CCR1 acts downstream of NFAT2 in osteoclastogenesis and enhances cell migration
CCR1 acts downstream of NFAT2 in osteoclastogenesis and enhances cell migration
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DOI：
10.1359/jbmr.051001
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发表时间：
2006-01-01
影响因子：
6.2
通讯作者：
Takeya, T
中科院分区：
文献类型：
--
作者：
Ishida, N;Hayashi, K;Takeya, T
我们发现趋化因子受体基因CCR 1在RANKL刺激的RAW 264和骨髓细胞中作用于NFAT 2的下游。CCR 1上游调控区具有RANKL依赖性和CsA抑制性启动子活性。下调CCR 1的表达和功能抑制细胞migration.Introduction：我们以前报道，表达的NFAT 2诱导RANKL是一个关键的过程，在体外破骨细胞生成系统中的多核细胞的进展。因此，鉴定NFAT 2的靶基因将提供有关分化过程的信息。我们集中在这里的趋化因子和趋化因子受体基因，在RAW 264细胞以及破骨细胞前体的NFAT 2下游的行为从骨髓cells.Materials和方法：RAW 264小鼠单核细胞/巨噬细胞系细胞培养或不环孢菌素A（CsA）在RANKL或谷胱甘肽S-转移酶（GST）的存在下。从骨髓细胞制备破骨细胞前体。通过微阵列分析搜索RANKL诱导和CsA抑制基因，并通过定量RT-PCR证实表达。通过荧光素酶基因报告基因测定来测量启动子活性。将针对CCR-1的短干扰（si）RNA引入RAW 264细胞中。使用Boyden室检测细胞迁移活性。结果和结论：我们确定了趋化因子受体基因CCR 1作为一个基因，在存在与不存在的CsA，NFAT的抑制剂的情况下，破骨细胞生成显示显着的差异表达谱。在RAW 264和骨髓细胞中检测的趋化因子和趋化因子受体基因中，这种特性是CCR 1所独有的。从CCR 1中分离出上游调控区，并证实了其RANKL依赖性和CsA抑制性启动子活性。通过监测细胞在transwell迁移试验中的迁移来评估CCR 1的功能意义，并且当使用CsA或CCRIsiRNA处理的细胞时，该活性被消除。此外，用Gu抑制剂百日咳毒素（PTX）或甲基化调节的活化、表达和分泌的正常T细胞（MetRANTES）（CCR 1的拮抗剂）治疗抑制骨髓细胞系统中的多核细胞形成。总之，这些结果表明，CCR 1信号级联是在NFAT 2的控制下，似乎增强分化破骨细胞的迁移。
We found that a chemokine receptor gene, CCR1, acts downstream of NFAT2 in RANKL-stimulated RAW264 and bone marrow cells. The upstream regulatory region of CCR1 showed RANKL-dependent and CsA-suppressible promoter activity. Downregulation of the expression and function of CCR1 suppressed cell migration.Introduction: We previously reported that the expression of NFAT2 induced by RANKL is a key process for progression to multinucleated cells in an in vitro osteoclastogenesis system. Identifying the target genes of NFAT2 would thus be informative about the differentiation process. We focused here on chemokine and chemokine receptor genes that act downstream of NFAT2 in RAW264 cells as well as osteoclast precursors prepared from bone marrow cells.Materials and Methods: RAW264 mouse monocyte/macrophage line cells were cultured with or without cyclosporin A (CsA) in the presence of RANKL or glutathione S-transferase (GST). Osteoclast precursors were prepared from bone marrow cells. RANKL-inducible and CsA-suppressible genes were searched for by microarray analysis, and expression was confirmed by quantitative RT-PCR. Promoter activity was measured by luciferase gene reporter assay. Short interfering (si)RNA for CCR-1 was introduced in RAW264 cells. Cell migration activity was examined using a Boyden chamber assay.Results and Conclusions: We identified the chemokine receptor gene CCR1 as a gene showing significant differential expression profiles in osteoclastogenesis in the presence versus the absence of CsA, an inhibitor of NFAT. This property was unique to CCR1 among the chemokine and chemokine receptor genes examined in both RAW264 and bone marrow cells. The upstream regulatory region was isolated from CCR1, and its RANKL-dependent and CsA-suppressible promoter activity was confirmed. The functional significance of CCR1 was assessed by monitoring the migration of cells in a transwell migration assay, and this activity was abolished when either CsA- or CCRI siRNA-treated cells were used. Moreover, treatment with a Gu inhibitor pertussis toxin (PTX) or methiolynated-regulated on activation, normal T cells expressed and secreted (MetRANTES), an antagonist of CCR1, suppressed multinucleated cell formation in the bone marrow cell system. Together, these results suggest that the CCR1 signaling cascade is under the control of NFAT2 and seems to enhance the migration of differentiating osteoclasts.