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

脂多糖（LPS）是革兰氏阴性菌外膜的主要成分，在细菌引起的炎症性疾病中被认为是一种有效的骨吸收刺激物。细菌脂蛋白/脂肽也是病原体特异性的分子模式。最近，toll样受体4 （TLR4）被确定为LPS的信号受体。此外，TLR6与TLR2结合，TLR6和TLR2的复合物可识别二酰基化的支原体脂肽。TLR的信号级联被认为与IL-1受体（IL- 1R）的信号级联相似，因为TLR和IL-1R都使用髓样分化因子88 （MyD88）作为共同的细胞质信号分子。然而，越来越多的证据也证明了myd88独立通路的存在，这可能解释了个体TLR和IL-1R的独特生物学反应。使用myd88缺陷（-/-）小鼠，我们探索了myd88介导的信号在破骨细胞形成中的作用。LPS、合成脂素（FSL-1）、IL-1a和1.25(OH)_2D_3均能刺激野生型小鼠原代成骨细胞和骨髓细胞共培养的破骨细胞形成。骨保护素是RANKL的诱饵受体，在共培养中完全抑制破骨细胞的形成。相比之下，LPS、脂肽和IL-1a在Myd88（-/-）小鼠来源的成骨细胞和骨髓细胞共培养中未能诱导破骨细胞的形成，尽管1.25(OH)_2D_3甚至在Myd88（-/-）共培养中也能刺激破骨细胞的形成。RT-PCR分析显示，野生型和MyD88（-/-）小鼠获得的原代成骨细胞表达TLR2、TLR4、TLR6和IL-1R mrna相似。LPS、脂肽和IL-1a可刺激野生型小鼠的成骨细胞在24小时内表达RANKL mRNA，而MyD88（-/-）小鼠的成骨细胞则无此作用。同样，LPS和IL-1a刺激了野生型成骨细胞中ERK的磷酸化，但对MyD88（-/-）成骨细胞没有作用。在RANKL和M-CSF的作用下，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。这些结果表明，myd88介导的信号通路本质上参与了LPS、脂肽和IL-1a通过成骨细胞表达RANKL诱导破骨细胞形成的过程。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