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Published ahead of print on September 15, 2005, doi:10.1165/rcmb.2005-0194OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 83-91, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0194OC

Proliferative Stimulus of Lung Fibroblasts on Lung Cancer Cells Is Impaired by the Receptor for Advanced Glycation End-Products

Babett Bartling, Nina Demling, Rolf-Edgar Silber and Andreas Simm

Clinic of Cardio-Thoracic Surgery, Martin Luther University Halle-Wittenberg, Halle/Saale; and Institute of Immunology, Technical University Dresden, Dresden, Germany

Correspondence and requests for reprints should be addressed to Dr. Babett Bartling, Klinik für Herz- und Thoraxchirurgie, Martin-Luther-Universität Halle-Wittenberg, Ernst-Grube-Str. 40, D-06120 Halle/Saale, Germany. E-mail: babett.bartling{at}medizin.uni-halle.de


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The receptor for advanced glycation end-products (RAGE) is highly expressed in lung tissue, especially at the site of the alveolar epithelium, but its expression is reduced in lung carcinomas. Because epithelial–mesenchymal interactions are suggested to contribute to cancer progression, we investigated the RAGE-dependent impact of fibroblasts on tumor cell growth. Cocultivation of human lung cancer cells (H358) with lung fibroblasts (WI-38) improved their proliferation in monolayer and spheroid culture models, the number of H358 cells in the S/G2 cell cycle phase increased, and there was less spontaneous cell death. Overexpression of full-length human RAGE reduced the proliferative stimulus of fibroblasts as seen in monolayers (cell number, cell cycle), spheroid cultures (spheroid size), and in a colony-forming assay compared with mock-transfected cells. Comparable results were observed by culturing H358 cells with and without RAGE overexpression in the presence of conditioned medium taken from WI-38 cells, or in response to selected growth factors, such as basic fibroblast growth factor. Moreover, we clearly showed that the fibroblast-induced proliferation correlates with activation of the p42/44 mitogen-activated protein kinase, but not with Akt kinase activation. On the basis of lung cancer as an age-related disease, we additionally proved the impact of senescent WI-38 fibroblasts. Here, we show that senescent fibroblasts improve H358 cell proliferation to the same extent as do presenescent fibroblasts. From our data, we conclude that re-expression of RAGE in lung cancer cells impairs the proliferative stimulus mediated by fibroblasts. Therefore, lung cancer progression may be enhanced by the RAGE downregulation in human lung carcinomas.

Key Words: coculture • fibroblast • lung cancer • receptor for advanced glycation end-products • senescence


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
It is well established that tumor initiation and progression is profoundly regulated by an appropriate tissue microenvironment. In particular, there is growing evidence that fibroblasts are involved in this process as an important microenvironmental factor (1, 2). Fibroblasts express several growth-promoting factors and cytokines, but their expression profile is strongly modulated by localization and functional status of the cells. However, the release of growth factors and cytokines from fibroblasts can change during the cellular life span in response to chronic inflammation or microenvironmental impacts, such as irradiation. Also, stromal fibroblasts of solid tumors exhibit a divergent phenotype associated with multiple modifications that additionally support tumor growth and propagation (3). In mice, irradiation of stromal cells greatly potentiated the tumor-forming ability of transplanted cells (4). Moreover, fibroblasts undergoing senescence might even have the potential to trigger the oncogenic transformation under some conditions as shown for premalignant epithelial cells (5). Because epithelial tumors, such as those in lung cancer, are closely associated with age, this observation significantly contributes to a better understanding of tumorigenesis in the elderly.

Lung tissue highly expresses the receptor for advanced glycation end-products, a member of the Ig superfamily of cell surface proteins (6). Although the high expression and biological function of the receptor for advanced glycation end-products (RAGE), especially in lung, is still elusive, this receptor is strongly located in the alveolar epithelium, where it has been identified as a basolateral marker protein for type I lung alveolar cells (7). Several ligands for the engagement of RAGE have been found, including advanced glycation end-products, members of the S100 family of calcium-binding proteins, and amphoterin. In this context, it has been shown that activation of RAGE contributes to perturbation of the cell homeostasis in pathophysiologic conditions (8), as well as cell migration and differentiation in nonpathophysiologic conditions (9, 10). Many of these responses are mediated by intracellular signaling pathways induced by RAGE, which include the activation of small GTPases (Cdc42, Rac) (11), p38 mitogen-activated protein kinase (MAPK), and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) (12). However, knowledge of the signal transduction pathways activated by engagement to RAGE is still fragmented, and intracellular factors binding to the cytoplasmic region of RAGE are not yet identified. Moreover, we recently showed that the localization of RAGE at the cell surface is completely independent of the intracellular domain, suggesting RAGE-dependent effects, which might be exclusively mediated by the extracellular region (13).

