Genome-Wide Transcriptional Responses to Acrolein - American

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Articles Genome-Wide Transcriptional Responses to Acrolein Colin A. Thompson and Philip C. Burcham* Pharmacology and Anaesthesiology Unit, School of Medicine and Pharmacology, The UniVersity of Western Australia, Perth WA 6009, Australia ReceiVed May 30, 2008

The lipid peroxidation product and environmental pollutant acrolein participates in many diseases. Because of its formation during tobacco combustion, its role in various smoking-related respiratory conditions including lung cancer has received increasing attention. As a reactive electrophile, acrolein seems likely to disrupt many biochemical pathways, but these are poorly characterized on a genomewide basis. This study used microarrays to study short-term transcriptional responses of A549 human lung cells to acrolein, with cells exposed to 100 µM acrolein for 1, 2, or 4 h prior to RNA extraction and transcription profiling. Major pathways dysregulated by acrolein included those involved in apoptosis, cell cycle control, transcription, cell signaling, and protein biosynthesis. Although HMOX1 is a widely used marker of transcriptional responses to acrolein, this gene was the sole upregulated member of the Nrf2-driven family of antioxidant response genes. Transcript levels of several members of the metallothionein class of cytoprotective metal-chelating proteins decreased strongly in response to acrolein. Other novel findings included strong and persistent upregulation of several members of the early growth response (EGR) class of zinc finger transcription factors. Real-time PCR and Western blotting confirmed strong upregulation of a key member of this family (EGR-2), the DNA damage response gene GADD45β, the heat shock response participant Hsp70, and also HMOX1. Consistent with changes in Nur77 mRNA levels during the microarray study, Western blotting confirmed strong Nur77 induction at the protein level, raising the possibility that this death-inducing protein contributes to the loss of cell viability during acrolein exposure. Collectively, the transcriptional response to acrolein is complex and dynamic, with future work needed to determine whether acrolein-responsive genes identified in this study contribute to cell and tissue injury in the smoke-exposed lung. Introduction Acrolein is a noxious 3-carbon R,β-unsaturated aldehyde formed on combustion of a diverse range of organic matter including plastics and polymers, fossil fuels, timber, vegetation, and cooking oils (1). The combustion of sugar-containing cigarette additives (e.g., corn syrup, honey, etc.) also ensures that high levels of acrolein are present in tobacco smoke (2). Recent findings suggest a significant role for acrolein in p53 mutagenesis and lung tumorigenesis in tobacco smokers (3), although this association is controversial since other findings suggest that acrolein-induced DNA damage is weakly mutagenic (4–6). In contrast, acrolein plays a less ambiguous role in the induction of life-threatening pulmonary edema in fire victims (7). In addition to foreign sources, acrolein forms endogenously via the oxidation of lipids, amino acids, and polyamines (1). Increased levels of either free acrolein or acrolein-adducted macromolecules occur in numerous degenerative diseases (8). Possessing two electrophilic centers in close proximity (carbonyl and olefin groups), acrolein is a strong electrophile that reacts rapidly with soft nucleophiles, particularly favoring thiol groups possessed by glutathione and cysteine residues in proteins (9). Although less favored on thermodynamic grounds, * To whom correspondence should be addressed. Tel: 61-8-9346 2986. Fax: 61-8-9346 3469. E-mail: [email protected].

acrolein also reacts with harder nucleophilic centers within DNA to form adducts and cross-links (1). During protein damage, adduction primarily occurs via Michael additions to form carbonyl-retaining adducts (10). The use of classical antibodybased approaches to detect carbonylated proteins in acroleinexposed cells revealed that many cell proteins are targeted by the electrophile (10, 11). Little is known concerning the identity of acrolein targets on a proteome-wide scale, although new work in mice that examined protein damage in myocardial tissue following intravenous acrolein administration found that at a dose inducing contractile dysfunction, acrolein primarily damaged proteins in two broad classes, namely, sarcomere/cytoskeletal proteins and those involved in energy metabolism (12). Because these pathways are crucial to myocardial function, such studies highlight the valuable insights that accompany the use of global technologies to study toxicological responses to electrophiles. Genome-wide approaches for studying changes in mRNA transcript levels also promise to provide new insights into acrolein toxicity. In addition to direct adduction of protein and DNA targets, acrolein elicits other cellular perturbations that are expected to alter gene expression, including the induction of oxidative stress and disruption of the cellular thiol:disulfide

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redox state (13). Acrolein may also elicit both receptor- and mitochondrial-mediated apoptosis (14–16), together with activation of mitogen-activated protein kinases (MAPK)1 (17) and downstream caspases (18). The extent to which transcriptional events regulate these outcomes is largely unknown, since study of transcriptional responses to acrolein has typically focused on NF-E2-related factor 2 (Nrf2)-driven genes controlled by the antioxidant response element (ARE) such as heme oxygenase-1 (HMOX1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) (16, 19, 20). Given that potent electrophiles usually disrupt numerous cell pathways, any knowledge gained concerning transcriptional changes at non-ARE genetic loci is potentially valuable. As in a preliminary study conducted previously by others (21), our present study used microarray analysis to study transcriptional responses of human lung A549 cells to acrolein. The results indicate that acrolein dysregulated a broad range of cellular pathways including those involved in apoptosis, cell cycle control, transcription, cell signaling, and protein biosynthesis. We also report strong upregulation of several members of the early growth response (EGR) class of transcription factors, an intriguing finding since one member of this family, EGR-1, is a proinflammatory and proapoptotic mediator known to contribute to chronic obstructive pulmonary disease and acute lung injury (22).

Experimental Procedures Caution: Because of the pronounced inhalational hazards accompanying acrolein use, all preparation and handling of acrolein-containing solutions was performed in a fume hood. Materials. Agarose was purchased from Progen (Ipswich, Australia). F12 nutrient mixture (Kaighn’s modification), Dulbecco’s phosphate buffered saline, fetal bovine serum, Trypsin-EDTA, and gentamicin were manufactured by Gibco and purchased from Invitrogen Australia Pty. Ltd. (Mount Waverly, VIC, Australia). Acrolein was purchased from Alexis Biochemicals (Lausen, Switzerland). HMOX1 rabbit, GADD45β rabbit EGR-2 goat polyclonal antibodies, and donkey antigoat secondary antibody were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Hsp70 rabbit polyclonal antibody was obtained from Cell Signaling Technology (Danvers, MA), while mouse anticytochrome c monoclonal antibody and the HRP goat antimouse polyclonal antibody were supplied by BD Pharmingen (San Jose, CA). The bicinchoninic acid (BCA) Protein Assay kit was obtained from Pierce (Rockford, IL). iO SYBR Green Supermix and Nitrocellulose Trans-blot membrane were purchased from Bio-Rad (Hercules, CA). QuantiTect Reverse Transcription Kit and all QuantiTect real-time PCR primers RT for EGR-2, HMOX1, Hsp70, and GADD45β were purchased from Qiagen (Hilden, Germany). Human Genome U133 Plus 2.0 genechips and associated kits were purchased from Affymetrix (Santa Clara, CA). Cell Culture. Human A549 adenocarcinoma lung cells were grown to confluency in F12 nutrient mixture supplemented with 10% fetal bovine serum (FBS) (v/v) and gentamicin (100 mg/L) and then harvested by trypsin-EDTA digestion before final resuspension in F12 media supplemented with 0.5% FBS. The cell number was determined using a hemocytometer, and cells were plated in 1.4 mL volumes on six well plates at a density of 0.6 × 1 Abbreviations: AN/7-AAD, annexin V-PE and 7-amino-actinomycin D; ARE, antioxidant response element; BCA, bicinchoninic acid; BSA, bovine serum albumin; DBS, Dulbecco’s buffered saline; EGR, early growth response; FBS, fetal bovine serum; F12K, F12 nutrient mixture (Kaighn’s modification); HMOX1, heme oxygenase-1; 4-HNE, 4-hydroxy-2-nonenal; MAPK, mitogen-activated kinase; MT, metallothionein; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NQO1, NAD(P)H: quinone oxidoreductase 1; Nrf2, NF-E2-related factor 2; Nur77, orphan nuclear receptor NUR77; RT-PCR, real-time polymerase chain reaction.

