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

项目总结 哮喘的发病机制涉及驻留的呼吸道细胞的结构和功能的变化，包括气道平滑 肌肉(ASM)细胞。了解细胞和分子机制参与的动态平衡改变 呼吸道细胞为开发新的、有效的抗哮喘药物提供了机会。泛素化是一种 进化上保守且高度调控的动态平衡过程，确保关键信号的周转 中间体。E3连接酶的改变被认为与人类的病理有关，包括癌症、衰老、 神经退行性变。这一建议是基于我们从人类ASM细胞和 使用分子和药理学方法建立的小鼠模型，表明：a)E3连接酶环指 蛋白145(RNF145)在人肺和ASM细胞中表达，在 哮喘患者来源的纤维化/重塑的呼吸道和ASM细胞；B)沉默或药物抑制 RNF145通过调节PI3K和PI3抑制ASM细胞增殖和细胞外基质的分泌 AMPK信号转导；C)促纤维化介质转化生长因子b1治疗上调 RNF145在ASM细胞中的表达；D)抑制RNF145在体外减少ECM的分泌，细胞ATP水平， 并恢复AMPK信号转导；E)过表达RNF145促进ASM细胞生长和ECM合成 不依赖于有丝分裂原或促纤维剂；以及E)抑制RNF145可预防屋尘螨(HDM)- 诱导的气道高反应性、炎症和气道重塑的标志物。我们的数据得到了支持 根据合作者Frey博士的临床发现，他在RNF145上显示了单核苷酸多态 (Rs10076782)与新生儿肺功能、更多的呼吸道症状和学龄期的喘息有关。 ASM细胞在调节肺功能中起关键作用，并增加ASM质量和超收缩力。 与哮喘症状有关。然而，E3连接酶RNF145在ASM细胞或哮喘中的作用并不是 为人所知。RegulomeDB分析表明，RNF145调节与细胞代谢有关的信号分子 细胞周期、基质生成、细胞命运和氧化应激途径在哮喘发病机制中起着重要作用。 因此，我们假设RN145在调节ASM功能中起关键作用，从而抑制 RNF145将防止过敏原诱导的哮喘表型的发展。利用获得的ASM细胞进行研究 将描述RNF145在ASM细胞增殖中的作用(目标1)和 细胞外基质的产生(Aim 2)，并建立RNF145调节的细胞和分子通路。 为了进一步确定RNF145在呼吸道功能和哮喘发病机制中的作用，我们将使用野生型 以及受到变应原攻击的全局和平滑肌特异性条件性RNF145基因敲除小鼠(AIM 3)。我们还将测试E3连接酶的药理抑制剂SMER在小鼠模型中的有效性。 哮喘。总的来说，拟议研究的结果将为一项重要的和 以前未知的RNF145在调节ASM功能和哮喘发病机制中的作用。

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.