Regulation of PTH-induced RankL transcription in osteoblasts

PTH 诱导的成骨细胞 RankL 转录的调节

• 批准号: 10247514
• 负责人: Michael Joseph Mosca
• 金额: $ 4.6万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10247514
• 关键词: Aging; Binding; Biochemical; Biomechanics; Bone Resorption; Bone Tissue; Calvaria; Catabolism; Cell Nucleus; Cells; Co-Immunoprecipitations; Cyclic AMP; Disease; Drug Design; Effectiveness; Endocrinology; Enhancers; Enzymes; Equilibrium; G-Protein-Coupled Receptors; Gene Expression; Genetic Transcription; Goals; Histologic; Homeostasis; Hormonal; Immunofluorescence Immunologic; Immunoprecipitation; Individual; Inflammatory; Knowledge; Laboratories; Laboratory Research; Lead; Ligaments; Mass Spectrum Analysis; Messenger RNA; Methods; Modeling; Morbidity - disease rate; Muscle; Musculoskeletal; Nuclear Translocation; Osteoblasts; Osteoclasts; Osteoporosis; PTH gene; Parathyroid Hormone Receptor; Pathology; Pharmaceutical Preparations; Pharmacology; Phosphoric Monoester Hydrolases; Phosphotransferases; Play; Process; Production; Protein Dephosphorylation; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Proteins; Proteomics; Quantitative Reverse Transcriptase PCR; Regulation; Research; Research Personnel; Research Training; Role; Series; Serum Calcium Level; Signal Transduction; Small Interfering RNA; TNFSF11 gene; TRANCE protein; Techniques; Tendon structure; Therapeutic; Time; Tissues; Training; Transcription Coactivator; Transcription Initiation Site; Transcriptional Activation; Transfection; Work; analog; bZIP Domain; bone; bone quality; career; chromatin immunoprecipitation; cytokine; density; experience; graduate student; improved; interest; knock-down; mortality; new therapeutic target; novel; offender; osteoblast differentiation; receptor; response; salt-inducible kinase; transcription factor

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.

【摘要】：骨质疏松症是一种常见的老年性疾病,其特征是骨密度和质量下降。 骨组织并与大量发病率/死亡率相关。骨质疏松症的稳态过程 形成新骨和去除旧骨/受损骨的功能失调，促进过度吸收。甲状旁腺 激素（PTH）是这种体内平衡的关键调节剂，与其类似物一起被用于治疗 骨质疏松症。尽管 PTH 对骨骼具有积极的合成代谢作用，但它也可以通过以下方式刺激分解代谢： 核因子κ-β配体受体激活剂（RankL）的活性。通过PTH治疗骨质疏松症是 受到短暂的“合成代谢窗口”的限制，此后积极的影响会因由 兰克L。理论上，如果满足以下条件，则可能仅保留 PTH 衍生治疗的积极作用： RankL 的转录可以被抑制。 PTH 及其类似物与成骨细胞上的相同受体结合，激活 导致 RankL 转录的信号级联。最近的工作涉及一系列信使， 酶、激酶和磷酸酶调节 RankL 转录的两个共激活因子。确切的 导致这种调节的机制以及与共激活剂相关的转录因子启动 RankL 转录尚未建立。本研究的目的是确定具体的监管规定 激活 RankL 转录的机制和转录因子。在完成目标和训练中 本提案中概述的计划中，研究生迈克尔·莫斯卡 (Michael Mosca) 将在新的领域获得丰富的经验 生化技术，将获得内分泌学和药理学的具体知识，并将扩展他的 肌肉骨骼组织（包括骨骼）方面的专业知识。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 并将为他作为肌肉骨骼领域的独立研究者的研究生涯做好准备。 目标 1.1 将确定几种盐诱导因子的具体个体和/或组合调节作用 激酶和蛋白磷酸酶对 cAMP 调节的转录共激活因子 2/3 (CRTC2/3) 起作用。首先，一个 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 转录的每个因子。然后，击倒的影响 CRTC2/3 核易位将通过定量测定有或没有 PTH 处理 免疫荧光。目标 1.2 将评估 CRTC2/3 在成骨细胞中作为 RankL 共激活剂所发挥的作用 使用类似的方法进行区分。目标 2 旨在鉴定 CRTC2 和 CRTC3 所依赖的转录因子 每种方法都使用质谱、染色质免疫沉淀来激活 RankL 转录， 和 siRNA 敲低。 RankL 表达负责骨质疏松症治疗中的分解代谢 PTH 及其类似物。阐明调节和启动 RankL 转录的机制可能会确定 抑制RankL并提高PTH治疗骨质疏松症疗效的新治疗靶点。