Abnormal Bladder Epithelial Cell Gene Expression in BPS/IC

BPS/IC 中膀胱上皮细胞基因表达异常

• 批准号: 8965983
• 负责人: Susan F. Keay
• 金额: --
• 依托单位: BALTIMORE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-10-01 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8965983
• 关键词: ATP Receptors; Abnormal Cell; Address; Affect; American; Biopsy; Biopsy Specimen; Bladder; CBA/J Mouse; Cell Culture Techniques; Cell Line; Cell Proliferation; Cells; Chromatin; Chronic; Clinical Trials; Complement; Coupled; DTR gene; Data; Development; Diagnosis; Disease; E-Cadherin; Epigenetic Process; Epithelial; Epithelial Cells; Epithelium; Etiology; Frequencies; Gene Expression; Gene Expression Regulation; Genes; Glycopeptides; Goals; Growth; HDAC1 gene; HDAC2 gene; Health; Healthcare; Healthcare Systems; Histone Acetylation; Histones; Human; In Vitro; Increased frequency of micturition; Interstitial Cystitis; Laboratories; Lead; MMP2 gene; Mechanics; Metalloproteases; MicroRNAs; Modeling; National Institute of Diabetes and Digestive and Kidney Diseases; Neurotransmitters; Normal Cell; Nuclear; Pathogenesis; Patients; Permeability; Phenotype; Play; Polymerase Chain Reaction; Population; Production; Proteins; Proteoglycan; Regulation; Regulator Genes; Reporting; Research; Role; SWI/SNF Family Complex; Sampling; Small Interfering RNA; Staging; Stretching; Symptoms; Syndrome; TP53 gene; Techniques; Testing; Theophylline; Tight Junctions; Time; Toxin; Transcriptional Regulation; Transfection; UPK3 gene; Ulcer; United States; Up-Regulation; Urine; Urologic Diseases; Veterans; Vimentin; Western Blotting; alanylproline; base; bladder pain; chromatin immunoprecipitation; chromatin remodeling; effective therapy; epithelial to mesenchymal transition; experience; gain of function; genetic regulatory protein; histone modification; in vivo; inhibitor/antagonist; knock-down; micturition urgency; monolayer; occludin; protein biomarkers; protein expression; receptor; reduce symptoms; repaired; research study; response; transcription factor

DESCRIPTION (provided by applicant): Bladder pain syndrome/interstitial cystitis (BPS/IC) is a chronic painful bladder syndrome that affects the lives of approximately one million people in the United States. BPS/IC is also becoming increasingly important for the veterans population as it was reported in 2006 to be the fourth most common urologic disease as well as the primary diagnosis for 1.4% of all VA healthcare users (rate increased 13.7% and raw numbers increased 38% from 1999-2002, most recent data available). The etiology of BPS/IC is unknown, and there is currently no reliably effective treatment. It therefore continues to be important to study the pathogenesis of this debilitating disorder, in order to systematically devise effective, disease-specific therapies. Epithelial abnormalities are the most consistent findings in bladder biopsies from BPS/IC patients, including denudation, tears, or thinning of the epithelium, coupled with abnormal proteoglycan and protein expression (including tight junction and adherens proteins, vimentin, and uroplakin III). Explanted BPS/IC bladder epithelial cells also have decreased proliferation in vitro along with the same abnormal proteoglycan and protein expression as seen in patient bladder biopsies in vivo. In addition, BPS/IC cell explants secrete a Frizzled 8-related anti-proliferative factor (APF) (sialyl-Galb1-3GalNAcaO-Thr-Val-Pro-Ala-Ala-Val-Val-Val-Ala) that is not secreted by control cells, is found uniquely in urine from approximately 95% of BPS/IC patients (but not in control urine), and which induces the same abnormalities in proliferation and protein expression in normal bladder epithelial cells as seen in IC cells. The bladder pain plus increased urinary frequency and urgency experienced by BPS/IC patients may therefore result from aberrant bladder epithelial cell gene expression/differentiation that is unique to this syndrome and which results in bladder epithelial thinning or ulceration, leakiness, and abnormal cell protein expression. Because previous microarray experiments showed abnormal BPS/IC bladder epithelial cell expression of several differentiation proteins plus a chromatin remodeling protein, recent studies were performed which determined that BPS/IC cells indeed display an abnormal complement of certain gene regulatory proteins (including specific histone deacetylases (HDACs), transcription factors, and microRNAs) that could explain their abnormal gene expression and differentiation. Therefore, the proposed research will address the hypothesis that the gene expression abnormalities that we and others have described in BPS/IC bladder epithelial cells both in vivo and/or in vitro result from aberrant levels of specific gene regulatory factors. Our preliminary findings of abnormal HDACs, specific transcription factors, and microRNAs in explanted BPS/IC cells will be confirmed using explants from additional BPS/IC patients and controls, and the potential role for these and other regula- tory factors in abnormal specific gene expression determined using Western blot, chromatin immunoprecipita- tion (ChIP), microRNA microarray, and quantitative real-time polymerase chain reaction (qRT-PCR) techniques. The role of each regulatory factor in causing the BPS/IC cell phenotype will be confirmed via loss or gain of function experiments (including siRNA knockdown and transfection). Because APF can induce the same changes in proliferation and permeability, as well as many of the consistent gene expression abnormal- ities found in BPS/IC bladder cells in vivo and/or in vitro, the role of this toxin in regulation of these chromatin- related factors willalso be studied, and the ability of four APF inhibitors (shown previously to normalize proliferation, paracellular permeability, and tight junction protein expression in BPS/IC cells) to normalize chromatin regulatory factors in BPS/IC cells determined. Finally, the ability of a readily availabl, relatively nontoxic HDAC stimulator (theophylline) to normalize gene expression in BPS/IC cells will also be established. These studies should yield valuable information about the mechanism of aberrant gene expression/ differentiation in BPS/IC bladder cells, potentially leading to development of more effective therapies.

