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Published ahead of print on April 17, 2008, doi:10.1165/rcmb.2007-0244OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 337-345, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0244OC

Loss of RAGE in Pulmonary Fibrosis

Molecular Relations to Functional Changes in Pulmonary Cell Types

Markus A. Queisser1, Fotini M. Kouri2, Melanie Königshoff2, Malgorzata Wygrecka1, Uwe Schubert1, Oliver Eickelberg2 and Klaus T. Preissner1

Departments of 1 Biochemistry and 2 Medicine II, University of Giessen Lung Center, Justus-Liebig-University, Giessen, Germany

Correspondence and requests for reprints should be addressed to Klaus T. Preissner, PhD, Department of Biochemistry, University of Giessen Lung Center, Justus-Liebig-University, Friedrichstr. 24, 35392 Giessen, Germany. E-mail: klaus.t.preissner{at}biochemie.med.uni-giessen.de


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The receptor for advanced glycation end products (RAGE) is a transmembrane receptor of the Ig superfamily. While vascular RAGE expression is associated with kidney and liver fibrosis, high expression levels of RAGE are found under physiological conditions in the lung. In this study, RAGE expression in idiopathic pulmonary fibrosis was assessed, and the relationship of the receptor to functional changes of epithelial cells and pulmonary fibroblasts in the pathogenesis of the disease was investigated. Significant down-regulation of RAGE was observed in lung homogenate and alveolar epithelial type II cells from patients with idiopathic pulmonary fibrosis, as well as in bleomycin-treated mice, demonstrated by RT-PCR, Western blotting, and immunohistochemistry. In vitro, RAGE down-regulation was provoked by stimulation of primary human lung fibroblasts and A549 epithelial cells with the proinflammatory cytokines, transforming growth factor-β1 or TNF-{alpha}. Blockade of RAGE resulted in impaired cell adhesion, and small interfering RNA–induced knockdown of RAGE increased cell proliferation and migration of A549 cells and human primary fibroblast in vitro. These results indicate that RAGE serves a protective role in the lung, and that loss of the receptor is related to functional changes of pulmonary cell types, with the consequences of fibrotic disease.

Key Words: receptor for advanced glycation end products • pulmonary fibrosis • idiopathic pulmonary fibrosis • adhesion molecule • alveolar epithelial cells



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Our study provides evidence that the expression and regulation of receptor for advanced glycation end products (RAGE) in the pulmonary system differs from that in the vascular system. Here, a possible functional mechanism of RAGE in pulmonary fibrosis is described for the first time.

 
Idiopathic pulmonary fibrosis (IPF) is a progressive, degenerative disease of unknown etiology, for which no effective treatment exists. IPF is characterized histologically by unrestricted fibroblast proliferation, formation of fibroblast/myofibroblast foci, and excessive deposition of extracellular matrix (ECM) associated with abnormal wound healing (1–3).

Recently, the involvement of the receptor for advanced glycation end products (RAGE) in renal fibrosis during diabetes, as well as in liver fibrosis, was demonstrated (4, 5). In particular, although the kidney is dramatically affected by microangiopathy and fibrosis in patients with diabetes, the lung, which exhibits significantly higher baseline RAGE expression than the kidney, remains unaffected. The underlying mechanisms to explain such differences are not known.

RAGE is a member of the Ig superfamily, and is expressed as single-chain transmembrane receptor in several cell types, including neurons, endothelial and smooth muscle cells, and mononuclear cells (6). It recognizes a variety of ligands, including advanced glycation end products (AGE), amyloid β-peptides, amphoterin (HMGB-1), and members of the S100/calgranuline family (7). The ligand–RAGE interaction leads to the activation of several intracellular signaling cascades, including the mitogen-activated protein kinase pathway to the production of reactive oxygen species, and further activation of nuclear factor-{kappa}B (8, 9). Several studies have suggested that the activation of different pathways is ligand and cell type dependent (10). Apart from the full-length receptor, truncated, soluble RAGE (sRAGE), which lacks the transmembrane and cytoplasmatic regions, serves as a decoy receptor, and thereby inhibits several intracellular signaling pathways. Furthermore, RAGE can interact with ECM proteins (11) and function as a counter-receptor for the leukocyte adhesion molecule, Mac-1 (CD11b/CD18) (12).

