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Phosphorylation of p65 is required for zinc oxide nanoparticle-induced interleukin 8 expression in human bronchial epithelial cells.

BACKGROUND: Exposure to zinc oxide (ZnO) in environmental and occupational settings causes acute pulmonary responses through the induction of proinflammatory mediators such as interleukin-8 (IL-8).

OBJECTIVE: We investigated the effect of ZnO nanoparticles on IL-8 expression and the underlying mechanisms in human bronchial epithelial cells.

METHODS: We determined IL-8 mRNA and protein expression in primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line using reverse-transcriptase polymerase chain reaction and the enzyme-linked immunosorbent assay, respectively. Transcriptional activity of IL-8 promoter and nuclear factor kappa B (NFkB) in ZnO-treated BEAS-2B cells was measured using transient gene transfection of the luciferase reporter construct with or without p65 constructs. Phosphorylation and degradation of IkB[alpha], an inhibitor of NF-kB, and phosphorylation of p65 were detected using immunoblotting. Binding of p65 to the IL-8 promoter was examined using the chromatin immuno precipitation assay.

RESULTS: ZnO exposure (2-8 [micro]g/mL) increased IL-8 mRNA and protein expression. Inhibition of transcription with actinomycin D blocked ZnO-induced IL-8 expression, which was consistent with the observation that ZnO exposure increased IL-8 promoter reporter activity. Further study demonstrated that the kB-binding site in the IL-8 promoter was required for ZnO-induced IL-8 transcriptional activation. ZnO stimulation modestly elevated IkB[alpha] phosphorylation and degradation. Moreover, ZnO exposure also increased the binding of p65 to the IL-8 promoter and p65 phosphorylation at serines 276 and 536. Over expression of p65 constructs mutated at serines 276 or 536 significantly reduced ZnO-induced increase in IL-8 promoter reporter activity.

CONCLUSION: p65 phosphorylation and IkB[alpha] phosphorylation and degradation are the primary mechanisms involved in ZnO nanoparticle-induced IL-8 expression in human bronchial epithelial cells.

Key WORDS: bronchial epithelial cells, IL-8, interleukin-8, NFkB, p65, zinc oxide. Environ Health Perspect 118:982-987 (2010). DOI:10.1289/ehp.0901635 (Online 1 March 2010]

Inhalation of zinc oxide (ZnO) particles can provoke a number of clinical responses of which the best known is metal fume fever (Gordon et al. 1992; Kuschner et al. 1995). This is accompanied by changes in composition of bronchoalveolar lavage fluid, including early increase in tumor necrosis factor [alpha] (TNF[alpha]) followed by interleukin (IL)-8 and IL-6, and in numbers of polymorphonuclear leukocytes (Blanc et al. 1993; Kuschner et al. 1995, 1997).

ZnO particles in ambient air arise from incinerator emission and from wear and tear of vehicle tires (Adachi and Tainosho 2004; Bennett and Knapp 1982; Councell et al. 2004; Horner 1996; Lough et al. 2005). Previous studies have demonstrated that exposure to Zn-laden (Zn in its salt and oxidized forms) ambient particles contribute to the increase in bronchitis and asthma morbidity and in lung toxicity (Adamson et al. 2000, 2003; Hirshon et al. 2008; Pope 1989). It is noteworthy that engineered ZnO nano-particles (< 100 nm in diameter) are currently being produced in high tonnage (Xia et al. 2008). Exposure to these nanoparticles may occur in occupational, consumer, and environmental settings (Stone et al. 2007). Inhaled nanoparticles can deposit along the entire respiratory tract, including airways and alveolar regions (Yang et al. 2008). Recent in vitro studies have revealed that ZnO nanoparticles had a stronger effect on induction of cell damage to human alveolar epithelial cells and on IL-8 production from human bronchial epithelial cells and aortic endothelial cells compared with other metal oxide nanoparticles (Gojova et al. 2007; Park et al. 2007; Xia et al. 2008).