Among the binding partners for RAGE, amphoterin is highly expressed in immature and malignant cells (13). Beyond the nuclear identity of amphoterin as a major form of the high-mobility group box DNA-binding proteins, amphoterin can be secreted and can thus mediate extracellular function via RAGE (9). Whereas the passive secretion of amphoterin by dying cells triggers inflammation, the active secretion is well controlled and local, without inflammatory response. In tumor biology, it has been suggested that RAGE engagement through the binding of amphoterin localized outside the cell promotes tumor growth and metastasis (12). Although the level of RAGE expression correlates with the metastatic potential of other tumor cells as well (15, 16), there is some disagreement about the role of RAGE activation in carcinogenesis. Studies using neuroblastoma and melanoma cells clearly showed that binding of amphoterin to RAGE inhibits invasive migration in vitro and suppresses the formation of metastasis in vivo (17). Moreover, we recently demonstrated that overexpression of RAGE in lung cancer cells without additional application of amphoterin does not mediate an increased tumor growth in athymic mice (13). Overexpression of full-length human RAGE in lung cancer cells even showed a diminished in vitro proliferation in monolayer cell cultures compared with cells expressing the cytoplasmic deletion mutant of RAGE ({Delta}cytoRAGE) or mock-transfected cells. This observation also contributed to a better understanding of our clinical data, demonstrating that the expression level of RAGE is strongly reduced in non–small cell lung carcinomas compared with the corresponding normal lung tissues. Furthermore, we revealed that the downregulation of RAGE correlates with higher tumor stages, and can be found in other solid tumors and in metastatic tissues as well (13, 18).

However, our previous investigations did not involve the impact of mesenchymal cells on cell proliferation and tumor growth. Therefore, we studied the proliferative stimulus of lung fibroblasts on lung cancer cells dependent on the re-expression of RAGE in a bronchoalveolar cell line. On the basis of lung carcinomas as an age-related disease, we also analyzed the potential effects of senescent fibroblasts on tumor growth.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Culture Conditions and Reagents
The National Cancer Institute H358 lung cancer cell line used corresponds to non–small (bronchoalveolar) cell lung carcinoma. For generation of H358 cells overexpressing RAGE, full-length human RAGE cDNA (XM_004205) and {Delta}cytoRAGE cDNA (without cytoplasmic domain, amino acid 367–404) were cloned into the mammalian expression vector pIRES2-enhanced green fluorescent protein (EGFP) (Clontech, Palo Alto, CA). Recombinant plasmids or the vector alone were stably transfected into H358 cells as previously described (17). Moreover, WI-38 cells used were primary embryonic lung fibroblasts and undergo replicative senescence after multiple cell passages. Both H358 cells and WI-38 fibroblasts (ATCC Cell Bank, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen, Karlsruhe, Germany) at 37°C in a 10% CO2 atmosphere. Three days after culture, conditioned medium was taken from WI-38 and H358 cells corresponding to 3.5–4.0 x 105 cells/ml. Replicative senescence of WI-38 fibroblasts was assessed by reduction of the population doubling (PD), which is defined as (ln cell numberDay x – ln cell numberDay 0)/ln2 (19). Here, WI-38 cells were seeded at 7.5 x 103 per cm2 and PD was estimated at Day 4 after counting the total number of living cells using a Multisizer3 Coulter Counter (Beckman Coulter, Krefeld, Germany). Senescent WI-38 fibroblasts showed a PD < 1.3, whereas presenescent cells had a PD range of 1.9–2.2. Cellular senescence was also proved by positive cell staining for acid {beta}-galactosidase (20). In this case, cells were fixed in 2% formaldehyd/0.2% glutaraldehyde in PBS and incubated in X-gal staining solution (1 mg/ml in dimethylformamide, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM sodim chloride, 2 mM magnesium chloride, 100 mM citric acid/sodium phosphate buffer; pH 6) at 37°C. Characteristic blue staining was observed after 24 h.

Soluble RAGE peptide (sRAGEc [21]; 10 µg/ml) as well as mouse monoclonal antibodies against human RAGE (clone 4F1 and 9A11 [21]; 40 µg/ml), basic fibroblast growth factor (bFGF; 10 µg/ml clone bFM-1; Upstate Biotechnology, Lake Placid, NY) and insulin-like growth factor-1 (IGF-1; 20 µg/ml clone Sm1.2; Upstate Biotechnology) were applied for neutralizing conditioned media. Additionally, H358 cells were treated with several growth factors for 2 d. IGF-1 (30 ng/ml), bFGF (50 ng/ml), and platelet-derived growth factor variants AA, AB, and BB (50 ng/ml; Roche Diagnostics, Mannheim, Germany) were used to induce cell proliferation after synchronizing H358 cells in serum-free medium.

