Sundaram Kumaran, Mani Santhosh K, Kitatani Kazuyuki, Wu Kongming, Pestell Richard G, Reddy Sakamuri V
Charles P. Darby Children's Research Institute, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Cell Biochem. 2008 Apr 15;103(6):1747-59. doi: 10.1002/jcb.21561.
Receptor activator of NF-kappaB ligand (RANKL) is a critical osteoclastogenic factor that is expressed on bone marrow stromal/preosteoblast 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 co-repressor (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 microscopy 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.
核因子-κB受体激活剂配体（RANKL）是一种关键的破骨细胞生成因子，在骨髓基质/前成骨细胞上表达。大多数骨吸收刺激通过调节这些细胞中的RANKL表达来诱导破骨细胞形成。然而，关于调节RANKL基因表达的机制知之甚少。我们最近报道，热休克因子-2（HSF-2）是FGF-2信号传导的下游靶点，可增强骨髓基质/前成骨细胞中的RANKL基因转录。在本研究中，我们发现DACH1（果蝇腊肠犬基因的人类同源物）负向调节RANKL基因表达，并抑制这些细胞中FGF-2增强的RANKL基因表达。DACH1在N端区域包含一个保守的腊肠犬结构域（DS），它与核共抑制因子（NCoR）相互作用以抑制基因表达。在正常人骨髓来源的基质细胞中，DACH1与hRANKL启动子-荧光素酶报告质粒共表达显著降低（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DACH1对FGF-2增强的RANKL基因启动子活性的抑制作用。蛋白质印迹分析证实，DACH1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DACH1共免疫沉淀，并且FGF-2刺激显著增加（2.7倍）HSF-2与DACH1的结合。共聚焦显微镜分析进一步表明，FGF-2促进HSF-2在骨髓基质细胞中的核转运并与DACH1共定位。NCoR与DACH1共表达显著降低（5.3倍），并且在DACH1共转染细胞中对NCoR的siRNA抑制增加（3.6倍）RANKL启动子活性。此外，DACH1与NCoR共表达显著降低（7.5倍）骨髓基质细胞中RANKL mRNA表达。总体而言，这些研究表明NCoR参与DACH1对骨髓基质/前成骨细胞中RANKL基因表达的抑制作用。因此，DACH1在骨微环境中骨髓基质/前成骨细胞中RANKL基因表达的负调节中起重要作用。