IL-8, a member of the CXC chemokine family, is an important activator and chemoattractant for polymorphonuclear leukocytes and has been implicated in a variety of inflammatory diseases (Strieter 2002). IL-8 protein is secreted at low levels from nonstimulated cells, but its production is rapidly induced by a wide range of stimuli encompassing proinflammatory cytokines (Kasahara et al. 1991), bacterial or viral products (Hobbie et al. 1997; Johnston et al. 1998), and cellular stressors (Fritz et al. 2005; Hirota et al. 2008; Kafoury and Kelley 2005; Sonoda et al. 1997). Expression of the IL-8 gene is regulated primarily at the level of transcription, although contributions by posttranscriptional mechanisms such as mRNA stabilization have also been demonstrated (Holtmann et al. 1999, 2001; Roebuck 1999; Winzen et al. 1999). The IL-8 gene is located on human chromosome 4, q12-21, and consists of four exons and three introns. Its 5' -flanking region contains the usual CCAAT and TATA boxlike structures and a number of potential binding sites for several inducible transcription factors including nuclear factor kappa B (NFkB), activator protein-1 (AP-1), and CAAT/enhancer-binding protein (C/EBP) (Luster 1998; Roebuck 1999; Wu et al. 1997). Regulation of IL-8 gene transcriptional activation is stimulus and cell-type specific (Brasier et al. 1998; Kasahara et al. 1991; Medin and Rothman 2006; Roebuck et al. 1999; Strieter 2002), which requires a functional NFkB element in addition to either an AP-1 or a C/EBP (NF-IL-6) element under some conditions of transcriptional induction (Strieter 2002). Unlike the NFkB site, the AP-1 and C/EBP sites are not essential for induction but are required for maximal gene expression of the IL-8 gene (Hoffmann et al. 2002). Although ZnO induces IL-8 expression in bronchial epithelial cells and IL-8 plays a critical role in the pathogenesis of pulmonary disorders (Blanc et al. 1993; Kuschner et al. 1997, 1998; Standiford et al. 1993), the mechanisms underlying ZnO-induced IL-8 expression have not been well characterized. In this study, we investigated the regulatory mechanisms underlying ZnO-induced IL-8 expression in human bronchial epithelial cells.

Materials and Methods

Materials and reagents. We purchased ZnO (99% purity, 24-70 nm in diameter) from Alfa Aesar (Ward Hill, MA); Triton X-100 and polyacrylamide from Sigma Chemical Co. (St. Louis, MO); and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) supplies, such as molecular mass standards and buffers, from Bio-Rad (Richmond, CA). We obtained anti-human p65 polyclonal antibody from Cayman Chemical (Ann Arbor, MI); phospho-specific rabbit antibodies against human NFkB p65 [serine 276 (Ser276), serine 536 (Ser536)] and human IkB[alpha] [serine 32 (Ser32)] from Cell Signaling Technology (Beverly, MA); [beta]-actin antibody from USBiological (Swampscott, MA); and IkB[alpha] antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody from Santa Cruz Biotechnology (Santa Cruz, CA). Actinomycin D (Act D) was purchased from EMD Biosciences, Inc. (San Diego, CA); the IL-8 ELISA (enzyme-linked immunosorbent assay) kit and the recombinant TNF[alpha] were purchased from eBioscience (San Diego, CA); the FuGENE 6 transfection reagent was obtained from Roche Diagnostics Corporation (Indianapolis, IN); and chemiluminescence reagents were obtained from Pierce Biotechnology (Rockford, IL).

Cell culture and exposure. Primary human bronchial epithelial cells. According to a protocol approved by the University of North Carolina Institutional Review Board, cells were obtained by cytologic brushing during bronchoscopy from healthy nonsmoking adult volunteers who had given informed consent; cells were frozen in liquid nitrogen until use. After thawing, the human bronchial epithelial cells were expanded to passage 2 in bronchial epithelial growth medium (Cambrex Bioscience Walkersville, Inc., Walkersville, MD), then plated on collagen-coated filter supports with a 0.4-[micro]m pore size (Trans-CLR; Costar, Cambridge, MA) to undergo air liquid interface (ALI) culture in a 1:1 mixture of bronchial epithelial cell basic medium and Dulbecco's modified Eagle's medium-H with SingleQuot supplements (Cambrex), bovine pituitary extract (13 mg/mL), bovine serum albumin (1.5 [micro]g/mL), and nystatin (20 units). Upon confluency, all-trans retinoic acid was added to the medium, and ALI culture conditions (removal of the apical medium) were created to promote differentiation. ZnO nanoparticles were suspended in molecular-grade water. Because ZnO nano-particles were poorly dissolved in water, the ZnO suspension was sonicated before being added to the apical surface of the ALI culture for stimulation.

