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: 8597942
• 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/8597942
• 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; Complex; 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 Factor; HDAC1 gene; HDAC2 gene; 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; Sampling; Small Interfering RNA; Staging; Stretching; Symptoms; Syndrome; Techniques; Testing; Theophylline; Tight Junctions; Time; Toxin; Transcriptional Regulation; Transfection; UPK3 gene; Ulcer; United States; Up-Regulation; Urine; Urologic Diseases; Veterans; Vimentin; Western Blotting; alanylalanine; 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; micturition urgency; monolayer; occludin; prolylvaline; protein expression; public health relevance; receptor; repaired; research study; response; transcription factor; valyl-valyl-valine

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) 是一种慢性膀胱疼痛综合征，影响美国约一百万人的生活。 BPS/IC 对于退伍军人群体也变得越来越重要，因为据 2006 年报道，BPS/IC 是第四大常见泌尿系统疾病，也是所有 VA 医疗保健用户中 1.4% 的主要诊断对象（最新可用数据，从 1999 年到 2002 年，比率增加了 13.7%，原始数字增加了 38%）。 BPS/IC的病因尚不清楚，目前尚无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、针对疾病的治疗方法。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，以及蛋白聚糖和蛋白表达异常（包括紧密连接和粘附蛋白、波形蛋白和尿斑蛋白 III）。外植的 BPS/IC 膀胱上皮细胞在体外的增殖也有所减少，并且与体内患者膀胱活检中所见的异常蛋白聚糖和蛋白质表达相同。此外，BPS/IC 细胞外植体分泌卷曲 8 相关抗增殖因子 (APF) (sialyl-Galb1-3GalNAcaO-Thr-Val-Pro-Ala-Ala-Val-Val-Val-Ala)，该因子是对照细胞不分泌的，在约 95% 的 BPS/IC 患者的尿液中独特发现（但不在对照尿液中），并且它诱导 正常膀胱上皮细胞的增殖和蛋白质表达异常与 IC 细胞中所见相同。因此，BPS/IC 患者的膀胱疼痛以及尿频和尿急增加可能是由于膀胱上皮细胞基因表达/分化异常所致，这是该综合征特有的，导致膀胱上皮变薄或溃疡、渗漏和细胞蛋白表达异常。由于之前的微阵列实验显示 BPS/IC 膀胱上皮细胞异常表达多种分化蛋白和染色质重塑蛋白，最近的研究确定 BPS/IC 细胞确实表现出某些基因调节蛋白（包括特定组蛋白脱乙酰酶 (HDAC)、转录因子和 microRNA）的异常补充，这可以解释其异常基因表达和 差异化。因此，拟议的研究将解决这样的假设：我们和其他人在体内和/或体外的 BPS/IC 膀胱上皮细胞中描述的基因表达异常是由特定基因调节因子的异常水平造成的。我们对外植 BPS/IC 细胞中异常 HDAC、特定转录因子和 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证实，并使用蛋白质印迹、染色质免疫沉淀 (ChIP)、microRNA 微阵列和定量实时聚合酶确定这些和其他调节因子在异常特定基因表达中的潜在作用。 链式反应（qRT-PCR）技术。每个调节因子在引起 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）来证实。由于 APF 可以诱导相同的增殖和通透性变化，以及在体内和/或体外 BPS/IC 膀胱细胞中发现的许多一致的基因表达异常，因此还将研究该毒素在调节这些染色质相关因子中的作用，以及四种 APF 抑制剂的能力（之前已显示使增殖、细胞旁通透性和紧密连接正常化） BPS/IC 细胞中的蛋白质表达）以使 BPS/IC 细胞中的染色质调节因子正常化。最后，还将建立一种容易获得、相对无毒的 HDAC 刺激剂（茶碱）使 BPS/IC 细胞中基因表达正常化的能力。这些研究应该会产生有关 BPS/IC 膀胱细胞异常基因表达/分化机制的有价值的信息，从而有可能开发出更有效的疗法。