Asthma, a pathological condition of reversible airways obstruction, comprises both inflammation of the lung as well as 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) contractility may exist in this disease. The major substances that induce bronchial smooth muscle contraction are agonists of G protein coupled receptors (GPCRs). Allergic reactions are initiated by allergen crosslinking of of high affinity IgE receptors on lung mast cells MCs sensitized by IgE, and this allergic mechanism is probably the most common inciter of the pathophysiological cascade 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. 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 bind to the G protein alpha subunits Gi and Gq (but not Gs) through a conserved RGS domain and inactivate 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. We have established RGS9 expression by PCR, immunofluorescence, immunohistochemistry, and immunoblotting in alveolar type II pneumocytes (TIIPs) identified in mouse lung sections and in a cultured human cell line with a TIIP phenotype. RGS5 was shown to be expressed by PCR and immunoblotting in human and mouse bronchial smooth muscle. In human cultured airway smooth muscle (ASM) cells, RGS5 expression appears to be affected by physiological regulators of ASM function such as endothelin-1, acetylcholine, bradykinin, and isoproterenol. ? The second objective will be addressed by studying RGS knockout mice and by using RNA interference to knockdown gene expression in cultured human cells. To elucidate how RGS5 regulates the ASM function in normal lung and in asthma, we are studying RGS5 knockout (KO) mice. The tracheas of these mice are excised and their contraction determined in a physiological salt solution bath connected to a pressure transducer at baseline and after agonist stimulation or electric field stimulation. Preliminary data indicate a decrease in responses of wild type (WT) and KO tracheas to acetylcholine (Ach, a bronchoconstrictor) stimulation. Moreover, the mobilization of Ca in tracheal smooth muscle cells cultured from KO mice is substantially decreased whereas responses to other agonists (bradykinin, endothelin) are intact. Thus, future studies will examine tracheal contractility in these mice after allergen exposure or other treatments that upregulate Rgs5 levels as its expression appears to be low at baseline. In addition, airway responses of intact animals will be measured by whole body plethysmography before and after allergen exposure. We have also established siRNA duplexes that effectively reduce expression of endogenous RGS5 in cultured human ASM cells. We are currently examining the influx of intracellular calcium of siRNA-transfected human ASM cells and cultured mouse ASM from WT or KO mice. Contractility of cultured human airway smooth muscle cells will be studied in vitro by measuring distensibility of collagenn gel matrices connected to the pressure transducer. We also plan to study the proliferation and synthetic function (e.g. cytokine secretion) of cultured ASM cells directly.? To determine the function of RGS9 in type II pneumocytes (TIIP), we employ RNA interference against both RGS9-1 and G-beta-5, its obligate binding partner. The most important physiological function of TIIPs is to secrete surfactant. Surfactant is composed of phospholipids important for the maintenance of alveolar surface tension and proteins involved in host defense. Abnormalities in number and function of alveolar TIIP have been suggested in asthma. Surfactant secretion can be mediated by GPCRs and is in general dependent on increases in intracellular calcium. We are examining the calcium responses of cultured TIIP transfected with RGS9-1 or G-beta-5 siRNA in response to physiological GPCR agonists. Future plans involve studying surfactant secretion in the human TIIP cell line transfected with siRNA or in primary cells isolated from RGS9 or G-beta-5 knockout mice. ? Finally, axin, a protein with a conserved RGS domain but with previously unknown G protein partners, was shown to interact with Gs and Gq. Axin association with Gs was shown to mediate prostaglandin (PGE2)-induced activation of the canonical Wnt-beta catenin pathway, which is important for many physiological processes such as colon cancer cell growth. In addition, axin was shown to be expressed in human airway smooth muscle cells and to be required for full beta-adrenergic receptor, Gs-mediated cyclic AMP (cAMP) formation. These findings indicate a potential role for axin in bronchial smooth muscle relaxation andor contractility. We will determine the mechanism whereby axin modulates G-protein-dependent cAMP levels and establish whether axin affects Gq-mediated signaling pathways. Eventually, we will study how axin affects bronchial smooth muscle contraction or other functions of ASM cells by manipulating axin expression by RNA interference.
| Madigan, Laura A; Wong, Gordon S; Gordon, Elizabeth M et al. (2018) RGS4 Overexpression in Lung Attenuates Airway Hyperresponsiveness in Mice. Am J Respir Cell Mol Biol 58:89-98 |
| Rosenberg, Helene F; Druey, Kirk M (2016) Eosinophils, galectins, and a reason to breathe. Proc Natl Acad Sci U S A 113:9139-41 |
| Yang, Zhao; Balenga, Nariman; Cooper, Philip R et al. (2012) Regulator of G-protein signaling-5 inhibits bronchial smooth muscle contraction in severe asthma. Am J Respir Cell Mol Biol 46:823-32 |
| Liang, Genqing; Barker, Tolga; Xie, Zhihui et al. (2012) Naive T cells sense the cysteine protease allergen papain through protease-activated receptor 2 and propel TH2 immunity. J Allergy Clin Immunol 129:1377-1386.e13 |
| Iwaki, Shoko; Lu, Yunbiao; Xie, Zhihui et al. (2011) p53 negatively regulates RGS13 protein expression in immune cells. J Biol Chem 286:22219-26 |
| Yang, Zhao; Cooper, Philip R; Damera, Gautam et al. (2011) Beta-agonist-associated reduction in RGS5 expression promotes airway smooth muscle hyper-responsiveness. J Biol Chem 286:11444-55 |
| Bansal, Geetanjali; Druey, Kirk M; Xie, Zhihui (2007) R4 RGS proteins: regulation of G-protein signaling and beyond. Pharmacol Ther 116:473-95 |
| Castellone, Maria Domenica; Teramoto, Hidemi; Williams, Bart O et al. (2005) Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-beta-catenin signaling axis. Science 310:1504-10 |