BEAS-2B cell line. The BEAS-2B cell line was derived by transforming human bronchial cells with an adenovirus 12-simian virus 40 construct (Reddel et al. 1988). We obtained BEAS-2B cells from the American Type Culture Collection (ATCC, Manassas, VA). BEAS-2B cells (passages 70-80) were grown on tissue culture-treated Costar plates in keratinocyte basal medium supplemented with 30 [micro]g/ ml bovine pituitary extract, 5 ng/mL human epidermal growth factor, 500 ng/mL hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, and 5 ng/mL insulin. A suspension of ZnO was added to the surface of confluent BEAS-2B cells for stimulation. The doses of ZnO nanoparticles used in this study ranged from 2 to 8 [micro]g/mL.

Real-time reverse transcriptase/polymerase chain reaction (RT-PCR). Bronchial epithelial cells grown to confluence were exposed to ZnO. Cells were washed with ice-cold phosphate-buffered saline (PBS) and then lysed with TRIZOL reagent (Invitrogen Corporation, Carlsbad, CA). Total RNA (100 ng), 0.5 mM nucleoside triphosphate (Pharmacia, Piscataway, NJ), 5 [micro]M random hexaoligonucleotide primers (Pharmacia), 10 U/[micro]L RNase inhibitor (Promega, San Luis Obispo, CA), and 10 U/[micro]L Moloney murine leukemia virus RT (GIBCO-BRL Life Technologies, Gaithersburg, MD) were incubated in a 40[degrees]C water bath for 1 hr in 50 [micro]L 1x PCR buffer to synthesize first-strand cDNAs. The reverse transcription was inactivated by heating at 92[degrees]C for 5 min. Quantitative PCR of IL-8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specimen cDNA and standard cDNA was performed in a 50-[micro]L final volume mixture containing TaqMan master mix (PerkinElmer, Foster City, CA), 1.25 [micro]M probe, 3 [micro]M forward primer, and 3 [micro]M reverse primer. The probe annealed to the template between the two primers. This probe contained both a fluorescence reporter dye at the 5' end (6-carboxyfluorescein: emission [lamda.sub.max] = 518 nm) and a quencher dye at the 3' end (6-carboxytetramethyl rhodamine: emission [lamda.sub.max] = 518 nm). During polymerization, the probe was degraded by the 5' -'3 exonuclease activity of the Taq DNA polymerase, and the fluorescence was detected by a laser in the sequence detector (TaqMan ABI Prism 7700 Sequence Detector System; PerkinElmer). Thermal cycler parameters included 2 min at 50[degrees]C, 10 min at 95[degrees]C, and 40 cycles of denaturation at 95[degrees]C for 15 sec and annealing/extension at 60[degrees]C for 1 min. Relative amounts of IL-8 and GAPDH mRNA were based on standard curves prepared by serial dilution of cDNA from human BEAS-2B cells. The oligonucleotide primers and probes were purchased from Applied Biosystems (Foster City, CA).

ELISA. We assayed IL-8 protein in the cell culture supernatant using with a human IL-8 ELISA kit according to the manufacturer's instructions.

Immunoblotting. BEAS-2B cells were treated with ZnO suspension, washed twice with ice-cold PBS, then lysed in RIPA buffer [lx PBS, 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors (20 [micro]g/mL leupeptin, 20 [micro]g/mL aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 200 [micro]M sodium orthovanadate, and 20 mM sodium fluoride)]. Supernatants of cell lysates were subjected to SDS-PAGE. Proteins were transferred onto nitrocellulose membrane. The membrane was blocked with 5% nonfat milk, washed briefly, and incubated with primary antibody at 4[degrees]C overnight, followed by incubation with corresponding HRP-conjugated secondary antibody for 1 hr at room temperature. Immunoblot images were detected using chemiluminescence reagents and the Gene Gynome Imaging System (Syngene, Frederick, MD).

