Grantee Research Project Results
Final Report: Mechanisms of Air Pollutant-induced Pulmonary Inflammation; Effects of Zinc on EGFR Receptor Function
EPA Grant Number: R829214Title: 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: Particulate Matter , Air Quality and Air Toxics , Human Health , Air
Objective:
The overall objectives of this research project were to investigate the effects of zinc (Zn) on epidermal growth factor receptor (EGFR) function and to determine the consequences of Zn exposure on EGFR-regulated cell signaling pathways. Zn is a common component of airborne particulate matter (PM). Chronic or acute exposure to Zn has been shown to induce a wide-range of inflammatory responses. The primary emphasis of this investigation was to use human airway epithelial cells as cellular model systems to determine the effects of Zn exposure on protein kinase-mediated signaling pathways. Because our preliminary results that showed that Zn exposure activated the EGFR tyrosine kinase, we focused our studies on the effects of the Zn on the EGFR and its cognate signaling pathways. The specific objectives of the research project were to: (1) characterize the transactivation of the EGFR induced by Zn; (2) determine the role of the non-receptor kinases in Zn-induced activation of EGFR; and (3) examine the link between EGFR activation and Zn-induced expression of pro-inflammatory mediator proteins in human airway epithelial cells.
Summary/Accomplishments (Outputs/Outcomes):
We attempted to characterize the modulation of COX-2 expression in human airway epithelial cells exposed to Zn2+ and determine the involvement of intracellular signaling pathways and transcription factors in this event.
Rationale and Significance. Next to iron, Zn is the most abundant combustion-derived metal associated with respiratory ambient PM. Animal studies have shown that instillation of Zn2+ or Zn2+-containing particles caused lung injury and inflammation. Exposure to Zn2+-containing PM may be associated with or exacerbate pulmonary inflammatory diseases, such as asthma. Welders exposed to ZnO fumes develop acute neutrophilic airway inflammation and a flu-like syndrome. Airway inflammation is a common pathophysiological event and also an important biological mechanism of diverse lung diseases, which are mediated by inflammatory mediators including cytokines, COX-2, nitric oxide, and so on. COX-2 expression has been observed in various inflammatory diseases, but not all aspects of the expression and the role of COX-2 in conditions of allergic inflammation, such as asthma, are clear. Airway epithelial cells are the initial target for inhaled toxicants, as well as the target for factors released by infiltrating inflammatory cells. These cells can synthesize and release pro-inflammatory mediators, which can cause inflammatory cell differentiation, chemotaxis, and activation. In this research project, we have systemically investigated the regulation of COX-2 expression in human airway epithelial cells. The results may provide basic information for helping the design and development of new therapies for a variety of pulmonary disorders.
COX-2 enzymes catalyze the synthesis of prostaglandins (PGs) from arachidonic acid. The latter is a 20-carbon polyunsaturated fatty acid that is released from membrane phospholipids in a reaction catalyzed by phospholipase A2 (PLA2). There are three known isoforms of COX. COX-1 is constitutively expressed in most cell types and appears to be responsible for the production of PGs that control normal physiological functions. COX-2 has a low basal expression in most tissues but is induced by mitogenic and inflammatory stimuli, which results in the enhanced synthesis of PGs in neoplastic and inflamed tissues. Both COX-1 and COX-2 are the targets of nonsteroidal anti-inflammatory drugs. COX-3 is an alternatively spliced COX-1 and most abundant in the cerebral cortex and heart. It is selectively inhibited by analgesic/antipyretic drugs and is potently inhibited by some nonsteroidal anti-inflammatory drugs.
Research Approaches and Results. The research approaches have been performed as described below. First, we examined the effect of Zn2+ exposure on COX-2 expression at both mRNA and protein levels. Furthermore, the effect of Zn2+ on COX-2 transcriptional activity and mRNA stability was investigated. Second, we determined the effect of Zn2+ exposure on various signaling pathways and their association with COX-2 expression. At last, we defined the involvement of transcription factors AP-1 and NFkB in Zn2+-induced COX-2 expression and their regulation by intracellular signaling pathways. The following are the data achieved from the above approaches.
