J ALLERGY CLIN IMMUNOL FEBRUARY 2008
Protein Kinase A (PKA) Inhibition Renders Interleukin-1 Beta (IL-1b) Pro-mitogenic to Human Airway Smooth Muscle (ASM) Cells: Role of Induced GPCR Ligands A. M. Misior, H. Yan, R. M. Pascual, D. Deshpande, R. B. Penn; Wake Forest University Health Sciences, Winston-Salem, NC. RATIONALE: Increased ASM mass, a pathogenic feature of asthma, is presumed to be driven by inflammation. Pro-inflammatory cytokines such as IL-1b typically decrease the ASM proliferation stimulated by growth factors such as epidermal growth factor (EGF). Recently, we demonstrated PKA inhibition renders IL-1b a strong mitogen and an enhancer of the EGF-stimulated proliferation of human ASM. We explored the mechanisms behind this cooperativity by examining changes in gene expression. METHODS: HASM cells stably expressing GFP fused to PKA inhibiting peptide (PKI-GFP) or GFP (control) were generated by retroviral infection. [3H]-thymidine incorporation was assessed in cultures stimulated with vehicle, IL-1b, EGF, or EGF1IL-1b. In parallel, RNA was isolated from 8hstimulated cells and gene expression was assessed employing Affymetrix U133A microarrays. RESULTS: Growth assays confirmed our previous results that PKA regulates the mitogenic effect of IL-1b on EGF-stimulated human ASM cells. Microarray analysis indicated EGF1IL-1b stimulation induced a variety of GPCR ligands, including chemokines (IL-8, GROa) and proteases that directly activate GPCRs or generate matrix-derived GPCR agonists. Additional transcriptome and functional data suggest pro-mitogenic signaling by these ligands is augmented by suppression of intracellular PKA activity via either PKI-GFP expression or glucocorticoid treatment. CONCLUSION: IL-1b-induces the expression of multiple GPCR agonists with potential to synergize with growth factors to augment ASM growth. Our collective data suggest that induced PKA serves to suppress the mitogenic signaling by these agents, a phenomenon subject to biologic variability and reversible by treatment with steroids. Funding: HL58506
Extracellular Reactive Oxygen Species (ROS) Inhibit Glucocorticoid Receptor (GR) Translocation R. Ramirez, N. N. Bulayeva, W. J. Calhoun; University of Texas Medical Branch, Galveston, TX. RATIONALE: Oxidative stress is heightened in severe asthma and during exacerbations, which is also associated with steroid resistance. We hypothesized that ROS will inhibit GR function in cells. We investigated the effect of ROS on dexamethasone (dex)-induced GR nuclear translocation, a measure of GR function. METHODS: Lung fibroblasts were transfected with a GR-GFP construct, and exposed to a range of concentrations of H2O2 or superoxide anion (generated by Xanthine/Xanthine Oxidase - X/XO), for 30 min and 2 hrs. Following ROS exposure, dex (100 nM) was used to induce GR translocation to the nucleus. Cells were fixed, and GR distribution was quantified by the GFP fluorescence signal in nuclear and cytoplasmic regions using semi-automated software. Nuc:cyto ratio of GR-GFP was used as an index of GR translocation. RESULTS: Dex produced GR-GFP nuclear translocation in control cells. H2O2 exposure produced significant reductions in GR translocation at 1 and 10 uM concentrations, in a time- and dose-dependent manner. Dose ranging demonstrated both Ôno-effectÕ (0.1 uM) and ÔtoxicÕ doses >10 uM) of H2O2. Catalase blocked the inhibiting effects of H2O2 on GR signaling. The protective effect of catalase could be overcome with high concentrations of H2O2. X/XO treatment produced similar reductions in nuclear translocation of GR. CONCLUSIONS: ROS impair GR translocation into the nucleus. Oxidative inhibition of GR function may play an important role in the steroid resistance of asthma exacerbations and the severe asthma phenotype. When ROS metabolism is high, or anti-oxidant defenses are impaired, the resulting oxidant burden may impair GR signaling.
