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Receptor for Advanced Glycation End-Products Signals through Ras during Tobacco Smoke–Induced Pulmonary Inflammation Paul R. Reynolds1, Stephen D. Kasteler2, Robert E. Schmitt2, and John R. Hoidal2 1

Department of Physiology and Developmental Biology, Brigham Young University, Provo; and 2Department of Internal Medicine, Pulmonary Division, University of Utah Health Sciences Center, Salt Lake City, Utah

We previously demonstrated up-regulation of the receptor for advanced glycation end-products (RAGE) and its ligands by cigarette smoke extract (CSE) in rat R3/1 cells, a type I–like alveolar epithelial cell line. However, RAGE-mediated intracellular signaling pathways that lead to pulmonary inflammation remained unclear. Using ELISAs, we demonstrate that alveolar epithelial cell lines exposed to 25% CSE for 2 hours induce the activation of Ras, a small GTPase that functions as a molecular switch in the control of several intracellular signaling networks. Conversely, cells treated with siRNA for RAGE (siRAGE) resulted in decreased Ras activation. Furthermore, Ras was significantly diminished in lungs from RAGE null mice exposed to chronic tobacco smoke when compared with smokeexposed wild-type mice. The use of a luciferase reporter containing NF-kB binding sites also demonstrated elevated NF-kB activation in R3/1 cells after CSE stimulation and decreased NF-kB activation in cells transfected with siRAGE before CSE exposure. ELISA revealed an increase in the secretion of IL-1b and CCL5 by R3/1 cells, two cytokines induced by NF-kB and associated with leukocyte chemotaxis. Furthermore, real-time RT-PCR and ELISAs revealed decreased cytokine secretion in RAGE null mouse lung exposed to tobacco smoke compared with lungs from smoke-exposed wild-type animals. These results support the conclusion that CSE-induced RAGE expression functions in pathways that involve Ras-mediated NF-kB activation and cytokine elaboration. This RAGE-Ras–NF-kB axis likely contributes to inflammation associated with several smokingrelated inflammatory lung diseases. Keywords: tobacco; inflammation; Ras; RAGE; lung

Receptors for advanced glycation end-products (RAGE) are cell-surface receptors expressed in many cell types, including endothelial and vascular smooth muscle cells, fibroblasts, macrophages/monocytes, and epithelium (1). RAGE expression is most abundant in the lung, from which it was initially isolated, and it is selectively localized to well differentiated alveolar type (AT)I epithelial cells (2). Identification in respiratory epithelium has led to the implication of RAGE in important developmental processes, such as the spreading, thinning, and adherence that characterize the transitioning of ATII cells to squamous ATI cells. RAGE was first described as a progression factor in cellular responses induced by advanced glycation end products (AGEs) that accumulate in hyperglycemia and oxidant stress. Subsequent studies have distinguished RAGE as a pat(Received in original form June 9, 2010 and in final form November 15, 2010) This work was supported by a Parker B. Francis Fellowship in Pulmonary Research (P.R.R.) and a Young Clinical Scientist Award provided by the Flight Attendants Medical Research Institute (P.R.R.). Correspondence and requests for reprints should be addressed to Paul Reynolds, Ph.D., Department of Physiology and Developmental Biology, 375A Widtsoe Building, Provo, UT 84602. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 45. pp 411–418, 2011 Originally Published in Press as DOI: 10.1165/rcmb.2010-0231OC on December 3, 2010 Internet address: www.atsjournals.org

CLINICAL RELEVANCE Understanding the role of receptors for advanced glycation end-products in the context of tobacco smoke exposure and its specific intermediates in signaling pathways will clarify tobacco smoke–mediated pulmonary inflammation and avenues of treatment. Such insight into mechanisms of inflammation has the probability of alleviating disease progression in former smokers and in those voluntarily or involuntarily exposed to tobacco.