Thompson and Burcham 106 cells per well. Plates were maintained in 5% CO2 at 37 °C overnight prior to the commencement of exposure to acrolein. Immediately before use, stock dilutions of acrolein were prepared in Dulbecco’s phosphate-buffered saline (DBS) followed by a second dilution in culture medium to obtain desired final concentrations. Following acrolein addition, the plates were returned to the incubator for 1, 2, or 4 h. Cell ATP levels were measured as indicators of cell viability using a Promega CellTiter-Glo Luminescent Cell Viability assay kit (Madison, WI) according to the manufacturer’s protocols with luminescence readings taken using a PolarStar Optima microplate reader. Total RNA Extraction. Total RNA was isolated from 0.6 × 106 A549 cells using an RNeasy Mini Kit (Qiagen) following 1, 2, or 4 h of exposure to acrolein as per the manufacturer’s guidelines. Control cells were washed and incubated in fresh 0.5% FBScontaining medium for 4 h prior to RNA extraction. Microarray Analysis of Global Transcript Levels. RNA quality was confirmed using an Agilent Bioanalyzer and then subjected to cDNA synthesis and in vitro transcription as per kit instructions (Affymetrix Santa Clara, CA). The resulting cRNA was hybridized overnight to the U133 Plus 2 chip (Affymetrix, Santa Clara, CA) at the Lotterywest Microarray Research Facility (Perth, Western Australia). This chip is a human genome-wide array comprising 54000 probes. The hybridization solution was removed, and the chip was washed, stained, and scanned according to the manufacturer’s guidelines. Differential regulation of gene expression was analyzed using software obtained from Genesifter (www.genesifter.net). Probes marked present or marginal in at least two out of three replicates were selected for analysis. The statistical significance of genes differentially regulated by a factor of 2 or 3 in the three replicates was determined by a one-way ANOVA parametric test comparing replicate samples (p e 0.05). Data were filtered using the Bonferroni false discovery test. Relative Real-Time Polymerase Chain Reaction (RT-PCR). Total RNA (1.0 µg) from acrolein-treated and control cells was reverse transcribed using the Quantitect Reverse Transcription Kit (Qiagen). Real-time reactions were performed using the iO SYBR Green Supermix (Bio-Rad) in an iQ5 Multicolor Real-Time PCR Detection System thermal cycler (Bio-Rad). A 50 ng quantity of nucleic acid served as the template for each reaction. The parameters for the amplification were as follows: initial denaturation (95 °C for 3 min) and then amplification [95 °C for 10 s (denaturation) and 58 °C for 30 s (annealing/extension) for 40 cycles]. A melting curve was generated to ensure the amplification of a single product. The statistical significance of genes differentially regulated was determined by a one-way ANOVA parametric test comparing replicate samples (p e 0.001). Data were filtered using the Tukey false discovery test. Western Blot Analysis. Following acrolein exposure, the media were removed, and the cells were washed three times with DBS. Monolayers were lysed using RIPA buffer, and the protein concentration of the resulting lysate was determined using the BCA assay. One volume of (5×) SDS-PAGE reducing sample buffer was added to aliquots containing 50 µg of protein sample, and following heat denaturation, the samples were resolved on a 14% polyacrylamide gel for 45 min at 200 V (23). Following transfer to nitrocellulose (100 V, 30 min), the membrane was blocked in PBS/5% nonfat milk for 1 h followed by an overnight incubation in primary antibody solutions prepared in TBS/5% bovine serum albumin (BSA) (1/1000 dilution). Membranes were then washed in PBS and incubated with the secondary antibody (alkaline phosphatase-coupled antirabbit IgG) in TBS/5% BSA for 1 h. Finally, the membrane was washed in TBS and then incubated with substrate (Pierce Super Signal West Pico Chemiluminescent Substrate) before exposure to Kodak Biomax Light film. The relative intensities of protein bands were determined by scanning densitometry analysis using Kodak Molecular Imaging software (Version 4.0). Western blots shown in Figures 3 and 4 are typically representative of three independent experiments. Cytosolic Fractionation and Cytochrome c Detection. As per methods described by Berger et al. (24), the cytoplasmic fraction

Transcriptional Responses to Acrolein in A549 Cells

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Figure 1. (A) Lack of cell death in adherent cells after 4 h of exposure to 100 µM acrolein. Following acrolein exposure and rinsing with DBS, cells monlayers were harvested and stained with Annexin V-PE and 7AAD. Panel A shows the proportion of live (AN-/7AAD-), apoptotic (AN+/ 7AAD-), and necrotic (AN+/7AAD-) cells as determined via flow cytometry after 0 (solid bars) and 4 h (empty bars) (mean ( SE, n ) 3). Ten thousand cells were scored per treatment. (B) Overview of transcriptional responses to acrolein in A549 cells. Upregulated genes (2-fold or greater) are shown in red, and downregulated genes are represented in green.

Figure 2. RT-PCR confirmation of microarray findings for selected acrolein-responsive genes. Total RNA was isolated from A549 cells treated with acrolein for 0, 1, 2, or 4 h and reverse transcribed as described in the Experimental Procedures. RT-PCR was performed on cDNAs for the indicated transcripts and normalized to the β-actin loading control. Results are depicted as the mean ( SE from three independent experiments. A one-way ANOVA parametric test comparing replicate samples (p e 0.001) was performed, and data were filtered using the Tukey false discovery test.

