Nuclear Events in PTH Action on Bone Cells

PTH 对骨细胞作用的核事件

• 批准号: 6621451
• 负责人: Nicola C Partridge
• 金额: $ 27.21万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6621451
• 关键词: AP1 protein; JUN kinase; binding proteins; bone metabolism; collagenase; fos protein; gel mobility shift assay; gene induction /repression; genetic regulatory element; genetic transcription; genetically modified animals; hormone receptor; hormone regulation /control mechanism; laboratory mouse; osteoblasts; parathyroid hormones; protein protein interaction; tissue /cell culture; transcription factor; transfection; western blottings

DESCRIPTION (provided by applicant): Parathyroid hormone is an essential regulator of calcium homeostasis and also has a role as an anabolic hormone for bone. The hormone has multiple actions, including indirect activation of the osteoclast resulting in increased bone resorption, as well as many direct changes in the functions of the osteoblast. The latter involve a switch in the pheontype of the osteoblast from one of matrix synthesis to one of matrix degradation and active participation in the resorption process. An example of this response to PTH occurs in the rat osteoblastic cell line, UMR 106-01, where the hormone induces collagenase-3 gene transcription through a cAMP-dependent pathway requiring de novo protein synthesis. Thus, this is a secondary effect that we previously hypothesized involves the induction and activation of specific transcription factors acting on this gene. We identified the PTH-response elements as being the activator protein-I (AP-1) and the runt domain (RD) binding sites in the collagenase-3 promoter. In addition, we demonstrated a PTH-dependent cooperative interaction between the sites and the proteins binding to them. We also showed that PTH stimulated c-Fos and c-Jun protein abundance but no significant change in the level of Cbfal. These transcription factors are able to interact both in vitro and in vivo. Supporting our earlier work, the PKA pathway was shown to be the only pathway regulating the collagenase-3 promoter as a mediator of PTH action. The importance of this pathway was demonstrated by the fact that PTH stimulates the transactivation of activation domain-3 (AD3) in Cbfal through its PKA site. Thus, PTH regulates both transcription factors through this pathway, either by increasing their expression or altering their phosphorylation. Our hypothesis of the functions of these proteins is that they interact physically in a nucleosomal structure, recruiting other proteins such as coactivators, modifiers of the nucleosome and the general transcription factors. The long-term goals of this work are to delineate the mechanisms conveying PTH action to regulation of transcription of the collagenase-3 gene in osteoblasts. Consequently, the specific aims of this revised competing continuation proposal are to, 1) examine the in vivo phosphorylation of Cbfal following PTH treatment. 2) assess the PTH-regulated interaction of Fos and Jun and Cbfal, and identify the domains involved, 3) identify other PTH-regulated proteins interacting with Fos and Jun and Cbfal, 4) investigate the role of the AP-1 and RD sites and their binding proteins in the structure of the promoter and how PTH affects this. 5) use transgenic animals to verify that Cbfal regulates the collagenase-3 promoter in vivo. The results of this work will continue to contribute to our knowledge of how PTH exerts its nuclear effects on osteoblast function. In so doing, the data will also provide new perspectives into treatment of disorders of calcium metabolism.

描述（由申请人提供）：甲状旁腺激素是一种必需的 调节钙稳态，也有作为合成代谢激素的作用， 骨头这种激素有多种作用，包括间接激活 破骨细胞导致骨吸收增加，以及许多直接 成骨细胞功能的改变。后者涉及到一个开关， 成骨细胞由基质合成型向基质型转变 降解和主动参与再吸收过程。的示例 这种对PTH的反应发生在大鼠成骨细胞系，UMR 106-01， 其中该激素通过一种免疫调节剂诱导胶原酶-3基因转录， 需要从头蛋白质合成的cAMP依赖性途径。因此，这是一个 我们先前假设的次级效应涉及诱导， 激活作用于该基因的特定转录因子。我们确定 PTH反应元件为激活蛋白-I（AP-1）和矮小型 结构域（RD）结合位点。另外我们 证明了PTH依赖的网站之间的合作相互作用， 与之结合的蛋白质。我们还发现，PTH刺激c-Fos和c-Jun 蛋白质丰度，但Cbfal水平无显著变化。这些 转录因子能够在体外和体内相互作用。 支持我们早期的工作，PKA途径被证明是唯一的途径 调节胶原酶-3启动子作为PTH作用的介质。的 甲状旁腺激素刺激血管内皮细胞这一事实证明了这一途径的重要性。 Cbfal中激活结构域-3（AD 3）通过其PKA位点的反式激活。 因此，PTH通过这一途径调节两种转录因子， 增加它们的表达或改变它们的磷酸化。我们的假设 这些蛋白质的功能之一是它们以一种 核小体结构，募集其他蛋白质如共激活因子， 核小体的修饰物和一般转录因子。的 这项工作的长期目标是阐明PTH的传递机制 对成骨细胞中胶原酶-3基因转录的调节作用。 因此，这一经修订的竞争性续设提案的具体目标 1）检查PTH后Cbfal的体内磷酸化 治疗2)评估PTH调节的Fos和Jun和Cbfal的相互作用， 并鉴定所涉及的结构域; 3）鉴定其他PTH调节蛋白 与Fos和Jun和Cbfal相互作用，4）研究AP-1的作用， RD位点及其结合蛋白在启动子结构中的作用以及如何 PTH对此有影响。5)使用转基因动物来验证Cbfal调节 体内胶原酶-3启动子。这项工作的成果将继续 有助于我们了解PTH如何对成骨细胞发挥其核作用 功能在这样做的过程中，这些数据还将提供新的视角， 治疗钙代谢紊乱。