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Proteomic Analyses of Lung Lysates from Short-Term Exposure of Fischer 344 Rats to Cigarette Smoke Charleata A. Carter,*,† Manoj Misra,† and Steven Pelech‡ † ‡
A. W. Spears Research Center, 420 N. English Street, Lorillard Tobacco Company, Greensboro, North Carolina 27405, United States Kinexus Bioinformatics Corporation, Suite 1, 8755 Ash Street, Vancouver, BC, Canada V6P 6T3 and Department of Medicine, University of British Columbia, Vancouver, BC, Canada
bS Supporting Information ABSTRACT: A short-term 5 day mainstream cigarette smoke exposure study was conducted in Fischer 344 rats to identify changes in lung proteins. Groups of 10 male and female rats at 5 weeks of age were assigned to one of four exposure groups. Animals received either nose-only filtered air (Air Control) or 75, 200, or 400 mg total particulate matter (TPM)/m3 of diluted cigarette smoke. Exposures were conducted for 3 h per day, for 5 consecutive days. One lung per animal was frozen in liquid nitrogen and processed for proteomic analyses. Lung lysates from control verses treated animals were screened with 650 antibodies for changes in signaling protein levels and phosphorylation using antibody microarray technology, and then over 100 of the top protein hits were assessed by immunoblotting. The top smoke-altered proteins were further evaluated using reverse lysate microarrays. Major protein changes showed medium to strong bands on Western blots, depended on dose and gender, and included protein-serine kinases (Cot/Tpl2, ERK1/2, GSK3R/β, MEK6, PKCR/γ, RSK1), protein phosphatases (PP4/A0 2, PP1Cβ), and other proteins (caspase 5, CRMP2, Hsc70, Hsp60, Rac1 and STAT2). The most pronounced changes occurred with 75 mg TPM/m3 exposed females and 200 mg TPM/m3 exposed males. Smoke-altered proteins regulate apoptosis, stress response, cell structure, and inflammation. Changes in identified proteins may serve as early indicators of lung damage. KEYWORDS: proteomics, microarrays, biomarkers, cell signaling, protein kinases, apoptosis, cytoskeleton, inflammation
’ INTRODUCTION Cigarette smoke is a major cause of lung diseases including chronic obstructive pulmonary disease (COPD) and cancer. Long-term animal studies for assessment of toxicity/carcinogenesis are not ideal models, because of the requirement for longterm intensive smoke exposure, the length of time it takes to induce visible tumors, and the lack of an established robust animal model for cigarette smoke-mediated carcinogenesis. A newer approach has been suggested that focuses on induction of toxicological changes relevant to disease such as cell proliferation, chronic inflammation, and inhibition of apoptosis.1 Toxicoproteomics is currently being employed in order to evaluate the protein changes associated with these functional changes. Proteomics approaches may help define markers of disease for early diagnosis, which is critical for the treatment of many cancers including lung cancer as well as many other diseases. The identification of biomarkers for lung cancer, COPD, and other diseases associated with exposure to environmental toxins is an area of active research that may offer useful tools to complement radiological imaging, early diagnosis, prevention, and treatment of multiple diseases. Unlike the study of a single gene, protein, or pathway, proteomic technologies enable a more systematic r 2011 American Chemical Society
overview that provides the potential to improve our understanding of lung cancer.2 Various analytical proteomic analyses have been applied to exhaled breath condensate and lung tissues.3 New proteomics approaches have expanded the frontiers of medical research and demonstrate tremendous potential in the early detection, diagnosis, and staging, as well as provision of novel therapeutic targets for improved management of smokingrelated lung diseases.4 Cell signaling pathways play critical roles in biological alterations in human diseases, but detailed knowledge of altered signal transduction systems in response to toxins is still in its infancy. Elucidation of the regulatory networks in the various cells in the human body and the development of technologies to track changes in their protein components is a major challenge.5 Protein antibody microarrays are the most efficient way to analyze multiple samples or proteins at the same time.6 Antibody microarrays permit evaluation of a high number of proteins that can be tracked simultaneously and economically with automated high-throughput potential.7 These microarrays have the potential to facilitate systems proteomics research using limited Received: April 14, 2011 Published: May 31, 2011 3720
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Journal of Proteome Research amounts of biological material, a problem that confounds immunoblotting and mass spectrometry-based approaches. We used antibody microarrays to elucidate changes in proteins in response to short-term cigarette smoke exposure in F344 rats, which have been widely used in long-term toxicity studies. Western blot analysis was subsequently performed to further analyze key protein changes using denatured proteins. In this manner, protein hits from microarray analyses were validated. Lysate microarrays were further employed to evaluate biological variation in promising biomarker proteins. Assessment of toxicological changes and candidate biomarkers in short-term animal studies could provide a new approach for evaluation of the mechanism(s) of toxin action as well as new assays for product testing and disease diagnoses. Protein profiling in cigarette smoke-exposed rats has often involved long-term exposures. Proteomic analysis of lung tissue of male Wistar rats exposed to cigarette smoke and radon for 75 days revealed changes in proteins involved in signal transduction, metabolism, heat shock and stress, and cytoskeletal organization.8 Another study of male Wistar rats exposed to cigarette smoke for 2 months revealed by immunohistochemistry that NF-kB, MEK1, and ERK2 were increased in terminal bronchioles, suggesting that exposure to cigarette smoke results in oxidant stress promoting lung pathogenesis.9 Exposure of male SpragueDawley rats to cigarette smoke from 1 day to 34 weeks and gene expression profiling revealed that genes involved in the stress response and inflammatory pathways are increased by smoke.10,11 Proteomic analysis revealed that proteins changed by cigarette smoke from human lung in chronic smokers included GRP78, actin, albumin, gelsolin, transferrin, and annexin.12 The purpose of the present study was to evaluate changes in lung proteins in nose-only exposed Fischer 344 rats exposed to mainstream whole smoke generated from 3R4F Kentucky reference cigarettes at concentrations of 75, 200, or 400 mg total particulate matter (TPM)/m3 for 5 consecutive days. This study focused on the effects of short-term cigarette smoke exposure on changes in lung proteins in both genders of Fischer 344 rats. We hypothesized that the proteins that were most affected by shortterm smoke exposure would fit into pathways involved in inflammation, proliferation, stress responses, and changes in cell morphology. In this study we present the profiles of differentially expressed proteins in lung tissue of each exposure group and the biological networks/pathways involved and relate the changed biological processes to mechanisms of disease development.