Due to its involvement in inflammatory reactions, tissue fibrosis, and myoblast and tumor formation (4, 5), we hypothesized that RAGE expression in the lung, in contrast to blood vessels, could be related to protection of the pulmonary system against degenerative processes, such as IPF pathogenesis.

In this study, we demonstrated that the expression of RAGE was significantly decreased in the lungs as well as in alveolar epithelial type (AT) II cells of patients with IPF. Similar observations were made in bleomycin-induced lung fibrosis, a commonly employed mouse model for IPF. In vitro experiments revealed that RAGE acts in an adhesion-related manner, and its knockdown resulted in increased cellular proliferation and migration, suggesting an important protective function for RAGE in the lung.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
All materials were purchased from Sigma (St. Louis, MO) unless otherwise stated. Recombinant soluble mouse RAGE was expressed and purified as described previously (13). RAGE antibodies were purchased from Biologo (Kronshagen, Germany) and Affinity BioReagents (Golden, CO). Hepatocyte growth factor, TNF-{alpha}, transforming growth factor-β1 (TGF-β1), and IL-1β were purchased from R&D Systems (Minneapolis, MN), and keratinocyte growth factor (KGF) was purchased from Biomol (Hamburg, Germany). The monoclonal anti–β1-integrin antibody, P4C10, was purchased from Millipore (Billerica, MA).

Patient Population
Lung tissue was obtained from six subjects with IPF and six donor lungs rejected for transplantation (mean age, 45.6 ± 15.7 yr; three females/three males). The diagnosis of IPF was made in accordance with American Thoracic Society–European Respiratory Society criteria (14). All patients exhibited the typical usual interstitial pneumonia pattern (mean age, 52.4 ± 11.8 yr; two females/four males). The study protocol was approved by the ethics committee of the Justus-Liebig-University School of Medicine. Informed consent was obtained from each subject per the study protocol.

Animal Treatment
C57BL/6J mice were purchased from the Jackson Laboratory (Bar Habor, ME) and used for bleomycin challenge to induce pulmonary fibrosis. Bleomycin sulfate (Almirall Prodesfarma, S.A., Barcelona, Spain) was dissolved in sterile saline and applied by microspray as a single dose of 0.08 mg/mouse in a total volume of 200 µl. Control mice received 200 µl saline. Mice were killed at Days 7, 14, and 21 after bleomycin exposure. All experiments were performed in accordance with the guidelines of the ethics committee of the University of Giessen School of Medicine, and approved by local and national authorities.

Isolation and Culture of Human ATII Cells
Human ATII cells were isolated, as previously described (15), including tissue digestion and magnetic bead isolation. Briefly, the lung was digested and minced. The cell-rich fraction was filtered, layered onto a Percoll density gradient, and centrifuged. The cells were then incubated with magnetic beads coated with anti-CD14 antibodies. The remaining cell suspension was incubated in human IgG-coated tissue culture–treated dishes in a humidified incubator. The purity of isolated human ATII cells was examined by Papanicolaou staining. The purity and viability of ATII cell preparations was consistently between 90 and 95%.

Isolation and Culture of Human Pulmonary Fibroblasts
Fibroblasts were isolated from human donor lungs, as described previously (16). Briefly, the lungs were perfused via pulmonary artery and lavaged. Lung tissue was dissected from the airways, minced into 2-mm3 pieces, and placed in tissue culture flasks in a humidified incubator at 37°C under 5% CO2 atmosphere with a minimal volume of Dulbecco's modified Eagle medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. An appropriate volume of medium was then added to the flask, and the cells were maintained until fibroblasts began to migrate out from the tissue.

Identification of fibroblasts was based on the morphology and presence of vimentin staining. Passages 2 to 5 were used for experiments.

Cytokine Stimulation
Cells were cultured in Dulbecco's modified Eagle medium containing 0.5% (vol/vol) FBS for 24 and 48 hours in the absence or presence of TNF-{alpha} (10 ng/ml) or TGF-β1 (5 ng/ml).

Immunohistochemistry
IPF and donor lung sections were stained for RAGE (goat anti-RAGE; Biologo) using Histostain Plus kit (Zymed Laboratories, San Francisco, CA), as recommended by the manufacturer.

Immunofluorescence
Cells were fixed with methanol for 5 minutes at –20°C, blocked with 5% (vol/vol) FBS in PBS, and stained with goat anti-RAGE antibody (Biologo) and a rhodamine-conjugated anti-goat secondary antibody (Jackson ImmunoResearch, West Grove, PA) in 2.5% (vol/vol) FBS in PBS.