Coculture and Assessment of Cell Proliferation and Growth
Direct cocultivation was performed after seeding of three parts H358 cells with one part WI-38 fibroblasts at a total cell count of 7.5 x 103 cells/cm2. H358 cells without pIRES2-EGFP transfection (control H358 cells) replaced WI-38 fibroblasts in internal control experiments. Proliferation of the pIRES2-EGFP–transfected tumor cells was indirectly determined by measuring the EGFP signal after 3 d. EGFP was detected by immunoblotting of the total amount of lysed cells as described subsequently here. There is a linear relationship between the cell number and GFP signal detected within the analyzed range (data not shown). Indirect coculture assays were performed with the same cell ratio using 0.4-µm pore membrane inserts (Greiner Bio-One, Frickenhausen, Germany).

For spheroid culturing, a 5x stock solution of methyl-cellulose (25 mg/ml in DMEM; Sigma, Deisenhofen, Germany) was mixed with three parts of H358 cells and one part of WI-38 fibroblasts or control H358 cells, to a final number of 500 cells per spheroid, and seeded into a single well of a 96-well suspension culture plate. Twenty spheroids were cultured per experiment and medium was partially exchanged after 4 d. The diameter of each spheroid was estimated using the Zeiss Axiovert microscope (Zeiss, Jena, Germany) equipped with a SPOT Camera and Metamorph 4.6.5. software (Visitron Systems, Puchheim, Germany). Spheroid volumes were calculated as a product of 4/3{pi} · diameter3 and subsequently averaged. The colony forming assay was performed in 0.6% agar (Roth, Karlsruhe, Germany) dissolved in DMEM/10% FCS with 2 x 103 cells/cm2 after a standard protocol (22). For coculturing in soft agar, WI-38 fibroblasts or control H358 cells were seeded 1 d before (2 x 104 cells/cm2) and covered with a thin layer of 0.6% agar shortly before the experiment. The number of formed cell colonies was estimated by microscopy after 14 d.

Conditioned medium was used from WI-38 fibroblasts and control H358 cells, as described previously here, and mixed with one volume of fresh DMEM/10% FCS medium. Cell proliferation was determined spectrophotometrically after 2 d by measuring the amount of reduced alamarBlue reagent (Bioscource Europe, Nivelles, Belgium) according to the manufacturer's instruction. The linearity of cell number and alamarBlue staining was proved for the range of detection.

Generation of Recombinant RAGE Fragments and Anti-RAGE Antibodies
For generation of mouse monoclonal anti-RAGE antibodies, cDNA coding for sRAGE (corresponding to amino acids 22–342 of the mature protein) was amplified and cloned into the BamHI and EcoRI restriction sites of the mammalian expression vector pSecTag2B (Invitrogen, Carlsbad, CA), which allows the secretion of the protein into the culture medium. After stable overexpression in HEK293 cells, sRAGE was purified from the cell culture supernatant according to Hsieh and colleagues (23). A total of 100 µg of purified sRAGE was used to immunize BALB/c mice. Hybridomas were generated according to standard procedures and screened for reactivity as described previously (21). Anti-RAGE monoclonal antibodies (IgG1; designed 9A11 and 4F1) were obtained that are suitable for ELISA, immunoblot, precipitation, and fluorescence analyses. For RAGE-neutralizing experiments, another soluble RAGE construct (sRAGEc) was used which corresponds to the human RAGE sequence of sRAGE. On the basis of PCR cloning into the SfiI and EcoRI restriction sites of the vector pSecTag2B, it differs from sRAGE at the N-terminus due to the vector-specific sequences. The detailed protocol for generation of sRAGEc was described earlier (21).

Immunocytochemistry
For H358 immunofluorescence, cells grown on glass coverslips were fixed in 4% formaldehyde for 10 min at 4°C and permeabilized by 0.5% saponin. After blocking with 10% goat serum albumin (DAKO, Hamburg, Germany), they were stained for 1 h with the monoclonal mouse primary antibody 9A11 (1:400 in PBS) at room temperature. RAGE detection was performed with the AlexaFluor594 anti-mouse IgG secondary antibody (Molecular Probes Europe, Leiden, The Netherlands) and fluorescence microscopy. Actin cytoskeleton was visualized with AlexaFluor488 phalloidin (Molecular Probes Europe).

Cell Cycle Analysis
H358 cells were collected at Day 2 after seeding in coculture or in conditioned medium (i.e., start of the exponential growth). Nuclear DNA content was analyzed after staining with propidium iodide (PI; Sigma) and subsequent flow cytometry. Briefly, cells were fixed in ice-cold 70% ethanol and incubated with PI solution (50 µg/ml PI, 0.5 mg/ml RNase A, 0.1% sodium citrate, 0.1 mM disodium ethylene-dinitrilotetraacetic acid in PBS; pH 7.2) for 1 h at 4°C. DNA content was measured using the FACSCalibur flow cytometer equipped with CellQuest Pro software (Becton Dickinson, San Diego, CA). In the case of direct coculture with WI-38 fibroblasts, the pIRES2-EGFP–transfected H358 cells were gated by EGFP fluorescence. The number of proliferating cells in the S and G2 cell cycle phase was estimated using the MultiCycle software (Phoenix Flow Systems, San Diego, CA).

