Analysis of signal transduction of osteoclast differentiation factor (RANKL) in alveolar bone destruction

内毒素是革兰氏阴性菌外膜的主要成分，在由杆菌引起的炎症性疾病中被认为是一种有效的骨吸收刺激因子。细菌脂蛋白/脂肽也是病原体特有的分子模式。最近发现Toll样受体4(TLR4)是内毒素的信号受体。此外，TLR6与TLR2结合，TLR6与TLR2的复合体识别二酰化的支原体脂肽。由于TLR和IL-1R都使用髓系分化因子88(MyD88)作为共同的细胞质信号分子，TLR的信号级联被认为与IL-1R相似。然而，越来越多的证据也证明了MyD88非依赖通路的存在，这可能解释了单个TLR和IL-1R独特的生物学反应。利用MyD88基因缺陷(-/-)小鼠，我们探讨了MyD88介导的信号在破骨细胞形成中的作用。脂蛋白，合成脂蛋白…更多的IDE(FSL-1)、IL-1a和1.25(OH)_2D_3均能促进原代成骨细胞与野生型小鼠骨髓细胞共培养的破骨细胞形成。骨保护素是RANKL的诱骗受体，在共培养中完全抑制破骨细胞的形成。相反，在MyD88(-/-)小鼠成骨细胞与骨髓细胞共培养中，内毒素、脂肽和IL-1a均不能诱导破骨细胞的形成，而1.25(OH)_2D_3即使在MyD88(-/-)共培养中也能促进破骨细胞的形成。RT-PCR分析表明，野生型和MyD88(-/-)小鼠的原代成骨细胞表达TLR2、TLR4、TLR6和IL-1RmRNAs。脂多糖、脂肽和IL-1a可在24小时内刺激野生型小鼠成骨细胞RANKL mRNA的表达，而MyD88(-/-)小鼠的成骨细胞则不表达RANKL。类似地，内毒素和IL-1a刺激野生型成骨细胞ERK的磷酸化，但不能刺激MyD88(-/-)成骨细胞。在RANKL和M-CSF作用下，MyD88(-/-)小鼠和野生型小鼠的造血细胞在3天内类似地分化为破骨细胞。这些结果提示，MyD88介导的信号通路主要参与了内毒素、脂肽和IL-1a诱导的破骨细胞的形成，其机制是通过成骨细胞表达RANKL来实现的。较少

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, is proposed to be a potent stimulator of bone resorption in inflammatory diseases caused by bacreria. Bacterial lipoprotein/lipopeptides are also pathogen-specific molecular patterns. Recently, toll-like receptor 4 (TLR4) was identified as the signaling receptor for LPS. In addition, TLR6 associate with TLR2, and the complex of TLR6 and TLR2 recognizes diacylated mycoplasmal lipopeptides. The signaling cascade of TLR is believed to be similar to that of IL-1 receptors (IL- 1R), because both TLR and IL-1R use myeloid differentiation factor 88 (MyD88) as a common cytoplasmic signaling molecule. However, accumulating evidence also demonstrates the existence of MyD88-independent pathways, which may explain unique biological responses of individual TLR and IL-1R. Using MyD88-deficient (-/-) mice, we explored the involvement of MyD88-mediated signals in osteoclast formation. LPS, synthetic lipopept … More ide (FSL-1), IL-1a and 1.25(OH)_2D_3 all stimulated osteoclast formation in co-cultures of primary osteoblasts and bone marrow cells obtained from wild-type mice. Osteoprotegerin, a decoy receptor of RANKL, completely inhibited the osteoclast formation in the co-culture. In contrasts, LPS, lipopeptide and IL-1a failed to induce the osteoclast formation in the co-culture of Myd88 (-/-) mice-derived osteoblasts and bone marrow cells, though 1.25(OH)_2D_3 stimulated osteoclast formation even in the MyD88 (-/-) co-culture. RT-PCR analysis showed that primary osteoblasts obtained from both wild type and MyD88 (-/-) mice similarly expressed TLR2, TLR4, TLR6 and IL-1R mRNAs. LPS, lipopeptide and IL-1a stimulated expression of RANKL mRNA within 24 hr in primary osteoblasts obtained from wild-type mice but not in those from MyD88 (-/-) mice. Similarly, LPS and IL-1a stimulated phosphorylation of ERK in wild-type osteoblasts but not MyD88 (-/-) osteoblasts. Hemopoietic cells obtained from MyD88 (-/-) mice and those from wild-type mice similarly differentiated into osteoclasts within 3 days in response to RANKL and M-CSF. These results suggest that the MyD88-mediated signaling pathway is essentially involved in osteoclast formation induced by LPS, lipopeptide and IL-1a through the RANKL expression by osteoblasts. Less