Small Interfering RNA Knockdown
Cells were seeded and cultured in starvation medium (FBS free) for 4 hours before transfection. Cells were transfected using the transfection reagent, TransPass R1, purchased from New England Biolabs (Ipswich, MA) with scrambled small interfering RNA (siRNA) from Santa Cruz Biotechnology (Santa Cruz, CA) or siRNA SMARTpool purchased from Dharmacon (Lafayette, CO), with the following antisenses: 5'-ttccattcctgttcattgctt-3'; 5'-tactgctccaccttctggctt-3'; 5'-tgttccttcacagatactctt-3' and 5'-tttgaggagagggctgggctt-3'. The cells were transfected with 150 nM siRNA and incubated for 24 or 48 h.

RT-PCR
Total RNA was extracted from lung tissue and cells using the GenElute mammalian total RNA kit, following the manufacturer's instructions. A 1-µg sample of total RNA was used for each reverse transcription reaction. ImProm-II reverse transcriptase, random primers, RNasin RNase inhibitor (all from Promega, Madison, WI), and deoxyribonucleotide triphosphates (Finnzymes, Espoo, Finland) were used per the manufacturer's instructions.

Real-Time PCR
Expression levels of RAGE–messenger RNA (mRNA) transcripts from human lungs were quantified by real-time PCR. cDNAs were mixed with SYBR Green PCR master mix and primers (Invitrogen, Carlsbad, CA) (Table 1), and real-time PCR was performed using the Sequence Detection System 7500 (Applied Biosystems, Wellesley, ME). In addition to profiling all samples for the target sequence, samples were profiled for hydroxymethylbilane synthase expression as reference. For each single-well amplification reaction, a threshold cycle (Ct) was observed in the exponential phase of amplification, and the quantification of relative expression levels was achieved using standard curves for both the target and endogenous control samples. Relative transcript abundance of a gene is expressed in {Delta}Ct values ({Delta}Ct = Ctreference – Cttarget).


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  		TABLE 1. PRIMER SEQUENCES AND AMPLICON SIZES

 
Western Blot
Protein extraction from lung tissue samples was performed as described previously (17). Protein concentration was determined by the BCA Protein Assay Reagent kit (Pierce, Rockford, IL). For Western blotting, 20 µg of total lysate was resolved on a 10% SDS-PAGE gel and blotted onto a polyvinylidene fluoride membrane. A polyclonal goat anti-RAGE antibody (Biologo) was used for RAGE detection. The membrane was stripped and reprobed with an anti–β-actin antibody clone, AC-74.

ECM Preparation
Adherent fibroblast cells were washed three times with PBS containing 2% (wt/vol) BSA and 0.1 mM CaCl2, followed by incubation with 0.5% (vol/vol) Triton-X-100 in PBS for 15 minutes at 37°C. Plates were then washed with PBS containing 0.1 M NH4Cl to remove the cells. Cell-free ECM was blocked with PBS containing 3% (wt/vol) BSA for 30 minutes at room temperature.

Adhesion Assay
Cell adhesion to ECM, collagen (2 µg/ml) or BSA (as control) was tested, as described previously (18), in the absence or presence of an anti-RAGE antibody (5 µg/ml), control IgG, anti–β1-integrin antibody (10 µg/ml), or sRAGE (10 µg/ml). Adherent cells were fixed and stained with violet blue and quantified by absorbance at 405 nm.

Proliferation Assay
Cell proliferation was determined by cell counting using the CASY Cell Counter System (Model DT; Schaerfe Systems, Reutlingen, Germany). Cells were transfected with 150 nM siRNA under starvation conditions for 4 hours and cultured for a further 48 hours before assessing proliferation. KGF (10 ng/ml) and TGF-β1 (10 ng/ml) were used as positive controls for A549 and fibroblast cell proliferation, respectively.

Migration (Chemotaxis) Assay
The migration of cells was analyzed using a Boyden chamber (Neuro Probe, Gaithersburg, MD) as previously described (19). Cells were allowed to migrate toward different chemotactic stimuli, including hepatocyte growth factor (10 ng/ml) and TGF-β1 (10 ng/ml), or 5% FBS, and the extent of migration was measured by densitometric image analysis with Quantity One software (Bio-Rad Laboratories, Hercules, CA) and expressed as optical density per squeare millimeter.