Detection of Protein and mRNA Expression
For protein analysis, samples were homogenized in lysis buffer (10 mM Tris-[hydroxymethyl]-aminomethane [pH 7.4], 80 mM potassium chloride, 1 mM disodium ethylene-dinitrilotetraacetic acid, 2% SDS) containing complete protease inhibitor (Roche Diagnostics) and 1 mM sodium metavanadate (Sigma-Aldrich, Deisenhofen, Germany) as a phosphatase inhibitor. Protein concentration was measured by the bicinchoninic acid protein assay (Pierce, Rockford, IL). A total of 50 µg protein was separated by SDS-PAGE and blotted onto polyvinylidene-difluoride membranes (Roche Diagnostics). Thereafter, the membrane was blocked with 6% non-fat dry milk in Tris-buffered saline (TBS)-T buffer (200 mM Tris-[hydroxymethyl]-aminomethane [pH 7.5], 300 mM sodium chloride, 0.15% polyethylenesorbitan monolaurate) and incubated with the respective antihuman antibodies. Phospho-specific rabbit polyclonal antibodies against p-p42/44-MAPK/p-Erk-1/2 (Thr202/Tyr204), p-Akt (Ser473), and p-SAPK/cJNK (Thr183/Tyr185) (all from Cell Signaling Technology, Beverly, MA) or mouse monoclonal antibodies against EGFP (Roche Diagnostics) were applied in TBS-T buffer. Protein loading was controlled by using the polyclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (Abcam, Cambridge, UK). Bound antibodies were detected using a calf intestinal alkaline phosphatase–conjugated goat anti-mouse or anti-rabbit IgG antibody (Dianova, Hamburg, Germany) and the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium system (Sigma-Aldrich). The intensities of visualized signals were densitometrically quantified per glyceraldehyde-3-phosphate dehydrogenase signal (Aida software; Raytest, Straubenhardt, Germany).

For PCR, RNA was extracted using RNeasy columns (Qiagen, Hilden, Germany) and cDNA was synthesized with Superscript II reverse transcriptase (Invitrogen). PCR amplification for amphoterin (X_12597; sense: 5' GGA GAG ATG TGG AAT A 3'; anti-sense: 5' GGG AGT GAG TTG TGT A 3') was performed as previously described (17) and normalized per amplification of 18S rRNA.

Measurement of Cell Death and Mitochondrial Depolarization
Cell death was assessed by uptake of PI (50 µg/ml PI in PBS) and subsequent cytometric analysis using the FACSCalibur flow cytometer with CellQuest Pro software (Becton Dickinson, San Diego, CA). In the case of direct coculture with WI-38 fibroblasts, H358 cells were gated by EGFP fluorescence. Reduction of the mitochondrial membrane potential was determined by staining of cells with tetramethylrhodamine ethyl ester perchlorate (25 nM; Molecular Probes Europe), a cationic, lipophilic fluorochrome dye that is retained in the negatively charged mitochondrial matrix but lost during mitochondrial depolarization. The loss in mitochondrial membrane potential was determined by diminished tetramethylrhodamine ethyl ester perchlorate staining using the flow cytometer, as described previously (24).

Statistical Analysis
One-way ANOVA or the ANOVA on ranks for nonparametric groups was used for multiple comparisons followed by Dunnett's or Dunn's method as appropriate. Regression analyses were performed and the coefficients of each proliferation or growth curve were calculated according to the given formula, and subsequently subjected to multiple analyses using SigmaStat and SigmaPlot software (Jandel Corp., San Rafael, CA). All data are reported as mean ± SEM (n >= 3 in all cases), and P values < 0.05 were accepted as indicating a significant difference.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Proliferation and Growth in Response to Lung Fibroblasts
H358 lung cancer cells stably transfected with a pIRES-EGFP2 vector were used in all coculture experiments to ensure a reliable reanalysis of the cells by their EGFP fluorescence. The direct cocultivation of these EGFP-transfected (mock) cells with WI-38 lung fibroblast improved their proliferation in a spheroid culture model (Figure 1A), as well as in monolayers associated with an increased the number of cells in the S/G2 cell cycle phase (Table 1). The impact of fibroblasts on the cell cycle of H358 cells is mainly detectable by a significant increase of the S-phase and reduction of G1-phase cells. Moreover, we showed that senescent fibroblasts improved the proliferation and growth of H358 cells to the same extent as do presenescent fibroblasts (Figures 1A and 4B) in accordance with an increased number of cells in the S phase of the cell cycle (Table 1). The presence of fibroblasts also resulted in reduced spontaneous cell death and mitochondrial depolarization (Figure 2).




