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 bronchial 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 airway hyper-responsiveness (AHR), suggesting that underlying abnormalities in structural cells including 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), which has been designated "severe asthma with fungal sensitization" (SAFS). Current therapies for SAFS including antifungals or omalizumab monoclonal antibody (mAb) targeting IgE have 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 Alkaline protease 1 (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 extracellular matrix (ECM) components, leading to dysregulation of ASM contraction. Subsequently, we demonstrated that Alp1 induces AHR in mice devoid of eosinophils or the protease activated receptor 2 (PAR2) and that Alp1 directly increased contractile force of human ASM cells in vitro. In Fiscal Year (FY) 23, identified an inhibitor of Alp1 protease activity in a compound screen done in collaboration with Dr. Krishnan through an R21 grant. A critical therapeutic gap is to identify moieties with specific affinity for fungal Alp1 but without activity on endogenous serine proteases present in the human airway. The lead compound, which is used therapeutically for hepatitis C (HCV) infections, inhibits the effect of Alp on ASM contraction in vitro and in human airways of murine and human precision-cut lung slices (PCLS) treated with Alp1. We are currently studying the efficacy of the compound in mouse models of fungal asthma. We expect these studies to identify a novel therapeutic application for an HCV inhibitor for fungal asthma and provide novel insight into both ASM-intrinsic and allergen-protease-dependent mechanisms of bronchoconstriction. The second area of emphasis for this project is the study of Regulators of G protein signaling (RGS) proteins in the lung. RGSs bind 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.