tern recognition receptor that also binds S100/calgranulins, amyloid–b-peptide, and HMGB-1 (or amphoterin) to influence gene expression via activated signal transduction pathways (3–5). RAGE expression increases as a direct result of ligand accumulation (2), and RAGE–ligand interaction may contribute to pathological processes, including diabetic complications, neurodegenerative disorders, atherosclerosis, and inflammation (3, 4). Our previous work demonstrated that cigarette smoke extract (CSE) induces the expression of RAGE (6), its ligands (6), and Egr-1, a zinc-finger containing transcription factor, in pulmonary epithelial cells (7). These findings were confirmed in vivo using immunohistochemistry, which revealed elevated levels of RAGE and Egr-1 in the lungs of mouse models of cigarette smoke–induced emphysema (6, 7). A direct relationship between RAGE and Egr-1 is evidenced by data demonstrating cis-acting transcriptional control of RAGE by Egr-1 (6). The possibility of coordinated RAGE/Egr-1 functions in cigarette smoke–related injury also supports data that suggest RAGE null mice are protected from pulmonary inflammation induced by various stimuli. Despite the progression in the field of RAGE biology in the context of lung disease, the full extent of RAGE localization, the molecular mechanisms that control its expression, and its downstream effects have not been adequately evaluated. As an intracellular signaling molecule that regulates the fate of target cells, Ras oscillates between active guanosine triphosphate–bound and inactive guanosine diphosphate–bound conformations (8). Signaling involving active and inactive Ras has been associated with development, cellular proliferation, and differentiation (8–10) resulting from the transduction of signals coordinated by the Raf/MAP kinase, PI3K, JNK/p38, and Rho pathways (11, 12). Signal transduction involving these pathways is broadly implicated in the mechanisms of inflammation (13–15). Although the dysregulation of Ras is observed in many neoplastic malignancies (16), its direct role in inflammation and other cellular responses resulting from adverse stimuli requires further investigation. In the present study, we test the hypothesis that CSEinduced RAGE signals through Ras in signal transduction pathways that culminate in proinflammatory cytokine secretion. Through the use of siRNA specific for RAGE (siRAGE), we demonstrate that RAGE abrogation significantly diminishes Ras

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activation, NF-kB translocation to the nucleus, and proinflammatory cytokine elaboration. We further report that RAGE null mice exposed to chronic tobacco smoke experience less Ras activation and diminished cytokine secretion when compared with cigarette smoke–exposed wild-type mice. Collectively, these data offer novel insights into potential mechanisms whereby Ras functions as a central molecule in RAGE-mediated pulmonary inflammation elicited by tobacco smoke. Further research may demonstrate that RAGE and its specific downstream effectors are potential targets in the treatment or prevention of tobacco smoke–related pulmonary complications.

MATERIALS AND METHODS Cell Culture and Mouse Lung Samples ATI-like R3/1 (17) and ATII-like A-549 cells were exposed to media supplemented with CSE (n 5 7) or media alone. RAGE null and wildtype C57Bl/6 mice were divided into sham room air (n 5 10) and smokeexposed groups (n 5 10) at 3 months of age (7). Smoke-exposed animals were subjected to two standard research cigarettes (2R1), 5 days a week, for 6 months generated by a smoke exposure system (18). Lungs were immediately removed and frozen. Animal use followed approved IACUC protocols at the University of Utah and McGill University.

differences, Student t tests were used with Bonferroni correction for multiple comparisons. Results are representative, and those with P values , 0.05 were considered significant.

RESULTS RAGE Influences CSE-Induced Ras Activation

Active Ras, an intracellular molecular switch involved in many signal transduction pathways, was assessed in R3/1 and A-549 cells after exposure to a dilution series of CSE for 2 hours. There was a significant increase in Ras activation after acute exposure to 10% CSE, and there were further elevations in active Ras expression as the percentage of CSE increased (Figure 1A). After 2 hours of CSE exposure, trypan blue exclusion assays were performed and revealed no significant decrease in R3/1 or A-549 cell viability after exposure to 0% (99.2 6 0.6 and 99.3 6 2.3), 10% (97.1 6 2.3 and 97.0 6 3.1), or 25% (96.8 6 3.2 and 97.1 6 2.9) CSE. Trypan blue exclusion assays also revealed insignificant changes in viability in cell cultures exposed to 10 to 25% CSE for 2 hours followed by up to 8 hours of fresh media exposure (not shown). The viability of cells exposed to siRNA or Ras-DN was not significantly different when compared with control cells.

