DACH1 negatively regulates the human RANK ligand gene expression in stromal/preosteoblast cells
DACH1 negatively regulates the human RANK ligand gene expression in stromal/preosteoblast cells
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DOI：
10.1002/jcb.21561
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发表时间：
2008-04-15
影响因子：
4
通讯作者：
Reddy, Salkamuri V.
中科院分区：
文献类型：
--
作者：
Sundaram, Kumaran;Mani, Santhosh K.;Reddy, Salkamuri V.
NF-κ B配体受体激活因子（RANKL）是一种重要的破骨细胞生成因子，在骨髓基质/前骨B last细胞上表达。大多数骨吸收刺激通过调节破骨细胞中RANKL的表达诱导破骨细胞形成。然而，很少有人知道调节RANKL基因表达的机制。我们最近报道，热休克因子-2（HSF-2）是FGF-2信号转导的下游靶点，可增强骨髓基质/前成骨细胞中RANKL基因的转录。在这项研究中，我们表明，DACH 1（果蝇腊肠犬基因的人类同源物）负调控RANKL基因的表达，并抑制FGF-2增强RANKL基因在这些细胞中的表达。DACH 1在N端含有一个保守的腊肠犬结构域（DS），它与核辅阻遏物（NCoR）相互作用以抑制基因表达。在正常人骨髓基质细胞中共表达DACH 1与hRANKL启动子-荧光素酶报告质粒显著降低（3.3倍）FGF-2刺激的hRANKL基因启动子活性。DS结构域的缺失消除了DACH 1对FGF-2增强的RANKL基因启动子活性的抑制。Western blot分析证实，DACH 1抑制骨髓基质/前成骨细胞中FGF-2刺激的RANKL表达。我们发现HSF-2与DACH 1共免疫沉淀，FGF-2刺激显著增加（2.7倍）HSF-2与DACH 1的结合。共聚焦显微镜分析进一步证明FGF-2促进HSF-2核转运并与DACH 1在骨髓基质细胞中共定位。NCoR与DACH 1的共表达显著降低（5.3倍），DACH 1共转染细胞中NCoR的siRNA抑制增加（3.6倍）RANKL启动子活性。此外，DACH 1与NCoR共表达显著降低（7.5倍）骨髓基质细胞中RANKL mRNA的表达。总之，这些研究表明，NCoR参与骨髓基质/前成骨细胞中RANKL基因表达的DACH 1抑制。因此，DACH 1在骨微环境中骨髓基质/前成骨细胞中RANKL基因表达的负调控中起重要作用。
Receptor activator of NF-kappa B ligand (RANKL) is a critical osteoclastogenic factor that is expressed on bone marrow stromal/preosteob last cells. Most bone resorption stimuli induce osteoclast formation by modulating RANKL expression in these cells. However, little is known about the mechanisms regulating RANKL gene expression. We recently reported that heat shock factor-2 (HSF-2) is a downstream target for FGF-2 signaling to enhance RANKL gene transcription in marrow stromal/preosteoblast cells. In this study, we show that DACH1 (human homologue of Drosophila dachshund gene) negatively regulates RANKL gene expression and suppresses FGF-2-enhanced RANKL gene expression in these cells. DACH1 contains a conserved dachshund domain (DS) in the N-terminal region,which interacts with the nuclear corepressor (NCoR) to repress gene expression. Co-expression of DACH1 with hRANKL promoter-luciferase reporter plasmid in normal human bone marrow-derived stromal cells significantly decreased (3.3-fold) FGF-2-stimulated hRANKL gene promoter activity. Deletion of DS domain abolished DACH1 inhibition of FGF-2-enhanced RANKL gene promoter activity. Western blot analysis confirmed that DACH1 suppressed FGF-2-stimulated RANKL expression in marrow stromal/preosteoblast cells. We show HSF-2 co-immune precipitated with DACH1 and that FGF-2 stimulation significantly increased (2.7-fold) HSF-2 binding to DACH1. Confocal rnicroscopy analysis further demonstrated that FGF-2 promotes HSF-2 nuclear transport and co-localization with DACH1 in marrow stromal cells. Co-expression of NCoR with DACH1 significantly decreased (5.3-fold) and siRNA suppression of NCoR in DACH1 co-transfected cells increased (3.6-fold) RANKL promoter activity. Furthermore, DACH1 co-expression with NCoR significantly decreased (7.5-fold) RANKL mRNA expression in marrow stromal cells. Collectively, these studies indicate that NCoR participates in DACH1 repression of RANKL gene expression in marrow stromal/preosteoblast cells. Thus, DACH1 plays an important role in negative regulation of RANKL gene expression in marrow stromal/preosteoblast cells in the bone microenvironment.