Dissection of Alternaria-induced Asthma: Role of the PAR2-beta-arrestin Signaling Axis
The goal of this dissertation project was to understand how activation of Protease-activated receptor-2 (PAR2)-beta-arrestin-2-dependent signaling leads to promotion of inflammation in the allergic response to the airborne allergen Alternaria alternata (A. alternata) in the lung. Exposure to A. alternata spores is a major problem for people susceptible to the allergens, and is a leading cause of asthmatic symptoms. The current standard of care for treatment of acute asthma exacerbations is not sufficient for many patients, highlighting the need for alternative therapies. PAR2 is of great interest as a therapeutic target because it promotes both pro- and anti-inflammatory responses in the airway, through opposing G-protein and β-arrestin signaling pathways, and it is activated by proteases released by household pathogens such as Alternaria. These studies examine: 1) the molecular mechanisms underlying Alternaria-induced asthma, delineating the signaling pathways and identifying a new PAR2 activating protease (AASP) present in Alternaria extracts and 2) characterizing a novel PAR2 antagonist (C391) for potential β-arrestin bias, and investigating its ability to inhibit Alternaria –induced inflammation. In cultured cells, I examined key readouts of G-protein and β-arrestin-dependent PAR2 signaling and demonstrate that Alternaria and AASP promote both pathways. In vivo, Alternaria and AASP promote pro-inflammatory responses in the lung that are dependent on PAR2 and β-arrestin-2. However, the proinflammatory components of Alternaria also promote the protective PAR2 effects in isolated bronchioles. My studies indicate that the PAR2 antagonist, C391, inhibits Alternaria-induced airway inflammation in vivo. In cultured cells, C391 inhibits PAR2-β-arrestin signaling in cells at an IC50 100-fold lower than that for inhibition of G-protein signaling, suggesting it may represent a β-arrestin biased antagonist. Altogether, I have found that PAR2 activation via β-arrestin-2 plays a major role in mediating pro-inflammatory responses induced by several allergens in the airway, and that the use of the novel antagonist using C391 may be a new avenue for therapeutic intervention. Thus, PAR2-βarrestin-2 activation acts as a central switch that allergens turn on but can be turned off therapeutically.