was prepared by differential centrifugation. After acrolein exposure, the media were removed, and the cells were washed three times with DBS before they were harvested with trypsin/EDTA. After they were washed a further three times with DBS, cells were resuspended in 250 mM sucrose containing 20 mM Tris (pH 8.0), 1.0 mM EDTA, 10 mM KCl, 1.5 mM MgCl2, and 1 mM phenylmethanesulphonylfluoride. The cells were disrupted using 20 strokes of a Dounce tissue grinder, and the resulting lysates were centrifuged for 10 min at 700g to remove unbroken cells and cell debris. The supernatants were centrifuged for 20 min at 15000g to pellet the mitochondria. The protein content of the resulting postmitochondrial supernatants (which include cytoplasmic components) was determined before 40 µg quantities of protein were loaded onto a 14% polyacrylamide gel, and the proteins were resolved as outlined above. The immunoblots were processed as described above except that the primary and secondary antibody solution (containing a 1/500 dilution of mouse anticytochrome c

and a 1/10000 of goat antimouse serum, respectively) was prepared in PBS/5% BSA. Genomic DNA Fragmentation Assay. DNA fragmentation was assessed via agarose gel electrophoresis as outlined previously (25) with minor alterations. Briefly, cells were resuspended in lysis buffer [comprising 50 mM Tris (pH 8.0), 50 mM EDTA, 1% SDS, and 10 mM NaCl], and the DNA was extracted using a QIAquick PCR Purification Kit (Qiagen), which is optimized to purify DNA fragments between 100 bp and 10 kbp. Bromophenol blue loading buffer was added to the DNA samples (6 × 0.05% w/v bromophenol blue, 50 mM EDTA, and 15% w/v sucrose) to achieve a 1× concentration, and then, 0.5 µg of DNA was loaded onto a 1.0% agarose gel and resolved at 200 mA for 1 h. The gel was stained with ethidium bromide (100 µg/mL for 30 min) and washed with water for 5 min before visualization of the DNA banding pattern was performed using the Biorad Geldac gel imaging system, which has UV transilluminator capabilities. Flow Cytometry (Annexin-V Assay). Following subculture overnight in 0.5% FBS/F12 nutrient mixture (F12K) in 5% CO2 at 37 °C, A549 cells were incubated for 4 h with or without 100 µM acrolein as described above. Relative levels of apoptotic and necrotic cell death were then investigated using an Annexin V-PE Apoptosis Detection Kit (BD Pharmingen, San Jose, CA) according to the manufacturer’s instructions. Briefly, A549 cells were washed twice with DBS and resuspended in Annexin binding buffer (10 mM Hepes, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4). The cells were stained with annexin V-PE (AN) and 7-amino-actinomycin D (7AAD) for 15 min in the dark. Ten thousand cells were analyzed by flow cytometry (FACS Calibur), and three cell populations corresponding to live cells, early apoptotic cells, and both late apoptotic and necrotic cells (respectively, AN-/7AAD-, AN+/ 7AAD-, and AN+/7AAD+) were identified. The results presented represent means ( SE of three independent experiments.

Results Cell Culture Conditions and Cell Viability. Because acrolein is a strong electrophile, side reactions with nucleophilic media constituents complicate in vitro studies of its toxicity (16). In recent work exploring the effect of media composition on the toxicity of acrolein in A549 cells, we found that the combination of 100 µM acrolein in F12K media containing 0.5% FBS elicited a strong transcriptional response (HO-1 induction) while causing minimal cell death after 3.5 h [as assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

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Figure 3. Confirmation of acrolein-induced gene expression changes at the protein level. (A) Time-course of protein expression for four acroleinresponsive markers after exposure of A549 cells to 100 µM acrolein for up to 24 h. (B) Concentration-response of protein expression in cells treated with 0, 25, 50, and 100 µM acrolein for 24 h. (C) Time-course and (D) concentration-response of EGR-2 protein expression after shorter exposures to acrolein.

mide (MTT) cell viability assay] (16). Comparison of the concentration-response of HO-1 induction by acrolein in the presence of F12K/0.5% FBS revealed that a 10-fold higher concentration of acrolein (i.e., 100 µM) was needed to achieve comparable HO-1 induction to that elicited in nucleophile-free media by 10 µM acrolein (16). This suggests that extracellular media constituents scavenge approximately 90% of free acrolein under the experimental conditions used in our present study. The conclusion that cell viability was maintained during the course of acrolein exposure was reinforced in the present study by the finding that after a 4 h exposure to 100 µM acrolein under the abovementioned cell culture conditions, cell ATP levels were 117 ( 0.2% of controls (N ) 3, mean ( SE). Use of an Annexin V-based flow cytometry assay to characterize acrolein-treated cells revealed that for adherent cells, viability was maintained after a 4 h exposure to 100 µM acrolein (AN-/ 7AAD-, Figure 1A). When detached, nonadherent cells were included in the analysis, a higher proportion of cells were necrotic at 4 h (i.e., AN+/7AAD+ cells comprised 26 ( 0.3% of counted cells vs 15 ( 0.24% in adherent cells as in Figure 1A).2 Because the microarray study used RNA extracted from adherent cells only, overt cellular death is unlikely to have confounded transcriptional responses to acrolein. Acrolein-Induced mRNA Transcript Profiles. A complete list of genes that were up- or down-regulated more than 2-fold at one or more time points is shown in Supporting Information, Tables 1 and 2.3 The transcription profile map in Figure 1B summarizes genes that responded significantly (>2-fold) at each of the three time points and shows how A549 cells mounted a rapid, dynamic transcriptional response to acrolein. Consistent with damage to numerous cell targets, at 1 h, acrolein altered the expression of 617 genes, a higher number than at either 2 or 4 h (Figure 1B). The initial cellular response was to suppress the expression of many genes, with downregulation accounting for 77.5% of the altered transcripts at 1 h (Figure 1B). At 2 h, just 341 genes were altered, suggesting that many of the initially responsive genes had recovered by this time point. By the final 2

Thompson, C. A. Unpublished observation. Microarray data obtained in this study has been deposited in the ArrayExpress database in a MIAME-compliant format with the identification code E-MEXP-1599. 3

Figure 4. Acrolein induces Nur77 protein expression and cytochrome c release. (A) Nur77 expression and cytochrome c release after 0, 2, 6, and 24 h of exposure to 100 µM acrolein. (B) Dose-response of Nur77 expression and cytochrome c release after 24 h of exposure to acrolein. (C) DNA fragmentation in A549 cells following 24 h of exposure to a range of acrolein concentrations. Ten micrograms of DNA was loaded per well and separated on a 1.2% agarose gel before staining with ethidium bromide and visualization under UV.