’ MATERIALS AND METHODS Experimental Design
Cigarette smoke analysis and exposures of the animals, resultant gross changes in body weights and pathology have been described previously.13 The study protocol was also described in detail previously and was approved by the Institutional Animal Care and Use Committee at the Illinois Institute of Technology Research Institute, Chicago, IL.13 Briefly, groups of 10 male and female rats at 5 weeks of age were randomly assigned to one of four exposure groups. Depending upon the group, animals received either filtered air (Air Control) or 75, 200, or 400 mg TPM/m3 of diluted mainstream cigarette smoke. Exposures were conducted for 3 h per day, for 5 consecutive days. For tissue processing, the lungs were collected from all animals immediately after the exposure on Day 5. The right lung was dissected, frozen, and processed for further proteomics analyses
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Figure 1. Strategic approach for investigation of smoking effects on rat lung cell signaling.
as outlined below and in Figure 1. The left lung was analyzed for pathological changes.13 Proteomics Evaluation: Antibody-Based Protein Microarray
The Kinex Antibody Microarray KAM 1.0 analyses were performed with detergent-solubilized protein lysates as described previously.5 Briefly, protein lysates from control and treated rat lung tissues were labeled with a fluorescent dye, and unincorporated dye molecules were removed by ultrafiltration. Equal amounts of purified labeled proteins from the control and its correspondingly treated sample were incubated simultaneously on opposite ends of a Kinex KAM-1.0 antibody microarray (Kinexus Bioinformatics, Vancouver, B.C. Canada). Each Kinex antibody microarray is based on duplicate measurements with 2 identical fields of antibody grids containing antibodies recognizing 650 signaling proteins and phospho-sites. After probing, arrays were scanned using a ScanArray scanner (Perkin-Elmer, Wellesley, MA) with a resolution of 10 μm, and the resulting images were quantified using ImaGene 8.0 (BioDiscovery, El Segundo, CA). The values for duplicate measurements were averaged, and the mean value for the difference in duplicates from the average was (8.5%. Proteins that were selected as valid to move to the next screening step fit into at least one of the following categories: (1) proteins that changed in >1 microarray; (2) proteins that were altered in >1 exposure group of samples; (3) proteins that had highest % change from control (CFC) 3721
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Journal of Proteome Research values; and (4) proteins that were previously found in high quantity in rat tissues by searching the KiNET database (www. kinet.ca). Validation
The top 108 antibodies that demonstrated smoke-induced changes and fit at least 1 of the 4 criteria stated above moved to a prescreen status for prevalidation. The pooled samples were evaluated to assess which target proteins were detectable by immunoblotting in lung lysates. This step and the next step of Western blotting were necessary, because the proteins evaluated in the Kinex KAM 1.0 antibody microarrays were not denatured, which increases the opportunity for false positives and false negatives due to antibody cross-reactivity, proteinprotein interactions, and blocked epitopes in protein complexes. Western blotting was performed with pooled samples to determine the presence of the 108 target proteins using methods detailed previously.5 In this case, pooled samples corresponded to a mixture of both control and treated rat lung lysates. Samples were analyzed using Kinetworks KCPS multiprotein immunoblotting analyses with a combination of phospho-site and pan-specific antibodies (Kinexus). The immunoblotting analyses involved probing with mixes of in-house validated primary antibodies from commercial sources. Immunoblotting was performed using 300 μg of detergent-solubilized lysate proteins as described previously.5 The Kinetworks analysis involves resolution of proteins in a single lysate sample by SDS-PAGE and subsequent immunoblotting overnight at 4 °C with panels of up to 3 primary phospho-site-specific antibodies per channel in a 20-lane Immunetics multiblotter. Phospho-site antibodies were primarily obtained from InVitrogen (Carlsbad, CA), Cell Signaling Technologies (Beverly, MA), and Millipore (Temecula, CA). The antibody mixtures were carefully selected to avoid overlapping cross-reactivity with target proteins. Membranes were later rinsed with TBST buffer and then incubated with the relevant horseradish peroxidase conjugated secondary antibody for 45 min at room temperature. The immunoblots were developed with enhanced chemiluminescence (ECL) Plus reagent (Amersham, Arlington Heights, IL), and signals were captured by a Fluor-S MultiImager and quantified using Quantity One software (Bio-Rad, Hercules, CA). Background was less than 100 CPM for these analyses. Determination of Protein Changes Induced by Smoke
From the prescreen of 72 proteins, the 18 most prevalent proteins were identified and evaluated by Western blot as described above in control versus treated pooled rat lung lysates in order to question whether the exposures altered the amount or phosphorylation of each target protein. To assess whether the trend in pooled samples of 10 tissues is similar among the individual animals, the top 6 proteins from the 18 most prevalent proteins were assessed by Western blotting in 4 randomly selected individual animals, and the results of the individual animal data were averaged. Individual variability was examined more extensively in the follow-up reverse lysate microarray analyses. Protein Lysate Microarray Analyses
Each of the lung lysates from the 80 rats in the 8 groups were heat-denatured and individually printed at 3 dilutions in triplicate on nitrocellulose-coated microarray slides. The amount of protein in each spot was assessed by Sypro-Ruby staining followed by scanning the stained array in a microarray scanner at the
