Regulation of Normal and Asthmatic Lung Function by G-Protein-Coupled Receptors

National Institute Of Allergy And Infectious Diseases

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Abstract

Asthma, a pathological condition of reversible airway obstruction, is comprised of both inflammation of the lung and hyper-contractility of the bronchiolar smooth muscle. The major naturally occurring substances that induce bronchial smooth muscle contraction are ligands of G-protein-coupled receptors (GPCRs), such as allergen proteases, thrombin, and those contained in allergen-IgE activated mast cell granules (e.g. histamine, cysteinyl leukotrienes (LTD4), endothelin 1, adenosine, and bradykinin). In general, these agonists induce activation of the heterotrimeric G protein G-alpha q, which increases the concentration of intracellular calcium in smooth muscle cells, promoting actin-myosin interactions and muscle fiber shortening. In contrast, ligands acting on G-alpha-s-coupled receptors, such as albuterol, increase intracellular levels of cyclic AMP (cAMP), facilitating ASM relaxation. Although eosinophilic inflammation typifies allergic asthma, it is not a prerequisite for AHR, suggesting that underlying abnormalities in structural cells such as airway smooth muscle (ASM) contribute to the asthmatic diathesis. Dysregulation of procontractile, GPCR signaling in ASM could mediate enhanced contractility. 10-15% of people with asthma experience severe, life threatening attacks and even death despite aggressive treatment with bronchodilators and corticosteroids. Nearly half of these (10-20 million) are sensitized (i.e. have IgE-mediated allergy) to filamentous fungi (e.g. Aspergillus fumigatus, Af)designated severe asthma with fungal sensitization (SAFS). Current therapies for SAFS including antifungals or omalizumaba monoclonal antibody (mAb) targeting IgEhave not achieved uniform success. Utilizing a model of allergic airway inflammation in mice induced by respiratory Af exposure, we have 2 overarching aims for this project: 1) identify derangements in ASM contraction signaling downstream of inflammatory mediators; 2) examine the functions of allergen protease activity in AHR, particularly in relation to allergen-ASM interactions. Protease activity is a common and important feature of allergens capable of inducing asthma, most notably from ubiquitous fungi such as Af. Whether any allergens affect ASM contraction directly has never been explored. Previously we found a causal link between fungi and asthma occurring independently of allergenicity;in other words, host inflammatory response to the allergen. The secreted Af protease Alp1 was detected in the airways of asthmatic subjects but not controls. In mouse and in vitro studies, we found that Alp1 directly promoted AHR by degrading ECM components, leading to dysregulated ASM function. In FY17, we that amounts of Alp1 in the airways directly correlated with clinical asthma severity and parameters of respiratory impairment. A large family of Regulators of G protein signaling (RGS) proteins binds to the G protein alpha subunits Gi and Gq (but not Gs) through a conserved RGS domain and inactivates them by catalyzing their intrinsic GTPase activity and by blocking downstream effector interactions. Although they are generally considered to act as negative regulators of GPCR signaling pathways, the physiological function of RGS proteins in the lung is mostly unknown. We identified expression of several RGS proteins (RGS4, RGS5) in bronchial smooth muscle of humans and mice. In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung ASM mass and stiffness. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mittogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. We are currently examining the phenotype of with global, epithelial, and smooth muscle-specific Rgs4 gene deletion, as well as mice that overexpress RGS4, in models of acute and chronic allergic airway inflammation. In collaboration with Dr. Neubig at the University of Michigan, we will examine the effect of an RGS4-specific inhibitor on the development of the asthma phenotype and ASM hyperplasia and contraction in animal models and cell culture. This is a first-generation RGS inhibitory compound. In collaboration with Dr. Simeonov and colleagues at NCATS, we propose high throughput screening to identify inhibitors of Alp1 protease. Such compounds will be used to study the functions of protease allergens in AHR and elucidate ASM contraction mechanisms in fungal-associated asthma. Eventually, we hope to identify small molecules targeting Alp1 protease for the treatment of patients with SAFS. Toward this end, we are generating recombinant Alp1 protease in yeast to use in screens.