time point (4 h), cells had mounted a broad inductive response to acrolein with 492 genes differentially regulated, 58.1% of which were upregulated, twice the number of upregulated genes at 1 h (Figure 1B). In contrast, the number of genes downregulated at 4 h was less than half that observed at 1 h, indicating that the initial response to acrolein involved switching off a broad range of genes, followed by a quick recovery phase in which cells responded to “electrophile stress” by upregulating numerous genes. To identify strongly responsive genes, the microarray data were filtered using a more stringent 3-fold threshold of dysregulation, yielding 42 genes that are classified according to function in Tables 1-3. Many transcription factors are represented among this subset of strongly responsive genes, including early growth response-2 (EGR-2), the most acrolein-responsive gene observed during the microarray study (v 76-fold at 4 h) (Tables 1 and 3). The EGR family of pro-death transcription factors participates in the immediate early gene (IEG) response to stress, and other notable members of this gene family, EGR-1 and EGR-3, were also upregulated by acrolein (Table 1). Other immediate early genes upregulated by acrolein included V-fos, FOSB, activating transcription factor 3 (ATF3), inhibitor of DNA binding 2 (ID2), H3 histone, family 3B (H3.3B), nuclear receptor subfamily 4, group A, member 3 (NOR1), nuclear receptor subfamily 4, group A, member 1 [NR4A1/orphan nuclear receptor NUR77 (Nur77)], and cAMP early repressor (CREM). Genes associated with either cell cycle control or inhibiting the cell cycle after DNA damage were strongly upregulated by acrolein, including GADD45β, GADD35, ING1, BTG1, CLK1, and the IEG, DNA damage-inducible transcript 3 (DDIT3/ GADD153). Upregulation of these genes suggests an attempt to suppress cell proliferation and coincided with downregulation of key proliferative genes (CDK6, MKI67) as well as several cyclins [cyclin A, cyclin A2, cyclin F, cyclin D1, and cyclin B1 (Supporting Information, Table 2)]. In contrast to this trend, cyclin L (ania-A6) was upregulated (Table 1), although the impact of this change may be attenuated by downregulation of its interacting protein, CDC2L1 (Table 2). Acrolein disrupted the transcripts of many genes involved in apoptosis, altering participants in both the extrinsic and the intrinsic pathways (Tables 1 and 2). Activation of the former pathway was indicated by the increased expression of TNFSF10 (Table 1), a TNF family cytokine that induces receptor-mediated apoptosis (Table 1). Induction of the proapoptotic signaling protein Nur77 suggests activation of the mitochondrial pathway, although Nur77 also contributes to other proapoptotic pathways (vide infra) (Table 1). Consistent with the tight regulation of apoptosis in mammalian cells, these changes were accompanied by upregulation of TRAIL-R4, a decoy receptor with antiapoptotic efficacy, together with downregulation of the pro-apoptotic gene programmed cell death 5 (PDCD5) (Tables 1 and 2). Acrolein also downregulated IAP (MIHC), an antiapoptotic protein that suppresses apoptosis by ubiquitinating caspases-3, -7, and -9 (Table 2). Consistent with its ability to covalently modify numerous cell proteins, acrolein upregulated several pathways that respond to protein-damaging electrophiles, including heat shock response proteins such as Hsp70, BAG3, and DnaJ (HSP40/HSC70), together with proteins involved in protein turnover such as the ubiquitin-conjugating enzyme E2D3 (Table 1). The activation of these pathways may partly explain the time-dependent recovery of transcriptional activity during the experiment (Figure 1B).

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The ARE is a well-characterized battery of Nrf2-dependent genes (e.g., HMOX1 and NQO1) that are upregulated by oxidants and electrophiles. Acrolein produced modest 3.4-fold HMOX1 induction after 4 h in A549 cells (Table 1), concurring with the acrolein-mediated HMOX1 protein induction that we observed in A549 cells (16). Other ARE family transcripts were unaffected by acrolein. Concurring with recent findings concerning cyclooxygenase-2 upregulation by acrolein in cultured cells (26, 27), acrolein produced sustained upregulation of this mRNA transcript (Table 1). As arachidonic acid oxidation products feature strongly in the pulmonary inflammation that accompanies smoke inhalation, by inducing the cyclooxygenase-2 pathway, acrolein may be an important participant in this tissue response. Among the surprising findings from the microarray study was the strong and persistent downregulation of a range of metallothionein genes (MT1F, 1L, 1H, 1X, and 2A) (Tables 2 and 3). Because MT induction often occurs in cells subjected to oxidative stress (28), the downregulation of all members of the MT gene family further suggests that oxidative stress was not a defining feature of acrolein toxicity in A549 cells. Verification of Microarray Data. Relative RT-PCR (Figure 2) and Western blotting (Figure 3) confirmed induction of four key genes that responded strongly to acrolein in the microarray study (EGR-2, GADD45β, Hsp70, and HMOX1). Although the two technologies for assessing transcript abundance provided different fold-change estimates for individual genes, overall responses were similar. Thus, RT-PCR confirmed the transcription factor EGR-2 as the most acrolein-responsive target (360× induction at 4 h), while the fold changes for Hsp70 (6.2×), HMOX1 (2.8×), and GADD45β (29×) were also consistent with changes detected in the microarray study (Figure 2). Protein abundance for the four highlighted genes was studied at 2, 6, and 24 h in cells exposed to 100 µM acrolein (Figure 3A) and also after 24 h of exposure to several acrolein concentrations (Figure 3B). Densitometry of the resulting immunoblots revealed that 100 µM acrolein elicited modest (2-2.5-fold, normalized to β-actin) upregulation of Hsp70 and HMOX1 at 2 and 6 h, although levels returned to control after 24 h (Figure 3A). Lower concentrations of acrolein (25 and 50 µM) produced more sustained Hsp70 and HMOX1 induction (2-2.5-fold) that persisted after 24 h (Figure 3B). The proapoptotic cell cycle regulator GADD45β increased after 6 and 24 h (2.6- and 2.8-fold, respectively) in cells exposed to 100 µM acrolein (Figure 3A), although this DNA damage marker was less responsive to lower concentrations of acrolein after 24 h (Figure 3B). EGR-2 protein levels increased strongly but transiently on exposure to acrolein, peaking at 2 h (3.5-fold induction) and returning to baseline at 6 h (Figure 3A). No EGR-2 upregulation occurred at any acrolein concentration after 24 h (Figure 3B). Over a shorter time frame (Figure 3C), 100 µM acrolein increased EGR-2 levels at 90 and 120 min by 1.7- and 2.3fold, respectively (Figure 3C). In a concentration-response study conducted over 2 h, EGR-2 was strongly induced by 50 and 100 µM concentrations of acrolein (Figure 3C). Cell Death Signaling in Acrolein-Exposed Cells. The microarray study revealed that several proapoptotic genes were upregulated in acrolein-treated cells, including the nuclear transcription factor Nur77 (Table 1). A key activator of the intrinsic apoptotic pathway, Nur77, translocates to mitochondria to induce cytochrome c release (29). Because we recently observed cytochrome c release in acrolein-exposed A549 cells, we explored whether Nur77 upregulation accompanies cyto-

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Table 1. Genes Upregulated More Than 3-Fold by Acrolein at One or More Time Points fold expression gene/biological response

accession no.

1h

2h

4h

NM_002133

1.3

2.0

3.4

NM_173157

4.5

5.8

7.5

NM_003810 NM_003840

3.6 2.2

3.2 2.5

2.9 3.2

NM_003840

2.3

2.0

3.4

NM_198219 NM_006910 NM_001423 NM_001731 NM_004071 NM_020307 NM_004083 NM_015675 NM_014330 NM_006910 NM_013376 NM_003955

1.4 1.0 1.2 2.1 2.4 2.5 2.8 3.3 4.7 1.1 2.0 1.2

1.8 1.9 2.5 3.3 4.1 3.5 3.1 9.3 10.0 2.1 3.0 2.5

3.7 3.1 3.8 3.3 6.2 4.2 3.4 19.6 15.3 3.2 5.3 7.8

NM_000963

2.0

3.0

3.4

NM_015193 NM_013262

2.2 1.2

2.1 1.9

23.1 4.1

heat shock response BCL2-associated athanogene 3 (BAG3) NM_004281 heat shock 70 kDa protein 6 (HSP70B) (HSPA6) NM_002155 DnaJ (Hsp40) homologue, subfamily B, member 1 (DNAJB1) NM_006145 heat shock protein 70 testis variant (HSPA6) NM_005527

1.0 3.1 1.5 1.4

1.6 9.6 1.7 2.3

3.0 31.5 2.9 5.1

NM_031419

3.6

4.3

3.4

phosphoenolpyruvate carboxykinase 1 (soluble) (PCK1) ribokinase (RBKS) arginase II (ARG2)

