Regulation of cyclooxygenase-2 expression by cAMP response element and mRNA stability in a human airway epithelial cell line exposed to zinc

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Abstract

Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. Cyclooxygenase 2-derived eicosanoids are important modulators of airway inflammation. In this study, we characterized the transcriptional and posttranscriptional events that regulate COX-2 expression in a human bronchial epithelial cell line BEAS-2B exposed to Zn2+. Zn2+ exposure resulted in pronounced increases in COX-2 mRNA and protein expression, which were prevented by pretreatment with the transcription inhibitor actinomycin D, implying the involvement of transcriptional regulation. This was supported by the observation of increased COX-2 promoter activity in Zn2+-treated BEAS-2B cells. Mutation of the cAMP response element (CRE), but not the κB-binding sites in the COX-2 promoter markedly reduced COX-2 promoter activity induced by Zn2+. Inhibition of NFκB activation did not block Zn2+-induced COX-2 expression. Measurement of mRNA stability demonstrated that Zn2+ exposure impaired the degradation of COX-2 mRNA in BEAS-2B cells. This message stabilization effect of Zn2+ exposure was shown to be dependent on the integrity of the 3′-untranslated region found in the COX-2 transcript. Taken together, these data demonstrate that the CRE and mRNA stability regulates COX-2 expression induced in BEAS-2B cells exposed to extracellular Zn2+.

Introduction

Cyclooxygenase (COX) is a heme-containing enzyme that catalyzes two sequential enzymatic reactions: the bis-oxygenation of arachidonic acid leading to the production of prostaglandin G2 (PGG2) and the reduction of 15-hydroperoxide of PGG2, leading to the formation of PGH2, a precursor of all PGs, thromboxanes, and prostacyclins, in concert with a series of cell-specific isomerases (Smith et al., 2000). Three COX isoforms, COX-1, COX-2, and COX-3, have been identified in mammals (Chandrasekharan et al., 2002). COX-1 is expressed constitutively in most tissues and appears to be responsible for the production of PGs that modulate physiological functions. COX-3 is an alternatively spliced form of COX-1, expressed primarily in brain and heart as a constitutive enzyme. In contrast, COX-2 is expressed at low or undetectable levels in most tissues and cells under basal conditions, but is rapidly inducible by a variety of stimuli such as lipopolysaccharide (LPS), inflammatory cytokines, growth factors, ultraviolet radiation, and chemicals (Fu et al., 1990, Zhang et al., 1998, Subbaramaiah et al., 2000, Chang et al., 2003, Huh et al., 2003).

The COX-2 gene is mapped to human chromosome 1q25.2-q25.3, approximately 8.3 kb in length with 10 exons, and is transcribed as a 4.4 kb mRNA (Tanabe and Tohnai, 2002). The human COX-2 5′-flanking region contains a canonical TATA box and several functionally important enhancer elements including a cyclic AMP response element (CRE), E box and activator protein 1 (AP-1) regulatory element complex situated very close to TATA, a CCAAT/enhancer binding protein (C/EBP) site and two κB sites (Tazawa et al., 1994). The proinflammatory stimuli can induce binding of different transcription factors to their specific DNA-binding sites in a cell type- and stimulus-specific fashion. The transcription factors that bind and activate COX-2 transcription involve C/EBPβ and C/EBPδ for the nuclear factor interleukin-6 (NF-IL-6) elements, AP-1, activating transcription factor (ATF) and CRE-binding protein (CREB) for the CRE element, and upstream stimulatory factor 1 (USF-1) for the E box (Murakami and Kudo, 2004). Posttranscriptional events also play an important role in modulating COX-2 mRNA levels (Dannenberg et al., 2005). The first 60 nucleotides of the 3′-untranslated region (UTR) of COX-2 mRNA are highly conserved and contain multiple copies of the regulatory sequence AUUUA. These well-known AU-rich elements (AREs), present within the 3′-UTRs of many proto-oncogene and cytokine mRNAs, confer posttranscriptional control of expression by acting as a mRNA instability determinant or as a translation inhibitory element that can affect both mRNA and protein translation (Caput et al., 1986, Xu et al., 1997). An ARE element within the 3′-UTR of COX-2 mRNA has been identified that can control both mRNA decay and protein translation (Dixon et al., 2000, Dixon et al., 2001).

Increased COX-2 protein expression has been implicated in the pathogenesis of lung diseases characterized by chronic airway inflammation, including asthma, chronic bronchitis, cystic fibrosis, and bronchiectasis (Ermert et al., 1998, Oguma et al., 2002). Expression of the COX-2 gene has been shown in human airway epithelial cells exposed to exogenous stimuli, such as air-borne residual oil fly ash (Samet et al., 2000), hydrochloric acid (Bonnans et al., 2006), peroxisome proliferator-activated receptor-gamma agonists (Patel et al., 2005), respiratory syncytial virus and Streptococcus pneumoniae infection (Liu et al., 2005, N'Guessan et al., 2006). Zinc (Zn) is an essential micronutrient involved in structural and regulatory cellular functions of a large number of proteins (Vallee and Falchuk, 1993). Zn is also a ubiquitous contaminant in ambient and occupational settings. It exists as a combustion-derived metal associated with ambient particulate matter (PM) and may contribute to the adverse health effects of ambient PM inhalation (Horner, 1996, Adamson et al., 2000). In this study, the regulation of COX-2 expression was studied in a human bronchial epithelial cell line BEAS-2B exposed to Zn2+. We report here that Zn2+ exposure increases COX-2 expression through the CRE site located in the COX-2 promoter region and stabilization of COX-2 mRNA.

Section snippets

Materials and reagents

American Chemical Society-grade zinc sulfate, Triton X-100, and polyacrylamide were purchased from Sigma Chemical Co. (St. Louis, MO). SDS-PAGE supplies such as molecular mass standards and buffers were from Bio-Rad (Richmond, CA). Anti-human COX-2 polyclonal antibody was obtained from Cayman Chemical (Ann Arbor, MI). β-actin antibody was purchased from USBiological (Swampscott, MA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse IgG was obtained from Santa Cruz

Zn2+ exposure increases COX-2 mRNA and protein expression, and PGE2 production in BEAS-2B cells

As demonstrated in our previous study (Wu et al., 2003), exposure of BEAS-2B cells to 50 μM Zn2+ for 8 h did not result in significant alterations in cell viability, as assessed by assay of lactate dehydrogenase activity released into the culture medium. Exposure of BEAS-2B cells to 25 or 50 μM Zn2+ for 8 h caused a marked increase in COX-2 mRNA expression (Fig. 1A). The highest dose utilized (50 μM) induced elevations in COX-2 mRNA which reached about 11 fold at 4 h of exposure and remained

Discussion

COX-2 is an important pharmacological target for the treatment of diseases ranging from inflammation to cancer. The molecular mechanisms controlling expression of COX-2 are not fully defined. This study demonstrates that Zn2+ stimulation markedly increases COX-2 mRNA expression through a combination of transcriptional activation and mRNA stabilization in a human airway epithelial cell line, BEAS-2B. This results in a marked increase in expression of functional COX-2 protein. In the context of

Acknowledgments

We greatly appreciate the technical assistance from Lisa Dailey, Drs Ilona Jaspers and William Reed. This work was supported by the United States Environmental Protection Agency Cooperative Agreement CR83346301 awarded to the Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina. The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory and National Risk Management Laboratory, U.S. EPA, and

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