ACTION OF ESTROGEN RECEPTOR CO-REGULATORS IN OSTEOBLASTS

雌激素受体协同调节剂在成骨细胞中的作用

• 批准号: 6758328
• 负责人: THOMAS C SPELSBERG
• 金额: $ 19.45万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6758328
• 关键词: estrogen receptors; estrogens; genetic regulation; genetic transcription; high performance liquid chromatography; hormone regulation /control mechanism; intermolecular interaction; mass spectrometry; neoplastic cell culture for noncancer research; osteoblasts; protein isoforms; protein structure function; tissue /cell culture; western blottings

The cell-specific concentrations and interactions of the estrogen receptor (ER) isoforms and ER coregulators (activators and repressors) in target cells are now believed to explain the tissue specific actions of selective estrogen receptor (ER) modulators (SERMs). Estrogens (E) and SERMs modulate the structure of the ligand binding domain (LBD) of the ER which, in turn, regulates the interactions of the nuclear coregulators with the ER and determines the actions of the ER on gene expression and subsequent tissue responses. Studies supported by this grant over the past 3 1/2 years have yielded information on the actions of ERalpha or ERbeta (or combinations of the two) on gene expression in osteoblasts (OB) and their effects on OB function. Using these results and our new OB cell lines, derived from human and murine knockout skeletal tissues, we propose the following Aims for this project. Aim 1: Examine the synergism/antagonism of the ERalpha and ERbeta isoforms on gene transcription of selected endogenous genes [IL-6, alkaline phosphatase (AP), progesterone receptor (PR), TGFbeta inducible early gene (TIEG), and veriscan (Ver)] in novel human OB (U2OS) cell lines. Aim 2: Determine the effects of reduced/absent SRC-1 and SRC-2 co-activator levels on the ERalpha and ERbeta-regulated transcription by E and SERMs of the same endogenous genes (as in Aim 1) in human OB: a) Transfect Dharmacon siRNAs with oligofectamine in our newly created human OB (U2OS) cells containing tetracycline regulated expressions of ERalpha, or ERbeta, or both ERalpha/beta, and b) perform the same studies as in Aim 2-a using immortalized mouse OB from wild-type and SRC-1 and SRC-2 KO mice. Aim 3: Determine the molecular interactions of the ER (alpha and beta) and its co-regulators (SRC-1, -2, -3, p300, CBP, CARM1, N-CoR, RIP 140, and REA) using GST pull-down studies with GST-ERalpha- and GST-ERbeta-LBD fusion proteins when the LBD is bound with E or SERMs. Identify bound co-regulators by western blotting. Aim 4: Determine the complete profile of the OB-derived co-regulators bound to the GST-ERalpha and GST-ERbeta LBD fusion proteins, when bound with E or the SERMs, using 1D-PAGE as well as 3D nano-high performance liquid chromatography coupled directly with tandem mass spectrometry and use the data to search genomic and protein databases for characterization and identification.

雌激素受体（ER）亚型和ER辅调节因子（激活剂和抑制剂）在靶细胞中的细胞特异性浓度和相互作用现在被认为可以解释选择性雌激素受体（ER）调节剂（SERM）的组织特异性作用。雌激素（E）和SERMs调节ER的配体结合结构域（LBD）的结构，这反过来又调节核辅调节因子与ER的相互作用，并决定ER对基因表达和随后的组织反应的作用。在过去的3年半里，这项资助支持的研究已经获得了关于ER α或ER β（或两者的组合）对成骨细胞（OB）基因表达的作用及其对OB的影响的信息 功能使用这些结果和我们的新的OB细胞系，来自人类和小鼠敲除骨骼组织，我们提出了这个项目的以下目标。 目标1：在新型人OB（U2 OS）细胞系中检查ER α和ER β亚型对选定内源性基因[IL-6、碱性磷酸酶（AP）、孕酮受体（PR）、TGF β诱导早期基因（TIEG）和veriscan（Ver）]基因转录的协同/拮抗作用。 目标二：确定SRC-1和SRC-2共激活因子水平降低/缺失对相同内源性基因的E和SERMs调节的ER α和ER β转录的影响（如目标1）在人OB中：a）在我们新创建的含有四环素调节的ER α或ER β或ER α/β表达的人OB（U2 OS）细胞中，用寡转染胺转染Dharmacon siRNA，和B）进行与 目的2-a使用来自野生型和SRC-1和SRC-2 KO小鼠的永生化小鼠OB。 目标3：当LBD与E或SERM结合时，使用GST-ER α-和GST-ER β-LBD融合蛋白的GST下拉研究确定ER（α和β）及其辅助调节因子（SRC-1、-2、-3、p300、CBP、CARM 1、N-CoR、RIP 140和REA）的分子相互作用。通过蛋白质印迹法鉴定结合的共调节因子。 目标4：当与E或SERM结合时，使用1D-PAGE以及直接与串联质谱联用的3D纳米高效液相色谱法确定与GST-ER α和GST-ER β LBD融合蛋白结合的OB衍生辅助调节剂的完整谱，并使用数据搜索基因组和蛋白质数据库进行表征和鉴定。