Regulation of PTH-induced RankL transcription in osteoblasts

摘要：骨质疏松症是一种以骨密度和质量下降为特征的老年性疾病 骨组织的损伤，并与大量的发病率/死亡率相关。在骨质疏松症中， 形成新骨和去除旧骨/受损骨的细胞失调，促进过度再吸收。旁腺 激素（PTH）是这种体内平衡的关键调节剂，并且沿着其类似物已被用于治疗 骨质疏松虽然甲状旁腺激素对骨骼有积极的合成代谢作用，但它也可以通过 核因子κ-β配体（RankL）受体激活剂的活性。通过PTH治疗骨质疏松症是 受短的“合成代谢窗口”的限制，在此之后，积极的作用被由 RankL.从理论上讲，如果符合以下条件， RankL的转录可以被抑制。PTH及其类似物结合成骨细胞上的相同受体，激活 导致RankL转录的信号级联。最近的研究暗示了一系列信使， 酶、激酶和磷酸酶在调节RankL转录的两种共激活因子中的作用。的确切 导致这种调节的机制和与共激活因子相关的转录因子启动 RankL转录未建立。这项研究的目的是确定具体的监管 机制和转录因子激活RankL转录。在完成目标和培训方面 计划概述在这个建议的研究生，迈克尔莫斯卡，将获得大量的经验，在新的 生化技术，将获得内分泌学和药理学的具体知识，并将扩大他的知识范围 包括骨骼在内的肌肉骨骼组织的专业知识。这项研究培训将建立在他的生物力学和 与肌肉、肌腱/韧带病理的炎症和纤维化机制相关的组织学经验 并将准备他的研究生涯作为一个独立的调查员在肌肉骨骼领域。 目的1.1将确定特定的个别和/或联合的调节作用，几种盐诱导的 激酶和蛋白磷酸酶具有cAMP调节的转录辅激活因子2/3（CRTC 2/3）。一是 将用原代颅骨成骨细胞进行一系列siRNA转染，以探索 通过qRT-PCR敲低RankL转录上的每个因子。那么，击倒对手对 将通过定量分析确定CRTC 2/3核易位，包括有和无PTH治疗。 免疫荧光目的1.2将评估CRTC 2/3在成骨细胞中作为RankL共激活剂的作用 使用类似的方法进行区分。目的2旨在鉴定CRTC 2和CRTC 3表达的转录因子。 每一个都与激活RankL转录相关，使用质谱，染色质免疫沉淀， 和siRNA敲除。RankL的表达是导致骨质疏松症治疗中所见的catastrophic的原因， PTH及其类似物。阐明调节和启动RankL转录的机制可以鉴定 抑制RankL并提高PTH治疗骨质疏松症的疗效的新治疗靶点。

Abstract: Osteoporosis is a prevalent disease of aging characterized by a decrease in the density and quality of bone tissue and is associated with substantial morbidity/mortality. In osteoporosis the homeostatic processes that form new and remove old/damaged bone are dysregulated, promoting excessive resorption. Parathyroid hormone (PTH) is a key regulator of this homeostasis and along with its analogs has been used to treat osteoporosis. Although PTH has positive anabolic effects on bone it can also stimulate catabolism through activity of receptor activator of nuclear factor kappa-β ligand (RankL). Treatment of osteoporosis via PTH is limited by a short “anabolic window,” after which the positive effects are mitigated by the resorption initiated by RankL. Theoretically, it may be possible to retain just the positive effects of PTH-derived treatments if transcription of RankL can be inhibited. PTH and its analogs bind the same receptor on osteoblasts, activating a signaling cascade leading to RankL transcription. Recent work has implicated a cascade of messengers, enzymes, kinases, and phosphatases in regulating the two coactivators of RankL transcription. The exact mechanisms that cause this regulation and the transcription factor(s) the coactivators associate with to initiate RankL transcription are not established. The purpose of this research is to identify the specific regulatory mechanisms and transcription factor(s) that activate RankL transcription. In completing the aims and training plan outlined in this proposal the graduate student, Michael Mosca, will gain substantial experience in new biochemical techniques, will gain specific knowledge in endocrinology and pharmacology, and will expand his expertise in musculoskeletal tissues to include bone. This research training will build on his biomechanical and histological experiences related to inflammatory and fibrotic mechanisms of muscle, tendon/ligament pathologies and will prepare him for a research career as an independent investigator in the musculoskeletal field. Aim 1.1 will determine the specific individual and/or combined regulatory roles that several salt-inducible kinases and protein phosphatases have on cAMP-regulated transcriptional coactivators 2/3 (CRTC2/3). First, a series of siRNA transfections will be performed with primary calvarial osteoblasts to explore the effects of knocking down each factor on RankL transcription via qRT-PCR. Then, the effect the knockdowns have on CRTC2/3 nuclear translocation will be determined with and without PTH-treatment via quantitative immunofluorescence. Aim 1.2 will assess the roles CRTC2/3 play as RankL co-activators throughout osteoblast differentiation using similar methods. Aim 2 seeks to identify the transcription factor(s) that CRTC2 and CRTC3 each associate with to activate RankL transcription using Mass Spectrometry, Chromatin-immunoprecipitation, and siRNA knockdowns. RankL expression is responsible for catabolism seen in treatment of osteoporosis by PTH and its analogs. Elucidation of the mechanisms that regulate and initiate RankL transcription may identify novel therapeutic targets to inhibit RankL and improve the efficacy of PTH in the treatment of osteoporosis.