NM_002591 NM_022128 NM_001172

1.6 5.0 1.9

2.8 8.1 2.2

4.9 9.5 3.9

mRNA cleavage PCF11, cleavage and polyadenylation factor subunit, homologue (S. cerevisiae) (PCF11)

NM_015885

1.6

2.9

4.3

NM_006276

2.0

2.7

3.7

AL833001

1.0

3.1

3.0

mitogen-activated protein kinase kinase kinase 8 (MAP3K8) neuropeptide Y receptor (NPYR) chemokine receptor 4 (CXCR4) calmodulin 2 (phosphorylase kinase, δ) (CALM2) heparin-binding epidermal growth factor-like growth factor) (DTR) activator of G protein signaling (AGS1) regulator of G-protein signaling (RGS16) Mex-3 homologue B (C. elegans) (MEX3B) STAT-induced STAT inhibitor 3 (SSI-3) (SOCS3) GTP-binding protein overexpressed in skeletal muscle (GEM) glomerular epithelial protein 1 (GLEPP1) WD repeat and SOCS box-containing 1 (WSB1) adrenomedullin (ADM) CXCR4 gene encoding receptor CXCR4 (C-X-C motif)

NM_005204 NM_000909 NM_003467 NM_001743 NM_001945 NM_016084 NM_002928 NM_032246 NM_003955 NM_005261 NM_030667 NM_015626 NM_001124 AF147204

5.3 1.3 1.5 1.9 0.8 2.3 0.4 1.5 1.5 3.8 4.6 2.6 1.6 1.2

4.4 2.8 2.0 4.4 1.9 5.3 1.9 3.0 2.2 5.2 5.5 3.0 2.4 2.4

4.8 8.2 6.5 10.0 9.7 19.7 3.3 4.1 4.3 6.3 5.1 3.6 5.4 10.4

transcription p35srj (MRG1) chromobox homologue 4 (Pc class homologue, Drosophila) (CBX4) inhibitor of DNA binding 2 (ID2) sex comb on midleg (Drosophila)-like 1 (SCML1) SIX homeobox 4 (SIX4) snail 1 (Drosophila homologue), zinc finger protein (SNAI1) suppressor of hairy wing homologue 1 (Drosophila) (SUHW1) fragile X mental retardation, autosomal homologue 2 (FXR2) Kreisler (mouse) maf-related leucine zipper homologue (KRML) zinc finger, AN1-type domain 6 (ZFAND6)

NM_006079 NM_003655 NM_002166 NM_006746 NM_017420 NM_005985 NM_080740 NM_004860 NM_005461 NM_019006

1.5 1.1 1.2 1.0 0.7 2.7 1.6 1.5 1.3 1.4

1.9 1.6 1.6 1.6 1.4 6.8 1.9 3.7 2.3 2.1

4.5 3.0 4.6 3.4 3.1 15.5 3.2 5.5 10.6 4.1

antioxidant response heme oxygenase (decycling) 1 (HMOX1) apoptosis nuclear receptor subfamily 4, group A, member 2 (NGFI-Bnur77 β type transcription factor homologue) tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10) tumor necrosis factor receptor superfamily, member 10d, decoy with truncated death domain (TNFRSF10D) TRAIL-R4-B (TRAIL-R4) cell cycle tumor suppressor p33ING1 (ING1) retinoblastoma binding protein 6 (RBBP6) epithelial membrane protein 1 (EMP1) B-cell translocation gene 1, antiproliferative (BTG1) CDC-like kinase1 (CLK1) cyclin L ania-6a DNA damage-inducible transcript 3 (DDIT3/GADD153) growth arrest and DNA damage-inducible, β (GADD45B) growth arrest and DNA damage-inducible 34 (GADD34) retinoblastoma-binding protein 6 (RBBP6) CDK4-binding protein p34SEI1 (SERTAD1) suppressor of cytokine signaling 3 (SOCS3) cyclooxygenase cyclooxygenase 2b (PTGS2) cytoskeleton activity-regulated cytoskeleton-associated protein (ARC) myosin regulatory light chain interacting protein (MIR)

inflammation nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, ζ (NFKBIZ) metabolism

mRNA splicing splicing factor, arginineserine-rich 7 (35kD) (SFRS7) ribosomal similar to 40S ribosomal protein S16 signaling


Chem. Res. Toxicol., Vol. 21, No. 12, 2008 2251 Table 1. Continued fold expression

gene/biological response

accession no.

zinc finger, CCHC domain containing 12 (ZCCHC12) ATF3 mRNA for activating transcription factor 3∆Zip2 (ATF3∆Zip) hCREM (cyclic AMP-responsive element modulator) type 2 protein (CREM) early growth response-1 (EGR-1) early growth response-2 (EGR-2) early growth response-3 (EGR-3) FBJ murine osteosarcoma viral oncogene homologue B (FOSB) H3 histone, family 3B (H3.3B) mitogen induced nuclear orphan receptor (MINOR) (NR4A3) RPA-binding trans-activator (RBT1) v-fos FBJ murine osteosarcoma viral oncogene homologue (FOS) zinc finger CCCH-type containing 12A (ZC3H12A) cAMP early repressor (CREM) chromobox homologue 4 (Drosophila Pc class) (CBX4) cytoplasmic polyadenylation element binding protein 4 (CPEB4) enhancer of polycomb homologue 1 (Drosophila) (EPC1) family with sequence similarity 46, member A (FAM46A) Kruppel-like factor 4 (gut) (KLF4) nuclear receptor subfamily 4, group A, member 1 (NR4A1), (Nur77) nuclear receptor subfamily 4, group A, member 3 (NR4A3) novel MAFF LIKE protein activating transcription factor 3 (ATF3) MAX dimerization protein (MXD1)

1h

2h

4h

NM_173798 NM_004024 NM_182717

1.6 2.9 2.1

3.1 3.8 2.3

5.6 6.4 3.8

NM_001964 NM_000399 NM_004430 NM_006732 NM_005324 NM_173199 NM_013368 NM_005252 NM_025079 AB209533 NM_003655 NM_030627 NM_025209 NM_017633 NM_004235 NM_002135 NM_006981 NM_012323 NM_001674 NM_002357

2.0 20.4 2.1 3.9 2.8 4.5 2.6 4.0 2.0 1.4 1.2 1.4 2.1 1.1 1.6 1.8 3.7 1.1 4.4 1.0

3.1 34.5 4.2 14.1 3.8 9.7 3.0 5.6 3.0 2.1 2.1 2.2 2.3 2.4 3.5 2.9 8.2 1.5 6.5 2.1

3.3 76.0 6.3 39.6 5.8 12.0 3.6 8.1 3.0 3.2 3.8 3.0 3.7 4.4 4.9 5.5 10.4 3.9 12.1 4.1

