Mechanisms of Air Pollutant-induced Pulmonary Inflammation; Effects of Zinc on EGFR Receptor FunctionEPA Grant Number: R829214
Title: Mechanisms of Air Pollutant-induced Pulmonary Inflammation; Effects of Zinc on EGFR Receptor Function
Investigators: Graves, Lee M. , Wu, Weidong
Institution: University of North Carolina at Chapel Hill
EPA Project Officer: Chung, Serena
Project Period: November 19, 2001 through November 18, 2004
Project Amount: $874,125
RFA: Health Effects of Particulate Matter (2001) RFA Text | Recipients Lists
Research Category: Air Quality and Air Toxics , Health Effects , Particulate Matter , Air
Description:Evidence from numerous epidemiological, clinical, and toxicological studies indicates that airborne particulate matter (PM) can induce pulmonary inflammation. However, the biological mechanism by which PM air pollutants contributes to pulmonary inflammatory responses is presently unknown. Significant research efforts are focusing on the role of PM-associated transition metals in pulmonary inflammation. Zinc (Zn) is an abundant combustion-derived metal found in most airborne PM. Our previous studies have shown that exposure of airway epithelial cells to Zn triggers the activation of intracellular signaling pathways that lead to enhanced production of inflammatory cytokines. Elevated pulmonary cytokine production results in increased mucin hypersecretion which may contribute to the pathogenesis of asthma and the increased morbidity and mortality associated with exposure to air pollutants.
Identifying the signaling pathways that are acutely activated by toxic metals is essential to elucidating the mechanisms by which air pollutants disrupt normal cellular homeostasis. Recent studies have identified the epidermal growth factor receptor (EGFR) as one of the key mediators of this process. Activation of the EGFR results in further stimulation of "downstream" intracellular signaling pathways such as MAP kinase (MAPK) that may be linked to both inflammatory cytokine production and mucin hypersecretion. Therefore our hypothesis is that EGFR is a key mediator of Zn-dependent responses leading to increased cytokine production and mucin hypersecretion and the objective of this proposal is to investigate the biochemical mechanism(s) by which this occurs.
Approach:The overall aim of this proposal is to specifically determine the mechanism(s) by which Zn increases the phosphorylation and activation of the EGFR and the impact of this event on inflammatory cytokine production or mucin hypersecretion. In aim #1, we will use cellular model systems expressing EGFR proteins (and mutants thereof) to investigate the molecular aspects of the EGFR that are required for the action of Zn on both EGFR and MAPK signaling. The mutant receptors lack key tyrosine phosphorylation sites that are believed to be essential for receptor coupling to "downstream" signals such as MAPK. These cells will be exposed to various concentrations of Zn and the influence of these individual mutations on cell signaling will be evaluated using biochemical assays designed to measure MAPK or other components of this pathway (e.g., RAS). We are particularly interested in characterizing the effect of Tyr 845 mutation (Tyr/ Phe) that is predicted to block the effects of the Src tyrosine kinase on EGFR. In addition, we will use pharmacological inhibitors to help define the intracellular protein kinase signaling pathways required for Zn-dependent activation of EGFR and MAPK. Our preliminary studies demonstrate that inhibition of Src (PP1) uncouples the effects of Zn on the EGFR and MAPK; thus we will continue to use this and other protein kinase inhibitors (i.e., PI-3 kinase inhibitors) to dissect the Zn-dependent signaling pathways that lead to inflammatory cytokine production.
In aim #2, we will directly examine the influence of Zn on EGFR dimerization and activation in model systems and intact cells. We envision two potential mechanisms of Zn action that may or may not be related. Zn may act directly on the receptor itself, acting as a surrogate ligand to stimulate receptor signaling. Alternatively, Zn has previously been shown to inhibit tyrosine phosphatases that naturally oppose the EGFR; this may occur through an increase in reactive oxygen species (ROS) and we will measure this possibility. We will determine if blocking ROS production will prevent the effects of Zn on EGFR signaling. Moreover, we have begun to characterize the tyrosine phosphatases affected by Zn exposure and will attempt to purify and identify these targets through affinity purification. Thus the studies in this aim are expected to elucidate whether the effects of Zn are direct (i.e. inducing receptor dimerization) or indirect (inhibition of a phosphatase) on the EGFR.
Finally, in aim #3 we will use modern techniques of mass spectrometry and proteomics to isolate and identify tyrosine phosphorylated proteins that may be necessary elements for the mediation of Zn-dependent effects on cell signaling. Tyrosine phosphorylation is a rare event in normal cells; our studies have demonstrated a highly specific effect of Zn on the tyrosine phosphorylation of multiple proteins in airway epithelial cells. Thus using a combination of anti-phosphotyrosine antibody affinity purification and mass spectrometry methods we will attempt to identify these proteins. In addition, we will investigate whether specific proteins (e.g., GAB1) which are tyrosine phosphorylated in response to EGF, are also involved in mediating a Zn cellular response.