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Regulation of PTH-induced RankL transcription in osteoblasts

PTH 诱导的成骨细胞 RankL 转录的调节

基本信息

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
项目类别：
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

项目摘要

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质密度和质量下降为特征的老年常见病