To examine the effect of Zn2+ on COX-2 expression, BEAS-2B cells (a human airway epithelial cell line) were treated with Zn2+ under various conditions. The expression of COX-2 mRNA and protein was determined using real-time polymerase chain reaction and immunoblotting. The effect of Zn2+ on COX-2 enzyme activity was detected through measuring the release of PGE2 with enzyme-linked immunosorbent assay. Zn2+ exposure resulted in a time- and dose-dependent increase in COX-2 mRNA and protein expression in BEAS-2B cells. The regulation of COX-2 mRNA level was studied at both transcriptional and post-transcriptional levels. The transcription inhibitor, actinomycin D, markedly blocked Zn2+-induced COX-2 mRNA expression. In support of this observation, transient transfection was carried out using a luciferase expression construct containing the COX-2 promoter region. It demonstrated that Zn2+ exposure increased the activity of COX-2 promoter reporter in a time- and dose-dependent fashion in BEAS-2B cells. To further explore whether Zn2+ exposure affected COX-2 mRNA stability, luciferase expression constructs containing diverse COX-2 3’-untranslated region (UTR) mutants were transfected into BEAS-2B cells. It was found that the 3’-UTR of COX-2 mRNA, especially the AU-rich element in this region, was required for Zn2+-induced COX-2 mRNA stabilization. From the above, Zn2+ exposure increased COX-2 expression through the induction of COX-2 promoter activity, as well as COX-2 mRNA stabilization. In the presence of the PLA2 activator mellitin, Zn2+ potently induced PGE2 release from BEAS-2B cells. The effect of Zn2+ on PGE2 release or PLA2 activity is being confirmed or tested using high-performance liquid chromatography.
To investigate the involvement of intracellular signaling pathways in Zn2+-induced COX-2 expression, BEAS-2B cells were pretreated with or without kinase inhibitors or infected with constructs encoding dominant negative or constitutively active kinases. Phosphorylation of various kinases was measured. The results showed that Zn2+ stimulation induced phosphorylation of several kinases, including EGFR, ERK, JNK, p38, and Akt. Inhibition of EGFR, Src, ERK, p38, or PI3K, but not JNK, with suitable pharmacological inhibitors blocked Zn2+-induced COX-2 protein expression. In concert with these findings, overexpression of a construct encoding dominant negative Akt or p38 suppressed Zn2+-induced COX-2 promoter activity. Overexpression of construct encoding wild-type PTEN, the putative antagonist of PI3K/Akt signaling pathway, also blocked Zn2+-induced COX-2 expression. In contrast, overexpression of constitutively active PI3K (p110-CAAK) and p38 directly induced COX-2 promoter activation. The role of ERK in Zn2+-induced COX-2 promoter activation is being tested using a construct encoding a dominant negative ERK. Similar to previous observation in B82L cell lines, inhibition of Src kinase with a Src kinase inhibitor blocked Zn2+-induced EGFR phosphorylation in BEAS-2B cells. Inhibition of Src kinase also suppressed Akt phosphorylation induced by Zn2+. In addition, inhibition of p38 or EGFR but not JNK, decreased Zn2+-induced Akt phosphorylation. From the above, Src/EGFR, ERK, p38, and PI3K, but not JNK, are involved in Zn2+-induced COX-2 expression in human airway epithelial cells. The PI3K/Akt pathway appears to play a central role in this event.