Ras Activation ELISA Ras activation ELISA assay kits (Millipore, Temecula, CA) were used to assess active and inactive Ras. In vitro analyses involved 50 mg of total cell lysates quantified by bicinchoninic acid assay. Total membrane and soluble proteins were isolated by homogenizing mouse lungs in RIPA buffer with inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA), quantified by bicinchoninic acid assay, and screened for Ras in 50-mg aliquots. In vitro experiments were repeated three times, each in triplicate, and lungs from six mice per group were assayed.

Immunoblotting and Immunocytochemistry Cell lysates were subjected to immunoblotting with a polyclonal RAGE antibody (R&D Systems, Minneapolis, MN) as described previously (6). Immunostaining for active NF-kB involved a p65 mAb (Cell Signaling, Beverly, MA) at 1:50 and a donkey anti-rabbit secondary (Santa Cruz Biotechnology). Individuals blinded to the type of siRNA and presence or absence of CSE performed three counts of 100 cells in each experiment. Cells with greater than 50% immunoreactivity in the cytoplasm or nucleus were considered cytoplasmic or nuclear positive, respectively. Cells with an equal cytosolic/nuclear distribution were not counted. Total cells were averaged, and SDs were determined.

siRNA and Vector Transfection and Reporter Gene Assays The recommended transfection reagent mix (Santa Cruz Biotechnology) was used to transfect R3/1 cells with vectors, siRNA for rat RAGE (siRAGE), or scrambled control siRNA (siControl) (Santa Cruz Biotechnology) 24 hours before CSE or fresh media exposure. For reporter assays, cells were transfected with 500 ng pRSV-bgal to determine transfection efficiency, 100 ng pNF-kB–Luc (Stratagene, La Jolla, CA), 100 ng siRNA or pCMV-RasN17 (Ras dominant-negative vector; Clontech, Mountain View, CA), and pcDNA control vector to bring total DNA concentration to 1.0 mg (19).

Measurement of Cytokine Levels IL-1b (Ray Biotech, Norcross, GA) and Quantikine RANTES/CCL5 ELISA kits (R&D Systems) were used to quantify cytokines secreted by R3/1 cells and in whole lung lysates. Cytokine mRNA was detected in lung lysates from RAGE null and control mice exposed to room air or chronic tobacco smoke using the RT2 Profiler PCR Array System (SABiosciences, Fredrick, MD), and levels were normalized to room air–exposed wild-type mice.

Statistical Analysis Mean values 6 SD from three experimental replicates were assessed by one- and two-way ANOVA. When ANOVA indicated significant

Figure 1. Cigarette smoke extract (CSE)-induced Ras activation in alveolar epithelial cells. (A) Alveolar type (AT)I-like R3/1 and ATII-like A549 alveolar epithelial cells were exposed to increasing concentrations of CSE for 2 hours and assayed for active Ras. Positive controls (50 mg HELA cells stimulated with 5 mg/ml epidermal growth factor) and negative controls (no cell lysates) were included. Significant increases in active Ras were demonstrated after exposure to 10% CSE. (B) R3/1 and A-549 cells were exposed to 25% CSE, and a time course revealed significant Ras activation when exposure lasted 1, 2, 4, and 6 hours. *P < 0.05.

Reynolds, Kasteler, Schmitt, et al.: RAGE/Ras Signaling Is Induced by Tobacco Smoke

A time course was conducted in which 25% CSE was exposed to R3/1 and A-549 cells for up to 6 hours. Significant Ras activation resulted from cells exposed to CSE for 1, 2, 4, and 6 hours (Figure 1B). To determine whether increased Ras was attributed to CSE-induced RAGE expression (6), RAGE was inhibited with siRNA specific to RAGE (siRAGE) before CSE exposure. Immunoblotting revealed that siRAGE significantly decreased basal RAGE expression in R3/1 cells compared with cells transfected with scrambled control siRNA (siControl) (Figure 2A). Using this transfection strategy, R3/1 cells that incorporated siRAGE had significantly diminished active Ras expression after CSE exposure when compared with siControl transfected cells (Figure 2B). RAGE Null Mice Have Significantly Diminished Tobacco Smoke–Induced Ras Activation