0.6

2.9

3.2

0.7 0.5 1.3 1.2 1.9 2.4 3.4 1.3 1.2 0.7 2.0 1.4 2.2 3.0

2.0 3.7 1.9 1.7 1.9 2.2 3.5 2.3 2.1 2.4 3.7 2.5 2.8 4.0

2.9 8.6 3.0 3.5 3.0 3.0 2.6 3.0 4.9 5.2 2.9 4.3 3.1 6.7

ubiquitin pathway ubiquitin-conjugating enzyme E2D 3 (UBC4/5 homologue, yeast) AK127304 (UBE2D3) other F-box protein Lilina (LILINA) FGF2-associated protein GAFA1 (GAFA1) headcase homologue (Drosophila) (HECA) Josephin domain containing 1 (JOSD1) phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) GABA(A) receptor-associated protein like 1 (GABARAPL1) KIAA1199 protein similar to JH0148 nucleolin (LOC153222) hypothetical protein FLJ20037 phosphatidylserine receptor (JMJD6) MUC5AC mRNA for mucin (MAR11) vacuolar protein sorting 37 homologue B (S. cerevisiae) (VPS37B) pTM5 mariner-like transposon clone 24659

chrome c release under these conditions (Figure 4). Western blot analysis revealed Nur77 upregulation following 6 and 24 h of exposure (4.5- and 5.0-fold, respectively) to 100 µM acrolein (Figure 4A), while after 24 h, concentration-dependent Nur77 upregulation was elicited by all concentrations of acrolein (Figure 4B). The time course and concentration dependence of Nur77 upregulation closely coincided with those for cytochrome c release (Figure 4A,B). Study of DNA fragmentation revealed concentration-dependent DNA fragmentation after 24 h, with maximal DNA cleavage elicited by 50 and 100 µM acrolein.

Discussion Because our laboratory has an interest in smoke inhalation injury, a condition involving acute respiratory epithelial damage due to high-dose inhalation of acrolein and other pulmonary irritants, we studied transcriptional responses of lung cells to a single subacutely toxic dose of acrolein. The change in cellular mRNA profile under these conditions was dynamic and multifaceted. At the 2-fold gating threshold, cells responded to acrolein by downregulating 478 genes after 1 h (Figure 1B). The mechanism underlying this response is unclear but may involve damage to proteins that act at the transcription complex to transcribe target genes. Alternatively, the downregulation may reflect DNA damage within the promoters of downregulated transcripts, a phenomenon known to cause downregulation of susceptible genes during aging and oxidative stress (30). The

NM_012308 AF190748 NM_016217 NM_014876 NM_021127 NM_031412 NM_018689 NM_153607 AL078599 NM_001081461 AJ001402 NM_024667 AF278605 AF070569

partial recovery after 2 h (when just 172 genes were downregulated) may reflect efficient DNA repair within damaged promoters to allow resumption of transcription or, alternatively, restoration of the functional capacity of nuclear transcriptional apparatuses. After 4 h, more genes were upregulated (286, 58.1% of total), further suggesting the recovery of transcriptional capability in stressed cells. Consistent with the ambiguity evident in the literature concerning the role of apoptosis vs necrosis as the predominant pathway of acrolein-induced cell death, our data suggest that acrolein elicited a complex pattern of cell death outcomes in A549 cells. Others have shown that in some cell lines, acrolein elicits changes suggesting activation of the mitochondrial apoptotic pathway, including loss of mitochondrial membrane potential, cytochrome c release, and caspase-7 and -9 activation (14). Our work strengthens this association by identifying the nuclear receptor Nur77 as a possible activator of this pathway (acrolein activated Nur77 at both the mRNA (Table 1) and the protein level, with the time-course of the latter coinciding with cytochrome c release (Figure 4). Nur77 has analogous actions to p53, helping maintain cell survival under normal conditions while undergoing activation by proapoptotic stimuli to a potent pro-death effector that translocates to mitochondria where it interacts with Bcl-2 to elicit cytochrome c release (31). A parallel proapoptotic pathway involves Nur77 translocation to the endoplasmic reticulum followed by CaII release and activa-

2252



Table 2. Genes Downregulated More Than 3-Fold by Acrolein at One or More Time Points fold expression gene/biological response

accession no.

1h

2h

4h

AK123482 NM_175622 NM_005949 BI835300 NM_005951 NM_005952 NM_005953

0.05 0.14 0.18 0.31 0.22 0.29 0.34

0.18 0.12 0.17 0.25 0.20 0.28 0.33

0.42 0.15 0.24 0.35 0.32 0.36 0.41

NM_001165 NM_003883

0.22 0.11

0.21 0.49

0.25 0.41

NM_000593

0.34

0.76

0.50

NM_001748

0.52

0.45

0.29

NM_001259 NM_033486 NM_017778 AJ278112 AK025846 NM_002417

0.33 0.28 0.35 0.25 0.16 0.30

0.53 0.84 1.00 0.38 0.50 0.53

0.56 0.68 0.69 0.38 0.54 0.39

complement activation NM_000716

0.42

0.19

0.39

NM_201380

0.26

0.85

0.32

NM_005415

0.35

1.08

1.24

NM_152776 NM_005911

0.31 0.37

0.61 0.35

0.51 0.24

protein biosynthesis ribosomal protein S16 (RPS16) NM_001020 eukaryotic translation initiation factor 4A, isoform 1 (EIF4A1) NM_001416 putative translation initiation factor (SUI1) NM_005801 ribosomal protein L37a (RPL37A) NM_000998 ribosomal protein L17 (RPL17) NM_000985 ribosomal protein S24 (RPS24) BX647260

0.37 0.32 0.18 0.26 0.18 0.24

0.76 1.16 0.37 0.32 0.73 0.56

0.82 1.05 0.32 0.37 0.40 0.46

NM_003090

0.24

0.34

0.52

stanniocalcin 2 (STC2) transmembrane 4 L six family member 1 (TM4SF1) DEP domain containing 1 (DEPDC1) A kinase (PRKA) anchor protein 10 (AKAP10)

NM_003714 NM_014220 NM_017779 NM_007202

0.57 0.05 0.47 0.28

0.31 0.20 0.38 0.49

0.66 0.19 0.36 0.44

transcription myeloidlymphoid or mixed-lineage leukemia 3 (MLL3) histone deacetylase 3 (HDAC3) homeodomain-interacting protein kinase 2 (HIPK2) zinc finger protein 551 (ZNF551) zinc finger protein 416 (ZNF416) zinc finger protein 106 (ZFP106) zinc finger protein 207 (ZNF207) V-myb avian myeloblastosis viral oncogene homologue-like 1 (MYBL1) suppressor of variegation 3-9 homologue 2 (Drosophila) (SUV39H2) RNA binding motif protein 39 (RBM39)

NM_021230 NM_003883 NM_022740 NM_138347 NM_017879 NM_022473 NM_003457 NM_001080416 NM_024670 CR749443

0.35 0.33 0.39 0.59 1.19 0.30 0.22 0.24 0.29 0.22

0.91 0.91 0.93 0.32 0.78 0.45 0.41 0.37 0.25 0.57

0.75 0.54 0.69 0.51 0.29 0.31 0.53 0.70 0.61 0.41

NM_198461

0.34

0.72

0.82

NM_032853 NM_020234 EF177381 NM_006643 NM_013275 NM_014086 AY894575 NM_020814 NM_017779 NM_001909 BF528878 NM_030938

0.29 0.30 0.24 0.32 0.32 0.25 0.50 0.19 0.36 0.21 0.38 0.04

0.98 0.45 0.97 0.57 0.72 1.17 0.65 0.34 0.39 0.22 0.35 0.14

0.55 0.60 0.55 0.58 0.79 0.78 0.26 0.31 0.37 0.25 0.27 0.13

antioxidant microsomal glutathione S-transferase 1 (MGST1) metallothionein 1J (pseudogene) (MT1JP) metallothionein 1F (MT-IF) metallothionein 1 L (MT1L) metallothionein 1H (MT1H) metallothionein 1X (MT1X) metallothionein 2A (MT2A) apoptosis IAP homologue C (MIHC) programmed cell death 5 (PDCD5) ATPase activity ATP-binding cassette, subfamily B (MDRTAP), member 2 (ABCB2) calcium binding calpain 2, (mII) large subunit (CAPN2) cell cycle cyclin-dependent kinase 6 (CDK6) cell division cycle 2-like 1 (PITSLRE proteins) CDC2L1 Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1) cell cycle control protein (SDP35 gene) growth arrest-specific transcript 5 (GAS5) antigen identified by monoclonal antibody Ki-67 (MKI67) complement component 4-binding protein, β (C4BPB)

cytoskeleton plectin 1, intermediate filament binding protein, 500kD PLEC1 κB cascade solute carrier family 20 (phosphate transporter), member 1 (SLC20A1) metabolism glycerol kinase 5 (putative) (GK5) methionine adenosyltransferase II, alpha (MAT2A)

