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ARTICLE Expression and subcellular localization of RAGE in melanoma cells Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Santa Cruz (UCSC) on 10/10/14 For personal use only.

Ioana Popa, Elena Ganea, and Stefana M. Petrescu

Abstract: The receptor for advanced glycation end products (RAGE) is involved in multiple stages of tumor development and malignization. To gain further knowledge on the RAGE role in tumor progression, we investigated the receptor expression profile and its subcellular localization in melanoma cells at different stages of malignancy. We found that RAGE clustered at membrane ruffles and leading edges, and at sites of cell-to-cell contact in primary melanoma cells (e.g., MelJuSo), in contrast with a more dispersed localization in metastatic cells (e.g., SK-Mel28). RAGE silencing by RNAi selectively inhibited migration of MelJuSo cells, whilst having no influence on SK-Mel28 cell migration, in a “wound healing” assay. Western blot detection of RAGE showed a more complex RAGE oligomerization in MelJuSo cells compared to melanocytes and SK-Mel28 cells. By competing the binding of antibodies with recombinant soluble RAGE, an oligomeric form running at approximately 200 kDa was detected, as it was the monomeric RAGE of 55–60 kDa. SDS-PAGE electrophoresis under reducing versus nonreducing conditions indicated that the oligomer of about 200 kDa is formed by disulfide bonds, but other interactions are likely to be important for RAGE multimerization in melanoma cells. Immunofluorescence microscopy revealed that treatment with two cholesterolchelating drugs, nystatin and filipin, significantly affected RAGE localization in MelJuSo cells. SK-Mel28 cells showed a reduced RAGE glycosylation and association with cholesterol-rich membranes and also a considerable downregulation of the soluble forms. Our results indicate that RAGE isoform expression and subcellular localization could be important determinants for the regulation of its function in tumor progression. Key words: RAGE, melanoma cells, cell migration, oligomerization, cholesterol-rich membranes. Résumé : Le récepteur RAGE (Receptor for advanced glycation end products) est impliqué dans plusieurs stades du développement tumoral et de la malignité. Afin d’en apprendre davantage sur le rôle de RAGE dans la progression tumorale, nous avons examiné le profil d’expression du récepteur et sa localisation subcellulaire dans des mélanomes a` différents stades de malignité. Nous avons trouvé que RAGE se groupait dans les replis membranaires et au front avant de la cellule, et aux sites de contact cellule-cellule dans les cellules de mélanome primaire (p. ex. MelJuSo), comparativement a` une localisation plus dispersée dans les cellules métastatiques (p. ex. SK-Mel28). Le silençage de RAGE par un ARNi inhibait sélectivement la migration des cellules MelJuSo, tandis qu’il n’avait pas d’influence sur la migration des cellules SK-Mel28, dans un essai de « cicatrisation ». La détection de RAGE par buvardage Western montrait une oligomérisation plus complexe de RAGE chez les MelJuSo comparativement aux mélanocytes et aux SK-Mel28. Lors d’un test de compétition de liaison d’anticorps avec une forme recombinante de RAGE, une forme oligomère migrant a` environ 200 kDa a été détectée, de même que le monomère de RAGE de 55–60 kDa. L’électrophorèse SDS-PAGE en conditions réductrice et non réductrice a indiqué que l’oligomère d’environ 200 kDa était formé par des ponts disulfures, mais que d’autres interactions étaient probablement importantes a` la multimérisation de RAGE dans les mélanomes. La microscopie en immunofluorescence a révélé qu’un traitement avec deux médicaments chélateurs de cholestérol, la nystatine et la filipine, affectait significativement la localisation de RAGE dans les cellules MelJuSo. Chez les SK-Mel28, la glycosylation de RAGE et son association avec les membranes riches en cholestérol étaient réduites, et les formes solubles étaient considérablement régulées a` la baisse. Nos résultats indiquent que l’expression et la localisation subcellulaire des isoformes de RAGE pourraient constituer des déterminants importants de la régulation de sa fonction dans la progression tumorale. [Traduit par la Rédaction] Mots-clés : RAGE, mélanomes, migration cellulaire, oligomérisation, membranes riches en cholesterol.