描述（由申请人提供）： 膀胱疼痛综合征/间质性膀胱炎（BPS/IC）是一种慢性疼痛性膀胱综合征，影响美国约100万人的生活。BPS/IC对退伍军人群体也变得越来越重要，因为据报道，2006年它是第四大最常见的泌尿系统疾病，也是所有VA医疗保健用户的1.4%的主要诊断（从1999年到2002年，比率增加了13.7%，原始数字增加了38%，最新数据可用）。BPS/IC的病因不明，目前没有可靠有效的治疗方法。因此，研究这种使人衰弱的疾病的发病机制仍然很重要，以便系统地设计有效的疾病特异性疗法。上皮异常是BPS/IC患者膀胱活检中最一致的发现，包括上皮剥脱、撕裂或变薄，以及异常蛋白多糖和蛋白表达（包括紧密连接和粘附蛋白、波形蛋白和尿斑蛋白III）。移植的BPS/IC膀胱上皮细胞在体外也具有降低的增殖，沿着与在体内患者膀胱活检中观察到的相同的异常蛋白聚糖和蛋白质表达。此外，BPS/IC细胞外植体分泌卷曲8相关的抗增殖因子（APF）（唾液酸-Galb 1 - 3GalNH 3 O-Thr-Val-Pro-Ala-Ala-Val-Val-Val-Ala）不被对照细胞分泌，在大约95%的BPS/IC患者的尿液中唯一发现（但在对照尿液中不存在），并且其在正常膀胱上皮细胞中诱导与IC细胞中所见相同的增殖和蛋白质表达异常。因此，BPS/IC患者经历的膀胱疼痛加上增加的尿频和尿急可能是由异常的膀胱上皮细胞基因表达/分化引起的，这是该综合征所特有的，并导致膀胱上皮变薄或溃疡、渗漏和异常细胞蛋白表达。由于先前的微阵列实验显示BPS/IC膀胱上皮细胞异常表达几种分化蛋白加上染色质重塑蛋白，最近的研究确定BPS/IC细胞确实显示某些基因调控蛋白（包括特定的组蛋白脱乙酰酶（HDAC），转录因子和microRNA）的异常互补，这可以解释它们的异常基因表达和分化。因此，拟议的研究将解决的假设，即我们和其他人已经描述的BPS/IC膀胱上皮细胞在体内和/或体外的基因表达异常的特定基因调控因子的异常水平的结果。我们对BPS/IC细胞中异常HDAC、特异性转录因子和microRNA的初步发现将使用来自其他BPS/IC患者和对照的外植体进行确认，并使用Western印迹、染色质免疫沉淀（ChIP）、microRNA微阵列、和定量实时聚合酶链反应（qRT-PCR）技术。每种调节因子在引起BPS/IC细胞表型中的作用将通过功能丧失或获得实验（包括siRNA敲低和转染）来确认。由于APF可诱导体内和/或体外BPS/IC膀胱细胞中发现的相同的增殖和通透性变化以及许多一致的基因表达异常，因此还将研究该毒素在这些染色质相关因子的调节中的作用，并将研究四种APF抑制剂的能力。（先前显示使BPS/IC细胞中的增殖、细胞旁通透性和紧密连接蛋白表达正常化）以使所测定的BPS/IC细胞中的染色质调节因子正常化。最后，还将建立容易获得的、相对无毒的HDAC刺激剂（茶碱）使BPS/IC细胞中的基因表达正常化的能力。这些研究将产生关于BPS/IC膀胱细胞中异常基因表达/分化机制的有价值的信息，可能导致开发更有效的治疗方法。