E3 ubiquitin ligase RNF145 in airway smooth muscle functions and in asthma

E3 泛素连接酶 RNF145 在气道平滑肌功能和哮喘中的作用

• 批准号: 10522211
• 负责人: Pawan Sharma
• 金额: $ 47.29万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10522211
• 关键词: 1-Phosphatidylinositol 3-Kinase; Adult; Affect; Aging; Alleles; Allergens; Allergic; Asthma; Attenuated; Autophagocytosis; Biochemical; Biological Assay; Cell Cycle; Cell Proliferation; Cell model; Cell physiology; Cells; Chronic; Chronic lung disease; Clinical; Collagen; Data; Deposition; Development; Drug Targeting; Ensure; Extracellular Matrix; Extracellular Matrix Proteins; Fingers; Functional disorder; Future; Gene Family; Genes; Genetic; Homeostasis; Human; Human Pathology; In Vitro; Individual; Infant; Infection; Knockout Mice; Laboratories; Lead; Lung; Malignant Neoplasms; Mammals; Measures; Mediating; Mediator of activation protein; Metabolic; Metabolism; Mitogens; Modeling; Molecular; Mus; Muscle function; Myofibroblast; Neonatal; Nerve Degeneration; Oxidative Stress Pathway; Pathogenesis; Pathway interactions; Persons; Pharmaceutical Preparations; Pharmacology; Phenotype; Physiological; Physiology; Play; Process; Production; Profibrotic signal; Prophylactic treatment; Proteins; Pulmonary Inflammation; Pulmonary Pathology; Pyroglyphidae; Regulation; Respiratory Signs and Symptoms; Ring Finger Domain; Role; Scheme; School-Age Population; Signal Transduction; Signaling Molecule; Single Nucleotide Polymorphism; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; Solid; Specificity; Symptoms; Testing; Transforming Growth Factors; Ubiquitination; Up-Regulation; Wheezing; airway hyperresponsiveness; airway inflammation; airway remodeling; asthma model; asthmatic; asthmatic airway; base; cell growth; cytokine; drug discovery; effective therapy; effectiveness testing; genome wide association study; human disease; in vivo; inflammatory marker; inhibitor; insight; lentiviral-mediated; microscopic imaging; mouse model; muscle form; new therapeutic target; novel; obstructive airway disease; overexpression; prevent; protein expression; protein protein interaction; pulmonary function; respiratory smooth muscle; ubiquitin-protein ligase

PROJECT SUMMARY Asthma pathogenesis involves structural and functional changes in resident airway cells including airway smooth muscle (ASM) cells. Understanding the cellular and molecular mechanisms involved in altered homeostasis in airway cells provides opportunities to develop newer and effective anti-asthma drugs. Ubiquitination is an evolutionarily conserved and highly regulated homeostatic process that ensures turnover of key signaling intermediates. Alterations in E3 ligases have been implicated in human pathologies including cancer, aging, neurodegeneration. This proposal is based on our solid preliminary data derived from human ASM cells and mouse models using molecular and pharmacological approaches that suggest: A) an E3 ligase Ring Finger Protein 145 (RNF145) is expressed in the human lung and ASM cells, with expression significantly increased in fibrotic/remodeled airways and ASM cells derived from asthmatics; B) silencing or pharmacological inhibition of RNF145 reduces ASM cell proliferation and extracellular matrix secretion through regulation of PI3 kinase and AMPK signaling, respectively; C) profibrotic mediator transforming growth factor (TGF) b1 treatment upregulates expression of RNF145 in ASM cells; D) inhibition of RNF145 reduces ECM secretion in vitro, cellular ATP levels, and restores AMPK signaling; E) over-expression of RNF145 drives both ASM cell growth and ECM synthesis independent of mitogen or profibrotic agent; and E) inhibition of RNF145 prevents house dust mite (HDM)- induced airway hyperresponsiveness, inflammation and markers of airway remodeling. Our data are supported by clinical findings from collaborator Dr. Frey who shows a single nucleotide polymorphism in RNF145 (rs10076782) is associated with neonatal lung function, more respiratory symptoms and wheezing at school age. ASM cells play a pivotal role in the regulation of lung function, and increased ASM mass and hypercontractility are associated with asthma symptoms. However, the role of E3 ligase RNF145 in ASM cells or in asthma is not known. RegulomeDB analysis suggests that RNF145 regulates signaling molecules involved in metabolic, cell cycle, matrix production, cell fate and oxidative stress pathways, all of which play a role in asthma pathogenesis. Therefore, we hypothesize that RN145 plays a key role in regulating ASM functions such that inhibition of RNF145 will prevent development of the allergen-induced asthma phenotype. Studies using ASM cells obtained from healthy and asthmatic lung donors will delineate the role of RNF145 in ASM cell proliferation (Aim 1) and extracellular matrix production (Aim 2) and establish the cellular and molecular pathways regulated by RNF145. To further establish the role of RNF145 in airway functions and in asthma pathogenesis, we will use wild type and global and smooth muscle-specific conditional RNF145 knockout mice subjected to allergen challenge (Aim 3). We will also test the effectiveness of a pharmacological inhibitor of E3 ligase, SMER in murine model of asthma. Collectively, findings from the proposed studies will provide novel insights into an important and previously unrecognized role of RNF145 in regulating ASM functions and in the pathogenesis of asthma.