Introduction RAGE binds different classes of molecules, including advanced glycation end products, S100 proteins, high mobility group box-1 protein, ␤2-integrin Mac-1, amyloid ␤ peptides (for review see Yan et al. (2009)). N-glycosylation and oligomerization of the receptor are believed to be pre-requisites for binding to at least some of its ligands, followed by conformational changes within the complexed oligomer that trigger signal transduction (Srikrishna et al. 2010; Xie et al. 2008). Several receptor variants, N- or C-terminally truncated forms, are generated through alternative splicing or

proteolytic cleavage (Hudson et al. 2008a), yet additional receptor mutated variants (with intron insertions and alternative splicing of exons) have been relatively recently found in healthy and tumor samples or cell lines (Sterenczak et al. 2009). RAGE expression appears to be species- and tissue specific, which may constitute a mechanism to control receptor binding and modulate its function within a certain physiological or pathological environment. Upregulation of RAGE in cells that are challenged with an accumulation of receptor ligands leads to a sustained cellular activation, contributing to the instauration and (or) pro-

Received 10 June 2013. Revision received 9 January 2014. Accepted 19 January 2014. I. Popa, E. Ganea, and S.M. Petrescu. Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, Bucharest 060031, Romania. Corresponding author: Ioana Popa (e-mail: [email protected]). Biochem. Cell Biol. 92: 127–136 (2014) dx.doi.org/10.1139/bcb-2013-0064

Published at www.nrcresearchpress.com/bcb on 31 March 2014.

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gression of a pathological condition (Bierhaus and Nawroth 2009). Involvement of RAGE–ligand interactions in diabetic complications, inflammatory, and neurodegenerative disorders has been documented in a large number of studies (Sims et al. 2010; Yan et al. 2009). The expression of RAGE and some of its ligands is upregulated in several types of cancer (Hiwatashi et al. 2008; Ishiguro et al. 2005). It has been proposed that RAGE is one of the elements that connects chronic inflammation to neoplastic transformation, promoting carcinogenesis, tumor growth, and invasion (Sparvero et al. 2009; Turovskaya et al. 2008). Soluble receptor is believed to act as a decoy to attenuate receptor signalling. Such soluble receptor forms, either exogenously delivered or overexpressed in tumor cells, proved to be effective both in vitro and in vivo in inhibiting tumorigenesis (Kalea et al. 2010; Taguchi et al. 2000). It is therefore evident that an additional specificity in cell activation is acquired through the modulation of RAGE isoform expression and receptor processing. We hypothesize that RAGE function in cancer cells would rely as well on its subcellular localization and specific intracellular traffic. Given the various cellular processes associated with the malignant phenotype that RAGE can influence, we sought to identify differences in RAGE expression and localization in primary versus metastatic melanoma cells. In spite of a similar level of total RAGE expression in different types of melanoma cells, we found differences in the receptor isoform expression and processing to the soluble form and identified a distinct pattern of RAGE localization in these cells. The mechanisms regulating RAGE function in melanoma cells at different stages of malignancy could involve the extent of the receptor oligomerization, the N-glycosylation of a subset of RAGE forms that associated with specialized membrane domains, such as cholesterol-rich/lipid rafts, as well as a significant difference in the generation of the soluble forms of the receptor.

Materials and methods Cell culture Human melanoma cell lines MelJuSo, A375, SK-Mel28 (Baruthio et al. 2008; Filimon et al. 2012; Fimmel et al. 2007) were grown in RPMI 1640 medium (EuroClone or Gibco, Life Technologies) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Biochrom AG), and 2 mmol/L L-glutamine (Biochrom AG). MNT-1 cell line was maintained in DMEM with 10% AIM-V medium (Invitrogen) in the presence of 20% FBS and 2 mmol/L L-glutamine (Invitrogen). Human epidermal melanocytes were purchased from Lonza and cultured as recommended. Cloning of pcDNA3.1-hRAGE and pGEX-6P-1-sRAGE We obtained pcDNA3.1-hRAGE by PCR amplification of the receptor sequence from RZPDDo839C09122-pdEYFP-C1amp clone (ImaGenes, Germany) and insertion into EcoR1/Xho1 sites of pcDNA3.1(+) plasmid (Invitrogen). The construct was checked by sequencing. To obtain GST-soluble RAGE (GST-sRAGE), the region of aminoacids 23–327 of the extracellular part of the receptor was amplified and inserted into EcoR1/Xho1 sites of pGEX-6P-1 (GE Healthcare). Expression and purification of GST-sRAGE Escherichia coli BL21 (DE3) were transformed with pGEX-6P-1-sRAGE, and cells were grown as previously described (Allmen et al. 2008). Cells were lyzed in PBS containing 0.1% Triton-X-100, protease inhibitors and 5 mmol/L ␤-mercaptoethanol by sonication. After centrifugation at 100 000g for 60 min at 4 °C, bacterial lysate was incubated with glutathione (GSH)-Sepharose 4B beads (GE Healthcare) for 3 h in cold. Beads were extensively washed and GSTsRAGE was eluted with 10 mmol/L GSH in 50 mmol/L Tris-HCl, pH = 8.0. sRAGE was cleaved off the beads by incubating the bound fusion protein with PreScission protease. The final concentration