RNA splicing small nuclear ribonucleoprotein polypeptide A′ (SNRPA1) signaling

ubiquitination LON peptidase N-terminal domain and ring finger 2 (LONRF2) other melanoma associated antigen (mutated) 1 (MUM1) DTW domain containing 1 (DTWD1) metastasis associated lung adenocarcinoma transcript 1 (MALAT) serologically defined colon cancer antigen 3 (SDCCAG3) nasopharyngeal carcinoma susceptibility protein (LZ16) PRO1073 protein neuroblastoma breakpoint family, member 1 (NBPF1) membrane-associated ring finger (C3HC4) 4 hypothetical protein FLJ20354 cathepsin D (CTSD) testis-specific protein kinase (LOC91461) transmembrane protein 49 (TMEM49)


Chem. Res. Toxicol., Vol. 21, No. 12, 2008 2253 Table 2. Continued fold expression

gene/biological response

accession no.

1h

2h

4h

BRI3 binding protein (BRI3BP) Rho GTPase activating protein 11A (ARHGAP11A) WW and C2 domain containing 1 (WWC1) trophoblast-derived noncoding RNA (TncRNA) pleckstrin homology-like domain, family B, member 2 (PHLDB2) nexilin (F Actin binding protein) (NEXN) S100 calcium binding protein A10 (S100A10)

NM_080626 NM_014783 NM_015238 AF001893 NM_145753 NM_144573 NM_002966

0.33 0.48 0.35 0.27 0.50 0.53 0.17

0.45 0.25 0.36 0.87 0.33 0.34 0.27

0.34 0.35 0.52 0.34 0.40 0.29 0.29

Table 3. Genes Up- or Downregulated More Than 3-Fold by Acrolein at All Time Points fold expression gene/biological response upregulated at all time points

accession no.

1h

2h

4h

NM_001674 NM_000399 NM_006732 NM_173199 NM_006981 NM_005252

4.4 20.4 3.9 4.5 3.7 4.0

6.5 34.5 14.1 9.7 8.2 5.6

12.1 76.0 39.6 12.0 10.4 8.1

NM_005261 NM_030667

3.8 4.6

5.2 5.5

6.3 5.1

NM_022128

5.0

8.1

9.5

mitogen-activated protein kinase kinase kinase 8 (MAP3K8)

NM_005204

5.3

4.4

4.8

inflammation nuclear factor of κ-light polypeptide gene enhancer in B-cells inhibitor, ζ (NFKBIZ)

NM_031419

3.6

4.3

3.4

NM_002155

3.1

9.6

31.5

NM_015675 NM_014330

3.3 4.7

9.3 10.0

19.6 15.3

NM_173157

4.5

5.8

7.5

NM_003810 NM_003810

3.6 3.6

3.2 3.1

2.9 2.9

AF070569

3.0

4.0

6.7

transcription activating transcription factor 3 (ATF3) early growth response 2 (EGR-2) FBJ murine osteosarcoma viral oncogene homologue B (FOSB) mitogen-induced nuclear orphan receptor (MINOR) (NR4A3) nuclear receptor subfamily 4, group A, member 3 (NR4A3) V-fos FBJ murine osteosarcoma viral oncogene homologue (FOS) signaling GTP-binding protein overexpressed in skeletal muscle (GEM) glomerular epithelial protein 1 (GLEPP1) metabolism ribokinase (RBKS) kinase

heat shock response heat shock 70 kDa protein 6 (HSP70B) (HSPA6) cell cycle growth arrest and DNA damage-inducible, β (GADD45β) growth arrest and DNA damage-inducible 34 (GADD34) apoptosis nuclear receptor subfamily 4, group A, member 2 (NGFI-Bnur77 β-type transcription factor homologue) tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10) tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10) other clone 24659

fold expression gene/biological response downregulated at all time points

1h

2h

4h

apoptosis NM_001165

0.22

0.21

0.25

antioxidant NM_005949 NM_005951 NM_175622

0.18 0.22 0.14

0.17 0.20 0.12

0.24 0.32 0.15

S100 calcium binding protein A10 S100A10

cell cycle NM_002966

0.17

0.27

0.29

transmembrane 4 L six family member 1 TM4SF1 transmembrane protein 49 TMEM49 cathepsin D (CTSD) hqp0376 protein

signaling NM_014220 NM_030938 NM_001909 AF078844

0.05 0.04 0.21 0.15

0.20 0.14 0.22 0.17

0.19 0.13 0.25 0.19

IAP homologue C (MIHC) metallothionein 1F hMT-If metallothionein 1H (MT1H) metallothionein 1J (pseudogene) MT1JP

accession no.

tion of endoplasmic reticulum-specific caspase-4 (32). In addition, growing evidence suggests that Nur77 can activate the receptor-mediated apoptotic pathway (33, 34). Our finding that acrolein upregulated the extrinsic or death receptor pathway participant TNFSF10 is consistent with recent findings suggesting a role for the extrinsic pathway in acrolein toxicity (15). The finding that acrolein upregulated multiple proapoptotic genes, while causing DNA fragmentation, maintaining ATP levels, and promoting cytochrome c release all suggested that apoptosis was a major pathway of cell death during our

experiments, but other possibilities emerged upon using Annexin V-PE/7-AAD staining and flow cytometry to characterize acrolein-induced cell death. Annexin-V detects externalized phosphatidylserine (PS) residues on apoptotic cells, while 7-AAD reveals permeabilization of cellular membranes, a feature of necrotic cell death. Using this method to score 10000 adherent cells, we were unable to identify any differences in the frequencies of apoptotic (AN+/7AAD-) and necrotic (AN+/ 7AAD+) cells between acrolein-exposed cells and controls (Figure 1A). When detached cells were included in the analysis,