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wavelength of 546 nm. Selected antibodies with validated reactivity by Western blotting were then applied onto separate protein lysate microarrays individually. The binding of the antibodies was detected by the horseradish peroxidase (HRP)conjugated secondary antibodies via a tyramide-mediated 2-stage signal amplification approach. The arrays were scanned at the wavelength of 647 nm, and the resulting images were captured and quantified as described above for the Kinex antibody microarrays. The intensity of each spot on the array was normalized to the amount of total protein from the same spot as revealed by Sypro-Ruby staining for further data analyses. Protein Identification by Mass Spectrometry
Rat lung tissue lysates were prepared using the standard lysis buffer and procedures described in the Kinex Antibody Microarray Customer Information Package available from the Kinexus Web site (www.kinexus.ca). Tissue lysates were subsampled and pooled from 80 individual animal preparations to yield a protein concentration of 40 mg/mL. For each immunoprecipitation 10 μg of MKP1 rabbit polyclonal antibody was cross-linked to 10 μL of protein AþG agarose beads in a spin column using the Pierce Cross-link Immunoprecipitation Kit (ThermoFisher, Rockford, IL). Then, 100 μL of tissue lysate containing 4 mg of total protein was incubated with the antibody-agarose at 4 °C overnight with spin column rotation. The column was then washed and eluted according to the manufacturer’s instructions. The resulting 40 μL of eluate was mixed with 10 μL of 4X SDS Sample Buffer and heated to approximately 95 °C before loading onto an SDS-PAGE gel. After electrophoresis and Coomassie blue staining, the protein bands that were visible in the antibodyplus immunoprecipitate but not in the antibody-minus immunoprecipitate were excised and frozen at 70 °C. The gel bands were subjected to in-gel trypsin digestion was followed by HPLC and MS/MS on a Thermo Electron LTQ-Orbitrap to give accurate mass MS/MS data for parent and daughter ion combinations. These data were subject to a MASCOT database MS/ MS ion search looking for matching predicted parent-daughter ion masses from all known rat proteins. Bioinformatics Analyses
The potential interactions among the antibody-reactive proteins that were singled out in this study were investigated by individual queries of the Search Tool for the Retrieval of Interacting Genes and Proteins (STRING) database (www. string.embl.de) and the PhosphoNET knowledgebase (www. phosphonet.ca). For the STRING analyses, only experimentally obtained data from human, rat and mouse studies were used that had a confidence level of 0.15 or greater. The mRNA expression levels of these 28 proteins were also examined for changes in other studies of human cancers and the effects of cigarette smoke exposure by query of the National Centre for Biotechnology Information (NCBI) Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geoprofiles). The queries were based on the gene name of the target protein. The mean expression values and their standard deviations were calculated for each group of related cell or tissue samples in each separate study. Statistical Analyses
Protein amount is expressed as normalized counts per minute (CPM). CPM represents the trace quantity of the band corrected to a scan time of 60 s. Normalized CPM is the CPM adjusted to 3722
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Figure 2. Effect of mainstream cigarette smoke on protein expression and phosphorylation in rat lung as determined by antibody microarray analyses. Values shown are the averages of duplicate measurements with the ranges shown by the error bars. Each measurement was determined with pooled lysates from 10 gender- and exposure-matched rats: (O) male rats; (b) female rats.
correct for differences in protein amount. Statistical significance for changes was evaluated with the Student's t test.
’ RESULTS Antibody Microarray Analyses
Proteomic analysis of rat lung lysates using antibody microarray technology was performed whereby hundreds of signaling proteins and phosphorylation sites were screened using 650 panand phospho-site-specific antibodies with lysates from control and cigarette smoke-exposed tissues. Supplemental Table 1 provides a complete listing of all of the recorded signal intensity measurements of the proteins that were captured on the antibody microarrays from the 8 distinct groups of pooled lung tissue lysates. Supplemental Figure 1 reveals the most striking gender and smoke concentration-specific alterations in protein expression (Figure S1A and S1B) and phosphorylation (Figure S1C and S1D) evident with 57 of these antibodies. Gender-similar changes by microarray analysis in proteins from pooled rat lung lysates included ERK2, GSK3R, MEK6, ROKR, STAT2, STK33, and TAK1 (Figure S1A) as well as PKCγ Thr-514 (Figure S1C). Antibodies that revealed marked gender-distinct alterations with
smoke exposures included 14-3-3-ζ, caspase 5, CDK2, HSC70, NME6, and RSK4 (Figure S1B). Evaluations of the general protein trend changes in antibody microarrays while comparing all treated lysates to controls showed that protein levels of Abl, casein kinase 2R, CAMK4, Hsp60, MEK2, PKCδ, and phosphorylation of Hsp27 and Jun decrease in both genders (Figures S1A, S1B, S1C). Proteins that increase in both genders are PKCγ 514, ROKR, TEK, and Yes (Figures 2, S1A, S1C). Through a selection process detailed above under Materials and Methods and outlined in Figure 1, over 100 antibodies and their target proteins and phospho-sites were subjected to further analyses. Figure 2 shows the antibody microarray results for the top 18 of the 57 antibodies that could be confirmed by subsequent Western blot analyses to detect smoke-induced changes in target proteins. Immunoblotting Analyses
It was critical to perform immunoblotting analyses of the rat lung lysates to confirm that antibodies that demonstrated smokeinduced changes with the antibody microarray were in fact specific for their intended target proteins. Antibody microarrays use non-denatured native proteins, which can exist in complexes 3723
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Figure 3. Effects of cigarette smoke exposure on protein expression and phosphorylation in rat lung as determined by immunoblotting with specific antibodies. Values shown are the percent change with each concentration relative to the gender matched unexposed controls. Each measurement was determined with pooled lysates from 10 gender- and treatment-matched rats.
