Study on mechanism of the coupling between bone resorption and bone formation

研究目的：RANKL的发现阐明了破骨细胞分化的机制及其受成骨细胞调控的功能。骨保护素（Osteoprotegerin， OPG）是RANKL的可溶性诱饵受体，可抑制破骨细胞的分化和功能。OPG缺陷（OPG-/-）小鼠表现出由破骨细胞骨吸收增强引起的严重骨质疏松症。人类缺乏OPG已被证明可导致少年佩吉特病。形态学研究表明，OPG-/-小鼠成骨细胞骨形成被激活。这些结果表明破骨细胞骨吸收通过一种未知因子（偶联因子）巧合地诱导成骨细胞骨的形成。在本研究中，我们研究了成熟破骨细胞产生影响骨代谢的细胞因子的可能性，使用LPS处理成熟破骨细胞。使用OPG -/-小鼠，我们还探索了这种偶联因子是否存在于OPG -/-小鼠的骨骼中。结果：(1)p38MAP激酶主要参与骨髓巨噬细胞（破骨细胞前体）向破骨细胞的分化。p38MAP激酶信号通路在成熟破骨细胞中全部死亡。(2)成熟破骨细胞在LPS作用下产生成骨活化因子，而不产生骨吸收细胞因子如IL-1和IL-6。(3) OPG-/-小鼠血中骨钙素和ALP水平明显升高。骨组织形态学研究表明，OPG-/-小鼠的骨吸收和骨形成均被激活。(4) OPG-/-小鼠血清可溶性RANKL水平也升高。(5) 1,25(OH)_2D_3灌胃OPG-/和野生型小鼠后，血清RANKL浓度显著升高，而野生型小鼠无显著升高。(6)OPO -/-成骨细胞在培养基中大量释放RANKL，加入OPG后RANKL被完全抑制。(7)在OPG -/-小鼠体内注射二膦酸盐（利塞膦酸盐）后，处理小鼠的骨形成相关参数和骨吸收相关参数急剧下降。(8)利塞膦酸钠治疗OPG -/-小鼠可降低血清骨钙素和ALP水平，但未降低血清RANKL水平。(9) OPG -/-小鼠不加速BMP诱导的异位骨形成。结论：(1)成熟破骨细胞产生偶联因子，而不产生骨吸收因子；(2)骨吸收发生部位与骨形成紧密偶联。少

Aim of the study :The discovery of RANKL elucidates the mechanism of osteoclast differentiation and function regulated by osteoblasts. Osteoprotegerin (OPG), a soluble decoy receptor of RANKL, inhibits both differentiation and function of osteoclasts. OPG-deficient (OPG-/-) mice exhibited severe osteoporosis caused by enhanced osteoclastic bone resorption. Deficiency of OPG in human has been shown to result in juvenile Paget's disease. The previous morphological study showed that osteoblastic bone formation was activated in OPG-/-mice. These results suggest that osteoclastic bone resorption coincidentally induces osteoblastic bone formation by an unknown factor (called coupling factor). In the present study, we examined a possibility that mature osteoclasts produce cytokines which influence bone metabolism using mature osteoclasts treated with LPS. Using OPG -/-mice, we also explored whether such a coupling factor is present in bone in OPG -/-mice.Results :(1)p38MAP kinase was essentia … More lly involved in the differentiation of bone marrow macrophages (osteoclast precursors) into osteoclasts. The p38MAP kinase-signaling pathway was all dead in mature osteoclasts. (2)Mature osteoclasts were shown to produce an osteoblast-activating factor but not bone-resorbing cytokines such as IL-1 and IL-6 in response to LPS. (3)Blood levels of osteocalcin and ALP of OPG-/-mice were markedly increased in OPG-/-mice. Bone histomorphometric studies showed that both bone resorption and bone formation were activated in OPG-/-mice. (4)Serum levels of soluble RANKL were also elevated in OPG-/-mice. (5)When 1,25(OH)_2D_3 was administered into OPG -/-and wild-type mice, serum RANKL concentrations were markedly increased in OPG-/-mice but not in wild-type mice. (6)OPO -/-osteoblasts released a large amount of RANKL in the culture medium, which was completely inhibited by adding OPG (7)When bisphosphonate (risedronate) was injected into OPG -/-mice, bone formation -related parameters as well as bone resorption-related parameters were sharply decreased in the treated mice. (8)Treatment of OPG -/-mice with risedronate decreased serum osteocalcin and ALP levels but not the serum RANKL level. (9)Ectopic bone formation induced by BMP is not accelerated in OPG -/-mice.Conclusion :These results suggest (1)that mature osteoclast produce a coupling factor but not bone resorbing factors, and (2)that bone resorption is tightly coupled with bone formation at the sites where borne resorption takes place. Less