NFAT and MEF-2 Choreograph Bladder Wall Remodeling Following Partial Outlet Obstr

NFAT 和 MEF-2 编排部分出口阻塞后的膀胱壁重塑

基本信息

批准号：
7943043
负责人：
Stephen Anthony Zderic
金额：
$ 32.5万
依托单位：
CHILDREN'S HOSP OF PHILADELPHIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7943043
关键词：
Address Affect Age Alternative Splicing Anatomy Animals Antibodies Area Autoradiography Back Binding Binding Sites Bladder Bladder Tissue Calcineurin Calcineurin Pathway Calcium Cell Culture Techniques Cell Fraction Cell Line Cell Nucleus Cell Wall Cells Cellular Stress Child Childhood Clinical Clinical Trials Complement Complex Critical Pathways Cultured Cells Cyclosporine DNA biosynthesis Data Detection Development Etiology Fibroblasts Fibrosis Gene Family Gene Targeting Genes Genetic Transcription Homeostasis Human Human Cell Line Hyperplasia Hypertrophy Implant In Situ In Vitro Kidney Kidney Failure Lead Left Luciferases Measures Mechanics Mediating Medical Messenger RNA Methods Microtomy Modeling Molecular Molecular Analysis Monitor Morbidity - disease rate Morphology Mus Muscle Muscle Cells Myocardium Myosin Heavy Chains N-terminal Neurogenic Bladder Normal Range Nuclear Nuclear Envelope Nuclear Translocation Obstruction Organ Oryctolagus cuniculus Pathway interactions Patients Pattern Performance Physiological Plasmids Population Process Protein Isoforms Proteins Publishing Pump Reporter Role Run-On Assays Screening procedure Secondary to Signal Transduction Site Smooth Muscle Smooth Muscle Myocytes Smooth Muscle Myosins Spinal Spinal Dysraphism Staging Staining method Stains Stretching System Techniques Testing Thymidine Time Transgenic Mice Up-Regulation Ureter Urethra Urinary Incontinence Urine Urothelium Western Blotting Work Workload activating transcription factor base calcineurin phosphatase chromatin immunoprecipitation enhancing factor gel mobility shift assay human MYH11 protein human disease improved in vitro Model in vivo inhibitor/antagonist male mouse model muscle hypertrophy nuclear factors of activated T-cells pressure prevent promoter public health relevance randomized trial research study response therapeutic target therapy design tissue/cell culture transcription factor transcription factor NF-AT c3 uptake urologic

项目摘要

DESCRIPTION (provided by applicant): In response to partial outlet obstruction, the urinary bladder is capable of undergoing hypertrophy via a complex remodeling process that allows it to adapt to its new workload. This remodeling is driven by a number of signaling cascades and their corresponding transcription factors. This proposal focuses on the role of the calcineurin pathway and its associated transcription factors the Nuclear Factor of Activated T Cells (NFAT) and Myocyte Enhancing Factor 2 (MEF-2). Preliminary data support our central hypothesis that partial bladder outlet obstruction is associated with dysregulation of intracellular calcium homeostasis and activation of calcineurin and the subsequent nuclear importation of NFAT. With administration of cyclosporine A (CSA), the resulting bladder hypertrophy is diminished, and there is a reversal of the myosin heavy chain (MHC) mRNA isoforms back towards a normal expression pattern. We propose to further study these observations in a murine model of partial bladder outlet obstruction using a transgenic mouse containing an NFAT-luciferase reporter construct in the presence and absence of CSA administered of varying time points. End points will include bladder mass, in vitro determinations of contractility and shortening velocity, morphology, and a molecular analysis of MHC isoform expression. We also seek to localize in situ, which bladder wall cell population(s) show evidence of calcineurin activation. We also seek to localize the sites where DNA synthesis is taking place within the bladder wall and determine whether these sites of DNA synthesis represent fibroblasts, smooth muscle cells, or both. These in vivo experiments are complemented by in vitro experiments using cultured cells to assess the impact of mechanical deformation upon NFAT and MEF- 2 translocation to the nucleus. This approach will allow for in vitro screening of compounds that can prevent calcineurin activation, and will allow for study of the interaction between NFAT, MEF-2 and the smooth muscle MHC promoter. Using microarray methods we propose to identify the NFAT and MEF-2 responsive target genes within two cell populations; bladder smooth muscle and bladder fibroblasts. The relevance of this proposed work to human disease is apparent when one considers the socially devastating sequelae of urinary incontinence and renal failure that may result from untreated bladder wall hypertrophy. PUBLIC HEALTH RELEVANCE: Human Relevance - Two conditions can result in severe bladder wall hypertrophy in children, posterior urethral valves, and spinal bifida. If left untreated bladder wall hypertrophy leads to the socially devastating sequelae of urinary incontinence and in extreme cases renal failure may result as the bladder loses its ability to store urine at low pressures. New therapy to preserve and enhance native bladder function will help avoid the development of the end stage bladder.

描述（由申请人提供）：响应部分出口障碍物，膀胱能够通过复杂的重塑过程进行肥大，从而使其适应其新的工作量。这种重塑是由许多信号级联反应及其相应的转录因子驱动的。该建议的重点是钙调神经蛋白途径及其相关转录因子的作用。活化T细胞（NFAT）和心肌细胞增强因子2（MEF-2）的核因子。初步数据支持我们的中心假设，即部分膀胱出口阻塞与细胞内钙稳态的失调和钙调蛋白的激活以及随后的NFAT核进口有关。随着环孢菌素A（CSA）的施用，所得的膀胱肥大会减少，并且肌球蛋白重链（MHC）mRNA同工型逆转向正常表达模式。我们建议在存在和不存在不同时间点的CSA的情况下，使用含有NFAT-荧光酶报告构建体的转基因小鼠在部分膀胱出口阻塞的鼠模型中进一步研究这些观察结果。终点将包括膀胱质量，在体外确定收缩力和缩短速度，形态以及对MHC同工型表达的分子分析。我们还寻求原位定位，膀胱壁细胞种群显示出钙调蛋白激活的证据。我们还试图将DNA合成发生在膀胱壁内的位点，并确定DNA合成的这些位点是否代表成纤维细胞，平滑肌细胞或两者兼而有之。这些体内实验是通过使用培养细胞进行体外实验来补充的，以评估机械变形对NFAT和MEF-2转运对细胞核的影响。这种方法将允许在体外筛选可以防止钙调蛋白激活的化合物，并可以研究NFAT，MEF-2和平滑肌MHC启动子之间的相互作用。使用微阵列方法，我们建议在两个细胞群体内识别NFAT和MEF-2响应式靶基因；膀胱平滑肌和膀胱成纤维细胞。当人们认为未经治疗的膀胱壁肥大可能导致的尿失禁和肾衰竭的社会毁灭性后遗症时，这项提出的工作与人类疾病的相关性显而易见。公共卫生相关性：人类相关性 - 两个条件可能导致儿童，后尿道瓣和脊柱裂的儿童严重膀胱壁肥大。如果未治疗的膀胱肥大会导致尿失禁的社会毁灭性后遗症，并且在极端情况下，肾衰竭可能会导致肾脏失效，因为膀胱失去了将尿液储存在低压下的能力。保存和增强本地膀胱功能的新疗法将有助于避免终端膀胱的发展。