Role of Integrins in Bladder Urothelium

整合素在膀胱尿路上皮中的作用

• 批准号: 8037784
• 负责人: Warren G. Hill
• 金额: $ 37.04万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8037784
• 关键词: Abbreviations; Ablation; Actins; Adaptor Signaling Protein; Adenosine; Affect; Alleles; Animals; Apical; Apoptosis; Ataxia; Attenuated; Basement membrane; Behavior; Binding; Biochemical; Biological Assay; Biotinylation; Bladder; Bladder Control; Bladder Diseases; Bladder Urothelium; Cations; Cell Adhesion Molecules; Cell membrane; Cell surface; Cell-Cell Adhesion; Cells; Chemicals; Complement; Complex; Confocal Microscopy; Connective Tissue; Cystitis; Cytoskeleton; Data; Defect; Diabetes Mellitus; Electric Capacitance; Electron Microscopy; Enzymes; Epithelial; Epithelium; Exhibits; Exocytosis; Extracellular Matrix; Family; Fill-It; Focal Adhesion Kinase 1; Focal Adhesions; Frequencies; GAG Gene; Glycosaminoglycans; Hemidesmosomes; Heterozygote; Human; Hydrostatic Pressure; Hyperreflexia; Immunoprecipitation; Incontinence; Integrin Binding; Integrin Signaling Pathway; Integrins; Interstitial Cystitis; Ion Channel; Ions; Israel; Kinetics; Knock-out; Knockout Mice; Knowledge; Lamina Propria; Ligands; Link; Measures; Mechanics; Mediating; Mediator of activation protein; Medical center; Membrane; Membrane Protein Traffic; Membrane Proteins; Molecular; Monitor; Mouse Strains; Mus; Nerve Fibers; Nomenclature; Output; Overactive Bladder; PTK2 gene; Pain; Participant; Pathway interactions; Phenotype; Phosphate Buffer; Phosphotransferases; Play; Prostaglandins; Prostatic hypertrophy; Proteins; Protocols documentation; Publishing; Purinoceptor; Quality of life; Receptor Protein-Tyrosine Kinases; Reflex action; Regulation; Relaxation; Resistance; Role; Saline; Second Messenger Systems; Sensory; Signal Pathway; Signal Transduction; Signaling Molecule; Sodium Channel; Spastic; Sphincter; Spinal cord injury; Stimulus; Stress; Stretching; Surface; Symptoms; Technology; Testing; Tight Junctions; Transmission Electron Microscopy; UPK2 gene; Urethral sphincter; Urine; Urodynamics; Urothelial Cell; Urothelium; Viscosity; Work; adhesion receptor; afferent nerve; apical membrane; autocrine; cell motility; cell type; epithelial Na+ channel; extracellular; in vivo; inhibitor/antagonist; insight; integrin-linked kinase; knockout animal; novel therapeutics; paracrine; public health relevance; receptor; recombinase; response; second messenger; sensor

DESCRIPTION: Integrins are a broad family of cell surface adhesion and signaling molecules which link the extracellular matrix (ECM) to the cytoskeleton of the cell. Integrins bind directly to ECM ligands on the extracellular surface and are linked to the actin cytoskeleton through multiple adaptor proteins and receptor tyrosine kinases on the plasma membrane. They play critical roles in all cell types and regulate cell migration, differentiation, proliferation and apoptosis. They are also force sensors and transduce mechanical stimuli into biochemical signals. This project explores the role of integrins and integrin-signaling pathways within the epithelium lining the bladder - the urothelium - where almost nothing is known of their function. Our primary hypothesis is that integrins represent the initial upstream mechanical sensor in the bladder and detect bladder filling through membrane stretch. In order to investigate this hypothesis we have created a conditional knockout of 21-integrin within the urothelium (21-cKO). Loss of 21-integrin knocks out all integrins within the umbrella and intermediate cells making the superficial layers of the epithelium integrin-null. Preliminary data indicates that the bladders of these mice release significantly less ATP into the lumen upon stretch, hyperactivate ion conductances upon stretch and exhibit detrusor hyperreflexia and detrusor-sphincter dyssynergia when measured by cystometry. These findings are strongly indicative of an obstructed bladder phenotype possibly due to inadequate urethral sphincter relaxation. Taken together these symptoms indicate a mechanical signaling deficit originating in the urothelium which leads to an obstructed bladder phenotype. An understanding of the signaling mechanisms employed by the bladder as it fills is essential in understanding many different bladder disorders, from overactive bladder to interstitial cystitis. Specific aim 1 of our study will examine the repertoire of integrin binding partners in umbrella cells in vivo and will explore the mechanisms by which integrins regulate ATP secretion. ATP has been show to be an extremely important autocrine and paracrine mediator in the bladder and signals through purinergic receptors on both the luminal surface and in suburothelial layers e.g. on afferent nerve fibers. Specific aim 2 will investigate how integrins interact with and regulate mechanosensory ion channels within the urothelium using selective inhibitors and ion substitution protocols to determine which ion channels regulated by integrins. The role of ion fluxes in regulating membrane trafficking and ATP release will also be studied using (i) capacitance changes in response to stretch to monitor membrane trafficking; and (ii) short circuit currents on bladders mounted in Ussing chambers. Specific aim 3 will use cystometry in the presence of various pharmacological modulators of ion channels and receptors to investigate at the molecular level how integrins regulate filling and voiding behavior. PUBLIC HEALTH RELEVANCE: There are large numbers of people who for many different reasons - spinal cord injury, diabetes, cystitis - suffer from disorders of the bladder which can broadly be characterized as overactive bladder. They suffer greatly from embarrassing and debilitating symptoms of incontinence, urgency, pain and frequency which profoundly affect quality of life. This project will provide new knowledge of the mechanisms by which the bladder senses filling and responds and in so doing may suggest new therapeutic options for bladder disease.

