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

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。利用OPG-/-缺失（OPG-/-）小鼠、MyD88-缺失（MyD88-/-）小鼠和TRIF-缺失（TRIF-/-）小鼠，我们研究了炎症诱导骨吸收的信号转导和细胞间通讯。PGE_2已被证明能促进rankl诱导的前细胞向破骨细胞分化。我们研究了PGE2如何直接促进破骨细胞祖细胞的分化。用EP4表达载体检测PGE_2受体在破骨细胞中的作用。结果：(1)LPS通过TLR4信号刺激纯化破骨细胞的存活。(2)p38MAP激酶本质上参与了骨髓巨噬细胞（破骨细胞前体）向破骨细胞的分化。p38MAP激酶通路在成熟破骨细胞中全部死亡。(3)使用OPG-/-小鼠，我们发现IL-1和LPS通过两个平行事件刺激破骨细胞生成：直接增强RANKL表达和抑制OPG表达。(4)使用MyD88缺失（-/-）小鼠和TRF-/-小鼠，我们发现在IL-1和LPS处理的成骨细胞中，MyD88信号而非TRIF信号主要参与RANKL的表达。MyD88信号对于LPS和IL-1支持的破骨细胞的存活也是必不可少的。(5)消骨毒素、双膦酸盐和锶化合物S12911-2抑制破骨细胞骨吸收。破骨毒素可抑制破骨细胞的窝形成活性，但不影响其分化和存活。v - atp酶诱导的破骨细胞皱边下的酸化触发了双磷酸盐与破骨细胞的结合。S12911-2可抑制破骨细胞褶边的形成。(6)Muramyl dipeptide （MDP）是革兰氏阳性和阴性菌壁肽聚糖免疫佐剂活性的重要结构。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。nod2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。PGE2通过EP2和EP4协同促进raw264.7细胞的破骨分化。相反，成熟破骨细胞不表达功能性EP2或EP4。EP4 cDNA转染成熟破骨细胞后，PGE_2明显抑制了破骨细胞的骨吸收活性。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