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of GST-sRAGE was approximately 400 ␮g/mL and of cleaved sRAGE was 25 ␮g/mL. Antibodies Antibodies against RAGE (N-16), transferrin receptor (TfR), flotillin1, calnexin, early endosome autoantigen 1 (EEA1) were purchased from Santa Cruz Biotechnology. Anti-actin and anti-␤1-integrin antibodies were from BD Biosciences, anti-caveolin-1 was purchased from Cell Signaling Technology, while anti-RAGE antibody (clone DD/A11) was from Chemicon, Millipore. Horse radish peroxidase (HRP)- and fluorescently labeled secondary antibodies were from Molecular Probes (Life Technologies). RAGE secretion by melanoma cells Cells were transfected with pcDNA3.1-hRAGE using Lipofectamine 2000 (Invitrogen). Twenty hours later, cells were incubated with medium containing 2% FBS and 2 mmol/L L-glutamine for 6 h. Where indicated, nystatin (Sigma-Aldrich) was added to the medium to a final concentration of 25 ␮g/mL. Cell supernatants were centrifuged and subjected to Western blot. RNAi and cell migration MelJuSo and SK-Mel28 cells were transfected upon seeding and again after 24 h with 40 nmol/L pre-designed Silencer siRNA #9627 or #110859 (Ambion, Life Technologies, denoted here siRNA#1 and siRNA#2, respectively), using Lipofectamine RNAiMAX (Invitrogen). Both siRNAs target sequences within RAGE ectodomain. The next day, cells were trypsinized and re-seeded in 6-well plates. Cells were analyzed for RAGE expression by Western blot and for cell migration by “wound healing” assay after 48 h. Scratches were made with a pipet tip, and cells were allowed to migrate towards the scratch for 7 or 9 h (MelJuSo cells), and 11 or 19 h (SK-Mel28 cells). A shorter migration time in the case of MelJuSo cells was chosen because of the higher proliferation rate of this cell line compared with SK-Mel28. Photomicrographs of cells were taken using a Nikon Eclipse TS100 microscope, with the 10× objective. Cell migration was quantified using Image J (NIH, USA), by measuring the area of the scratch in at least 4 different places per condition, in 3 independent experiments. As controls we used cells treated with the transfection reagent or transfected with Silencer Negative Control siRNA#1 (Ambion, Life Technologies), for which we detected similar levels of RAGE expression and cellular migration behavior. The degree of migration was calculated from the ratio of the final area and the one at time zero (T 0 h, immediately after the scratch was made), and expressed as a percentage of the control. Statistical analysis was done using Student’s t-test. Modifications were considered statistically significant at p < 0.05. Cell lysate preparation and Western blot detection of RAGE Melanocytes, MelJuSo, and SK-Mel28 cells were lysed in 25 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, containing 1% Triton X-100, cocktail of protease inhibitors (Roche or Santa Cruz Biotechnology), and 1 mmol/L PMSF. Cell lysates were passed 20 times through an insulin syringe, further incubated on ice for 30 min, and finally were centrifuged at 19 500g for 15 min at 4 °C. We aimed to obtain similar protein concentrations in the cell lysates. The amount of protein was finally adjusted by the addition of lysis buffer. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were further solubilized in sample buffer by boiling at 96 °C for 5 min. In some cases samples were incubated at 65 °C for 5 min. Five times concentrated sample buffer contained 15% SDS and 250 mmol/L dithiothreitol (DTT). Alternatively, DTT was freshly added to the samples to a final concentration of 50 or 100 mmol/L. For the electrophoresis under nonreducing conditions, DTT was replaced by water. Proteins in sample buffer were run on a 10% or 8% SDS-PAA gel, subjected to SDS-PAGE, then transferred to PVDF membrane (Millipore) at 225 mA for 1 h and 25 min. Membranes were blocked overnight in Published by NRC Research Press

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Fig. 1. Overexpression of full-length RAGE in melanoma cells. MelJuSo and SK-Mel28 cells were transfected with pcDNA3.1-hRAGE or empty vector. Twenty-four hours later, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, and blocked with 0.5% BSA. Cells were labeled for RAGE with DD/A11 antibody and secondary donkey anti-mouse coupled with Alexa594; nuclei were stained with DAPI. Cells were analyzed using Axio Imager.M2 microscope. Scale bar, 20 ␮m.

