Signal transduction and cell-to-cell communication in the bone resorption induced by inflammation

本研究的目的：RANKL的发现阐明了破骨细胞分化和功能受成骨细胞调控的机制。本研究采用OPG缺陷型（OPG-/-）、MyD 88缺陷型（MyD 88-/-）和TRIF缺陷型（TRIF-/-）小鼠，研究炎症诱导的骨吸收过程中的信号转导和细胞间通讯。我们研究了PGE 2如何直接促进破骨细胞祖细胞的分化。结果：（1）LPS通过TLR 4信号刺激破骨细胞的存活，并通过LPS刺激破骨细胞的增殖，使破骨细胞的增殖和分化受到抑制。（2）p38 MAP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。成熟破骨细胞p38 MAP激酶通路全部死亡。（3）OPG-/-小鼠实验表明，IL-1和LPS通过两个途径刺激破骨细胞的生成， 关于我们 平行事件：RANKL表达直接增强和OPG表达抑制。（4）使用MyD 88缺陷（-/-）小鼠和TRF-/-小鼠，我们发现MyD 88信号而不是TRIF信号基本上参与了IL-1和LPS处理的成骨细胞中RANKL的表达。MyD 88信号也是LPS和IL-1支持的破骨细胞存活所必需的。（5）破坏素、二膦酸盐和锶化合物S12911-2抑制骨细胞骨吸收。破坏素显示抑制破骨细胞的陷窝形成活性而不影响其分化和存活。V-ATP酶诱导的破骨细胞皱褶边界下的酸化触发双膦酸盐掺入破骨细胞。S12911-2显示出抑制破骨细胞中的皱褶边缘形成。（6）胞壁酰二肽（MDP）是革兰氏阳性菌和革兰氏阴性菌壁肽聚糖免疫佐剂活性的基本结构。MDP通过RANKL表达协同增强LPS、IL-1α和TNF-α诱导的破骨细胞形成。Nod 2介导的信号似乎参与了MDP诱导的成骨细胞RANKL表达。(7)PGE_2可促进RANKL诱导的前体细胞向破骨细胞的分化。PGE 2通过EP 2和EP 4协同促进RAW264.7细胞向成骨细胞分化。相反，成熟破骨细胞不表达功能性EP 2或EP 4。将EP 4 cDNA转染成熟破骨细胞后，其骨吸收活性明显受到PGE_2的抑制。少

Aim of the study :The discovery of RANKL elucidates the mechanism of osteoclast differentiation and function regulated by osteoblasts. Using OPG-deficient (OPG-/-) mice, MyD88-deficient (MyD88-/-) mice, and TRIF-deficient (TRIF-/-) mice, we examined signal transduction and cell-to-cell communication in the bone resorption induced by inflammation PGE_2 has been shown to enhance RANKL-induced differentiation of the precursor cells into osteoclasts. We examined how PGE2 directly enhance differentiation of osteoclast progenitors. The role of PGE_2 receptors in osteoclasts was examined, using an EP4 expression vector.Results :(1)LPS stimulated the survival of purified osteoclasts through TLR4 signals. (2)p38MAP kinase was essentially involved in the differentiation of bone marrow macrophages (osteoclast precursors) into osteoclasts. The p38MAP kinase pathway was all dead in mature osteoclasts. (3)Using OPG-/-mice, we showed that IL-1 as well as LPS stimulated osteoclastogenesis through two … More parallel events : direct enhancement of RANKL expression and suppression of OPG expression. (4)Using MyD88-deficient (-/-) mice and TRF-/-mice, we showed that MyD88 signals but not TRIF signals were essentially involved in RANKL expression in osteoblasts treated with IL-1 and LPS. MyD88 signals were also essential for the survival of osteoclasts supported by LPS and IL-1. (5)Destruxin, bisphosphonates, and strontium compound S12911-2 inhibit osteoclastic bone resorption. Destruxin was shown to inhibit pit-forming activity of osteoclasts without affecting their differentiation and survival. V-ATPase induced acidification beneath the ruffled borders of osteoclasts triggers the incorporation of bisphosphonates into osteoclasts. S12911-2 was shown to inhibit ruffled border formation in osteoclasts. (6)Muramyl dipeptide (MDP) is the essential structure responsible for the immunoadjuvant activity of peptidoglycan of gram-positive and -negative bacterial walls. MDP synergistically enhanced osteoclast formation induced by LPS, IL-1α and TNF-α through RANKL expression in osteoblasts. Nod2mediated signals appear to be involved in the MDP-induced RANKL expression in osteoblasts. (7) PGE_2 has been shown to enhance RANKL-induced differentiation of the precursor cells into osteoclasts. PGE2 synergistically enhanced osteoclastic differentiation of RAW264.7cells through EP2 and EP4. In contrast, mature osteoclasts did not express functional EP2 or EP4. When EP4 cDNA was transfected into mature osteoclasts, the bone-resorbing activity was markedly inhibited by PGE_2. Less