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：骨质疏松症是一种普遍的衰老疾病，其特征是密度和质量降低 骨组织的，与大量发病/死亡率有关。在骨质疏松症中，稳态过程 形成新的和去除旧/受损的骨骼的情况失调，从而促进过量的分辨率。甲状旁腺 Horseone（PTH）是这种体内稳态的关键调节剂，并且与其类似物一起用于治疗 骨质疏松症。尽管PTH对骨骼具有积极的合成代谢作用，它也可以通过 核因子Kappa-β配体（RANKL）受体活化剂的活性。通过PTH治疗骨质疏松症 受一个简短的“合成代谢窗口”的限制，之后，通过该分辨率启动的分辨率来减轻积极影响 Rankl。理论上，如果 RANKL的转录可以抑制。 PTH及其类似物在成骨细胞上结合相同的受体，激活 信号级联导致RANKL转录。最近的工作已经实施了一系列的使者， 调节的酶，激酶和磷酸酶是RANKL转录的两个共激活因子。确切的 引起该调节和转录因子的机制，同时激活因子与启动相关的机制 RANKL转录尚未建立。这项研究的目的是确定特定的监管 激活RANKL转录的机制和转录因子。完成目标和培训 该提案中概述的计划研究生迈克尔·莫斯卡（Michael Mosca）将获得新的经验 生化技术，将获得内分泌学和药理学方面的特定知识，并将扩大他的 肌肉骨骼组织的专业知识包括骨骼。这项研究培训将以他的生物力学和 与肌肉，肌腱/韧带病理学的炎症和纤维化机制有关的组织学经验 并将为他做好研究职业的准备工作，担任肌肉骨骼领域的独立研究者。 AIM 1.1将确定特定的个体和/或合并的调节作用，几个可诱导的盐 激酶和蛋白质磷酸酶具有cAMP调节的转录共激活因子2/3（CRTC2/3）。首先，a 一系列siRNA转染将使用原发性颅骨成骨细胞进行探索 通过QRT-PCR击倒RANKL转录的每个因素。然后，敲低对 CRTC2/3核转运将通过定量确定有没有PTH处理的情况 免疫荧光。 AIM 1.2将评估CRTC2/3的角色在整个成骨细胞中扮演RANKL共激活因子 使用类似方法的分化。 AIM 2试图确定CRTC2和CRTC3的转录因子 每个与质谱，染色质免疫沉淀一起激活RANKL转录， 和sirna敲低。 RANKL表达负责通过治疗骨质疏松症的分解代谢 PTH及其类似物。阐明调节和启动RANKL转录的机制可以识别 新型的热靶标抑制RANKL并提高PTH治疗骨质疏松症的效率。