Wound Healing Assay
Wound healing assay was performed as previously described (19). Briefly, cells were seeded overnight in Lab-Tek chamber wells (Nalge Nunc, Rochester, NY) and transfected 48 hours before scratching. Each coverslip was then scratched with a sterile 200-µl pipette tip, washed with PBS, and placed into fresh medium with 5% FBS. After 24 hours, cells were fixed with 4% paraformaldehyde and cell nuclei were stained with 4',6'-diamidino-2-phenylindole. Pictures were captured by fluorescent microscopy under a x10 objective on a Leica DMR microscope (Leica Microsystems, Bensheim, Germany) at 0 and 24 hours after scratching, and the number of cells that had migrated into the same-sized square fields (marked in Figure 7) were counted with Methamorph software 7.0 (Molecular Devices, Downingtown, PA).

Statistical Analysis
All data were expressed as mean ± SD (n ≥ 3) unless otherwise indicated. Experimental conditions were compared by using Student's t test for single measurements or multiple comparisons were analyzed using ANOVA. Differences were considered significant at P < 0.05. All {Delta}Ct values obtained from quantitative RT-PCR were analyzed for normal distribution using the Shapiro-Wilk test. Data were assumed to be normally distributed when P > 0.05.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Differential Expression of RAGE in Mouse Tissue
The expression and distribution of RAGE was analyzed in different mouse organs. RAGE was abundantly expressed in the lung, in comparison with other organs, such as the brain or heart, where substantially lower levels of the protein were detected (Figure 1A). The anti-RAGE antibody detected multiple bands of different molecular mass in the lung, which resulted from post-translational modifications of RAGE (20).


Figure 1
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  		Figure 1. Abundant receptor for advanced glycation end products (RAGE) expression in the lung. RAGE expression was appreciably high in the lung and localized to the epithelium. (A) Mouse organ homogenates were prepared and analyzed by Western blot analysis. RAGE exhibited a tissue-specific expression pattern, and was highly expressed in the lung. Three variants were detected (55, 50, and 45 kD). (B) Human lung sections were stained for RAGE (red) and counterstained with H&E (blue). RAGE was localized to the alveolar and bronchial epithelium, as well as to fibroblasts. In comparison to donor lungs, idiopathic pulmonary fibrosis (IPF) lungs exhibited a weak RAGE staining in alveolar epithelium, as well as in fibroblasts. Magnification: top panel, x20; middle panel, x40; bottom panel, x63.

 
Distribution of RAGE in Donor and IPF Lung Tissue
In donor lung tissue, RAGE was localized to bronchial epithelial cells, alveolar epithelial cells, and pulmonary fibroblasts. In IPF lung sections, RAGE staining was clearly weaker in the bronchial and alveolar epithelium, as well as in pulmonary fibroblasts (Figure 1B).

RAGE Expression in Donor, IPF Lungs, Alveolar Type II Cells, and Fibroblasts
RAGE expression at the mRNA and protein level was investigated in IPF (n = 6) and donor lung samples (n = 6). Although the quantitative PCR amplified RAGE transcript in a high amount in all donor samples, it was highly down-regulated in the IPF lung homogenates (Figure 2A). Furthermore, down-regulation of RAGE was detected at the protein level in IPF lung homogenates, as shown by Western blotting. Two major isoforms (55 kD and 45 kD) were identified in donor lung homogenates. In contrast, little RAGE was discernable in IPF lung extracts (Figure 2B).


Figure 2
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  		Figure 2. RAGE down-regulation in IPF. (A) RAGE expression was analyzed by real-time PCR in human donor (n = 6) and IPF (n = 6) lung homogenates. The RAGE transcript was largely down-regulated in IPF lung homogenates. (B) Proteins from lung homogenates were resolved by SDS-PAGE and analyzed by Western blotting for RAGE detection. RAGE was hardly discernable in all samples from patients with IPF. Two bands of 55 and 45 kD were detected in donor lung homogenates. (C) RAGE expression was evaluated in isolated ATII cells derived from donor and IPF lungs. RAGE mRNA expression was significantly decreased in IPF isolated ATII cells. (D) RAGE expression in isolated pulmonary fibroblasts from donor and IPF lungs did not show any significant differences. Data represent mean ± SD from at least three separate experiments; *P ≤ 0.01.