with other proteins. Thus protein changes may be related to changes in protein expression, phosphorylation, protein protein interactions, cross-reactivity of the antibody with nontarget proteins, or any number of combinations of these. Antibody microarrays are also much more sensitive for protein detection than standard Western blotting. When antibody validation was performed with 108 different antibodies with pooled rat lung lysates, 47 of these visualized little or no detectable appropriately sized target protein bands on immunoblots (Supplemental Figure S1). In some instances, strong crossreactive off target proteins were also observed and are further described below. The most promising of these antibodies were subjected to additional testing for their ability to show smokeinduced changes in Western blots (Supplemental Figure S2). Figure 3 provides quantitation of these changes as revealed by immunoblotting of the pools of lung lysates from separate groups of control and smoke-exposed male and female rats. Figure 3 shows the percent changes in top affected proteins between control and smoke-exposed lung lysates based on Western blot analysis. Proteins and phospho-sites that demonstrate smoke-induced changes greater than 90% by Western blotting analyses include those detected with MEK6, PKCR
Ser-657, PKCγ Thr-514, STAT2, and vinculin Tyr-822 antibodies. With 75 mg TPM/m3 exposed female rats, MEK6, PKCR S657, and PKCγ T514 are reduced by more than 90%, while ERK2 and Rac1 increase by 70% or more. Over 100% elevated protein levels are observed for STAT2 and vinculin Tyr-822 in 200 mg TPM/m3 exposed male rats. Western blotting shows that there are striking differences in both up regulation and down regulation of protein expression and phosphoryation between the female and male rats with respect to their sensitivity to cigarette smoke concentration (Figure 3). Female rats show much higher sensitivity to smoke-induced changes for about twothirds of 30 proteins with 75 mg TPM/m3, but 200 mg TPM/m3 was usually necessary to evoke similar responses in the male rats. However, at 200 mg TPM/m3 in the female rats, many of the smoke-induced changes at 75 mg TPM/m3 are reversed. Such reversions and further changes in the opposite direction required 400 mg TPM/m3 in the male rats. Such profound differences in cigarette smoke sensitivities did not appear to reflect genderdifferences in the basal expression or phosphorylation states of the monitored proteins (Supplemental Figure S3). Four of the antibodies revealed smoke-induced down regulation of different PKC isoforms. While the classic PKC family 3724
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Figure 4. Individual animal variation for rat lung protein biomarkers of cigarette smoke exposure as determined by tissue lysate microarray analyses. Values are the averages of immunoblot signal intensities for the indicated proteins or phospho-sites from triplicate measurements at 3 different concentrations each with individual lung lysates from unexposed and cigarette smoke-exposed male and female rats. Within each group of 10 animals, the data has been sorted with increasing intensities in the x-axis. Values shown in the boxes are the mean ( SD (n = 10) for each condition. Statistical analysis was performed with the two-tailed Student's t test with paired data.
members PKCR and γ exhibited reductions in phosphorylation at Ser-657 and Thr-514, respectively, the novel PKCδ and atypical PKCζ exhibited decreases in total protein at the 75 mg TPM/m3 dose in females and at the 200 mg TPM/m3 dose in males (Supplemental Figure S3 and Figure 3). While it is possible that a global decrease in PKC levels, similar to what is seen after prolonged PKC activation in experimental systems, may account for the decreases in detection of classic PKC phosphorylation rather than a stochiometric decrease of phosphorylation at the indicated sites, the lack of inhibition of PKCβ1 phosphorylation would suggest the effect is not universal to all PKCs. Alternatively, this effect may be less universal than indicated. High structural and sequence homology among the classic PKC isoforms in the phosphorylation regions makes it possible that both of the phosphorylation signals originate from a single PKC, which is further supported by the identical molecular masses of the PKC bands with each antibody and a common 42 crossreactive protein as well as the common trends in smoke-induced changes. However, even if cross-reactivity explains the similar trend among the classic PKCs, the novel and atypical isoforms are structurally distinct and their likelihood of cross-reactivity is low.14,15 Finally, the PKCs may be undergoing catalytic processing to a different form that was not quantified because of its shift in molecular weight, as has been demonstrated in tissues exhibiting increased cell death through apoptosis.16,17 The rise in level of the 42 kDa protein detected with the PKCR Ser-657 antibody with 200 mg TPM/m3 in the female rats shown in Figure 4D supports this contention, because this corresponds to the size of the catalytic fragment of PKC known as PKM.18
Also supporting this idea are the bands of 43, 40, and 43 kDa detected by the Thr-514 PKCγ, the pan-PKCδ, and the panPKCζ antibodies, respectively. Thus, a smoke-induced shift in molecular weight subsequent to cleavage to the catalytic fragment seems the most likely scenario that explains the commonality of response among the PKC families. Two of the cross-reactive proteins of 52 and 60 kDa that were detected with a polyclonal antibody preparation originally developed to recognize the MAPK phosphatase 1 (MPK1) also demonstrate smoke-induced changes (Supplemental Figure S4). Both proteins show increased expression at 75 mg TPM/m3 in the females and with 200 mg TPM/m3 in the males. At higher smoke concentrations, these increases are reversed for the 60 kDa protein and decline to below untreated levels. Attempts were made to identify both cross-reactive proteins following immunoprecipitation with the MPK1 antibody and tandem MS/MS mass spectrometry. The 60 kDa protein was conclusively identified as dihydropyrimidinase-like 2 (DPYSL2), which is the cognate of the human collapsin response mediator protein 2 (CRMP2) (Supplemental Figure S5AC). Analysis of Individual Rat Lung Lysates
Up to this point in the study, the proteomics analyses were undertaken with pooled lysates from 10 gender-matched animals for each of the control and exposure groups. To explore the biological variation among individual animals, we initially used Western blotting as this is the most reliable method for specific quantification of target proteins. Six of the top smokeaffected proteins from the previous antibody microarray and 3725
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Figure 5. Individual animal variation with rat lung protein biomarkers of 200 mg TPM/m3 cigarette smoke exposure as determined by multi-immunoblotting analyses. Values are the means of immunoblot signal intensities for the indicated proteins or phospho-sites from 4 individual lung lysates from unexposed and cigarette smoke-exposed rats. Standard deviations are shown. Statistical analysis was performed with the two-tailed Student's t test with paired data.
