Zirngibl Ralph A, Chan Janet S M, Aubin Jane E
Department of Molecular Genetics, Faculty of Medicine, University of Toronto, 1 Kings College Circle, Medical Sciences Building Room 6230, Toronto, Ontario M5S 1A8, Canada.
J Mol Endocrinol. 2008 Feb;40(2):61-73. doi: 10.1677/JME-07-0114.
We previously demonstrated that the orphan nuclear receptor, estrogen receptor-related receptor alpha (ERRalpha) is highly expressed in osteoblasts and osteoclasts, regulates osteogenesis and expression of osteoblast-associated markers in the rat calvaria cell differentiation system, and is dysregulated in the rat ovariectomy model of postmenopausal osteoporosis. There are conflicting published data on the transcriptional regulation by ERRalpha of the gene for osteopontin (OPN), an extracellular matrix protein required in bone remodeling, and a potential direct target mediating ERRalpha effects in bone. We therefore readdressed OPN gene regulation by ERRalpha in both osteoblastic (rat osteosarcoma ROS17/2.8 cells) and non-osteoblastic (HeLa) cell lines using a mouse proximal 2 kb OPN promoter fragment. A minimal OPN promoter fragment spanning from -56 to +9 bp is activated in HeLa cells but repressed it in ROS17/2.8 cells. Adenine scanning mutagenesis revealed the presence of a non-canonical ERRalpha response element in this minimal promoter. Surprisingly, prototypical inactivating mutations in the activation function 2 (AF2) domain or a naturally occurring allelic variant of ERRalpha (ERRalphaH408) were all better activators than wild-type ERRalpha in HeLa cells, activities that were generally paralleled by repression in ROS17/2.8 cells. Finally, we found that the N-terminus of ERRalpha harbors a repressor domain that acts in a cell context-dependent manner. We conclude that OPN is an ERRalpha target gene whose promoter is regulated by ERRalpha in a cell context-dependent manner and that a predicted silencing mutation in AF2 or a more flexible helix 12 increases ERRalpha transcriptional activity, effects with implications for ERRalpha as a therapeutic target in bone.
我们之前证实，孤儿核受体雌激素相关受体α（ERRα）在成骨细胞和破骨细胞中高表达，在大鼠颅骨细胞分化系统中调节成骨作用及成骨细胞相关标志物的表达，且在绝经后骨质疏松症的大鼠卵巢切除模型中表达失调。关于骨桥蛋白（OPN）基因的转录调控存在相互矛盾的已发表数据，骨桥蛋白是骨重塑所需的细胞外基质蛋白，也是ERRα在骨中发挥作用的潜在直接靶点。因此，我们使用小鼠近端2 kb的OPN启动子片段，在成骨细胞系（大鼠骨肉瘤ROS17/2.8细胞）和非成骨细胞系（HeLa细胞）中重新研究了ERRα对OPN基因的调控。一个跨度为-56至+9 bp的最小OPN启动子片段在HeLa细胞中被激活，但在ROS17/2.8细胞中受到抑制。腺嘌呤扫描诱变揭示了该最小启动子中存在一个非典型的ERRα反应元件。令人惊讶的是，激活功能2（AF2）结构域中的典型失活突变或ERRα的天然等位基因变体（ERRαH408）在HeLa细胞中都是比野生型ERRα更好的激活剂，这些活性在ROS17/2.8细胞中通常伴随着抑制作用。最后，我们发现ERRα的N端含有一个以细胞背景依赖方式起作用的抑制结构域。我们得出结论，OPN是ERRα的靶基因，其启动子以细胞背景依赖的方式受ERRα调控，并且AF2中预测的沉默突变或更灵活的螺旋12会增加ERRα的转录活性，这些效应对于ERRα作为骨治疗靶点具有重要意义。