Chromatin immunoprecipitation (ChIP) assay. We conducted the ChIP assay using a ChIP kit (Upstate, Lake Placid, NY). Briefly, BEAS-2B cells growing in 100-mm dishes were treated with ZnO for 2 hr before being subjected to cross-linking with 1% formaldehyde at 37[degrees]C for 10 min. After washing with PBS, the cells were resuspended in 300 [micro]L lysis buffer [50 mM Tris-HCl (pH 8.1), 10 mM EDTA, 1% SDS, protease inhibitor cocktail]. DNA was sheared to 200-1,000 base pair small fragments by sonication. The supernatant was recovered, diluted, and precleared using salmon sperm DNA/protein A agarose. The recovered supernatant was incubated with anti-p65 antibody or an isotype control IgG for 2 hr in the presence of salmon sperm DNA was retrieved from the beads with 1% SDS and 0.1 mM NaHC[O.sub.3] solution at 65[degrees]C for 4 hr, then purified with a QIAquick spin column (Qiagen, Valencia, CA). The PCR was conducted on the extracted DNA using IL-8 promoter-specific primers at 95[degrees]C for 2 min, followed by 35 cycles of 95[degrees]C for 30 sec, 55[degrees]C for 30 sec, and 72[degrees]C for 30 sec. The PCR products were separated on a 1.4% agarose gel and stained with ethidium bromide.

Transient gene transfection. BEAS-2B cells grown to 40-50% confluence were transfected with the specific constructs for 24 hr using FuGENE 6 transfection reagent according to manufacturer's instructions. These constructs included luciferase-conjugated IL-8 promoter (pl.5IL-8-luc), kB binding-site-mutated IL-8 promoter (pl.5IL-8-kB-luc), the five tandem repeat of NFkB response element (pNFkB-luc), and Flag-p65, Flag-p65 S276A, and FLAG-p65 S536A (Kim et al. 2007). The pSV-[beta]-galactosidase construct was co-transfected with the individual construct depicted previously as an internal control (Kim et al. 2007). After transfection, the cells were incubated with keratinocyte basal medium overnight and then treated with ZnO before being lysed with lysis buffer. We detected luciferase and [beta]-galactosidase activities using the Dual-Light chemiluminescent reporter gene assay system (Tropix, Bedford, MA) and an AutoLumat LB953 luminometer (Berthold Analytical Instruments, Nashua, NH). Luciferase activity was estimated as luciferase count/[beta]-galactosidase count (luc/gal).

Statistical analysis. Data are presented as mean [+ or -] SE. Data were evaluated using non-parametric paired t-tests with the overall [alpha] level set at 0.05. One-way analysis of variance was used to analyze the time- and dose-dependent trends of IL-8 mRNA and protein expression.


ZnO exposure increases IL-8 expression in human bronchial epithelial cells. To examine the effect of ZnO nanoparticles on IL-8 expression in human bronchial epithelial cells, we used ALI-cultured primary human bronchial epithelial cells and BEAS-2B cells. As shown in Figure 1A, exposure of ALI-cultured primary human bronchial epithelial cells to 8 [micro]g/mL ZnO for 4 hr induced a significant increase in IL-8 mRNA levels. In BEAS-2B cells, ZnO stimulation (8 [micro]g/mL) induced a time-dependent increase in IL-8 mRNA expression (F = 47.24; p < 0.01). Exposure of BEAS-2B cells to ZnO for 4 hr caused a dose-dependent increase in IL-8 mRNA expression (Figure 1C; F = 41.83, p < 0.01). In addition, ZnO exposure resulted in a dose-dependent increase in IL-8 protein release from BEAS-2B cells after a 6-hr exposure (Figure 1D; F= 96.14,p [lessthan] 0.01). These results indicate that ZnO exposure up-regulates IL-8 expression at both mRNA and protein levels in human bronchial epithelial cells.