 
Moreover, there was a significant down-regulation of RAGE mRNA levels in IPF-derived ATII cells (n = 4) as compared with donor ATII cells (n = 2) (Figure 2C). No significant changes were observed between IPF- (n = 3) and donor (n = 3) -isolated fibroblasts (Figure 2D).

RAGE Expression in the Bleomycin Mouse Model of Lung Fibrosis
To study possible mechanistic relationships between pulmonary fibrosis and the down-regulation or loss of RAGE, an established mouse model was employed in which pulmonary fibrosis was provoked by bleomycin inhalation. In bleomycin-treated mice, no significant decrease in RAGE expression was noted at the mRNA level (Figure 3A). In contrast, at the protein level, RAGE was significantly down-regulated in bleomycin-treated mice, exemplified by the appearance of very weak protein bands upon Western blotting (Figure 3B). These data are consistent with the observations made on RAGE protein expression in the lungs of patients with IPF. In contrast to the human studies, an additional 50 kD RAGE variant was detected.


Figure 3
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  		Figure 3. RAGE down-regulation in the bleomycin model. Lung homogenates from saline- and bleomycin-treated mice were analyzed for RAGE expression at the mRNA and protein levels. (A) RNA samples from saline- and bleomycin-treated mice (n = 3) were subjected to RT-PCR. The RAGE mRNA was amplified in all samples, independent of the time period of bleomycin exposure. No significant changes at the mRNA level were observed after bleomycin treatment. (B) RAGE was significantly down-regulated at the protein level in the bleomycin-treated mice (n = 3) in comparison with saline-treated mice, as demonstrated by Western blotting.

 
Influence of Cytokines on RAGE Expression
Because RAGE expression is controlled by cytokines (21), the influence of TNF-{alpha} and TGF-β1 at RAGE mRNA and protein levels was tested in an alveolar epithelial cell line, A549, and primary human pulmonary fibroblasts after 24- and 48-hour stimulation. RAGE expression was significantly altered at the mRNA and protein level by both TNF-{alpha} and TGF-β1 in A549 cells after 48 hours, as analyzed by quantitative PCR and Western blotting (Figures 4A and 4B). In pulmonary fibroblasts, RAGE down-regulation at the mRNA level was provoked by TNF-{alpha}, and, to a lesser extent, by TGF-β1 after 24 hours stimulation (Figure 4C). RAGE expression was decreased at the protein level after 48 hours by both cytokines (Figure 4D), whereby full-length RAGE was mainly affected.


Figure 4
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  		Figure 4. Cytokine-dependent RAGE down-regulation in A549 cells and pulmonary fibroblasts. The influence of cytokines on RAGE expression was tested in the alveolar epithelial cell line, A549, as well as in primary human fibroblasts. RAGE expression was analyzed by immunofluorescence and Western blot analysis after 24- and 48-hour stimulation with different cytokines. (A) Cytokine-treated A549 cells exhibited no changes after treatment with TGF-β1 or TNF-{alpha} after 48 hours. (B) Cytokine-treated fibroblasts were analyzed after 24 hours. After stimulation with TNF-{alpha} and TGF-β1, the expression of RAGE was decreased. (C) TGF-β1 and TNF-{alpha} exposure significantly decreased RAGE expression after 48 hours. (D) Data represent means ± SD from at least three separate experiments; *P ≤ 0.05.

 
Relation between RAGE and Cell Adhesion, Migration, and Proliferation
To further elucidate the mechanism by which a decrease or loss of RAGE would affect cellular functions, we studied cell adhesion, migration, and proliferation of A549 cells and pulmonary fibroblasts. To examine the role of RAGE–collagen interaction, an adhesion assay with collagen was performed. Blockade of RAGE inhibited cell adhesion on collagen (Figure 5A) and on intact ECM (Figure 5B), suggesting a potential role of RAGE in cell–matrix adhesion. The inhibitory effect of anti-RAGE antibody was diminished by preincubation with sRAGE. In contrast, control IgG and sRAGE had no influence on cell adhesion, indicating that the full-length RAGE is essential for its adhesive function. In addition, knockdown of RAGE by specific siRNA led to increased proliferation of epithelial cells (A549) and fibroblasts, as demonstrated by cell counting (Figures 6B and 6C).