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  		Figure 1. (A) H358 cells and WI-38 fibroblasts were cocultured as spheroids in single wells (n = 20 each experiment). In control spheroids, the same number of control H358 cells replaced WI-38 cells. Spheroid growth was evaluated by estimating the mean volume at given days for mock-, RAGE-, and cytoplasmic deletion mutant of RAGE ({Delta}cytoRAGE)–expressing spheroids in response to fibroblasts. *P < 0.05 versus fibroblast-free spheroids for the mean coefficient a calculated from the regression for each experiment (n = 6). (B) Increased spheroid size in the presence of WI-38 fibroblasts compared with a fibroblast-free spheroid can be shown for {Delta}cytoRAGE- but not for RAGE-expressing H358 cells. Spheroids are demonstrated by enhanced green fluorescent protein (EGFP) fluorescence at Day 9 of coculture. (C) Fibroblasts also enhanced the formation of H358 tumor cell colonies in a soft agar assay by coculturing WI-38 fibroblasts at the bottom of the soft agar overlay. *P < 0.05 versus colony-forming efficacy assessed by coculture with control H358 cells instead of fibroblasts (n = 3).

 


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  		Figure 2. Direct coculturing of H358 tumor cells with WI-38 fibroblasts resulted independent of the expression of RAGE or {Delta}cytoRAGE in a reduced amount of spontaneous cell death, as detected by membrane leakage (propidium iodide uptake) and mitochondrial depolarization (loss of tetramethylrhodamine ethyl ester perchlorate uptake). *P < 0.05 versus coculturing with control H358 cells instead of fibroblasts in a monolayer cell culture model; #P < 0.05 as indicated (n = 6).

 


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  		Figure 3. Indirect coculturing of H358 cells in pore membrane inserts with WI-38 fibroblasts improved (A) the proliferation (increased optical density of the reduced amount per oxidized amount of alamarBlue reagent), as well as (B) the p42/44-MAPK activation (Thr202/Tyr204 phosphorylation), which is less pronounced in the RAGE- and {Delta}cytoRAGE-transfected H358 cells. *P < 0.05 versus indirect cocultivation with control H358 cells; #P < 0.05 as indicated (n = 3).

 

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  		TABLE 1. IMPACT OF WI-38 LUNG FIBROBLASTS ON THE CELL CYCLE OF H358 LUNG CANCER CELLS IN RESPONSE TO COCULTIVATION OR CONDITIONED MEDIUM DERIVED FROM FIBROBLASTS

 
In indirect monolayer cocultures using pore membrane inserts, we performed analyses to determine if the release of fibroblast-associated mitogens contributes to this growth-promoting effect in cancer cells. Indeed, indirect cocultivation of mock-transfected H358 cells with WI-38 fibroblasts revealed a better proliferation of H358 cancer cells compared with indirect cocultures using H358 cells alone (Figure 3A). This observation correlated with an increased number of H358 cells in the S cell cycle phase (Table 1) as well as with an activation of the p42/44-MAPK (Figure 3B). Therefore, we additionally verified the impact of conditioned medium taken from WI-38 fibroblasts on the proliferation of H358 cells. Corresponding with the indirect fibroblast cocultures, we also found a better proliferation of H358 cells (cell number, Figure 4C; cell cycle, Table 1) in the presence of conditioned medium taken from presenescent or senescent WI-38 fibroblasts compared with conditioned medium from H358 tumor cells. Moreover, we studied the impact of fibroblasts on the colony-forming capacity of the mock-transfected H358 cells. Through coculturing WI-38 fibroblasts or control H358 cells in soft agar, we demonstrated that the formation of tumor colonies from a single cell was significantly improved by the presence of fibroblasts (Figure 1C).




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  		Figure 4. (A) Direct coculturing of RAGE-expressing with control H358 cells or WI-38 fibroblasts. Immunocytochemistry of RAGE-overexpressing H358 cells indicates the membrane localization of RAGE, particularly at the intercellular contact sites. Cocultured control H358 cells and WI-38 fibroblasts are visualized by actin staining. (B) Direct coculturing of EGFP-expressing H358 cells with WI-38 fibroblasts improved the proliferation in a monolayer model, as assessed by increased EGFP detection. *P < 0.05 versus cocultivation with EGFP-negative control H358 cells (n = 6). This effect was less induced in the RAGE- and {Delta}cytoRAGE-transfected cells, as observed by culturing of H358 cells in conditioned medium and from WI-38 fibroblasts as well (C). Adding monoclonal anti-RAGE antibody (clone 4F1) or soluble RAGE peptide (sRAGEc) improved the proliferation of RAGE-expressing H358 cells in conditioned medium from WI-38 fibroblasts related to H358 cells (D). *P < 0.05 and (*)P = 0.06 versus equally treated conditioned medium from control H358 cells; #P < 0.05 as indicated (n = 6).

