Nuclear Events in PTH Action on Bone Cells

描述(申请人提供)：甲状旁腺激素是必需的 钙稳态的调节器，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 成骨细胞从基质合成到基质合成的表型 降级和积极参与再吸收进程。一个例子 这种对甲状旁腺素的反应发生在大鼠成骨细胞系UMR 106-01中， 其中激素通过一种途径诱导胶原酶3基因转录 需要从头合成蛋白质的cAMP依赖途径。因此，这是一个 我们先前假设的次要效应涉及诱导和 作用于该基因的特定转录因子的激活。我们确认了 PTH反应元件为激活蛋白-I(AP-1)和矮小 胶原酶-3启动子中的结构域(RD)结合位点。此外，我们 展示了依赖甲状旁腺素的位点和 与它们结合的蛋白质。我们还发现，甲状旁腺素刺激c-Fos和c-Jun 蛋白质丰富，但Cbfal水平无明显变化。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启动子作为甲状旁腺素作用的中介。这个 这一途径的重要性从以下事实得到证明：甲状旁腺激素刺激 通过其PKA位点反式激活Cbfal中的激活结构域3(AD3)。 因此，甲状旁腺激素通过这一途径调节两种转录因子，或者通过 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在物理上相互作用 核小体结构，招募其他蛋白质，如辅助激活因子， 核小体和一般转录因子的修饰物。这个 这项工作的长期目标是描绘甲状旁腺素的传递机制 胶原酶-3基因在成骨细胞转录调控中的作用。 因此，这项修订的竞争性延续提案的具体目标是 目的：1)检测甲状旁腺激素后Cbfal的体内磷酸化 治疗。2)评估甲状旁腺激素对Fos、Jun和Cbfal的相互作用， 3)鉴定其他甲状旁腺激素调节蛋白 与Fos、Jun和Cbfal相互作用，4)研究AP-1和 启动子结构中的RD位点及其结合蛋白及其如何 PTH会影响这一点。5)使用转基因动物来验证Cbfal调节 胶原酶-3的体内启动子。这项工作的成果将继续 有助于我们了解甲状旁腺素如何在成骨细胞中发挥其核作用 功能。通过这样做，这些数据还将提供新的视角 治疗钙代谢紊乱。

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.