Figure 5
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  		Figure 5. Impairment of cell adhesion on collagen and ECM by RAGE blocking A549. Epithelial cells and primary fibroblasts were incubated with a blocking anti-RAGE antibody and assessed for adhesion to different adhesive substrates. (A and B) The adhesion assay was performed on collagen- and ECM-coated plates. Cells treated with the anti-RAGE antibody exhibited significantly decreased adhesion in comparison with control cells. Control IgG and sRAGE had no significant influence on the adhesion. Impaired adhesion was restored by neutralization of anti-RAGE by sRAGE. As negative control, cells were plated on BSA-coated plates. Data represent means ± SD from at least three separate experiments; *P ≤ 0.05; filled bars, A549; open bars, fibroblasts.

 

Figure 6
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  		Figure 6. Increased cell proliferation and migration due to siRNA-mediated RAGE knockdown. A549 cells and primary human pulmonary fibroblasts were transfected with specific RAGE siRNA and assessed for cell proliferation and migration. (A) Western blot analysis demonstrated RAGE siRNA knockdown on the protein level in A549 cells and pulmonary fibroblasts. (B) A549 cells transfected with RAGE siRNA exhibited an increased proliferation rate in comparison with scrambled control siRNA. KGF was used as a positive control. (C) Pulmonary fibroblasts transfected with RAGE siRNA exhibited an increased proliferation rate in comparison with scrambled control siRNA. TGF-β1 was used as a positive control. (D) Transfected A549 cells and pulmonary fibroblasts were assessed for chemotactic migration. In more detail, RAGE knockdown with specific siRNA induced a migratory effect as compared with scrambled siRNA in both A549 cells and pulmonary fibroblasts. Data represent means ± SD from at least three separate experiments; *P ≤ 0.05; filled bars, A549; open bars, fibroblasts.

 
KGF and TGF-β1 served as positive controls for cell proliferation of A549 and pulmonary fibroblasts, respectively. RAGE knockdown resulted in increased migration of fibroblasts and epithelial cells (A549), as shown by chemotaxis migration assay (Figure 6D). Hepatocyte growth factor (for epithelial cells) and TGF-β1 (for fibroblast) served as positive controls. Undirected chemokinesis was tested by a scratch wound healing assay with siRNA-transfected A549 cells and pulmonary fibroblasts. Cells transfected with RAGE-specific siRNA migrated faster, inducing closure of the scratch, in comparison with scrambled siRNA-transfected cells after 24 hours (Figure 7).


Figure 7
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  		Figure 7. RAGE knockdown induced wound closure. A549 cells and primary human pulmonary fibroblasts were transfected with RAGE-specific siRNA and assessed for wound healing assay. (A) A549 cells transfected with RAGE siRNA exhibited increased migration and wound closure in comparison with scrambled siRNA-transfected cells. (B) Pulmonary fibroblasts exhibited increased migration and wound closure in comparison with scrambled siRNA-transfected cells. Data represent means ± SD from at least three separate experiments. Scale bar = 100 µm; *P ≤ 0.05.

 
siRNA knockdown of RAGE in A549 cells affected mainly cell proliferation, whereas RAGE knockdown in pulmonary fibroblasts resulted in both elevated cell migration and proliferation, but the latter to a lesser extent.

These data indicate that RAGE is an important component, related to cell adhesion, migration, and proliferation of alveolar epithelial cells and pulmonary fibroblasts.


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the present report, the relationship between RAGE down-regulation in fibrotic lungs and the loss of control of cell adhesion, migration, and proliferation (alterations believed to be involved in fibrosis development) was described. These data provide new mechanistic insight into the regulatory role of RAGE in cell communication in the lung, and are supported by recent findings of other investigators (22, 23). Decreased RAGE expression in diseased lungs of patients with IPF and in ATII cells in vitro are in accordance with the observed alterations in the animal model of bleomycin-induced lung fibrosis (22).

As demonstrated in this study, down-regulation and/or loss of RAGE expression by TNF-{alpha} and TGF-β1 in primary human pulmonary fibroblasts and A549 cells, as well as its involvement in cell adhesion, appear to be processes linked to the onset and/or progression of fibrosis pathogenesis. Here, the profibrotic cytokine, TNF-{alpha}, plays a critical role, possibly driving the inflammatory phase into fibrosis (24, 25). Another hallmark in the pathogenesis of pulmonary fibrosis is the alterations in cellular phenotype and functions, accompanied by changes in cell adhesion and communication, of lung epithelial cells. Although it remains to be further established whether RAGE is a specific marker for ATI or ATII cells (26, 27), our results demonstrate RAGE protein localization mainly on ATI cells, but also on ATII cells, which contain high amounts of RAGE mRNA. Whether these differences are related in any way to the transition of cells from ATII to ATI remains to be elucidated.