 
Impact of RAGE and {Delta}cytoRAGE on Fibroblast-Induced Cell Proliferation
To address a possible effect of RAGE, we stably overexpressed full-length human RAGE in H358 cells and cocultured them with WI-38 fibroblasts (Figures 1B and 4A). In comparison to mock-transfected cells, RAGE expression resulted in a diminished proliferation and growth mediated by fibroblasts. This has been shown for coculturing in direct and indirect monolayers (cell number, Figures 3A and 4B; cell cycle, Table 1), in spheroid cultures (spheroid volume, Figures 1A and 1B), as well as in a colony-forming assay (Figure 1C). Comparable results were observed by culturing lung cancer cells with and without RAGE overexpression in the presence of conditioned medium from WI-38 cells (Figure 4C). Blockade of RAGE using anti-RAGE clone 4F1 antibodies, which are directed to the extracellular domain, improved the proliferation of RAGE-expressing cells in conditioned medium from WI-38 fibroblasts relative to equally treated medium from H358 cells (Figure 4D). Similar results were observed in the presence of the antibody clone 9A11 (data not shown) or by adding sRAGEc (Figure 4D), but to a lesser extent.

The proliferative stimulus of fibroblasts was associated with an increased activation of the p42/44-MAPK, as demonstrated for indirect cocultures (Figure 3B) and by use of conditioned medium from WI-38 fibroblasts (Figure 5A). In both cases, we found a less pronounced activation of p42/44-MAPK in RAGE-expressing H358 cells. In contrast, the Akt kinase was less induced than p42/44-MAPK in response to conditioned medium from fibroblasts, and was independent of the RAGE expression (Figure 5A).



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  		Figure 5. (A and B) Culturing of H358 cells with conditioned medium from WI-38 fibroblasts enhanced the signal of active p-p42/44-MAPK and p-Akt. This induction was less detectable by overexpression of RAGE and {Delta}cytoRAGE in H358 cells for p-p42/44-MAPK, but not for p-Akt. *P < 0.05 versus conditioned medium from control H358 cells; #P < 0.05 as indicated (n = 5).

 
In this context, we additionally determined the impact of lung fibroblasts on the expression of amphoterin in H358 lung cancer cells. These studies showed that neither indirect cocultivation with WI-38 cells nor treatment with conditioned medium altered the mRNA expression of amphoterin in H358 cells (1.10-fold ± 0.12 versus control cultivation; n = 3). In addition, overexpression of RAGE did not impair the mRNA level of amphoterin (1.07-fold ± 0.04 versus mock-transfected H358; n = 6).

Overexpression of the cytoplasmic deletion mutant {Delta}cytoRAGE in H358 cells also affected the fibroblast-induced cell proliferation depending on the coculture model studied. Using these cells in monolayer models, the proliferation, as well as the p42/44-MAPK stimulation, was more related to RAGE-transfected cells than to H358 control cells. {Delta}cytoRAGE-expressing cells often tended to a higher basic stimulation of p42/44-MAPK in the presence of control-conditioned medium (data not shown), which might partially explain the lower responsiveness to conditioned medium from fibroblasts. However, in three-dimensional models, {Delta}cytoRAGE-expressing cells behaved like mock-transfected cells, and even formed the largest spheroids in response to WI-38 fibroblasts (Figures 1A and 1B). Moreover, we analyzed the impact of fibroblasts on the spontaneous cell death of H358 cells with and without overexpression of RAGE. Here we observed a slightly increased cell death of H358 cells expressing RAGE, which is reduced in the presence of WI-38 fibroblasts (Figure 2). An activation of the stress-activated protein kinase, p-SAPK/cJNK, was not seen at detectable levels.

RAGE-Dependent Proliferation in Response to Growth Factors
Fibroblasts and other cells release a panel of mitogenic factors. Therefore, we analyzed the impact of RAGE on the cell propagation in response to selected growth factors. Here we showed that bFGF and IGF-1 stimulate the proliferation of H358 cells (Figure 6A), but the variants of platelet-derived growth factor (AA, AB, and BB), have little or no influence (data not shown). In accordance with the reduced growth promotion in the presence of fibroblasts, RAGE expression also impaired the growth stimulation mediated by IGF-1 and bFGF, which has been demonstrated for cell proliferation (Figure 6A) as well as for p42/44-MAPK activation (Figures 6B and 6C). These data have been verified for conditioned medium from fibroblasts by application of neutralizing antibodies for IGF-1 and bFGF. As demonstrated in Figure 6D, anti-bFGF antibodies significantly reduced the proliferative stimulus of conditioned medium from WI-38 fibroblasts in relation to control-conditioned medium from H358 tumor cells. This has been determined for mock-transfected but not for RAGE-transfected H358 cells (Figure 6D). The direct impact of IGF-1 in WI-38–conditioned medium was less pronounced (Figure 6D).




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  		Figure 6. (A) Specific stimulation of synchronized H358 cells with IGF-1 and basic fibroblast growth factor (bFGF) indicates cell proliferation (reduction of alamarBlue reagent) and (B and C) activation of p-p42/44-MAPK after 2 d of growth factor treatment, which is more pronounced for mock- than for RAGE-expressing cells. *P < 0.05 versus growth factor-free medium (n = 3). (D) The presence of anti–IGF-1 and -bFGF antibodies in conditioned medium from WI-38 fibroblasts also affected the proliferation of H358 cells, depending on RAGE. Relative data are given per H358 conditioned medium treated in the same way. *P < 0.05 versus antibody-free medium (n = 4).

