Ishida Norihiro, Hayashi Koji, Hattori Asuka, Yogo Keiichiro, Kimura Toru, Takeya Tatsuo
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
J Bone Miner Res. 2006 Jan;21(1):48-57. doi: 10.1359/JBMR.051001. Epub 2005 Oct 10.
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.
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.
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 CCR1 was introduced in RAW264 cells. Cell migration activity was examined using a Boyden chamber assay.
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 CCR1 siRNA-treated cells were used. Moreover, treatment with a Galpha inhibitor pertussis toxin (PTX) or methiolynated-regulated on activation, normal T cells expressed and secreted (Met-RANTES), 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.
我们发现趋化因子受体基因CCR1在RANKL刺激的RAW264和骨髓细胞中作用于NFAT2的下游。CCR1的上游调控区域表现出RANKL依赖性和环孢素A（CsA）可抑制的启动子活性。CCR1表达和功能的下调抑制了细胞迁移。
我们之前报道过，在体外破骨细胞生成系统中，RANKL诱导的NFAT2表达是向多核细胞进展的关键过程。因此，鉴定NFAT2的靶基因将有助于了解分化过程。我们在此聚焦于在RAW264细胞以及从骨髓细胞制备的破骨细胞前体中作用于NFAT2下游的趋化因子和趋化因子受体基因。
RAW264小鼠单核细胞/巨噬细胞系细胞在有或无环孢菌素A（CsA）的情况下，于RANKL或谷胱甘肽S -转移酶（GST）存在时进行培养。破骨细胞前体从骨髓细胞制备。通过微阵列分析寻找RANKL诱导且CsA可抑制的基因，并通过定量RT - PCR确认表达。通过荧光素酶基因报告测定法测量启动子活性。将针对CCR1的短干扰（si）RNA导入RAW264细胞。使用博伊登小室测定法检测细胞迁移活性。
我们鉴定出趋化因子受体基因CCR1是一个在存在与不存在NFAT抑制剂CsA的破骨细胞生成过程中显示出显著差异表达谱的基因。在RAW264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CCR1独有的。从CCR1中分离出上游调控区域，并确认了其RANKL依赖性和CsA可抑制的启动子活性。通过监测跨膜迁移测定中细胞的迁移来评估CCR1的功能意义，当使用经CsA或CCR1 siRNA处理的细胞时，这种活性被消除。此外，用Gα抑制剂百日咳毒素（PTX）或CCR1拮抗剂甲硫氨酸化调节激活正常T细胞表达和分泌的趋化因子（Met - RANTES）处理可抑制骨髓细胞系统中的多核细胞形成。总之，这些结果表明CCR1信号级联受NFAT2控制，并且似乎增强了分化中破骨细胞的迁移。