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5% nonfat dry milk (Fluka or Santa Cruz Biotechnology), containing 0.2% Tween-20 in Tris buffer saline, pH = 7.5 (TBS1x) (blotto). Blocked membranes were incubated at room temperature (RT) for 1 h 30 min with blotto containing primary antibodies (N-16 at 1:500 or DD/A11 at 1:3000). Membranes were washed 3 times, for 15 min, with TBS1x + 0.1% Tween-20. Secondary antibodies (Molecular Probes, Invitrogen), coupled with HRP were used at a concentration of 1:2000 or 1:4000 in blotto, by incubating with membranes for 1 h at RT. Blots were further washed 3 times, for 15 min, with TBS1x + 0.1% Tween-20, then shortly with TBS1x, before detection with enhanced chemiluminescent substrate (Thermo Scientific or Santa Cruz Biotechnology) or Super Signal West Femto Chemiluminescent Substrate (Pierce, Thermo Scientific). For competition Western blotting, we pre-incubated 800 ng of N16-antibody in blotto with 3200 ng GST-sRAGE in 2 mL blotto (or 1750 ng cleaved sRAGE) for 40 min at RT, under gentle mixing. As a control, we used the same volume of elution buffer. These mixtures (2 or 4 mL, depending of the blot dimensions) were further incubated with the blots for 1 h 30 min, and the standard Western blot detection was followed exactly in the same manner for all the blots. Subcellular fractionation of melanoma cells Subcellular fractionation of melanoma cells (MelJuSo and SK-Mel28) on a 4%–25% OptiPrep density gradient (Sigma-Aldrich) was performed using a method previously described (Macdonald and Pike 2005). Cells from 4 175 cm2 flasks of cultured MelJuSo and SK-Mel28 were homogenized in 25 mmol/L Tris-HCl pH 7.8, 250 mmol/L sucrose (base buffer), containing 1 mmol/L CaCl2, 1 mmol/L MgCl2, and protease inhibitors cocktail (Roche) by passing 20 times through a 22-gauge needle. After 1000g centrifugation, the pellet was homogenized again in a similar way. The two post-nuclear supernatants were combined and mixed with 50% OptiPrep made in base buffer and placed at the bottom of a centrifuge tube. A 4%–20% OptiPrep gradient was laid on top. After centrifugation at 52 000g for 90 min in SW41Ti Beckman rotor, 15 fractions were collected from the top and analyzed by SDS-PAGE and Western blot using detection with SuperSignal West Femto Chemiluminescent Substrate (Pierce) (for anti-RAGE and anti-TfR antibody detection) or with enhanced chemiluminescent substrate (Thermo Scientific or Santa Cruz Biotechnology) (for ␤1-

integrin, flotillin-1, caveolin-1, and EEA1 detection). Treatment of fraction 6 with EndoH or PNGase F (New England Biolabs) was done in accordance with the manufacturer’s protocol. Immunofluorescence microscopy Cells were fixed with 3% paraformaldehyde, permeabilized with 0.1% saponin for 1 h or 0.2% Triton X-100 for 4 min, blocked with 0.5% BSA, and labeled for RAGE (with DD/A11 antibody), followed by incubation with secondary antibodies conjugated with Alexa594. Where specified, cells were treated before fixation with drugs (50 ␮g/mL nystatin for 45 min or 5 ␮g/mL filipin for 12 min in cell medium). Cells were analyzed with a Nikon Eclipse E600, Axio Imager.M2, or Zeiss LSM 710 microscopes.

Results and discussion To investigate RAGE isoforms in tumor progression, we determined their expression profile in MelJuSo and SK-Mel28, cell lines established from a primary melanoma lesion and a metastasis, respectively. We first analyzed the morphology and migration of these cells upon overexpression or downregulation of RAGE. By immunofluorescence microscopy, we showed that both types of melanoma cells transfected with cDNA for the full-length, membranebound RAGE (lung isoform) had increased cellular connections and filopodia-like structures, compared with cells transfected with empty vector (Fig. 1, arrowheads). RAGE was labeled with the monoclonal anti-RAGE (DD/A11) antibody and secondary Alexa594coupled anti-mouse. The overexpressed receptor was localized intracellularly and at regions of plasma membrane as well as within those cell extensions, which had a pronounced tropism for neighboring cells. RAGE was previously found to promote neurite outgrowth (Huttunen et al. 2002). Moreover, RAGE interacts with components of the ECM and can function as an adhesion receptor (Bierhaus et al. 2006; Mizumoto and Sugahara 2013). The overexpression phenotype we obtained suggests that RAGE could play a primary role in cell-to-cell interactions in melanoma cells. A role for RAGE in cell motility has been previously documented (Hudson et al. 2008b). We evaluated RAGE involvement in the migration of the above mentioned tumor cells by silencing RAGE expression with two siRNAs. Cell migration was then assayed by wound healing. Silenced MelJuSo cells retained only 30% of the control Published by NRC Research Press