LynB Kinase Plays a Distinct Role in RBL-2H3 Mast Cell Spreading and a Redundant Role with LynA Kinase in FceRImediated LTC4 Secretion B. M. Vonakis, J. Tano, D. Chen, M. McLane; Johns Hopkins University, Baltimore, MD. RATIONALE: The two forms of Lyn kinase (A and B) partition both into and outside of the membrane rafts important for compartmentalizing signaling. A regulated process of cell spreading and retraction is critical for mast cell chemotaxis into inflamed tissues. We investigated the requirement for raft localization of Lyn in inducing LTC4 secretion and spreading on fibronectin. METHODS: We stably transfected the RBL-2H3 mast cell line with raftexcluded chimeras consisting of the extracellular and transmembrane domains of the IL-2 receptor alpha-subunit (Tac) fused to the unique domain of LynA, LynB or the cytoplasmic domain of Tac (TacWT). Cells were sensitized with IgE, stimulated with antigen or A23187 and secretion of LTC4 quantified in the supernatants by EIA. Additionally, cells were plated onto fibronectin-coated dishes for 60 min, 37C and examined by light microscopy. RESULTS: LTC4 secretion after antigen stimulation was dose-dependently inhibited in Tac LynA unique-RBLs (73-93%) and Tac-LynB unique-RBLs (59-83%) compared to TacWT-RBLs. All clones released > 300 pg/ml LTC4 upon 2 micromolar A23187 stimulation. After 60 min. on fibronectin, the majority of both Tac-LynA unique-RBLs and TacWTRBLs had flattened, spread out considerably and produced broad cellular projections. However, Tac-LynB unique-RBLs had spread poorly with the majority of cells remaining rounded. CONCLUSIONS: One role of membrane rafts is to compartmentalize the positive (raft-excluded) and negative (raft-mediated) functions of Lyn kinase in FceRI signaling and secretion. LynB has a distinct role in spreading on fibronectin but a redundant role with LynA in positively regulating LTC4 secretion and as shown previously, histamine release. Funding: NIH
The Type I Il-4 Receptor Complex Selectively Activates The Tyrosine Phosphorylation Of IRS-2 N. M. Heller1, X. Qi1, I. Junttila2, W. E. Paul2, A. D. Keegan1; 1The University of Maryland Baltimore, Baltimore, MD, 2NIAID, NIH, Bethesda, MD. RATIONALE: IL-4 and IL-13 participate in the development of allergic inflammation. They elicit many similar functions and share a receptor subunit (IL-4Ra). However, there are reports of differential responses to these cytokines in vitro and in vivo. Therefore, we compared signaling between these cytokines. METHODS: Human monocytic cell lines or primary mouse macrophages derived from WT or gCKO mice were stimulated with each cytokine; signal transduction was analyzed by western blotting and receptor subunit expression by FACS. RESULTS: IL-4 induced highly efficient tyrosine phosphorylation of IRS-2 in U937 which bear type I IL-4 receptors (gC-positive cells), but not in THP-1 which lack gC. IL-13 did not activate this pathway strongly in either cell type. STAT6 was phosphorylated to maximal levels in both cell types. Enforced gC expression in THP-1 restored phosphorylation of IRS2 after IL-4 stimulation. IL-4 treatment of primary mouse bone marrow macrophages (BMM) stimulated efficient tyrosine phosphorylation of IRS2 while BMM from gCKO mice showed minimal tyrosine phosphorylation (30% of wildtype, P < 0.05, n 5 4); STAT6 was phosphorylated in both. mRNA for arginase I was highly induced in wildtype BMM after 6 hours of IL-4 treatment (88003); this was reduced in knockout BMM (10003). IL-13 induced expression of arginase I mRNA less effectively (5003) at this time point. CONCLUSIONS: IL-4 signaling through type I IL-4 receptors selectively induces efficient tyrosine phosphorylation of IRS-2. The gC subunit appears to be responsible for this selectivity, which may influence functional outcomes. Funding: NIH