Involvement of PU.1 in NFATc1 promoter function in osteoclast development
Involvement of PU.1 in NFATc1 promoter function in osteoclast development
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DOI：
10.1016/j.alit.2015.01.006
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发表时间：
2015-07-01
影响因子：
6.8
通讯作者：
Nishiyama, Chiharu
中科院分区：
文献类型：
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作者：
Ishiyama, Kentaro;Yashiro, Takuya;Nishiyama, Chiharu
背景：转录因子NFATc1和PU.1在破骨细胞发育中起重要作用。NFATc1和PU.1反激活破骨细胞特异性基因表达，由于破骨细胞发育不足，NFATc1或PU.1基因缺失导致骨质疏松。然而，NFATc1和PU.1之间是否存在交叉调控在很大程度上是未知的。本研究探讨了PU.1在NFATc1表达中的作用。方法：利用小鼠骨髓细胞制备破骨细胞。引入siRNA进行PU.1敲低。采用定量RT-PCR检测转染sirna细胞的mRNA水平。通过染色质免疫沉淀（ChIP）试验和报告基因试验分析了PU.1在NFATc1启动子中的作用。采用逆转录病毒载体强制表达PU.1。结果：将PU.1 siRNA引入骨髓源性破骨细胞，导致NFATc1 mRNA水平降低。ChIP实验表明，PU.1在破骨细胞中与NFATc1启动子结合。NFATc1启动子活性在PU.1敲低的细胞中降低。PU.1 siRNA的引入也下调了破骨细胞特异性基因的表达和酒石酸抗性酸性磷酸酶（TRAP）的活性。使用逆转录病毒载体强制表达PU.1可增加NFATc1的表达和TRAP活性。当使用siRNA敲除NFATc1表达时，破骨细胞特异性基因和trap阳性细胞的诱导受到抑制，但不影响PU.1的表达水平。结论：这些结果表明，PU.1通过直接结合NFATc1启动子激活NFATc1表达，参与破骨细胞的发育。版权所有，日本过敏症学会。制作和托管由爱思唯尔B.V.
Background: The transcription factors NFATc1 and PU.1 play important roles in osteoclast development. NFATc1 and PU.1 transactivate osteoclast-specific gene expression and a deficiency in NFATc1 or PU.1 genes causes osteopetrosis due to an insufficient development of osteoclasts. However, the existence of cross-regulation between NFATc1 and PU.1 is largely unknown. In the present study, the role of PU.1 in NFATc1 expression was investigated.Methods: Osteoclasts were generated from mouse bone marrow cells. PU.1 knockdown was performed with siRNA introduction. The mRNA levels in siRNA-introduced cells were determined by quantitative RT-PCR. The involvement of PU.1 in the NFATc1 promoter was analyzed by using a chromatin immunoprecipitation (ChIP) assay and a reporter assay. Retrovirus vector was used for enforced expression of PU.1.Results: Introduction of PU.1 siRNA into bone marrow-derived osteoclasts resulted in a decrease in NFATc1 mRNA level. A ChIP assay showed that PU.1 bound to the NFATc1 promoter in osteoclasts. NFATc1 promoter activity was reduced in PU.1 knockdown cells as assessed by a reporter assay. PU.1 siRNA introduction also downregulated the expression of osteoclast-specific genes and tartrate resistant acid phosphatase (TRAP) activity. Enforced expression of PU.1 using a retrovirus vector increased NFATc1 expression and TRAP activity. When NFATc1 expression was knocked down by using siRNA, the induction of osteoclast-specific genes and TRAP-positive cells was suppressed without affecting the expression level of PU.1.Conclusions: These results indicate that PU.1 is involved in osteoclast development by transactivating NFATc1 expression via direct binding to the NFATc1 promoter. Copyright (C) 2015, Japanese Society of Allergology. Production and hosting by Elsevier B.V.