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Fig. 2. Effect of RAGE silencing by RNAi on melanoma cell migration. Melanoma cell lines MelJuSo and SK-Mel28 were transfected with two RAGE siRNAs, siRNA#1 and siRNA#2, and were analyzed for cell migration in a “wound healing assay”, performed as described under Materials and methods. Cells were photographed at time 0 h and at indicated times after the scratch was made (A), and their migration was measured using ImageJ software and quantified from 3 independent experiments. Results for the migration of RAGE silenced cells were expressed as a percentage of the control (ctrl) cell migration. MelJuSo cells transfected with RAGE siRNA#1 or siRNA#2 displayed an inhibition of 70% of their migration. SK-Mel28 cell migration was similar to that of the control. Each bar represents the mean +/− SD. *p < 0.05 vs. ctrl and **p < 0.01 vs. ctrl. (B) RAGE knock-down was checked by Western blot of cell lysates with anti-RAGE antibodies DD/A11 and N-16. (C) Cell lysates and culture supernatants of cells transfected or not with pcDNA3.1-hRAGE were analyzed by immunoblotting for RAGE (DD/A11 antibody). SK-Mel28 cells have a much lower capacity of generating sRAGE through the shedding of the transfected receptor than MelJuSo cells. (D) Micrographs of MelJuSo cells showed that silencing of soluble RAGE by siRNA#1 had a specific effect on plasma membrane integrity (arrow), while downregulation of full-length receptor induced cell elongation, especially in SK-Mel28 cells.

cell migration capacity, while SK-Mel28 cell migration was similar to that of the control (Fig. 2A). This result points to a different regulation of RAGE function in the two cell types. Western blotting with anti-RAGE antibodies (mouse monoclonal DD/A11 and goat polyclonal N-16, both antibodies directed against the extracellular part of the receptor) was used to check RAGE isoform downregulation in MelJuSo and SK-Mel28 cells (Fig. 2B). The two siRNAs reduced the expression of RAGE isoforms, full-length or soluble variants (with molecular weights in the range 47–60 kDa). However, the two siRNA oligos appeared to target RAGE isoforms differently, siRNA#1 most efficiently downregulating a 50 kDa form (marked with *) of the receptor in primary melanoma cells, which may represent an alternatively spliced, soluble variant of RAGE (Fig. 2B, Western blot with DD/A11). An endogenous soluble RAGE expressed in HEK cells was previously reported to be detected by the DD/A11 monoclonal antibody (Quade-Lyssy et al. 2013). Interestingly, in case of MelJuSo cells targeted with siRNA#1, another striking effect was the loss of plasma membrane integrity, as observed by light microscopy (Fig. 2D, indicated by

arrow), while downregulation of the full-length protein caused cell elongation (MelJuSo and SK-Mel28) (Fig. 2D). RAGE ectodomain is cleaved at the membrane sites through proteolytic activity of ADAM10 and MMP9, being thus released as a soluble form of the receptor (Raucci et al. 2008; Zhang et al. 2008). We readily detected soluble RAGE (sRAGE) in culture supernatants of hRAGE-transfected MelJuSo cells by immunoblotting (Fig. 2C). This form has a similar electrophoretic mobility with a protein of approximately 50 kDa in the cell lysate, most likely representing a soluble endogenous isoform of the receptor. While full-length RAGE (which runs at 55–60 kDa, indicated by N in Fig. 2B) was higher expressed in metastatic SK-Mel28 cells compared to primary MelJuSo cells, soluble RAGE variants appeared to be significantly downregulated in SK-Mel28 (Figs. 2B and 2C). This result would be in line with previous reports on the decrease of soluble RAGE transcripts in melanoma lesions (Leclerc et al. 2009) or of the endogenous soluble RAGE in other various tumors (Kalea et al. 2010). Based on the results of siRNA experiments, we propose that RAGE and sRAGE are needed for melanoma cell migration. Published by NRC Research Press

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Yet, there are melanoma types where this process is much less dependent on a RAGE function by itself, requiring additional factors and interactions with microenvironment to sustain RAGEinvolvement in metastasis. Previous studies showed an increased RAGE expression in several tumors as well as in melanoma cells (Abe et al. 2004; Sparvero et al. 2009). We confirmed the upregulation in melanoma cells compared to melanocytes by Western blot with N-16 anti-RAGE antibody. In addition to the 3 major bands of 50–60 kDa, higher molecular complexes above 120 kDa showed reactivity to the antibody (Fig. 3A). N-16 antibody has been previously shown to specifically recognize various RAGE isoforms in cell lines and tissues in the range 40–70 kDa (Gefter et al. 2009). Higher molecular weights RAGE forms (in the range 72–100 kDa), detected with other anti-RAGE antibodies were found to be upregulated in regions of rat brains following an ischemic event (Greco et al. 2012). In our study, as presented in Fig. 2B, the two antibodies we tested detected the full-length protein of approximately 55–60 kDa (double arrow), but also a specific set of RAGE forms. The high molecular mass proteins containing N-16 epitope are likely to represent the oligomerized receptor, since downregulation of RAGE by siRNA#2 consequently reduced Western blot signal for at least one of these complexes corresponding to the most upper band, above 260 kDa (indicated with arrow, see also next figure for a molecular weight estimate), as shown in Fig. 3B. Our results indicate that such an oligomeric complex of RAGE was found only in MelJuSo cell lysate, and not in melanocyte and SK-Mel28 lysates (Fig. 3A). RAGE has been previously shown to use several different strategies of dimerization and multimerization through the 3 subdomains of the extracellular region, which can involve some of its interacting partners, as a mean to transduce signalling of RAGE various ligands and (or) perform its role in adhesion (Wei et al. 2012; Xie et al. 2007; Xu et al. 2013; Yatime and Andersen 2013).

