Functional analysis of TNF signaling in bone metabolism using knocked-out mice of type I and II TNF receptors.

肿瘤坏死因子信号在炎症条件下的骨吸收中起着至关重要的作用。为了区分I型和II型肿瘤坏死因子受体的功能作用，对每种受体基因敲除的小鼠进行了组织学和骨形态计量学分析。在12周龄的股骨中，I型(-/-)小鼠皮质和松质骨中的骨体积、厚度和矿物质含量均减少，而II型(-/)小鼠的这些参数则增加。为探讨肿瘤坏死因子受体参与骨代谢的分子机制，分别从颅骨和骨髓巨噬细胞培养成骨细胞和破骨细胞。然而，用BMP-2或地塞米松处理成骨细胞并不改变其ALP表达和细胞增殖。相反，只有在M-CSF和RANKL处理的II型(-/-)巨噬细胞中才能诱导破骨细胞的形成，这表明暴露在细胞表面的某些分子可能对破骨细胞的形成起重要作用。为了鉴定这些分子，我们对每个培养的细胞进行了DNA末端分析，发现了3个新基因和5个已知基因，它们可能是肿瘤坏死因子受体激活的靶信号或下游信号。我们现在正在分析这些基因的功能作用和表达模式。

TNF signaling is known to play a crucial role in bone absorption under inflammatory condition. To distinguish the functional role of type I and type II TNF receptor, knock-out mice of each receptor were subjected to histological and bone morphometric analysis. In 12 wks old femurs, bone volume, thickness and mineral content in both cortical and cancellous bone were decreased in type I (-/-) mice, whereas these parameters were increased in type II (-/) mice. To investigate a molecular mechanism of TNF receptor involved bone metabolism, osteoblasts and osteoclasts were cultured from calvariae and macrophage from bone marrow, respectively. Treatment of osteoblasts with BMP-2 or Dexamethasone, however, did not alter their ALP expression as well as cell proliferation. In contrast, osteoclast formation was only induced in type II (-/-) macrophage by treating with M-CSF and RANKL, indicating that certain molecules secreted of exposed on cell surface could be important for osteoclasts formation. To identify these molecules, we applied DNA tip analysis to each cultured cell and found three novel and 5 known genes that could be target or down-stream signaling for TNF receptor activation. We are now analyzing the functional role and expression pattern of these genes.