We next sought to determine whether active Ras is increased by exposure to tobacco smoke in the lungs of mice and if tobacco smoke–induced Ras activity is influenced by RAGE in vivo. RAGE null mice and age-matched wild-type control mice were exposed to chronic cigarette smoke or room air for 6 months. Active Ras in whole lung homogenates was significantly elevated in cigarette smoke–exposed wild-type mice when compared with control mice exposed to room air (Figure 3). RAGE null mice exposed to cigarette smoke also induced higher active Ras levels; however, active Ras was significantly lower in

Figure 2. CSE-induced Ras activation is mediated by receptor for advanced glycation end-products (RAGE). (A) R3/1 cells were transfected with small interfering RNA for rat RAGE (siRAGE) 24 hours before cell lysis and RAGE detection by immunoblotting. RAGE expression was significantly decreased in R3/1 cells transfected with siRAGE. (B) Active Ras was significantly diminished in R3/1 cells transfected with siRAGE 24 hours before a 2-hour exposure to 25% CSE when compared with CSE-exposed scrambled control siRNA (siControl). *P < 0.05 (siRAGE 1 CSE versus siConrol 1 CSE). †P < 0.05 (siRAGE 1 CSE versus siControl or siRAGE 2 CSE).

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cigarette smoke exposed RAGE null mice when compared with cigarette smoke–exposed wild-type mice (Figure 3). CSE-Induced NF-kB Activity Is Decreased by RAGE Inhibition

To assess the consequences of RAGE and Ras signaling, R3/1 cells were examined for NF-kB activity. Cells were transfected with pNF-kB–Luc, a luciferase reporter that contains NF-kB binding sites, so that nuclear translocation and activation of NFkB could be assessed. A nearly 2-fold increase in NF-kB activity was detected in R3/1 cells exposed to CSE when compared with cells exposed to media alone (Figure 4A). When CSE exposure was preceded by transfection of siRAGE, there was a significant reduction in nuclear NF-kB activity such that NF-kB activity was reduced to no-CSE baseline levels observed in siControl transfected cells (Figure 4A). To test the hypothesis that Ras is involved in RAGEmediated NF-kB activation by CSE, we performed a series of experiments that used a Ras dominant-negative vector (RasDN) capable ofblocking Ras activation (see Figure E1 in the online supplement). CSE-induced NF-kB activation was significantly decreased in R3/1 cells transfected with Ras-DN or siRAGE before exposure to CSE (Figure 4B). Data further revealed that R3/1 cells had a highly significant reduction in CSEinduced NF-kB activation when RAGE and Ras were targeted by siRAGE and Ras-DN before CSE exposure (Figure 4B). Combined, these data suggest that RAGE and Ras function in NF-kB–mediated inflammation elicited by cigarette smoke. Supporting the observation that nuclear translocation and activation of NF-kB are inhibited by RAGE targeting are data derived by immunocytochemical staining for NF-kB in R3/1

Figure 3. Ras activation was decreased in RAGE null mouse lung exposed to chronic cigarette smoke. RAGE null and wild-type control mice were exposed to chronic cigarette smoke for 6 months, and lungs were immediately frozen for analysis. Smoke-induced Ras activation was significantly attenuated in RAGE null mice when compared with smoke-exposed control mice. *P < 0.05.

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 45 2011 Figure 4. CSE-induced NF-kB activity was mediated by RAGE in R3/1 alveolar epithelial cells. (A) R3/1 cells were transfected with a NF-kB– Luc vector and siRAGE or siControl 24 hours before 25% CSE or fresh media replacement. Exposure to 25% CSE for 2 hours significantly induced nuclear NF-kB activity in siControl transfected R3/1 cells. When siRAGE was incorporated, CSE-induced NF-kB activity was markedly decreased to no-CSE basal levels. (B) Individually and additively, siRAGE and Ras-DN significantly decreased CSE-induced NF-kB activity. *P < 0.05; **P < 0.01. Representative immunostaining for NF-kB revealed NF-kB localization in R3/1 cells (arrows). (C ) NF-kB was predominantly located in the cytosolic compartment in R3/1 cells. (D) Transfection with siControl 24 hours before exposure to 25% CSE resulted in detectable nuclear translocation of NF-kB. (E ) siRAGE incorporation was sufficient to prevent significant nuclear translocation of NF-kB after CSE exposure. (F ) Control immunostaining of R3/1 cells transfected with control siRNA and exposed to CSE was performed without primary antibody to demonstrate specific immunoreactivity. Original magnification, 403.

