Asthma, a pathological condition of reversible airway obstruction, is comprised of both inflammation of the lung and hyper-contractility of the bronchiolar smooth muscle. Such airway hyperresponsiveness (AHR) can exist in the absence of frank inflammatory infiltrates, however, suggesting that primary abnormalities in airway smooth muscle (ASM) number or contraction may exist in this disease. The major substances that induce bronchial smooth muscle contraction are natural ligands of 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. 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 accentuating 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. Using immunohistochemistry and immunoblotting with specific antibodies, we identified expression of several RGS proteins (RGS4, RGS5, RGS10) in bronchial smooth muscle. 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, G protein-coupled receptor (GPCR) signaling in ASM could mediate enhanced contractility. Loss of RGS5 promoted constitutive AHR due to enhanced GPCR-induced Ca2+ mobilization in ASM. Precision-cut lung slices (PCLS) from nave Rgs5-/- mice contracted maximally at baseline, independent of allergen challenge. RGS5 deficiency had little effect on parameters of allergic inflammation including cell counts in bronchoalveolar lavage fluid (BALF), mucin production, ASM mass, and subepithelial collagen deposition. Unexpectedly, IL-13 levels were much lower in BALF from Rgs5-/- mice relative to WT. These studies showed that deficiency of RGS5 confers spontaneous AHR in mice in the absence of allergic inflammation. 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 mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited GPCR-mediated bronchoconstriction, RGS4 was unexpectedly required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma. Current studies are examining the phenotype of Rgs4-/- mice in models of acute and chronic asthma. 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. A key regulator of airway relaxation and ASM proliferation is the cyclic AMP-protein kinase A (PKA)-CREB pathway. Previous studies in our laboratory indicated that some but not all RGS proteins regulate this signaling route. RGS10, which is highly expressed in the immune system and in a broad range of brain regions including the hippocampus, striatum, dorsal raphe, and ventral midbrain, is also detected in asthmatic ASM. In collaboration with Dr. Tansey at Emory University School of Medicine, we found that stable over-expression of RGS10 rendered a model dopaminergic neuronal cell line (MN9D) resistant to TNF-induced cytotoxicity through regulation of this signaling pathway at the level of PKA. These results identified PKA as a key mediator of the neuroprotective effect of RGS10 against inflammatory stress and suggest that RGS10 may regulate airway tone through this mechanism. Finally, we explored the regulation of protease receptor signaling in collaboration with Dr. Catalfamo (LIR/NIAID). Disruption of vascular integrity by trauma and other tissue insults leads to inflammation and activation of the coagulation cascade, which are linked by activation of protease-activated receptor 1 (PAR-1) by the serine protease thrombin. We found that peripheral blood effector memory CD4(+) and CD8(+) T lymphocytes expressed PAR-1 and that expression was increased in CD8(+) T cells from human immunodeficiency virus (HIV)-infected patients. Thrombin enhanced cytokine secretion in CD8(+) T cells from healthy controls and HIV-infected patients. In addition, thrombin induced chemokinesis, but not chemotaxis, of CD8(+) T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and phosphorylated ezrin-radexin-moesin proteins.

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Balenga, Nariman A; Klichinsky, Michael; Xie, Zhihui et al. (2015) A fungal protease allergen provokes airway hyper-responsiveness in asthma. Nat Commun 6:6763
Balenga, Nariman A; Jester, William; Jiang, Meiqi et al. (2014) Loss of regulator of G protein signaling 5 promotes airway hyperresponsiveness in the absence of allergic inflammation. J Allergy Clin Immunol 134:451-9
Hurley, Amanda; Smith, Mindy; Karpova, Tatiana et al. (2013) Enhanced effector function of CD8(+) T cells from healthy controls and HIV-infected patients occurs through thrombin activation of protease-activated receptor 1. J Infect Dis 207:638-50
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