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: 10466929
• 负责人: Michael Joseph Mosca
• 金额: $ 4.68万
• 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/10466929
• 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：骨质疏松症是一种以骨密度和骨质疏松症为特征的老年性疾病。 对骨组织的损害，并与相当大的发病率/死亡率有关。在骨质疏松症中的动态平衡过程 形成新的和移除旧的/受损的骨骼的功能失调，从而促进过度吸收。甲状旁腺 激素(PTH)是这种动态平衡的关键调节因素，与其类似物一起被用于治疗 骨质疏松。虽然甲状旁腺素对骨骼有积极的合成代谢作用，但它也可以通过以下途径刺激分解代谢 核因子kappa-β受体激活剂配基的活性。甲状旁腺素治疗骨质疏松症 受一个很短的“合成代谢窗口”的限制，在此之后，积极的影响会被由 兰克。从理论上讲，在以下情况下，可能只保留PTH衍生治疗的积极效果 RANKL的转录可被抑制。PTH及其类似物结合成骨细胞上的同一受体，激活 导致RANKL转录的信号级联。最近的研究发现了一连串的信使， 调节RANKL转录的两个辅活化子的酶、激酶和磷酸酶。完全相同的 导致这一调控的机制及其相关的转录因子(S)启动 RANKL转录未建立。这项研究的目的是确定具体的监管 激活RANKL转录的机制和转录因子(S)。在完成目标和培训方面 在这份提案中概述的计划研究生迈克尔·莫斯卡将在新的 生物化学技术，将获得内分泌学和药理学方面的专门知识，并将扩大他的 在肌肉骨骼组织方面的专业知识，包括骨骼。这次研究性训练将建立在他的生物力学和 肌肉、肌腱/韧带病理炎症和纤维化机制的组织学经验 并将为他作为肌肉骨骼领域的独立调查员的研究生涯做好准备。 目标1.1将确定几个盐诱导的特定个体和/或组合的调节角色 激酶和蛋白磷酸酶对cAMP调节的转录辅活化子2/3(CRTC2/3)具有调节作用。首先，一个 利用原代培养的颅骨成骨细胞进行一系列siRNA转染，以探讨其对成骨细胞的影响。 用qRT-PCR方法敲除RANKL转录中的各个因子。然后，击倒对 CRTC2/3核转位将通过定量测定PTH治疗前后的核转位 免疫荧光。AIM 1.2将评估CRTC2/3作为RANKL共激活因子在成骨细胞中扮演的角色 使用类似的方法进行区分。目的2鉴定CRTC2和CRTC3转录因子(S) 每一个都与使用质谱仪、染色质免疫沉淀、 和siRNA敲除。RANKL的表达与骨质疏松症治疗中的分解代谢有关 PTH及其类似物。阐明调节和启动RANKL转录的机制可能会识别 抑制RANKL和提高甲状旁腺素治疗骨质疏松症疗效的新治疗靶点。