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

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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.