NFATc1 regulation of the human β3 integrin promoter in osteoclast differentiation
NFATc1 regulation of the human β3 integrin promoter in osteoclast differentiation
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DOI：
10.1016/j.gene.2005.12.012
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发表时间：
2006-05-10
期刊：
影响因子：
3.5
通讯作者：
McHugh, Kevin P.
中科院分区：
文献类型：
--
作者：
Crotti, Tania N.;Flannery, Merrilee;McHugh, Kevin P.
转录因子NFATc I在破骨细胞分化中从RANKL转导信号中起重要作用。然而，迄今为止，NFATc 1的特异性转录靶点尚不清楚。133整合素的表达是正常破骨细胞功能所必需的。因此，我们研究了NFATc 1在破骨细胞分化中人β 3整合素表达中的作用。对小鼠和人β 3基因启动子的分析显示，转录起始位点（TSS）上游1.3 kb区域具有相当大的序列同源性，存在保守的NFAT结合元件。将区域-1242至+29（相对于TSS）克隆为荧光素酶报告构建体（pB 3 -1.3），并制备去除至-997的缺失构建体（pB 3 -1）。245 bp的5 '端缺失去除了三个保守的NFAT位点，包括一个共有的NFAT：AP-1位点。通过用2.5-40 ng/ml范围的RANKL处理诱导pB 3 -1.3报告基因构建体，并通过在RAW 264.7细胞中与人NFATc 1共转染剂量依赖性诱导。RANKL处理对pB 3 -1缺失构建体的诱导最小，并且对共转染的NFATc 1无反应。通过EMSA和抗NFAT抗体的超位移证明了NFAT与该245 bp 5'区域内的两个共有NFAT位点的直接结合。在-1242至-997片段中的两个保守NFAT位点的突变是防止结合所必需的。在全长启动子的背景下，双NFAT突变体对RANKL处理或共转染的NFAR I无反应。我们产生了可渗透细胞的TAT显性阴性（dn）NFATc 1融合蛋白，以评估阻断NFAT信号传导的效果。用dnNFAT转导抑制人1 - 3整合素启动子的RANKL诱导。使用钙调磷酸酶途径抑制肽11 R-VIVIT进一步证实了NFARc 1-钙调磷酸酶途径参与调节人M整联蛋白启动子。这些结果共同确立了M基因作为RANKL依赖性破骨细胞形成中NFATc 1的直接靶点。(c)2006 Elsevier B. V.保留所有权利。
The transcription factor NFATc I plays an essential role in transducing signals from RANKL in osteoclast differentiation. To date, however, the specific transcriptional targets of NFATc1 are unknown. Expression of the 133 integrin is required for normal osteoclast function. We therefore examined the role of NFATc1 in human beta(3) integrin expression in osteoclast differentiation. Analysis of the mouse and human beta(3) gene promoters revealed considerable sequence homology across a 1.3 kb region upstream of the transcription start site (TSS), with conserved NFAT binding elements present. The region - 1242 to + 29 (relative to the TSS) was cloned as a luciferase reporter construct (pB3-1.3) and a deletion construct removing to -997 (pB3-1) made. The deletion of 245 bp 5'removed three conserved NFAT sites including a consensus NFAT:AP-1 site. The pB3-1.3 reporter construct was induced by treatment with RANKL in the range 2.5-40 ng/ml and dose-dependently induced by co-transfection with human NFATc1 in RAW264.7 cells. The pB3-1 deletion construct was minimally induced with RANKL treatment and unresponsive to co-transfected NFATc1. Direct NFAT binding to two of the consensus NFAT sites within this 245 bp 5' region was demonstrated by EMSA and supershift with anti-NFAT antibodies. Mutation of two of the conserved NFAT sites in the - 1242 to - 997 fragment was required to prevent binding. The double NFAT mutant, in the context of the full-length promoter was unresponsive to RANKL treatment or co-transfected NFAR I. We generated cell-permeable TAT-dominant-negative (dn)NFATc1 fusion proteins to assess the effect of blockade of NFAT signaling. Transduction with dnNFAT inhibited RANKL induction of the human 1 3 integrin promoter. Involvement of the NFARc1-calcineurin pathway in regulating the human M integrin promoter was further confirmed using the calcineurin pathway inhibitory peptide 11R-VIVIT. Together these results establish the M gene as a direct target of NFATc1 in RANKL-dependent osteoclast formation. (c) 2006 Elsevier B.V. All rights reserved.