Research Project
9784463
PROJECT SUMMARY Increased airway smooth muscle (ASM) mass, a major feature of airway remodeling in asthma, profoundly contributes to asthma morbidity, mortality, and pathogenesis. This increased mass is due in large part to enhanced proliferative activity of ASM cells. Unfortunately, proliferation of these latter cells is unaffected by current asthma medications, as the process appears to be insensitive to glucocorticoid (GC) effects. While research aimed at understanding the basis of GC insensitivity (GCI) has focused on the role of immune cells, few investigators have studied GCI in ASM cells, the pivotal cell regulating bronchomotor tone. The long-term goal of this research is to identify factors that impair the sensitivity of the ASM proliferative response to GC in patients with asthma, with the ultimately goal of establishing therapeutic strategies to circumvent GCI. The current proposal will examine the cellular and molecular mechanisms by which growth factors (GFs) modulate ASM sensitivity to GCs. Using unique cellular models of ASM cells derived from patients with asthma, our exciting preliminary data supports the central hypothesis that GF-induced abnormal site-specific phosphorylation of glucocorticoid receptor (GR) impairs ASM sensitivity to GC. These data now suggest that (previously unappreciated) GR phosphorylation at serine 134 (ser134) residue inhibits GR signaling. These data also suggest that GF-induced activation of serine/threonine kinase protein kinase B (PKB/Akt) and serine/threonine protein phosphatase 2A (PP2A) regulates GR-ser134 phosphorylation. The rationale for the proposed research is that understanding the interplay among GR signaling and GF-induced kinases and/or phosphatases may uncover critical information for the development of novel therapeutics to overcome GCI in asthma. Our hypotheses will be tested by pursuing four specific aims: to identify the functional consequences of GF-induced abnormal GR site-specific phosphorylation on GR signaling (Aim 1); to characterize the contribution of kinases and phosphatases to GF-induced abnormal GR site-specific phosphorylation (Aim 2); and to elucidate the role of steroid-target genes in modulating GC anti-proliferative effects in ASM cells (Aim 3). To this end, we generated tools (antibody, mutated constructs) to examine the role of GR-ser134 phosphorylation. In all aims, pharmacological inhibitors, mutated constructs, siRNA, and expression vectors will be used to modulate the expression of steroid co-repressor, kinases, phosphatases, and steroid-target genes, after which GR site-specific phosphorylation, GR-mediated transactivation activities and sub-cellular localization, and ASM growth will be analyzed using state-of-the-art approaches already established in our laboratories. We will also determine the clinical significance of our in vitro observations by examining whether the aforementioned pathways are activated in tissues using endobronchial biopsies from subjects both with and without asthma (Aim 4). This integrated translational approach is expected to advance our understanding of the mechanisms that contribute to the development of GCI in some patients with asthma.