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The development and progression of epithelial malignancies are significantly supported by tissue homeostasis. In this context, stromal cells that surround and sustain the epithelia are a primary source of additional growth stimuli for epithelial tumors, such as lung carcinoma (25). Recently, we reported that RAGE is strongly reduced in lung tumors compared with normal lung tissue (13), where RAGE is normally expressed at very high level (26). Moreover, we showed that RAGE might have the potential to impair the proliferation of lung cancer cells in some conditions (13). This has been primarily seen studying polarized cells in a monolayer cell culture model. The proliferative impact of RAGE was still detectable when analyzing three-dimensional cell cultures in vitro and in vivo compared with cells expressing {Delta}cytoRAGE, but not when compared with normal lung cancer cells (13). However, the biological behavior of epithelial cells is strongly associated with their polarization as well as with an altered tissue microenvironment in multicellular spheroids. On the basis of the mesenchymal-epithelial interaction as another key factor in the regulation of normal (27) and transformed epithelial cells (28), we studied the proliferative stimulus of lung fibroblast on lung epithelial cancer cells in depending on the re-expression of RAGE in cancer cells.

In this study, we clearly show that direct cocultivation of human lung cancer cells (H358) with lung fibroblasts (WI-38) improves their proliferation in monolayer and three-dimensional cell culture models. This has been demonstrated for cell propagation and spheroid growth associated with changes of the cell cycle. Moreover, the indirect cocultivation with WI-38 fibroblasts or the application of conditioned media revealed an improved proliferation of H358 cells as well, suggesting the release of potent growth factors from lung fibroblasts. In particular, we show the involvement of bFGF in this process by blocking conditioned medium from WI-38 fibroblasts with neutralizing antibodies. Although IGF-1 is also an interesting mitogen, as it is closely associated with an increased risk of common cancers (29), blocking IGF-1 had less impact on fibroblast-mediated cell propagation. In addition, the proliferative stimulus of fibroblasts was less pronounced in H358 cells overexpressing full-length human RAGE, as demonstrated for monolayer and spheroid cultures compared with mock-transfected cells. Correspondingly, blocking bFGF did not impair the fibroblast-mediated proliferation of H358 cells overexpressing RAGE.

The development of epithelial tumors, such as lung carcinomas, is closely associated with human age (30). This clinical observation is supported by experimental studies demonstrating that premalignant (p53-mutated) epithelial cells undergo oncogenic transformation and tumor growth by treatment with senescent fibroblasts (5). In contrast, senescent primary fibroblasts are also reported to have a limited impact on cell proliferation, and also on the migration and adhesion of tumor cells (31). For this reason, we did not only analyze the impact of normal (i.e., presenescent) WI-38 lung fibroblasts, but also of senescent fibroblasts, on the proliferation of lung cancer cells. Here, we show that senescent fibroblasts can still support proliferation and growth of the H358 cells in almost the same manner as do presenescent fibroblasts. Moreover, we did not detect any age-dependent impact of RAGE on lung fibroblast–mediated cell propagation.

The major ligand of RAGE in the lung seems to be amphoterin, which is also highly expressed in human lung tissue. Amphoterin is a high-mobility group box chromosomal protein that can be released into the extracellular space during certain stages of development (32), or in necrotic cells where it triggers inflammation (33). Although it is unknown as to what extent amphoterin is located in the extracellular space of tumor cells, the interaction of amphoterin with RAGE has been suggested to contribute to tumor growth (12). Therefore, we proved the influence of lung fibroblast on the transcription of amphoterin in H358 lung cancer cells. However, we did not find an altered expression of amphoterin in indirect presence of fibroblasts or by treating cells with conditioned medium from lung fibroblasts. Although expression analyses do not determine the amount of secreted amphoterin, we can at least observe that amphoterin is not actively regulated at the transcriptional level.

Proliferation, differentiation, and cell survival are well controlled by an intracellular network of protein kinases and other signal messengers. Many receptor tyrosine kinases are known to activate intracellular protein serine/threonine kinases, termed MAPKs, after receptor activation by mitogens, including growth factors. The basic arrangement of this signal cascade involves the G-protein Ras, the MAPK kinase kinase Raf that activates the MAPK kinase MEK1/2, which in turn culminates in the activation of the p42/44-MAPK (also known as Erk1/2) (34). In indirect coculture models or in response to conditioned medium from lung fibroblasts, we found that the terminal kinase p42/44-MAPK is less activated in RAGE-expressing cancer cells compared with mock-transfected cells. This has been confirmed for the bFGF-induced signaling, demonstrating a less pronounced activation of the p42/44-MAPK in RAGE-expressing cells. Although comparable results were found for the MAPK stimulation in response to IGF-1, IGF-1 seems not to mediate major effects derived from lung fibroblasts. On the basis of higher spontaneous cell death of RAGE-expressing H358 cells, we then investigated whether an activation of SAPK might be one target influencing the MAPK pathway through insulin receptor substrate 1 phosphorylation (35). However, active SAPK has not been found at detectable levels in H358 cells with or without overexpression of RAGE. This further indicates that amphoterin might have less importance in our coculture model, because the stimulation of RAGE through interaction with amphoterin, has been demonstrated to stimulate SAPK (12).