cells exposed to CSE with or without prior transfection of siRAGE. Cells transfected with siControl had prominent cytosolic NF-kB localization (Figure 4C), and an anticipated shift to the nucleus was observed in siControl transfected cells after CSE exposure (Figure 4D). Although some nuclear staining was observed, cells transfected with siRAGE before CSE exposure maintained persistent NF-kB staining in the cytosol (Figure 4E). A no primary control (staining performed without primary antibody) involving R3/1 cells transfected with siControl and exposed to CSE revealed immunospecificity (Figure 4F). Quantitative analyses revealed that cells transfected with siRAGE before CSE exposure had significantly less nuclear localization and significant cytosolic sequestration of NF-kB when compared with CSE-exposed siControl transfected cells (Table 1). Proinflammatory Cytokine Secretion Is Mediated by RAGE

Because RAGE signaling influenced NF-kB activity during CSE exposure, we assessed the regulation of cytokine secretion by R3/1 cells while targeting RAGE by siRNA. Cell culture media was removed at various time points after the onset of CSE exposure and screened by ELISA. IL-1b significantly increased after just 2 hours of CSE exposure, and levels were constant when exposure persisted for 12 hours (Figure 5A). A significant increase in CCL5 secretion was also observed after 2 hours of CSE exposure, and the levels of CCL5 were unchanged through 12 hours (Figure 5B). Based on the immediacy of IL-1b

TABLE 1. PERCENTAGE OF CELLS WITH COMPARTMENTALIZED NF-kB LOCALIZATION

% Cytoplasmic % Nuclear

siControl

siControl 1 CSE

siRAGE

siRAGE 1 CSE

88.5 6 7.4 14.7 6 3.9

36.2 6 5.7 68.2 6 4.5

82.9 6 5.6 21.8 6 6.1

54.7 6 4.2* 48.3 6 6.4*

Definition of abbreviations: CSE 5 cigarette smoke extract; siControl 5 control siRNA; siRAGE 5 cells treated with siRNA receptor for advanced glycation end products. * P , 0.05 compared with siControl 1 CSE.

and CCL5 secretion during CSE exposure (Figures 5A and 5B), cells were transfected with siRAGE or siControl and exposed to CSE for 2 hours before cytokine quantification. In both instances, siRAGE was sufficient to significantly reduce CSEinduced IL-1b and CCL5 secretion (Figures 5C and 5D). Cytokine secretion was also significantly decreased in R3/1 cells that incorporated a Ras-DN before CSE stimulation (Figures 5C and 5D). Just as acute CSE exposure to cells with diminished RAGE expression resulted in significantly attenuated IL-1b and CCR5 secretion, chronic smoke exposure of RAGE null mice resulted in significantly reduced concentrations of IL-1b and CCL5 when compared with cigarette smoke–exposed wild-type mice (Figure 6). However, RAGE targeting did not completely prevent cigarette smoke–induced CCL5 mRNA or protein expression when compared with RAGE null mice exposed to room air (Figures 6C and 6D).