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Fig. 3. Analysis of melanoma RAGE isoforms by Western blotting. (A) Cell lysates of melanocytes and melanoma cells were run on a 10% SDS-PAA gel under reducing conditions (sample buffer contained 50 mmol/L DTT) and subjected to Western blot for RAGE detection with N-16 antibody. Several putative RAGE isoforms were found to be upregulated in MelJuSo and SK-Mel28 cells compared to melanocytes. Bands corresponding to higher molecular weight proteins (above 120 kDa) may correspond to oligomeric forms of the receptor. (B) Specificity of the N-16 antibody was tested on lysates of MelJuSo cells transfected with siRNA#2, compared with control cells. At least one oligomeric complex containing N-16 epitope was specifically downregulated in RAGE silenced MelJuSo cells (arrow), at the same time with the monomeric RAGE (arrowhead).

Association of RAGE in dimers and higher oligomeric forms is facilitated by the formation of disulfide bridges as well as various noncovalent interactions (Wei et al. 2012; Yatime and Andersen 2013). Oligomerization of the receptor, in its membrane or soluble forms, which was demonstrated by different approaches thus far, is seen as a crucial regulatory mechanism for defining high affinity binding regions to at least some of the RAGE ligands and (or) for the modulation of receptor function (Sarkany et al. 2011; Srikrishna et al. 2010; Xie et al. 2008). We looked into more detail at the mode of RAGE oligomerization in melanoma cells by performing SDS-PAGE under reducing and nonreducing conditions (Fig. 4A). MelJuSo and SK-Mel28 cells were lysed in a very similar manner (same volume of buffer, for which we obtained almost equal protein concentrations in the lysate), ensuring comparable solubilization of cell membranes. Proteins were reduced under treatment at 96 °C for 5 min with either 50 mmol/L or 100 mmol/L DTT, freshly added to the sample buffer, or water was used instead of DTT for the nonreducing condition. Proteins were further resolved on a 8% SDS-PAA gel and subjected to Western blot with anti-RAGE N-16 antibody. Even the high concentration of DTT of 100 mmol/L was not sufficient to completely reduce the oligomeric RAGE. However, the complexes were destabilized more efficiently, compared with 50 mmol/L DTT. Under nonreducing conditions, the detection of the monomeric RAGE forms was significantly reduced (arrowheads), we detected instead a complex of about 200 kDa, which is likely to represent the oligomeric RAGE stabilized by disulfide bridges. The nonreduced RAGE appeared at similar levels in MelJuSo and SK-Mel28 cell lysates (Fig. 4A, arrow on the right side of the blot). However, in MelJuSo, compared with SK-Mel28, there was a more complex RAGE association, as indicated by the smear and additional bands for the nonreduced sample, including a band above 260 kDa of the molecular marker. Published by NRC Research Press

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Fig. 4. Detection of the monomeric and oligomeric RAGE was inhibited upon pre-incubation of N-16 antibody with GST-sRAGE. (A) Total proteins from MelJuSo and SK-Mel28 cell lysates were incubated in sample buffer to which DTT was freshly added to a final concentration of either 50 or 100 mmol/L, and further resolved on a 8% SDS-PAA gel. For the nonreducing condition water was used instead of DTT. Proteins were transferred to PVDF membrane. N-16 antibody (800 ng) was incubated with 3200 ng GST-sRAGE (or buffer as a control) for 40 min, in 2 mL 5% milk/0.2% Tween-20. The mixtures were then incubated with the PVDF membranes for 1 h 30 min, and the usual Western blot protocol was followed. Actin detection was used as a control for total protein level in the lysates. (B) GST- sRAGE (23–327aa), expressed and purified from E. coli BL21(DE3) and PreScission cleaved sRAGE (23–327aa) were checked by SDS-PAGE electrophoresis. Recombinant proteins were quantified by Coomassie staining using a standard protein (BSA).

