Yagi Mitsuru, Ninomiya Ken, Fujita Nobuyuki, Suzuki Toru, Iwasaki Ryotaro, Morita Kozo, Hosogane Naobumi, Matsuo Koichi, Toyama Yoshiaki, Suda Toshio, Miyamoto Takeshi
Department of Cell Differentiation, The Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan.
J Bone Miner Res. 2007 Jul;22(7):992-1001. doi: 10.1359/jbmr.070401.
DC-STAMP is essential for fusion of osteoclasts and foreign body giant cells; however, it is not known whether dc-stamp expression in these two cell types is differentially regulated. Here, we show that dc-stamp expression and cell-cell fusion are regulated in a cell type-specific manner.
The transcription factors c-Fos and NFATc1 cooperate to regulate osteoclast differentiation, whereas PU.1 and NF-kappaB are activated in macrophages and osteoclasts or in both cell types. Thus, we asked what role c-Fos, NFATc1, PU.1, and NF-kappaB played in regulating dendritic cell-specific transmembrane protein (dc-stamp) expression and fusion of osteoclasts and macrophage giant cells.
Transcriptional activation by c-Fos and NFATc1 was examined by dc-stamp promoter analysis. Multinuclear cell formation was analyzed in cells from c-Fos-deficient mice or in wildtype cells treated with the NFAT inhibitor FK506. The role of DC-STAMP in cell fusion was examined in vitro in a macrophage giant cell formation assay using DC-STAMP-deficient cells. Recruitment of c-Fos, NFATc1, PU.1, and NF-kappaB to the dc-stamp promoter in osteoclasts and macrophage giant cells was analyzed by chromatin-immunoprecipitation analysis.
Both activator protein-1 (AP-1) and NFAT binding sites in the dc-stamp promoter were needed for dc-stamp expression after RANKL stimulation of osteoclasts. dc-stamp expression was induced in osteoclasts and macrophage giant cells, and cells from DC-STAMP-deficient mice failed to form either multinuclear osteoclasts or macrophage giant cells. In contrast, c-Fos is indispensable for dc-stamp expression and cell-cell fusion under conditions favoring in vitro and in vivo induction of osteoclasts but not macrophage giant cells. Consistently, an NFAT inhibitor suppressed multinuclear osteoclast formation but not macrophage giant cell formation. In addition, PU.1 and NF-kappaB binding sites were detected in the dc-stamp promoter, and both PU.1 and NF-kappaB were recruited to the dc-stamp promoter after granulocyte-macrophage colony stimulating factor (GM-CSF) + interleukin (IL)-4 stimulation.
dc-stamp expression is regulated differently in osteoclasts and macrophage giant cells. c-Fos and NFATc1, both of which are essential for osteoclast differentiation, are needed for dc-stamp expression and cell-cell fusion in osteoclasts, but both factors are dispensable for giant cell formation by macrophages. Because PU.1 and NF-kappaB are recruited to the dc-stamp promoter after stimulation with GM-CSF + IL-4, dc-stamp transcription is regulated in a cell type-specific manner.
DC-STAMP对于破骨细胞与异物巨细胞的融合至关重要；然而，尚不清楚这两种细胞类型中dc-stamp的表达是否受到不同的调控。在此，我们表明dc-stamp的表达和细胞间融合是以细胞类型特异性的方式进行调控的。
转录因子c-Fos和NFATc1协同调节破骨细胞分化，而PU.1和NF-κB在巨噬细胞和破骨细胞中或在这两种细胞类型中均被激活。因此，我们探究了c-Fos、NFATc1、PU.1和NF-κB在调节树突状细胞特异性跨膜蛋白（dc-stamp）表达以及破骨细胞与巨噬细胞巨细胞融合中所起的作用。
通过dc-stamp启动子分析检测c-Fos和NFATc1的转录激活情况。在来自c-Fos缺陷小鼠的细胞或用NFAT抑制剂FK506处理的野生型细胞中分析多核细胞的形成。在使用DC-STAMP缺陷细胞的巨噬细胞巨细胞形成试验中体外检测DC-STAMP在细胞融合中的作用。通过染色质免疫沉淀分析来分析破骨细胞和巨噬细胞巨细胞中c-Fos、NFATc1、PU.1和NF-κB与dc-stamp启动子的结合情况。
破骨细胞经RANKL刺激后，dc-stamp启动子中的激活蛋白-1（AP-1）和NFAT结合位点对于dc-stamp表达均是必需的。dc-stamp在破骨细胞和巨噬细胞巨细胞中被诱导表达，并且来自DC-STAMP缺陷小鼠的细胞无法形成多核破骨细胞或巨噬细胞巨细胞。相反，在有利于体外和体内诱导破骨细胞而非巨噬细胞巨细胞的条件下，c-Fos对于dc-stamp表达和细胞间融合是不可或缺的。一致地，一种NFAT抑制剂抑制了多核破骨细胞的形成，但未抑制巨噬细胞巨细胞的形成。此外，在dc-stamp启动子中检测到PU.1和NF-κB结合位点，并且在粒细胞-巨噬细胞集落刺激因子（GM-CSF）+白细胞介素（IL）-4刺激后，PU.1和NF-κB均与dc-stamp启动子结合。
dc-stamp在破骨细胞和巨噬细胞巨细胞中的表达调控方式不同。c-Fos和NFATc1对于破骨细胞分化均至关重要，它们是破骨细胞中dc-stamp表达和细胞间融合所必需的，但这两种因子对于巨噬细胞形成巨细胞并非必需。由于在GM-CSF + IL-4刺激后PU.1和NF-κB与dc-stamp启动子结合，dc-stamp转录是以细胞类型特异性的方式进行调控的。
