况。与野生型OPG（+/+）同窝小鼠相比，施加力后，OPG缺陷小鼠的PDL中TRAP阳性OC显著增加，牙槽骨严重破坏。在野生型和OPG缺陷小鼠中，施加力后成骨细胞和成纤维细胞中的RANKL表达均增强。这些体外和体内研究表明，RANKL和OPG不仅是OC终末分化的重要调节因子，也是其吸收功能的重要调节因子。为了确定OC的吸收功能，我们进一步研究了H(+) - ATP酶特异性抑制剂巴弗洛霉素A1和溶酶体半胱氨酸蛋白酶（组织蛋白酶）抑制剂E - 64对培养的OC的超微结构、这些酶的表达及其吸收功能的影响。在巴弗洛霉素A1处理的培养物中，OC缺乏皱褶缘，H(+) - ATP酶表达和吸收活性显著降低。E - 64处理不影响OC的超微结构和酶分子表达，但通过抑制组织蛋白酶活性显著减少吸收陷窝的形成。最后，我们研究了人乳牙生理性牙根吸收过程中破牙细胞（OdC）中H(+) - ATP酶、组织蛋白酶K和基质金属蛋白酶 - 9的表达，发现OC和OdC之间这些分子的表达没有差异。在正在吸收的牙本质表面的基质细胞中也检测到了RANKL。这表明在骨和牙齿等矿化组织的细胞吸收中存在共同机制。