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sRAGE

To get further proof for the existence of the RAGE oligomers in melanoma cells we performed at the same time Western blot detection with N-16 antibody in the presence of a recombinant, soluble form of the receptor. The extracellular part (aa 23–327) of RAGE was cloned in pGEX-6P-1 and expressed in E. coli BL21(DE3), which we finally obtained either as a GST-fusion protein (GSTsRAGE) or as soluble RAGE (sRAGE) after cleaving off the GST with PreScission enzyme (Fig. 4B). In Fig. 4A we present the result of the Western blot after a pre-incubation step of the N-16 antibody with GST-sRAGE. Compared to the control Western blot, the detection

with antibodies pre-incubated with GST-sRAGE showed a significant decrease in the signal of around 55–60 kDa, especially of the upper band (upper arrowhead), most likely corresponding to the monomeric RAGE. In addition, we obtained a reduction of the high molecular protein complexes (indicated by arrow). To exclude artifacts of the boiling during sample preparation, the same amount of total protein deriving from cell lysates of similar protein concentrations from MelJuSo and SK-Mel28 cells was incubated at 65 °C for 5 min in a sample buffer under reducing conditions, in parallel with the treatment at 96 °C (not shown). Published by NRC Research Press

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Fig. 5. RAGE isoforms distribution by ultracentrifugation in a density gradient. (A) Melanoma cells were fractionated on a 4%–25% OptiPrep gradient, and fractions (1–14) were analyzed on a 10% SDS-PAA gel, under reducing conditions (sample buffer contained 50 mmol/L DTT) and probed by Western blot for RAGE (N-16 antibody), and for the indicated organelle markers. A pool of a complex containing RAGE epitope was detected in the light fractions of the density gradient in the case of MelJuSo cellular fractionation. (B) Fractions 5, 10, and 11 of the density gradient were incubated in sample buffer in the presence or absence of freshly added DTT (100 mmol/L final concentration), at 96 °C for 5 min. Proteins were further resolved on a 8% SDS-PAA gel and transferred to PVDF membrane for Western blot with anti-RAGE. Binding of N-16 was competed in a parallel Western blot by using GST-sRAGE, as described in Fig. 4A. (C) Fractions 6 and 10 of a density gradient for MelJuSo and SK-Mel28 cell fractionations were run on a 10% SDS-PAA gel upon reducing with 50 mmol/L DTT and probed by Western blotting with N-16 antibody. Flotillin-1 content in these fractions is also shown by Western blot. (D) The 140 kDa RAGE complex was sensitive to EndoH and PNGase F treatments. Detection by Western blot was done with N-16 anti-RAGE antibody.

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Boiling samples under reducing conditions caused a decrease in the high oligomeric form, suggesting that the complexes have been partially broken. We nevertherless saw the same reduction of the oligomeric form after pre-incubation with GST-sRAGE or with cleaved sRAGE (data not shown). Altogether our results bring evidence for an increased and more complex oligomerization of RAGE in primary melanoma MelJuSo cells, compared to the metastatic SK-Mel28 cells. RAGE has been previously found to become localized to lipid rafts, especially upon ligand stimulation (Sbai et al. 2010). Lipid rafts are defined as dynamic assemblies of sphingolipid, cholesterol, and proteins, which constitute membrane platforms to function in signalling and trafficking, as well as for receptor oligomerization (Lingwood and Simons 2010; Shi et al. 2013). We therefore wanted to investigate whether RAGE, either in its monomeric or in oligomeric forms, was constitutively localized to lipid rafts. To this purpose we undertook subcellular fractionation

in which homogenates of MelJuSo and SK-Mel28 cells were fractionated by ultracentrifugation on a 4%–25% OptiPrep density gradient. The method used was designed as a simple, detergent-free method for lipid raft isolation (Macdonald and Pike 2005). Western blot analysis in Fig. 5A shows MelJuSo RAGE isoforms distribution in the gradient. While the bulk of RAGE (monomeric and oligomeric species) was separated in the middle and bottom regions of the gradient, an 140 kDa form (indicated with = > in Fig. 5A) partitioned into the lighter fractions, presumably containing lipid raft membranes but not exclusively (fractions had the well described lipid raft markers, caveolin-1 and flotillin-1, but also some TfR, and ␤1-integrin). We investigated RAGE oligomeric status through the gradient by SDS-PAGE, under reducing (100 mmol/L DTT) and nonreducing conditions, followed by Western blot detection with or without pre-incubation with GSTsRAGE (Fig. 5B). The result on the fractions showed once more that the oligomeric RAGE of approximately 200 kDa is formed through Published by NRC Research Press