RAGE knockdown in ATII-like A549 cells, as well as pulmonary fibroblasts, resulted in elevated proproliferative responsiveness to serum and increased cellular motility. Interestingly, RAGE knockdown had a more proliferative effect on A549 cells and a higher migratory effect on pulmonary fibroblasts, indicative of a cell type–specific role of RAGE as well. Moreover, the cell phenotype changes provoked by RAGE down-regulation in lung fibroblasts are reminiscent of morphological alterations of these cells in IPF, demonstrating increased proliferation and migration in comparison with healthy donor-derived fibroblasts (28–30). Together, RAGE down-regulation appears to be associated with both cytokine and adhesion-related cellular changes, and may thus be mechanistically linked to the switch of chronic inflammation to fibrosis in fibrotic lung disease.

The adhesive properties of RAGE in mediating cell–matrix contacts described here and in previous reports (11, 27) appear to be similar and comparable to integrin-mediated adhesion of lung epithelial cells, and loss of RAGE is associated with disturbed cellular contacts. Moreover, preliminary data from our laboratory indicate a tight linkage of RAGE to cytoskeletal elements in lung epithelial cells (M. A. Queisser and colleagues, unpublished data), suggesting that RAGE provides a regulatory adhesion function linked to cytoskeleton-related signaling systems, also characteristic for integrin functions. It may thus be hypothesized that signaling pathways that would lead to inside-out signaling to affect RAGE function may not be unlikely. Because RAGE was described by our group as the major inflammation-related counter-receptor on endothelial cells for recognition of β2-integrins on leukocytes (12), it remains to be investigated whether loss of RAGE on the lung epithelium may lead to disturbances in inflammatory cell interactions in the lung as well. Although the clarification of the cell-stabilizing role of RAGE in the lung requires further work, RAGE appears to serve an "opposite" role in the vasculature, where it becomes up-regulated upon inflammatory processes and promotes, for example, leukocyte recruitment into diseased tissue (12).

Although the bleomycin model of lung fibrosis used in this study may not necessarily reflect all alterations of fibrosis pathogenesis as observed in humans, data from this in vivo model are in accordance with our in vitro and ex vivo data. A major loss of RAGE expression was seen in these mice, which is supported by recent findings from Englert and colleagues (23), who showed that RAGE–/– mice developed more severe asbestos-induced lung fibrosis than wild-type control mice, and underlined our data using a different animal model for lung fibrosis. In contrast, He and colleagues (31) reported that RAGE–/– mice were protected from bleomycin-induced lung fibrosis. Furthermore, it has been shown that RAGE levels are decreased in the alveolar epithelium after in vitro treatment of rat lung sections with CdCl2 and TGF-β1 (32).

Our observations agree with, and further complement, the findings that RAGE is down-regulated in non–small cell lung carcinoma, and its expression impairs the proliferative stimulus of lung fibroblasts on lung cancer cells (33, 34). Thus, loss of RAGE leads to increased cellular proliferation and migration of pulmonary cells in association with different pathologies, and (therapeutic) prevention of RAGE down-regulation may serve as a potential antagonizing mechanism in the diseased lung.


   Acknowledgments
 
The authors thank Sven Geisler and Dr. Karin Hersemeyer (Department of Biochemistry, Justus-Liebig-University Giessen, Germany) for assistance in bioanalysis and microscopy, Dr. Rory Morty (Department of Internal Medicine, JLU Giessen) for helpful comments and critical evaluation of the manuscript, and Dr. Walter Klepetko (Department of Cardiothoracic Surgery, University of Vienna, Vienna, Austria), for kindly providing the lung samples for this study.


   Footnotes
 
This work was supported by the Deutsche Forschungsgemeinschaft, Graduate Program 1062 "Signaling Mechanisms in Lung Physiology and Disease", and the SFB 547.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0244OC on April 17, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form June 27, 2007

Accepted in final form February 20, 2008


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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M. J D Griffiths and D. F McAuley
RAGE: a biomarker for acute lung injury
Thorax, December 1, 2008; 63(12): 1034 - 1036.
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