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

项目摘要 哮喘的发病机制涉及气道细胞的结构和功能变化，包括气道平滑肌细胞的结构和功能变化。 肌细胞（ASM）。了解参与改变体内稳态的细胞和分子机制， 气道细胞为开发更新和有效的抗哮喘药物提供了机会。泛在化是一种 进化上保守和高度调节的稳态过程，确保关键信号的周转 中间体的E3连接酶的改变与人类病理学有关，包括癌症、衰老、 神经变性该建议基于我们从人ASM细胞获得的可靠的初步数据， 使用分子和药理学方法的小鼠模型表明：A）E3连接酶环指 蛋白质145（RNF 145）在人肺和ASM细胞中表达，其中在人肺和ASM细胞中表达显著增加。 来源于哮喘的纤维化/重塑气道和ASM细胞; B）沉默或药理学抑制 RNF 145通过调节PI 3激酶降低ASM细胞增殖和细胞外基质分泌， C）促纤维化介质转化生长因子（TGF）b1处理上调 RNF 145在ASM细胞中的表达; D）RNF 145的抑制降低体外ECM分泌，细胞ATP水平， E）RNF 145的过表达驱动ASM细胞生长和ECM合成 不依赖于有丝分裂原或促纤维化剂;和E）抑制RNF 145防止屋尘螨（HDM）- 诱导的气道高反应性、炎症和气道重塑的标志物。我们的数据支持 通过合作者Frey博士的临床发现，他在RNF 145中显示了单核苷酸多态性， （rs 10076782）与新生儿肺功能、学龄期更多呼吸道症状和喘息相关。 ASM细胞在肺功能调节中起着关键作用，并且增加ASM质量和过度收缩性 与哮喘症状有关。然而，E3连接酶RNF 145在ASM细胞或哮喘中的作用并不明显。 知道的RegulomeDB分析表明，RNF 145调节参与代谢、细胞增殖和细胞凋亡的信号分子。 循环、基质产生、细胞命运和氧化应激途径，所有这些都在哮喘发病机制中发挥作用。 因此，我们假设RN 145在调节ASM功能中起关键作用，从而抑制平滑肌细胞的增殖。 RNF 145将防止过敏原诱导的哮喘表型的发展。使用ASM细胞的研究获得 将描述RNF 145在ASM细胞增殖中的作用（Aim 1）， 细胞外基质的产生（目标2），并建立由RNF 145调节的细胞和分子途径。 为了进一步确定RNF 145在气道功能和哮喘发病机制中的作用，我们将使用野生型 以及进行过敏原攻击的整体和平滑肌特异性条件性RNF 145敲除小鼠（Aim 3）。我们还将测试E3连接酶的药理学抑制剂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个重要的、 RNF 145在调节ASM功能和哮喘发病机制中的作用以前未被认识。

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.