Transcriptional activation is involved in ZnO-induced IL-8 expression. To examine the involvement of transcriptional regulation in ZnO-induced elevation of IL-8 mRNA, we pretreated BEAS-2B cells with 10 [micro]g/mL Act D, a potent inhibitor of RNA polymerase, before treatment with ZnO. Pretreatment with Act D for 30 min ablated ZnO-induced IL-8 mRNA (Figure 2A), suggesting that transcriptional regulation was required for IL-8 expression in ZnO-exposed cells. To confirm this observation, we measured the IL-8 promoter activity through transient gene transfection of a luciferase-conjugated IL-8 promoter construct. As predicted by the Act D results, ZnO exposure (8 [micro]g/mL) significantly increased IL-8 promoter reporter activity (Figure 2B). These data indicate that ZnO-induced IL-8 gene expression in human bronchial epithelial cells occurs through a transcriptional mechanism.

ZnO exposure induces NFkB activation. Activation of the transcription factor NFkB is required for IL-8 gene transcription activation in many cell types (Villarete and Remick 1996). To examine whether ZnO stimulation increased NFkB activity, we determined phosphorylation and degradation of the NFkB inhibitory protein KB[alpha] (IkB[alpha]), an event indicative of the canonical NFkB-activating pathway (Ghosh and Karin 2002; Karin and Ben-Neriah 2000). In BEAS-2B cells exposed to 8 [micro]g/mL ZnO for 15, 30, 60, or 120 min, we measured phosphorylation of IkB[alpha], which peaked at 15-min exposure and declined thereafter. As expected, TNF[alpha] (100 ng/mL), the positive IkB[alpha] phosphorylation inducer, increased IkB[alpha] phosphorylation at 30 min exposure. In a manner consistent with this observation, ZnO stimulation in the absence of MG132 caused IkB[alpha] degradation at 30 min exposure to ZnO (Figure 3B). These data indicated that ZnO exposure can induce modest canonical NFkB activation in BEAS-2B cells. To further confirm ZnO-induced NFkB activation, BEAS-2B cells were transiently transfected with pNFkB-luc and p-SV-[beta]-galactosidase constructs prior to ZnO treatment. As shown in Figure 3C, ZnO exposure (8 [micro]g/mL) increased NFkB reporter activity at 6 hr of exposure, demonstrating that ZnO treatment increases NFkB-dependent transcriptional activity.

NFkB is required for ZnO-induced IL-8 expression. To further determine whether NFkB was involved in ZnO-induced IL-8 gene transcription, we transfected BEAS-2B cells with luciferase-conjugated IL-8 promoter (pl.5IL-8-luc) and kB binding site-mutated IL-8 promoter (pl.5IL-8-kB-luc) constructs, respectively, prior to treatment with 8 [micro]g/mL ZnO for 6 hr. As shown in Figure 4, the luciferase reporter activity induced by ZnO stimulation was significantly reduced in the cells expressing kB binding site-mutated IL-8 promoter compared with that in the cells expressing the intact (wild-type) IL-8 promoter, implying that NFkB is required for ZnO-induced IL-8 gene transcription.

Phosphorylation of p65 NFkB mediates ZnO-induced IL-8 mRNA expression. NFkB exerts its regulatory function through binding specific DNA sequences as homo- or heterodimers composed of members of the Rel/NFkB family (Hayden and Ghosh 2004). The most ubiquitous NFkB complex is the heterodimer p50/p65(RelA). We investigated whether p65 NFkB could bind to the IL-8 gene promoter in ZnO-treated BEAS-2B cells incubated with 8 [micro]g/mL ZnO for 2 hr. Cell lysates were then subjected to the ChIP assay using anti-p65 and isotype control antibodies. As shown in Figure 5A, ZnO stimulation resulted in a marked increase in the binding of p65 NFkB to the IL-8 gene promoter.

Phosphorylation of specific serine residues of the p65 NFkB subunit has been shown to be important for its transcriptional activity (Okazaki et al. 2003). Therefore, we used phospho-specific antibodies to measure the phosphorylation of p65 NFkB at ser276 and ser536 in BEAS-2B cells exposed to ZnO. We observed that ZnO treatment induced a rapid increase in phosphorylation of p65 at both serine residues (Figure 5B). Phosphorylation of p65 (Ser536) in ZnO-treated cells occurred as early as 15 min exposure and was decreased at 60 min. In contrast, phosphorylation levels of p65 (Ser276) went up more slowly but were still above the control level at 60 min. These data indicated that ZnO exposure increased p65 phosphorylation in human bronchial epithelial cells.