immunoblotting analyses were screened in four individual animals of the same gender from the 200 mg TPM/m3 and untreated groups to determine if the same trends occurred in individual animals as in pooled samples (Figure 5 and Supplemental Figure S6). The 200 mg TPM/m3 exposure was selected, because this dose has been equated to a heavy human smoking pattern.19 The same trend of protein changes generally occurred between the pooled samples versus the averages of 4 individual animals with the largest changes apparent at the 200 mg TPM/m3 concentration in males as observed in Figure 5. The costs of individually testing 80 different cell lysates against 20 different antibodies with multiple replicate measurements by immunoblotting is highly prohibitive. However, with the production of custom reverse lysate microarrays, it was feasible to perform similar analyses of the separate lung lysates from the normal and smoke-exposed rats to explore biological variation among individual animals. Figure 4 and Supplemental Figure S7 show that 75 mg TPM/m3 in female rats produced statistically significant declines in calnexin, caveolin 2, CK2R, ERK1/2, JNK, MEK6, p38R MAPK, PKCR Ser-657, PKCγ Thr-514, PKCδ, PKCζ, PP4/A0 2, RSK1 Ser-380, and STAT2 as well as increases in GSK3R/β and Hsp60. At 200 mg TPM/m3 in male rats, smoke similarly evoked statistically significant declines in calnexin, CK2R, ERK1/2, MEK6, PKCR Ser-657, PKCγ Thr-514, and PKCζ. At higher TPM/m3 concentrations, most of these changes were reversed, and in some cases the decline was reduced below (e.g., PKCζ) basal levels. At 400 TPM/m3 in both female and male rats, caveolin 2, ERK2, and GSK3R/β levels declined below basal values. Supplemental Table S1 shows antibody microarray analysis of pooled lung tissue lysates from smoke-exposed rats. Supplemental Table S2 shows percent changes from gender controls for protein target hits using various proteomics methods employed in this study and compares data between antibody microarray, immunoblotting, and lysate analyses. Signaling Pathway Analysis and Linkage to Human Cancer
The combined data from these 3 proteomics methods employed in this study when subjected to STRING and PhosphoNET analyses permitted construction of a diagram showing potential connections between signaling proteins in the rat lung that are affected by cigarette smoke (Figure 6). Generally, the pattern of cigarette smoke-induced changes in specific proteins in this study at 75 mg TPM/m3 in females and 200 mg TPM/m3 in
males reflected a balanced mix of stimulation (e.g., caspase 5, Chk2, Cot, Hsp60, MEK6, PKCR, ROKR, RSK1, STAT2, ZIPK) and inhibition of (e.g., CK2R, Cot, GSK3R, Hsc70, PKCR, PKCγ, PKCζ, PP1Cβ) of growth signaling pathways and a generalized suppression of apoptotic pathways (e.g., caspase 5, CHK2, CK2R, GSK3R, Hsc70, MEK6, PKCR, PKCδ, PKCγ, TAK1, ZIPK). At higher smoke concentrations, these changes were usually reversed consistent with slightly more induction of growth stimulatory responses (e.g., ERK2, GSK3R, JNK1, PTP1C, Rac1, RSK1) than growth inhibitory responses (e.g., CK2R, Hsc70, ROKR, vinculin) and more of a balance of pro-apoptotic responses (e.g., 14-3-3-ζ, GSK3R, JNK1, PTP1C, Rac1) and antiapoptotic responses (e.g., CK2R, ERK2, Hsc70, JNK1, vinculin). To explore the relationship between common smoke-induced changes in gene expression in the top 28 proteins identified in this study with lung cancer and other human cancers, we undertook an extensive analysis the NCBI GEO database to identify whether these proteins were similarly affected in human cancers when compared to normal tissues (Figure 7). Supplementary Table S4 specifically details GEO mRNA expression analyses of human cancers and the effects of cigarette smoke on selected signaling proteins. The top changed proteins in our study that showed changes above 50% in the GEO database analysis are Cot (decreased 53% in mouse lung tumors), Hsc70 (increased 56% and 89% in mouse and human lung tumors, respectively), MEK 6 (increased 62% in human lung tumors), and Hsp60 (increased 104% in human lung tumors). Further analyses are shown in Supplementary Table S3 whereby STRING analysis and PhosphoNET analyses of potential interactions between rat lung signaling proteins regulated in response to smoke are shown. Proteins that scored highest with direct interactions with other top hit proteins are in descending order: PKC-R, PKC-δ, GSK3B, and RSK1. Many of the proteins that are altered in expression and phosphorylation in response to 75 mg TPM/m3 in females and 200 mg TPM/m3 in males are linked to the development of cancer.
