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

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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 G protein coupled receptors (GPCRs). Allergic reactions are initiated by allergen crosslinking of high affinity IgE receptors on lung mast cells sensitized by IgE, and this is considered the most common pathophysiological mechanism in asthma. Many of the compounds contained in mast cell granules or synthesized by mast cells act on procontractile GPCRs to induce bronchoconstriction. Examples include 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 isoproterenol, 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. The physiological function of RGS proteins in the lung is unknown. The principal objective of this project is to determine which RGS proteins are expressed in specific cell types in the lung and to enumerate their functions in this organ. The first objective is accomplished primarily by immunohistochemistry and immunoblotting using specific antibodies. RGS5 was shown to be expressed by PCR and immunoblotting in human and mouse bronchial smooth muscle cells. Although short-acting and long-acting inhaled beta2-adrenergic receptor agonists (SABA and LABA, respectively) relieve asthma symptoms, use of either agent alone without concomitant anti-inflammatory drugs (corticosteroids) may increase the risk of disease exacerbation in some patients. We found previously that pretreatment of human precision-cut lung slices (PCLS) with SABA impaired subsequent beta2-agonist-induced bronchodilation, which occurred independently of changes in receptor quantities. In current work, we provided evidence that prolonged exposure of cultured human airway smooth muscle (HuASM) cells to beta2-agonists directly augments procontractile signaling pathways elicited by several compounds including thrombin, bradykinin, and histamine. Such treatment did not affect expression of surface receptor, G protein, or downstream effector (phospholipase C-beta and myosin light chain) pathway components, but rather induced a dramatic reduction in RGS4 and RGS5 proteins, which are inhibitors of G-protein-coupled receptors (GPCR). Knockdown of RGS5 in HuASM increased intracellular calcium flux and myosin light chain (MLC) phosphorylation in response to these ligands, which are prerequisites for contraction, while overexpression of RGS5 inhibited these responses. Precision-cut lung slices from RGS5-deficient mice contracted more to carbachol than WT slices. These results indicate that RGS5 controls GPCR-evoked G-alpha-q-dependent signaling pathways in ASM. Repetitive beta2-agonist use may not only lead to reduced bronchoprotection but also to sensitization of excitation-contraction pathways as a result of reduced RGS5 expression. To determine whether RGS5 also regulates Ca2+ signaling in other cell types, we examined the function of RGS5 in parathyroid in collaboration with Dr. Olson. We found that (1) RGS5 is highly expressed in parathyroid cells; (2) parathyroid adenomas express elevated levels of RGS5 compared to matched pair normal tissue; (3) RGS5 can inhibit calcium-induced IP3 production in response to Calcium sensing receptor (CaSR) stimulation; and (4) mice nullizygous for RGS5 have abnormally depressed plasma PTH levels but normal serum calcium. A second project in collaboration with Dr. Panettieri identified the expression of RGS4 in human and mouse bronchial smooth muscle. In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through an unknown mechanism characterized by increased lung airway smooth muscle (ASM) mass and stiffness. Mitogens such as platelet-derived growth factor (PDGF) induce such plasticity of ASM in part by activating the phosphoinositide-3-OH kinase (PI3K) signaling pathway. We showed that specific upregulation of RGS4 by mitogens induces a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle bundles of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited GPCR-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained PI3K activation in cultured human ASM cells. These studies support a model in which increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in severe asthmatics. In collaboration with Dr. Neubig, 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 the first RGS inhibitory compound that has emerged.