RAGE is composed of an extracellular region containing three Ig domains, followed by a transmembrane domain, and a short cytoplasmic region (27). However, precise signal transduction pathways activated by interaction with RAGE are still elusive, basically because intracellular factors binding to the cytoplasmic domain of RAGE are not yet identified. Therefore, it is difficult to discuss the mechanism(s) by which the mitogen-induced MAPK signaling is impaired. Moreover, lung fibroblasts generate a panel of mitogenic and other factors that might induce numerous intracellular signal transduction pathways, which co-interact with themselves—and a direct receptor stimulation of RAGE by the interaction with ligands, such as S100P (36), can still trigger p42/44-MAPK activity. In an even more complex manner, H358 lung cancer cells expressing {Delta}cytoRAGE are also diminished in their fibroblast-mediated p42/44-MAPK activation and proliferation. Interestingly, the diminished responsiveness of {Delta}cytoRAGE-expressing cells was only observed for polarized cells, but not for H358 cells cocultured with fibroblasts as a multicellular spheroid. This observation might be strongly associated with subcellular changes in the receptor complex and alterations of the surrounding micromilieu in three-dimensional cell culture models. Despite the discrepancy between two- and three-dimensional culturing of {Delta}cytoRAGE-expressing cells, there is some suggestion that the extracellular region of RAGE is also essential for RAGE-mediated signal transduction. In this context, it was recently shown that the S100B-induced production of nitric oxide in microglia cells is independent of RAGE-transducing activity, but dependent on the extracellular domain of RAGE, whereas the activation of nuclear factor –{kappa}B relies on the presence of the cytoplasmic region (37). Moreover, we have previously demonstrated that the intercellular localization of RAGE also depends on the expression of the extra- but not intracellular RAGE domain (13). Altogether, these observations suggest that RAGE does not only act as an individual surface receptor, but in complex with other surface membrane compounds, which might also explain those differences between the polarized and nonpolarized cells expressing {Delta}cytoRAGE.

To further investigate the possible role of RAGE on cell growth, we blocked RAGE by application of neutralizing antibodies, resulting in an increased propagation of the RAGE-expressing H358 cells in response to lung fibroblasts. However, this was not observed for {Delta}cytoRAGE-expressing H358 cells, additionally indicating some importance of the cytoplasmic region of RAGE. Moreover, improvements of the fibroblast-induced proliferation of RAGE-expressing cells were also much more marked after the use of specific antibodies compared with sRAGE. This result seems to depend on the specificity of the RAGE blockade rather than on the concentration applied, which has recently been shown to be sufficient to impair RAGE-dependent effects (38).

In addition to the mitogen-induced MAPK signaling, the engagement of receptor tyrosine kinases by growth factors activates the phosphatidyl inositol 3-kinase that, in turn, stimulates the serine/threonine kinase Akt via 3-phospho-inositide-dependent kinase (PDK)1-mediated phosphorylation (39). The stimulation of Akt was also increased in H358 lung cancer cells in response to conditioned medium taken from lung fibroblasts when compared with conditioned medium from tumor cells. However, the fibroblast-induced activation of Akt occurred at a lower level than observed for p42/44-MAPK, and was not impaired by overexpression of RAGE. Altogether, these data indicate that the growth-promoting effect of the WI-38 lung fibroblasts on lung cancer cells is mainly related to the MAPK signal transduction pathway. However, growth factors still contribute, via Akt signaling, to cell growth and proliferation. This might partially explain the less pronounced impact of RAGE on the cell proliferation after treatment with bFGF, whereas the bFGF-induced activation of p42/44-MAPK was more changed in response to RAGE over-expression.

Recently, we have shown that the expression of the surface receptor RAGE in lung is sharply reduced in human lung carcinomas associated with an impact on growth, mainly on polarized cells in two-dimensional cell cultures, but also to a lesser extent in three-dimensional models in vitro and in vivo (17). Based on the results of the present study, we can conclude that the re-expression of RAGE in lung cancer cells impairs the proliferative stimulus by lung fibroblasts, independent of cell polarization. Thus, epithelial-mesenchymal interactions in lung cancer progression might be dependent on RAGE downregulation in human lung carcinomas.


   Acknowledgments
 
The authors are grateful to Renate Donath for her technical assistance.


   Footnotes
 
This work was supported by the Wilhelm Roux grant of the Bundesministerium für Bildung und Forschung (FKZ7/04) and, in part, by the Deutsche Forschungsgemeinschaft (SFB 598/TPA5).

Originally Published in Press as DOI: 10.1165/rcmb.2005-0194OC on September 15, 2005

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 May 23, 2005

Accepted in final form September 5, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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