Because airway smooth muscle (ASM) plays a crucial role in driving the excessive airway obstruction and inflammation seen in severe asthma, failure of ASM to properly respond to drug therapy entails serious consequences for asthmatic patients. While much research aimed at understanding the basis of insensitivity to glucocorticoid (GC) therapy in severe asthmatics focused on the role of immune cells, little has been done to clarify the role of ASM cells. The long-term goal of this research is to define the molecular mechanisms mediating inflammation-associated GC insensitivity in clinically relevant non-immune lung cells, with the hope of identifying novel targets to restore steroid responsiveness in severe asthmatics. The current proposal aims to systematically investigate the mechanisms by which inflammatory cytokines control ASM sensitivity to GCs. The proposal relies on exciting preliminary data supporting the central hypothesis that abnormal phosphorylation of the GC receptor (GR) at specific residues provides a mechanism by which inflammatory cytokines impair ASM sensitivity to GCs. Our preliminary data further suggest that the phosphorylation state of the GR at specific residues is regulated by cytokine-mediated activation of Mitogen-Activated Protein Kinases (MAPKs) concomitant with select serine/threonine protein phosphatases (PPs) present in ASM cells. The rationale for the proposed research is that understanding the interplay between GR signaling and inflammatory kinases and/or protein phosphatases may uncover critical information for the development of novel therapeutics to overcome GC insensitivity in severe asthma. This hypothesis will be tested by pursuing three specific aims: to identify the relative contribution of GR phosphorylation-dependent mechanisms in the differential responsiveness of steroid-target genes to GCs in control and GC-insensitive cells and tissues (Aim 1), to clarify the role of MAPKs (Aim 2) and the contribution of PPs (Aim 3) in cytokine-induced abnormal site- specific phosphorylation of the GR. In the first aim, mutated constructs will be used to modulate site-specific phosphorylation of the GR, after which the transcriptional regulation of 10 different steroid-target genes will be analyzed. In the second and third aims, siRNA, mutated constructs, and expression vectors will be used to modulate the expression of MAPKs and PPs, after which site-specific GR phosphorylation, GR-mediated transactivation activities, GR sub-cellular localization, steroid co-factor recruitment to steroid-target gene promoters, and quantitative expression of steroid-target genes will be analyzed using state-of-the-art molecular and biochemistry approaches already established in our laboratories. We will also evaluate the clinical significance of our in vitro observations by examining whether the aforementioned pathways are activated in tissue biopsies from normal subjects and asthmatic patients and correlate the research findings with asthma severity. This integrated translational approach is expected to advance our understanding of the cellular and molecular mechanisms that contribute to the development of steroid insensitivity in severe asthmatic patients.
The public health relevance of the proposed research derives from the potential of uncovering the link between the activities of asthma-related inflammatory molecules and glucocorticoid receptor dysfunction seen in severe asthmatics. Clarifying the nature and the mechanisms underlying this link could provide new therapeutic targets to overcome steroid insensitivity in severe asthmatics. Thus, the proposed research is relevant to the NIH mission that pertains to developing fundamental knowledge that will help to reduce the burdens of illness.
|Bouazza, Belaid; Debba-Pavard, Manel; Amrani, Yassine et al. (2014) Basal p38 mitogen-activated protein kinase regulates unliganded glucocorticoid receptor function in airway smooth muscle cells. Am J Respir Cell Mol Biol 50:301-15|
|Chachi, Latifa; Shikotra, Aarti; Duffy, S Mark et al. (2013) Functional KCa3.1 channels regulate steroid insensitivity in bronchial smooth muscle cells. J Immunol 191:2624-36|