DISCUSSION CSE-Induced RAGE Expression and Ras Activation

We previously demonstrated that the expression of RAGE, a multiligand pattern recognition receptor, is increased in pulmonary epithelial cells after exposure to CSE (6). Simultaneously conducted studies revealed that RAGE ligands were also elevated by CSE exposure, suggesting that epithelial cells respond acutely to CSE by stimulating molecules required in the initial stages of RAGE signaling. The current investigation adds significantly to the understanding of RAGE biology in the context of tobacco smoke exposure by seeking to understand likely events involved downstream of RAGE–ligand interaction. Our discovery that Ras is activated in epithelial cells exposed to CSE led to the hypothesis that RAGE signals through Ras-mediated mechanisms during a cell’s acute response to tobacco smoke. Through the use of siRNA targeting, we discovered that Ras activity is directly influenced by RAGE. A link between RAGE and Ras in signaling pathways is supported by the work of Lander and colleagues, which reveals AGE-induced activation

Reynolds, Kasteler, Schmitt, et al.: RAGE/Ras Signaling Is Induced by Tobacco Smoke

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Figure 5. RAGE/Ras signaling mediated CSE-induced proinflammatory cytokine secretion. (A) R3/1 cells exposed to 25% CSE revealed significant increases in IL-1b secretion in cell culture media after 2, 6, and 12 hours of exposure. (B) CCL5 was also significantly up-regulated by R3/1 cells exposed to CSE. (C ) CSE-induced IL-1b expression was significantly decreased by siRAGE or Ras-DN transfection 24 hours after CSE exposure. (D) siRAGE and Ras-DN significantly decreased CCL5 elaboration by R3/1 cells exposed to CSE. *P < 0.05.

of p21(ras) and subsequent downstream regulation of mitogenactivated protein kinase (20). Other groups have also attempted to clarify the potential roles for RAGE and its ligands in Rasrelated processes associated with neurite outgrowth and the inflammatory profile of neuroblastoma cells and microglia (21, 22). Our research expands current RAGE understanding by demonstrating a role for the receptor in Ras-mediated signaling in pulmonary cells exposed to cigarette smoke. Although the data in Figures 2 and 3 support the likelihood that additional pathways are involved in the coordination of Ras-mediated responses to cigarette smoke, it is evident that RAGE is important in such responses. Other studies suggest a high probability that RAGE is induced by pulmonary epithelial cells and that newly abundant RAGE interacts with elevated ligand availability, most notably AGEs prominently detected in tobacco smoke (23, 24). Therefore, the current data link RAGE–ligand interactions with the activation of Ras to perpetuate signaling cascades induced by tobacco smoke. RAGE, Ras, and Carcinogenesis

Elevated Ras activity has been identified as a characteristic of pulmonary carcinogenesis (25). Cigarette smoking causes over 90% of all lung cancers, and there is epidemiologic evidence that chronic inflammation influences lung epithelial carcinogenesis (26). Furthermore, isoforms and assorted mutations of p21(ras) have been implicated as biomarkers for smoking-related chronic obstructive pulmonary disease (COPD) and various stages of cancer in patients with COPD (27, 28). Accordingly, it is possible that cigarette smoke exposure elicits chronic inflammation, orchestrated in part by RAGE and mediated by activated Ras, which leads to lung cancer. Although there is evidence that RAGE expression is downregulated in select types of lung cancer (29), the current data suggest that RAGE may function in Ras-mediated inflammation as part of an early trigger in the progression of carcino-

genesis. Supporting this notion are data demonstrating that RAGE null mice exhibit severe defects in sustaining inflammation during the promotion stage of carcinogenesis, which leads to resistance to epithelial malignancies (30). Furthermore, because RAGE ligands, such as HMGB-1 and AGEs, are upregulated in tumors (31), endogenous RAGE ligands may combine with constituents in tobacco smoke to promote a proinflammatory microenvironment preceding tumorigenesis. Complicating the several proposed mechanisms involving RAGE is the fact that different RAGE isoforms have been identified (32), perhaps influencing the variable susceptibilities to inflammatory diseases regardless of the quantity or form of smoke inhaled. Therefore, it is plausible that RAGE/Ras signaling combines with other pathways and contributes to early proinflammatory events associated with tobacco exposure and that these pathways influence advanced stages of disease. NF-kB Activation and Inflammatory Cytokine Secretion Mediated by RAGE during Tobacco Exposure

In cells with differentially regulated RAGE expression, NF-kB was assessed after CSE exposure so that contributions by RAGE/Ras signaling to NF-kB–mediated inflammation could be evaluated. NF-kB was initially identified as a transcription factor in B cells and has since been detected in the cytoplasm of all cell types (33). When stimulated, NF-kB translocates to the nucleus where it regulates the expression of more than 200 genes that influence cell growth, survival, and inflammation (34). Because NF-kB activation during inflammation has been associated with the initiation and progression of several human cancers (35, 36), it is commonly considered a molecular link between inflammation and carcinogenesis (36, 37). Our in vitro findings revealing that RAGE influences NF-kB activation and proinflammatory cytokine elaboration support the concept that tobacco smoke–induced inflammation involves the activation of cancer-causing molecular pathways, including NF-kB (38, 39).