2254


higher populations of necrotic cells but not apoptotic cells were detected after a 4 h exposure to acrolein, indicating that both cellular PS externalization and loss of cell membrane integrity had occurred. These findings are consistent with reports implicating necrosis as the dominant pathway of acroleinmediated cell death (18, 35). However, during studies of acrolein toxicity in murine FL5.12 proB lymphocytes, others suggested that low yields of apoptotic cells detected with the Annexin-V assay might reflect reactivity of acrolein with PS residues in cell membranes, thereby diminishing Annexin-V binding (18). Clearly, more work is needed to define the mechanisms of cell death prevailing under conditions of acute acrolein exposure, particularly within the in vivo setting where little is known concerning the molecular events triggered by acrolein that mediate the epithelial damage accompanying smoke inhalation injury. The strong upregulation of the Egr family of zinc finger transcription factors at the mRNA transcript (Table 1 and Figure 2) and protein level (Figure 3, EGR-2 only) is intriguing given that these proteins are a convergence point for many signaling pathways (22, 36, 37). The best studied family member, EGR1, seems particularly important in the pathogenesis of acute lung injury, undergoing strong upregulation following a range of noxious pulmonary stimuli, including hypoxia, hyperoxia, and high tidal volume ventilation (22). Recent work also implicated EGR-1 in the pathogenesis of chronic obstructive pulmonary disease in smokers and also during activation of the heat shock response and matrix metalloproteinase-2 in fibroblasts exposed to cigarette smoke extracts (38–40). In contrast to EGR-1, the pulmonary functions of EGR-2 are obscure, although EGR-2 levels in alveolar macrophages were shown to increase on exposure to fibrous titanium oxide (41). EGR-2 plays important roles during brain development, with mutations in this gene contributing to a range of hereditary myelinopathies (42). The strong upregulation of Egr genes by acrolein may be related to the Nur77 activation discussed above, since specific upregulation of EGR-1/EGR-2/EGR-3 was found to accompany Nur77 activation in HEK293 kidney cells (43). A common event preceding both outcomes is activation of various mitogenactivated protein (MAP) kinases, namely, MEK, ERK, p38, and CREB (43–47). Because acrolein toxicity in CHO cells was recently shown to involve rapid activation of several MAP family kinases (17), a similar outcome may explain the Nur77/ EGR-1/EGR-2/EGR-3 upregulation seen in the present study. However, the conclusion that Nur77 induction causes the EGR-2 activation by acrolein is hard to support from our present findings since the temporary EGR-2 upregulation seen at the protein level occurred early in comparison to the 24 h incubation required for maximal Nur77 induction (Figure 4). This may suggest that the modest Nur77 upregulation evident at 2 h (Figure 4) was sufficient to drive activation of the Egr genes. Another possibility, in light of new findings concerning the role of MAP kinase-catalyzed Nur77 phosphorylation in apoptosis induction, is that Nur77 phosphorylation occurs in the early stages of acrolein exposure and that phosphorylated Nur77 activates apoptosis without strong changes in the absolute amount of Nur77 protein (48). Strong dysregulation of genes associated with cell cycle arrest and cell proliferation accompanied exposure to acrolein (Tables 1 and 2). mRNA transcripts for three members of the growth arrest and DNA damage family, GADD153, GADD45β, and GADD34, were strongly upregulated by acrolein (Table 1 and Figure 2), and this was confirmed at the protein level for GADD45β (Figure 3). The latter suppresses cell growth by


inhibiting the kinase activity of the Cdk1/CyclinB1 complex (49). The downregulation of the mRNA transcript for cyclin B1 over the course of the experiment is likely to amplify the effects of acrolein on cell growth (Supporting Information, Table 2). Under comparable conditions to those used in this study, we found that acrolein elicits extensive protein carbonylation and cross-linking in A549 cells (16, 50). Such macromolecular damage by electrophiles frequently activates the heat shock response, presumably in an effort to minimize the cellular impact of misfolded, abnormal proteins (51). Early work suggested that acrolein could activate Hsp70 expression in A549 cells, although the contribution of other heat shock participants was not defined (52). Our present work indicates that acrolein stimulates several heat shock genes, including Hsp70, Hsp70B, and DnaJB1 (Table 1 and Figures 2 and 3). Intriguingly, the range of heat shock proteins induced by acrolein was more limited than that identified during comparable studies of transcriptional responses to 4-hydroxy-2-nonenal (4-HNE) in RKO colorectal carcinoma cells (53). Although it is less reactive with cellular nucleophiles, as an electrophilic R,β-unsaturated aldehyde 4-HNE shares a number of chemico-toxicological properties with acrolein (54). Heat shock proteins responding to 4-HNE included a series of DnaJ homologue family proteins as well as heat shock 110 kDa protein family (APG-1) and heat shock 105/110 kDa protein 1 (HSPH1) (53). Although the distinct cell types used in these studies may contribute to these differences, since 4-HNE-derived protein adducts are bulkier than their acrolein-derived counterparts, they may more efficiently activate the heat shock response. A further unexpected difference between the transcriptional responses to acrolein and 4-HNE concerns the extent of upregulation of Nrf2-driven ARE genes (53). A 6 h exposure to 20 µM 4-HNE upregulated multiple Nrf2-driven genes, including HMOX1, GCLM, NQO1, SLC7A11, and TXNRD1 (53). In contrast, HMOX1 was the only ARE family member upregulated by acrolein (Table 1). An explanation for this discrepancy could be that overproduction of reactive oxygen species is not conspicuous during acrolein toxicity, a conclusion supported by recent work in S. cereVisiae, which negated a primary role for antioxidant proteins in mediating acquired resistance to acrolein (55). Alternatively, the upregulation of ARE genes might have been restricted by the comparatively high acrolein concentration used in our study. In the abovementioned study of transcriptional responses to 4-HNE, the widest range of Nrf2-driven genes was altered at a low concentration of 4-HNE, which did not elicit extensive cell death (20 µM) (53). Moreover, while our study focused on transcriptional responses directly elicited by acrolein, the paucity of transcriptional changes indicative of oxidative stress need not extend to the in vivo setting where tissue damage by acrolein may conceivably promote tissue infiltration by immune cells capable of eliciting the oxidative burst. Other novel findings during the microarray study included the sustained downregulation of several metallothionein (MT) transcripts by acrolein (Table 1). This was unexpected given that others reported modest MT induction in HepG2 cells after a 12 h exposure to 10 µM acrolein (56). MTs are small, cysteinerich proteins with strong affinity for a range of toxic metals while also possessing strong antioxidant activity (57). The strong MT downregulation by acrolein at the gene level may be a consequence of the role of MT proteins in cellular ZnII homeostasis (58–60). MT gene promoters typically contain multiple copies of metal response elements (MRE) that are targets for transcriptional activators such as metal-responsive


transcription factor-1 (MTF-1). Because acrolein upregulated many Zn finger transcription factors (Table 1), synthesis of these proteins may create a strong demand for Zn, with cells responding by downregulating MT genes that normally buffer intracellular Zn. Strong MT downregulation has also been observed in cells treated with cell-permeable Zn chelators (61, 62). In conclusion, this study provides new insights into the molecular events underlying cellular responses to the environmental pollutant and endogenous electrophile acrolein. Although more work is needed to determine whether transcriptional responses observed in A549 tumor cells are extendable to primary lung cells (e.g., epithelial cells, macrophages) or the in vivo setting, a number of pathways previously unknown to be disrupted by acrolein seem deserving of further attention. In particular, the work identified EGR-2 and Nur77 as novel transcriptional markers of acrolein toxicity. Such insights may assist future mechanistic studies of the contribution of acrolein to a wide range of human health conditions (8). Supporting Information Available: Genes induced by acrolein in excess of 2-fold at one or more time points during the experiment (Table S1) and genes downregulated by acrolein in excess of 2-fold at one or more time points during the experiment (Table S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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