项目摘要 哮喘发病机理涉及居民气道电池的结构和功能变化，包括气道光滑 肌肉（ASM）细胞。了解与改变改变的细胞和分子机制 气道细胞提供了开发更新和有效的抗窒息药物的机会。泛素化是一个 进化保守和高度调节的稳态过程，可确保关键信号的营业额 中间人。 E3连接酶的改变已与人类病理有关，包括癌症，衰老， 神经变性。该建议基于我们从人ASM细胞和 使用分子和药理学方法的小鼠模型建议：a）E3连接酶环手指 蛋白质145（RNF145）在人肺和ASM细胞中表达，表达在表达中显着增加 来自哮喘患者的纤维化/重塑气道和ASM细胞； b）沉默或药理抑制 RNF145通过调节PI3激酶和 AMPK信号分别； c）纤维化介质转化生长因子（TGF）B1处理上调 RNF145在ASM细胞中的表达； d）抑制RNF145可在体外降低ECM分泌，细胞ATP水平， 并恢复AMPK信号传导； e）RNF145的过表达驱动ASM细胞生长和ECM合成 独立于有丝分裂原或纤维化剂； e）抑制RNF145可防止房屋尘螨（HDM） - 诱导气道高反应性，炎症和气道重塑标记。我们的数据得到支持 合作者Frey博士的临床发现，他在RNF145中显示了单个核苷酸多态性 （RS10076782）​​与新生儿肺功能，更多的呼吸道症状和学龄前喘息有关。 ASM细胞在肺功能的调节中起关键作用，并提高ASM质量和超额收入 与哮喘症状有关。但是，E3连接酶RNF145在ASM细胞或哮喘中的作用不是 已知。调节性B分析表明，RNF145调节与代谢，细胞相关的信号分子 循环，基质产生，细胞命运和氧化应激途径，所有这些途径在哮喘发病机理中起作用。 因此，我们假设RN145在调节ASM函数中起关键作用，从而抑制 RNF145将防止过敏原诱导的哮喘表型的发展。使用获得的ASM细胞的研究 从健康和哮喘的肺供体中，将描述RNF145在ASM细胞增殖中的作用（AIM 1）和 细胞外基质产生（AIM 2），并建立由RNF145调节的细胞和分子途径。 为了进一步确定RNF145在气道功能和哮喘发病机理中的作用，我们将使用野生型 以及受到过敏原挑战的全球和平滑肌特异性有条件RNF145敲除小鼠（AIM 3）。我们还将测试E3连接酶的药理抑制剂的有效性，在鼠模型中 哮喘。总的来说，拟议的研究的发现将为重要和 RNF145在调节ASM功能和哮喘发病机理中的先前未识别的作用。