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Figure 7. RAGE/Ras/NF-kB signaling in pulmonary epithelial cells exposed to tobacco smoke. The data support the model that RAGE functions in tobacco smoke–induced inflammation involving Ras activation and subsequent NF-kB–mediated proinflammatory cytokine secretion. It is also clear that other pathways compliment RAGE-Ras– NF-kB signaling during the coordination of an inflammatory response elicited by tobacco smoke exposure. Figure 6. Proinflammatory cytokines were decreased in RAGE null mice exposed to tobacco smoke compared with smoke-exposed wildtype mice. Total concentrations of cytokines were detected by real-time RT-PCR and ELISA in whole lung lysates. IL-1b and CCL5 mRNA (A, C ) and protein (B, D) expression were significantly decreased in RAGE null mice exposed to 6 months of tobacco smoke when compared with wild-type smoke-exposed mice. ns 5 no smoke exposure. n 5 6 per group. *P < 0.05.

Our data identify the likelihood of other factors induced by CSE in pathways that lead to NF-kB activation (Figure 4B). Because a highly significant decrease in NF-kB activity was achieved when RAGE and Ras were both targeted, it appears that Ras may be a juncture through which RAGE and other parallel pathways involving MAP kinase converge. The current investigation identified significant decreases in the acute secretion of COPD-related proinflammatory molecules, such as IL-1b and CCL5, by cells with diminished RAGE expression and in lungs from RAGE null mice exposed to chronic tobacco smoke. IL-1b is an early proinflammatory cytokine that induces the release of many additional cytokines, enhances leukocytosis, and increases MMP expression during an inflammatory response (40). Produced by a multitude of cell types (41), IL-1b increases the expression of endothelial adhesion molecules necessary for the transmigration of leukocytes (42). CCL5 is a small CC family cytokine expressed by many cell types, including respiratory epithelium, which functions as a monocyte chemoattractant (43). CCL5 promotes leukocyte diapedesis in the lungs of patients with rhinosinusitis or COPD exacerbated by tobacco smoke (44–46). IL-1b and CCL5 are elevated in induced sputum and BAL from smokers with COPD when compared with nonsmokers and are recognized as biomarkers of COPD pathogenesis (47–54). Additional inflammatory mediators may also lead to the amplification of the initial trigger commenced by IL-1b, CCL5, and other early modula-

tors. Despite observations that pathways in addition to RAGE influence CCL5 mRNA and protein expression, it is possible that diminished secretion of inflammatory molecules in RAGE null mice prevent efficient feed-forward signals that create proinflammatory foci (30, 55, 56). Conclusions

The current investigation provides support for a working model wherein RAGE-mediated signal transduction pathways influence cigarette smoke–induced inflammation. Such a pathway exists in parallel with other pathways and may involve AGEs present in tobacco smoke (23, 24) that combine with smokeinduced RAGE (6), receptor/ligand interactions that cause the activation of Ras, and NF-kB–mediated cytokine secretion (Figure 7). As such, our findings have important implications for elucidating the mechanism of RAGE-mediated inflammation via the involvement of intracellular Ras activation. Additional research into the important role of RAGE signaling and the resulting inflammatory response may lead to strategies for blocking this proinflammatory axis in individuals exposed to voluntary or involuntary tobacco smoke. Author Disclosure: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgments: The authors thank Dr. Manuel Cosio and Kevin Whittaker (McGill University, Montreal, Canada) for kindly providing the mouse lung samples after cigarette smoke exposure and Anne Sturrock (University of Utah Health Sciences Center) for offering valuable advice.

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