’ DISCUSSION General Smoke-Induced Changes in Lung Protein Regulation
Protein profile and cell signaling changes often precede or occur concurrently with cellular dysfunction. Our screen of smoke-altered proteins in short-term exposed rat lungs revealed 3726
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Figure 6. Effect of cigarette smoke on cell signaling pathways in rat lung. Connections between signaling proteins that demonstrated mainstream cigarette smoke-induced changes in protein expression or phosphorylation as revealed by immunoblotting. Orange bars indicate reductions and green bars indicate increases as evident with 75 mg TPM/m3 (L) in female rats and often in 200 mg TPM/m3 (M) in male rats (appearance of a “p” indicates that phosphorylation rather than the protein level was monitored). Protein kinases are shown in dark ovals, whereas non-kinases are indicated in light ovals. Phosphorylations that stimulate the functional activities of target proteins are indicated with closed triangles with “plus” symbols, whereas phosphorylations that inhibit the functional activities of target proteins are shown with closed boxes with “negative” symbols. Direct proteinprotein interactions are indicated with at least one closed circle connected with bars to a closed circle, triangle (indicates activation of function), or box (indicates inhibition of function). Indirect interactions are shown with dashed lines. Detailed information on these proteinprotein interactions and references are provided in Supplemental Table 3.
several specific trends. It is noteworthy that 200 mg TPM/m3 has been equated to a heavy human smoking pattern,19 and many of the major changes in protein amount in smoke-treated male rats occur at this dose. In the present study, many of the major protein changes depend on dose and gender and include such diverse proteins as protein-serine kinases (Cot/Tpl2, ERK1/2, GSK3R/ β, MEK6, PKCR and γ, RSK1); protein phosphatases (PP4/A0 2, PP1Cβ, PTP1B); focal adhesion proteins (Rac1and vinculin); and others (caspase 5, CRMP2, Hsc70, Hsp60, and STAT2). Top altered proteins affected by smoke are involved in apoptosis, disease, stress activation, inflammation, cell growth, structure, adhesion and motility, cytoskeletal and F-actin organization. Indeed cigarette smoke affects phosphoproteins involved in endothelial cell migration and actin in endothelial cells20 and decreases cell spreading and motility in human bronchial cells21 and fibroblasts.22,23 The primary analytical methods used in this study were antibody microarrays, immunoblotting, lysate microarrays, and mass spectrometry. To our knowledge, these methods have not been previously applied successively in a single major proteomics study for biomarker identification. The antibody microarray is a powerful method to reveal key altered proteins from hundreds of evaluated proteins. However, additional studies were necessary to further characterize the nature of the observed protein changes. Therefore, key protein changes from the antibody microarray were tested by Western blotting, which relies on denatured proteins. With phospho-site-specific antibody probing of Western blots, it is possible that apparent changes in
phosphorylation arise from altered expression in addition to differences in the stoichiometry of phosphorylation. In view of the long durations of smoke exposures, it is likely that apparent phosphorylation differences with exposures may primarily reflect changes in protein levels. Hereafter, “protein level” refers to quantification by Western blotting, which is the gold standard for protein quantitation. Smoke-Affected Lung Proteins
PKC isoforms are often activated in diverse diseases including cancer, atherosclerosis, diabetes, and respiratory disorders including non-small cell lung cancer, asthma, and COPD24,25 and regulate proliferation, apoptosis, differentiation, motility, and inflammation.26 In our study, PKCR levels dramatically decrease (93%) in 75 mg TPM/m3 female exposed rats but increase (31%) in 75 mg TPM/m3 exposed males and then decrease (80%) in 200 mg TPM/m3 exposed males. Likewise, PKC-R staining decreases in the 75 mg TPM/m3 exposed female rats but is similar to control staining levels in the 75 mg and 200 mg TPM/m3 exposed male rats from these same exposed animals.13 Cigarette smoke exposure activates PKC in cultured rat tracheal epithelial cells27 and in ciliated tracheal epithelium from C57BL mice.28 PKCR inhibition decreases tumor formation in nude mice.29 Cigarette smoke condensate activates PKCR in human bronchial cells.21 Although PKCR activation is generally associated with disease, a significant decrease may also lead to altered cell function. An inverse relationship exists between PKCR activity and its effect on cationic amino acid transporter-1 3727
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Figure 7. Expression regulation of cigarette smoke-regulated proteins in human cancers. The Gene Expression Omnibus (GEO) of the U.S. National Centre for Biotechnology Information (NCBI) was searched for mRNA expression data from gene microarray analyses of human cancers for 28 proteins that were previously observed to undergo regulation in rat lung in response to exposure to cigarette smoke. Eleven separate studies were selected that involved comparisons of normal (control) tissues and human cancers. As provided in Supplemental Table S4 (Supporting Information), the mRNA expression levels of the 28 genes were separately averaged, and the standard deviations were determined. Statistical analyses were performed with a twotailed Student's t test. The percent change from control (%CFC) was calculated and colorized as shown only if the %CFC was determined to have a p value of 0.05 or less.