To determine the functional importance of p65 phosphorylation in ZnO-induced IL-8 gene transcription, we co-transfected BEAS-2B cells with pl.5IL-8-luc and either a wild-type p65 construct or a mutated version in which either Ser276 or Ser536 in p65 was mutated. As expected, ZnO exposure induced increased IL-8 promoter reporter activity in cells expressing wild-type p65 (Figure 5C). In comparison with the cells expressing wild-type p65, the cells that expressed mutated p65 showed a significant reduction in IL-8 promoter reporter activity after ZnO treatment. These data strongly suggest that phosphorylation of p65 is required for ZnO-induced IL-8 gene transcription.

Discussion and Conclusion

Cellular responses to environmental stimuli require rapid and accurate transmission of signals from cell-surface receptors to the nucleus (Karin and Hunter 1995). These signaling pathways rely on protein phosphorylation and, ultimately, lead to the activation of specific transcription factors that induce the expression of appropriate target genes. In the present study using human bronchial epithelial cells, we have demonstrated that exposure to the ZnO nanoparticles induces IL-8 gene expression by activating NFkB through a bimodal mechanism that involves p65 NFkB phosphorylation as well as IkB[alpha] phosphorylation and degradation.

Increased expression of IL-8 protein is largely dependent on transcriptional activation of the IL-8 gene (Wickremasinghe et al. 1999). This is corroborated by the results of the present study, which show that pretreatment of BEAS-2B cells with the transcriptional inhibitor Act D abrogated ZnO-induced IL-8 mRNA expression as well as the activation of IL-8 promoter activity in ZnO-treated cells. The NFkB family of transcription factors is essential for inflammation, immunity, and cell proliferation. Five members of the NFkB family have been identified: NFkB1 (p50/pl05), NFkB2 (p52/pl00), RelA (p65), RelB, and c-Rel. They share a highly conserved Rel homology domain at the N-terminal end that is responsible for specific DNA binding, dimerization, and interaction with IkB. In addition, some Rel proteins such as p65 (RelA) contain one or two C-terminal transactivating domain. In mammals, the NFkB transcription factor consists of two subunits of either homo-or heterodimers of RelA/p65, c-Rel, and p50. The p50/RelA (p65) heterodimer is the major Rel/NFkB complex in most cell types (Gilmore 1999). In resting cells, NFkB complexes are sequestered in an inactive form in the cytoplasm of the cells through its association with an inhibitory protein belonging to the IkB family, IkB[alpha] being the prototype. Upon cell stimulation, IkB[alpha] is phosphorylated by one of a number of IkB kinases, ubiquitinylated, and degraded, thereby allowing the NFkB complex to translocate into the nucleus and regulate the expression of its target genes, such as those coding for cytokines, adhesion molecules, and chemokines that have a crucial role in both immune and inflammatory responses (Hayden and Ghosh 2004; Karin and Ben-Neriah 2000; Rossi and Zlotnik 2000). Our data show that ZnO exposure induces rapid phosphorylation and degradation of IkB[alpha], consistent with an increase in NFkB transcriptional activity and ensuing IL-8 gene transcription. However, the modest degree of phosphorylation and degradation of IkB[alpha] induced by ZnO stimulation implied that other events might also participate in ZnO-induced NFkB transcriptional activation.