描述：整合素是细胞表面粘附和信号分子的广泛家族，它们将细胞外基质（ECM）与细胞的细胞骨架联系起来。整联蛋白直接与细胞外表面的ECM配体结合，并通过多种衔接蛋白和质膜上的多种衔接蛋白和受体酪氨酸激酶与肌动蛋白细胞骨架有关。它们在所有细胞类型中起关键作用，并调节细胞迁移，分化，增殖和凋亡。它们也是力传感器，并将机械刺激转导到生化信号中。该项目探讨了整联蛋白和整联蛋白信号途径在膀胱内衬里 - 尿皮细胞内的作用 - 几乎什么都不知道它们的功能。我们的主要假设是整联蛋白代表膀胱中的初始上游机械传感器，并通过膜伸展检测膀胱填充。为了研究这一假设，我们在尿路上皮（21-CKO）内创建了有条件的21-整合素敲除。 21-紧密蛋白的损失会在伞和中间细胞内击落所有整合素，从而使上皮整联蛋白无效。初步数据表明，这些小鼠的膀胱在拉伸时释放出较小的ATP，在拉伸时过度活化的离子电导量在拉伸时过度活化的离子电导，并在囊肿测量时表现出逆变器超反射性和弯曲器 - 丝状性抑制性。这些发现强烈表明膀胱表型可能是由于尿道括约肌松弛不足所致。这些症状在一起表明尿皮细胞中的机械信号赤字，导致膀胱表型受阻。对膀胱填充时采用的信号传导机制的理解对于了解从过度活跃的膀胱到间质膀胱炎的许多不同的膀胱疾病至关重要。我们研究的特定目的1将检查体内伞状细胞中整合素结合伴侣的曲目，并将探索整联蛋白调节ATP分泌的机制。 ATP已被证明是膀胱中极其重要的自分泌和旁分泌介质，并通过腔表面和层层层上的嘌呤能受体（例如关于传入的神经纤维。具体目标2将使用选择性抑制剂和离子替代方案来调查整联蛋白如何与尿皮细胞内的机械感应离子通道相互作用，以确定由整联蛋白调节的离子通道。还将使用（i）电容变化来响应拉伸以监测膜贩运方面的电容变化来研究离子通量在调节膜贩运和ATP释放中的作用； （ii）安装在Ussing Chambers中的膀胱上的短路电流。特定的目标3将在存在离子通道和受体的各种药理学调节剂的情况下使用囊肿，以在分子水平进行整合素调节填充和空隙行为上进行研究。 公共卫生相关性：有很多人出于多种不同的原因 - 脊髓损伤，糖尿病，膀胱炎）患有膀胱疾病，可以广泛地被描述为过度活跃的膀胱。他们遭受了令人尴尬和使人衰弱的症状，紧迫性，疼痛和频率的症状，这深刻影响着生活质量。该项目将提供新的知识，了解膀胱感官充满和反应的机制，这样做可能暗示了膀胱疾病的新治疗选择。