To define the involvement of transcription factors in Zn2+-induced expression of pro-inflammatory mediators, phosphorylation and activation of AP-1 and NFkB were investigated in Zn2+-treated BEAS-2B cells. Zn2+ exposure induced a pronounced phosphorylation of nuclear c-jun. Data from a transient transfection assay using construct containing AP-1 activity reporter consistently indicated that Zn2+ exposure induced a time- and dose-dependent increase in reporter activity. Preliminary studies demonstrated, however, that silencing of c-jun with c-jun siRNA failed to block Zn2+-induced COX-2 expression. A stably transfected BEAS-2B cell line overexpressing the dominant negative c-jun (TAM67) was just established in our laboratory. We plan to test whether the overexpression of TAM67 suppresses Zn2+-induced COX-2 expression. Zn2+ exposure did not induce IkBα phosphorylation and degradation. It caused, however, phosphorylation of nuclear NFkB p65 and time-dependent increase in NFkB reporter activity. Knockdown of p65 with NFkB p65 siRNA partially blocked Zn2+-induced COX-2 expression in BEAS-2B cells. The association of Src/EGFR, ERK, p38, or PI3K with transactivation of NFkB is being investigated. These data suggest that NFkB may play a role in Zn2+-induced COX-2 expression.
From the above data, we propose the working diagram of Zn2+-induced COX-2 expression in BEAS-2B cells (Figure 1).
Figure 1. Regulation of Zn2+-induced COX-2 Expression
Journal Articles on this Report : 8 Displayed | Download in RIS Format
| Other project views: | All 13 publications | 8 publications in selected types | All 8 journal articles |
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Gardner OS, Dewar BJ, Earp HS, Samet JM, Graves LM. Dependence of peroxisome proliferator-activated receptor ligand-induced mitogen-activated protein kinase signaling on epidermal growth factor receptor transactivation. Journal of Biological Chemistry 2003;278(47):46261-46269. |
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Gardner OS, Shiau CW, Chen CS, Graves LM. Peroxisome proliferator-activated receptor gamma-independent activation of p38 MAPK by thiazolidinediones involves calcium/calmodulin-dependent protein kinase II and protein kinase R: correlation with endoplasmic reticulum stress. Journal of Biological Chemistry 2005;280(11):10109-10118. |
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Samet JM, Dewar BJ, Wu W, Graves LM. Mechanisms of Zn2+-induced signal initiation through the epidermal growth factor receptor. Toxicology and Applied Pharmacology 2003;191(1):86-93. |
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Wu W, Graves LM, Gill GN, Parsons SJ, Samet JM. Src-dependent phosphorylation of the epidermal growth factor receptor on tyrosine 845 is required for zinc-induced Ras activation. Journal of Biological Chemistry 2002;277(27):24252-24257. |
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Wu W, Jaspers I, Zhang W, Graves LM, Samet JM. Role of Ras in metal-induced EGF receptor signaling and NF-κB activation in human airway epithelial cells. American Journal of Physiology-Lung Cellular and Molecular Physiology 2002;282(5):L1040-L1048. |
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Wu W, Wang X, Zhang W, Reed W, Samet JM, Whang YE, Ghio AJ. Zinc-induced PTEN protein degradation through the proteasome pathway in human airway epithelial cells. Journal of Biological Chemistry 2003;278(30):28258-28263. |
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Wu W, Samet JM, Silbajoris R, Dailey LA, Sheppard D, Bromberg PA, Graves LM. Heparin-binding epidermal growth factor cleavage mediates zinc-induced epidermal growth factor receptor phosphorylation. American Journal of Respiratory Cell and Molecular Biology 2004;30(4):540-547. |
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Wu W, Silbajoris RA, Whang YE, Graves LM, Bromberg PA, Samet JM. p38 and EGF receptor kinase-mediated activation of the phosphatidylinositol 3-kinase/Akt pathway is required for Zn2+-induced cyclooxygenase-2 expression. American Journal of Physiology-Lung Cellular and Molecular Physiology 2005;289(5):L883-L889. |
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Supplemental Keywords:
epidermal growth factor receptor, EGFR, airborne particulate matter, zinc exposure, human airway epithelial cells, HAEC, RFA, Air, Scientific Discipline, Geographic Area, Waste, Health Risk Assessment, EPA Region, State, particulate matter, Incineration/Combustion, Toxicology, air toxics, epidermal growth factor receptor (EGFR), pulmonary, region 4, combustion-derived metal, pulmonary inflammation, PM, ambient air, Zinc, North Carolina (NC), air pollutant-induced pulmonary inflammation, ambient air quality, human health effectsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.