Increasing evidence from biochemical and genetic experiments strongly suggests that optimal induction of NFkB target genes also requires posttranslational modifications of NFkB p65 (Perkins 2006; Schmitz et al. 2004). For example, the acetylation of p65 has been proposed to facilitate the retention of the NFkB complex in the nucleus (Ashburner et al. 2001; Chen et al. 2001). The phosphorylation of p65 can result in a conformational change that increases its DNA binding activity and ability to recruit histone acetyltransferases such as cAMP response element-binding (CREB)-binding protein and p300 and to displace p50--histone deacetylase-1 complexes from DNA, leading to increased transcriptional activity (Ashburner et al. 2001; Chen et al. 2001). The NFkB p65 can be phosphorylated at multiple sites either in the N-terminal Rel homology domain or C-terminal transactivating domain (Viatour et al. 2005). The best-characterized phosphorylated residues on p65 are Ser276 and Ser536. In the present study, we observed that ZnO exposure increased the binding of p65 to the IL-8 gene promoter and also increased the phosphorylation of p65 at Ser276 and Ser536 in BEAS-2B cells. Previous studies have shown that p65 can be phosphorylated by a variety of cytoplasmic and nuclear kinases in a stimulus-and cell type-specific manner (Jamaluddin et al. 2007; Schmitz et al. 2004; Viatour et al. 2005; Zhong et al. 2002). Further study will be required to elucidate the mechanisms responsible for ZnO-induced phosphorylation of p65.

The role of particle dissolution in ZnO-induced toxic effects has been investigated; however, the results differ with particle size and cell types. A study using human aortic endothelial cells showed that ZnO nano-particles (20--70 nm diameter) can be internalized into cells and that ZnO-induced inflammatory response is due to the presence of the particles rather than ZnO-released [Zn.sup.2+] (Gojova et al. 2007). In contrast, a recent study using Raw264.7 cells and BEAS-2B cells proposed that the toxicity of ZnO nano-particles (13 nm) in both cell types is related to particle dissolution that could happen in culture medium and intracellular endosomes (Xia et al. 2008). In separate experiments (data not shown) we observed that ZnO particles were poorly dissolved in water and that the phagocytosis inhibitor cytochalasin D partially blocked ZnO-induced IL-8 expression in BEAS-2B cells, implying that ZnO particle internalization and subsequent dissolution may be involved in ZnO-induced IL-8 expression.

Human inhalation studies have shown that exposure to ZnO in welding fumes induced early increase in TNF[alpha] protein concentration and subsequent elevation of IL-8 and IL-6 protein levels in bronchoalveolar lavage fluid (Blanc et al. 1993; Kuschner et al. 1997). Moreover, TNF[alpha] has been shown to induce IL-8 production from BEAS-2B cells (Fujisawa et al. 2000). These observations lead to an assumption that ZnO-induced IL-8 expression may be mediated through an autocrine mechanism that involves TNF[alpha]. However, we have observed that TNF[alpha]-neutralizing antibody had minimal inhibitory effect on ZnO-induced IL-8 mRNA and protein expression even though it blocked TNF[alpha]-induced IL-8 mRNA expression by> 90% (data not shown), implying that TNF[alpha] is not involved in ZnO-induced IL-8 expression in BEAS-2B cells.

ZnO usually exists in the form of ultra-fine particles in ambient and workplace air; thus, exposure to ZnO particles is associated with adverse effects in environmental and occupational settings. Characterization of the underlying mechanisms of ZnO toxicity may provide helpful information in the prevention and treatment of pulmonary and systemic disorders related to inhalation of ultrafine ZnO particles.


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Address correspondence to W. Wu, Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina at Chapel Hill, 104 Mason Farm Rd., Chapel Hill, NC 27599 USA. Telephone; (919) 843-2714. Fax: (919) 966-9863. E-mail:

We greatly appreciate technical assistance from R. Silbajoris, W. Zhang, and L. Dailey.

This work was supported by U.S. Environmental Protection Agency (EPA) 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 approved for publication. Approval does not signify that the contents necessarily reflect (he views and policies of the U.S. EPA, nor does mention of trade names constitute endorsement of recommendation for use.

The authors declare they have no actual or potential competing financial interests.

Received 29 October 2009; accepted 1 March 2010.

Weidong Wu, (1) James M. Samet, (2) David B. Peden, (1) and Philip A. Bromberg (1)

(1) Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina, Chapel Hill, North Carolina, USA; (2) Human Studies Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA
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Title Annotation:Research
Author:Wu, Weidong; Samet, James M.; Peden, David B.; Bromberg, Philip A.
Publication:Environmental Health Perspectives
Article Type:Report
Geographic Code:1USA
Date:Jul 1, 2010
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