(CAT-1) transport activity.30 CAT-1 is essential for cell survival during stress31 and is elevated in carcinomas.32 Smoke-induced decreases in PKCR activity may alter transporter functions in lung cells, while increases in PKCR activity may alter cell function and contribute to disease. PKCγ is a regulator of cytokine IL8 response33 and IL1B-induced COX2.34 Mammary epithelial cells overexpressing PKCγ display increases in ERK 1/2 and are tumorigenic and metastatic.35 Mercury vapor exposure of rats enhances expression of genes encoding PKCR and γ in the lung.36 PKCγ levels decrease 94% in 75 mg TPM/m3 females and 66% in 200 mg TPM/m3 males but increase 80% at higher 400 mg TPM/m3 exposed males. Lower PKCγ levels may be due to lower macrophage infiltration in the lung at Day 3 while proteins associated with inflammation and macrophage function become highly expressed during longer smoke exposure.11 Likewise, the inflammatory protein COX2 levels are elevated only at high smoke exposure (400 mg TPM/m3) in lung tissues from these animals.13 Actin-binding proteins are altered by cigarette smoke.21,23 The actin-binding proteins PKCR, vinculin, and Rac1 are among the top altered proteins in this study. PKCR binds to vinculin.37 Interestingly, there is a 103% increase in vinculin Tyr-822
phosphorylation in 200 mg TPM/m3 exposed males, while PKCR Ser-657 phosphorylation decreases 80%. Fibroblast exposure to mainstream whole smoke results in F-actin and vinculin increases, while focal adhesions are markedly elevated resulting in decreased cell migration.22 Cytoskeletal proteins are down-regulated in lungs from male Wistar rats exposed to smoke for 1, 2, and 4 months.38 Lower cigarette smoke doses increase vinculin in BEAS-2B lung cells, while vinculin decreases at higher exposures likely due to apoptosis.21 Rac1 increases 70% in 75 mg TPM/m3 exposed females, but in males Rac1 decreases 19% at 75 mg TPM/m3 exposure and then increases 23% at 200 mg TPM/m3 and 43% at 400 mg TPM/m3. Rac1 is necessary for neutrophil migration into the lungs. Loss of Rac1 neutrophil activity is associated with significant decreases in neutrophil recruitment into lung alveoli.39 Rac1 indirectly activates MEK6, which directly phosphorylates and activates p38 MAPK isoforms. Smoke exposed rats show large decreases in MEK6 levels with a 92% decrease in 75 mg TPM/m3 exposed females and a 68% decrease in 200 mg TPM/m3 exposed males. An “unknown” MPK1 antibody cross-reactive 60 kDa protein increases at 75 mg TPM/m3 in female rats and with 200 mg TPM/m3 in males. At higher smoke concentrations, these 3728
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Journal of Proteome Research increases are reversed in both genders and decline to below untreated levels. Using tandem MS/MS mass spectrometry this protein was conclusively identified as CRMP2. CRMP2 is necessary for signaling by class 3 semaphorins and subsequent cytoskeletal remodeling. CRMP2 binds to tubulin and promotes microtubulin assembly. Class 3 semaphorins are secreted proteins that alter the cytoskeleton and organization of actin filaments and microtubules and play a role in tumor formation or prevention.40 Cigarette smoke increases ERK1/2 phosphorylation in airway lining cells and alveolar macrophages in mice exposed for 10 days and 6 months.41 ERK1/2 also significantly increases in lung tissues from emphysema patients compared to controls.41 ERK2 increases 79% in 75 mg TPM/m3 females and 41% in 200 mg TPM/m3 males. The ERK1/2 pathway is constitutively active in human alveolar macrophages and contributes to cellular survival42 through JNK inhibition and activation of PP1 leading to protein translation and cell survival. In males, PP1Cβ increases 30% with 75 mg TPM/m3 exposure but decreases 84% in 200 mg TPM/m3 exposed rats. In females, PP1Cβ decreases 86% at 75 mg TPM/m3. PP1Cβ may play a role in extending lung macrophage life span. TGF β-activated protein-serine kinase (TAK1), a member of the mitogen-activated protein kinase kinase (MAPKK) superfamily, is elevated in TPM exposed male lung lysates with the largest increase of 52% at 200 mg TPM/m3. TAK1 activates MEK6 and JNK and participates in cell shape and apoptotic regulation.43 Protein phosphatase 4 is a PP2A-related protein serine/threonine phosphatase with important roles in apoptosis, tumor necrosis factor-alpha (TNFR) signaling, and activation of JNK and NF-kB.44 PP4A0 2, a regulatory subunit that associates with the catalytic subunit of protein-serine phosphatase 4, increases over 65% in 200 mg TPM/m3 exposed males and 56% in 75 mg TPM/m3 exposed females, but decreases 52% in 400 mg TPM/m3 exposed females. Cigarette smoke exposure activates inflammatory proteins. The MAPK kinase kinase Cot (Tpl2) plays a role in the production of pro-inflammatory cytokines such as TNF and IL1β in macrophages.45 Cot decreases in female rat lysates but increases in male rat lysates, by 82% with 200 mg TPM/ m3exposure. Cot is activated in breast,46 thyroid, and colon tumors47 and indirectly activates MAPK and SAPKs such as JNK leading to transcription pathway activation such as those with activator protein-1 and NF-kB.48 The transcription factor STAT2 is involved in cytokine receptor signal transduction. STAT2 increases in smoke-exposed males with the largest elevation being 109% at the 200 mg TPM/m3 exposure. STAT2 dimerizes with STAT-1. STAT-1 is a common gene target in environmental tobacco smoke-exposed mice.49 The inflammatory caspase 5 is regulated by lipopolysaccharide and interferon γ.50 Caspase 5 levels decrease 55% in 75 TPM/m3 exposed females and 63% in 200 TPM/m3exposed males. GSK3R/β are related protein-serine/threonine kinases that are negatively regulated by PI-3 kinase/Akt, through phosphorylation at Ser-21 and Ser-9, respectively. GSK3β phosphorylates substrates such as β-catenin and is involved in energy metabolism, diabetes, tumorigenesis, and cell death.51 GSK3R/β levels decrease 30% in 200 mg TPM/m3 exposed males but increase 81% in 400 mg TPM/m3 exposed males. A mouse inhalation study with a 2 week cigarette smoke exposure showed increases in GSK3β phosphorylation in cardiovascular tissue.52 GSK3R/β phosphorylation requires PKC, and PKC inhibition abolishes PDGF-induced GSK-3 phosphorylation and cell proliferation.53
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GSK3β is required for TNF activation of JNK, ERK1/2, and Akt.51 GSK3β activates NF-kB. NF-kB activation induced by lipopolysaccharide, interleukin-1β, or cigarette smoke condensate is completely suppressed in GSK3β (/) cells.51 Hsp60 overexpression or down-regulation is associated with various cancers. Loss of Hsp60 tissue staining is related to development and progression of bronchial cancer in smokers.54 Thus, it is noteworthy that there is an 83% decrease in Hsp60 in 75 mg TPM/m3 exposed females and 200 mg TPM/m3 exposed males. Limitations and Implications
One of the confounding factors for interpretation of the findings from this study is the heterogeneity of cell types in the rat lung. However, the use of an in vivo model containing numerous cell types is also one of the strengths of this study. In analyzing lung lysates, one must assume that the population of cells is reflective of the makeup of the lung and thus contains a combination of epithelial and endothelial cells, fibroblasts, smooth muscle, elastic fibers, and immune cells including macrophages, lymphocytes, Clara, and goblet cells. The state of inflammation and toxic response will dictate how many immune cells may be invading the area. Thus protein assessment in lung lysates represents measurements in a mixture of these types of cells. In future studies, it will be insightful to explore the biomarkers identified here in isolated lung cell lines as well as other tissues in airway passages that are exposed to cigarette smoke. Another trend in this study was the variation among individual animals within the same treatment groups. Our reverse lysate microarray analyses revealed up to 3-fold differences in analyzed proteins among individual rats within the same groups matched for gender and smoke exposure concentration. Such heterogeneity reveals the huge biological variation that likely exists in humans and may account partly for the large differences in individual disease susceptibilities. Biomarker identification such as the ones revealed in this study may allow the development of screening assays to reveal individuals most susceptible to the adverse effects of toxins. The use of pooled samples appears plausible in these types of proteomics screening studies. Analysis of one time point reveals changes in specific proteins in our study, but further validation of these potential biomarkers from this and other short-term studies may require assessment of proteins along the path to disease progression to validate disease-specific irreversible protein changes. In this manner, biomarkers that are activated early and translate into disease states should be elucidated.
’ SUMMARY Cigarette smoke-induced protein changes vary by gender and dose in this study. Proteins evaluated in female rat lung lysates often show a general trend whereby they increase or decrease at the 75 mg TPM/m3 dose, but some proteins return to control levels at higher doses. By contrast, protein changes in male lung lysates show the greatest changes at 200 mg TPM/m3. The return to control values at higher doses may represent a change that has occurred at an earlier time point at higher doses. The 5-week-old rats used in this study are at the beginning of puberty and thus circulating hormone levels may be responsible for some of the gender differences in protein expression. Similar to the increased responsiveness of the rats to smoke exposure, epidemiologic evidence indicates that women are more susceptible to tobacco-induced carcinogenesis than men.55 This may in part be regulated by estrogen and progesterone alterations in protein 3729
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Journal of Proteome Research expression. Likewise female mice have an exaggerated response to lung injury relative to male mice because of female sex hormones, which have direct fibrogenic activity on lung fibroblasts.56 Toxins can induce protein changes that induce transcription of genes that are not usually expressed in cells, thus interfering with normal cell functions. Generally cigarette smokeinduced protein changes in this study causes decreases in apoptosis, changes in actin-binding proteins and cell structure, decreases in cell migration, changes in adhesion, increased inflammation, and stress response. If toxins prevent apoptosis, they may allow the survival of a cell that contains defects leading to altered cell function and disease. Cells that exhibit decreased migration may lead to hyperplasia, while prolonged inflammation is associated with various diseases.
’ ASSOCIATED CONTENT
bS
Supporting Information Supplemental figures and tables. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Ph: 336-335-6940. Fax: 336-335-6640. E-mail: charleatacarter@ lortobco.com.
’ ACKNOWLEDGMENT We thank Drs. Jane Shi, Hong Zhang, and Arthur Yee as well as Litsa Blanis, Sara Marrello, and Catherine Sutter from Kinexus for their technical support and comments. Thanks to Dr. William W. Polk for helpful scientific discussion. ’ ABBREVIATIONS AP-1, activator protein-1; CAT-1, cationic amino acid transporter-1; CSC, cigarette smoke condensate; CO, carbon monoxide; COPD, chronic obstructive pulmonary disease; COHb, carboxyhemoglobin; CPM, counts per minute; CRMP2, collapsin response mediator protein 2; DMSO, dimethylsulfoxide; DHPL2, dihydropyrimidinase-like 2; ETS, environmental tobacco smoke; GEO, Gene Expression Omnibus; GSK3, glycogen synthase-serine kinase 3; IL1β, interleukin 1-beta; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKK, mitogen-activated protein kinase kinase; MPK1, MAPK phosphatase-1; NCBI, National Centre for Biotechnology Information; NBF, neutral buffered formalin; NF-kB, nuclear factor-kappa B; PKC, protein kinase C; PP1/Cβ, protein-serine phosphatase 1-catalytic subunitbeta isoform; PP4/A0 2, protein-serine phosphatase 4-regulatory subunit; SAPK, stress-activated protein kinase; STAT-2, Signal Transducer and Activator of Transcription-2; STRING, Search Tool for the Retrieval of Interacting Genes and Proteins; TAK1, transforming growth factor-beta (TGFβ)-activated proteinserine kinase; TNF, tumor necrosis factor; TPM, total particulate matter ’ REFERENCES (1) Battershill, J. M. The Multiple Chemicals and Actions Model of carcinogenesis. A possible new approach to developing prevention strategies for environmental carcinogenesis. Hum. Exp. Toxicol. 2005, 24 (11), 547–58.
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