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Fig. 6. Different melanoma cells showed distinct RAGE localization patterns by immunofluorescence microscopy. (A) Melanoma cells were fixed with 3% paraformaldehyde, incubated in blocking buffer containing 0.1% saponin and labeled for RAGE with DD/A11 antibody (red). Cells were analyzed with a Nikon Eclipse E600 microscope. In MelJuSo and A375 cells RAGE was found clustered at the plasma membrane sites, while in SK-Mel28 and MNT-1 cells the receptor had a more homogenous distribution throughout the cells. Scale bar, 20 ␮m. (B) Integrity of the cholesterol-rich membranes is important for RAGE localization and function in primary melanoma cells. MelJuSo and SK-Mel28 cells were treated with 50 ␮g/mL nystatin for 45 min. RAGE re-localization was then analyzed by immunofluorescence confocal microscopy, after staining with DD/A11 antibody. Scale bar, 10 ␮m. (C) pcDNA3.1-hRAGE-transfected MelJuSo cells were treated with 25 ␮g/mL nystatin for 6 h. sRAGE was detected by Western blot in culture supernatants. Calnexin detection was used as a control for total protein in the cell lysates. Nystatin treatment increased the level of sRAGE. The results are representative for 3 independent experiments.

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disulfide bonds, whereas the other two putative RAGE complexes (the one of 140 KDa and the complex above 260 kDa) are most likely based on other types of interactions, either involving selfassociation or association through ligands or other partner molecules. One cannot exclude, however, the possibility that these are nonspecific bands. SK-Mel28 fractions 6 and 10, resolved by SDSPAGE under reducing condition, and Western blot, were found to likely contain less of the high mass RAGE species, in comparison with same fractions separated from MelJuSo cells (Fig. 5C). The 140 kDa protein exhibited sensitivity to both EndoH and PNGase F treatment, meaning that it is N-glycosylated. A similar glycosylation pattern with complex and high mannose glycans has been previously described for RAGE (Srikrishna et al. 2010; Turovskaya et al. 2008). In addition, glycan modifications of RAGE molecule were found to have implications for the pathophysiological function of RAGE in tumorigenesis (Turovskaya et al. 2008). By employing several approaches it was demonstrated that sRAGE forms tetramers (of 140–150 kDa, (Xie et al. 2007)) even when the protein is fully glycosylated (Srikrishna et al. 2010). Although it is tempting to speculate at this moment, since we have detected only a partial de-glycosylation with PNGase F of the monomeric endog-

enous RAGE in the lysate, while the whole transfected RAGE was sensitive to the glycosidase treatment (not shown), the fact that an N-glycosylated oligomer of RAGE or sRAGE could be localized to lipid rafts requiers additional investigation. To further look into RAGE localization in melanoma cells, we used immunofluorescence microscopy. In spite of a similar level of the overall fluorescent signal for RAGE (measured also by flow cytometry, not shown), distinct patterns of the receptor localization were identified by microscopy (Fig. 6A). In MelJuSo and A375 cells, RAGE had an obvious polarized distribution. In these cells, the receptor was found intracelullarly and in patches at the plasma membrane or subplasmalemal, at leading edges and membrane ruffles, and sometimes at cell-to-cell contact sites, while in SK-Mel28 and MNT-1 cells, its localization was more dispersed, with an accumulation at cell protrusions. Two permeabilization methods preceding the immunolabeling (0.1% saponin versus 0.2% Triton X-100) gave similar results in terms of RAGE staining (data not shown). Since labeling with N-16 antibody was poor, we do not know whether the more clustered localization of RAGE in MelJuSo cells detected with DD/A11 could be linked to a more complex oligomerization of the receptor in these cells or to a Published by NRC Research Press

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Popa et al.

soluble RAGE form, expressed at a higher level compared to the metastatic SK-Mel28 cells. Nystatin and filipin, based on their property to bind membrane cholesterol, are routinely used to disrupt lipid rafts (Murai 2012). Immunofluorescence confocal microscopy on nystatin-treated cells showed that in MelJuSo cells the drug clearly re-localized RAGE from the prominent patch-like structures to more dispersed punctuate structures, while in SK-Mel28 cells changes were less evident (Fig. 6B). Same results were obtained upon cell exposure to filipin (not shown). We have also checked the specificity of nystatin treatment towards a membrane raft associated protein, namely caveolin-1, which showed a more diffused pattern upon incubation with the drug, while at the same time transferrin steady-state distribution was not altered (data not shown). Based on the result of subcellular fractionation in which we saw the majority of RAGE outside the lightest fractions (which most likely contain the lipid rafts), we are cautious to say that the re-localization of RAGE is directly linked to lipid rafts microdomains destruction. Nevertherless, RAGE localization seems to be dependent on the integrity of the cholesterol-rich membranes in primary melanoma cells. To check the influence of the cholesterol-rich/lipid raft destabilizing drug, nystatin, on sRAGE formation, we looked by Western blot at the level of sRAGE in culture supernatants of hRAGE transfected MelJuSo cells, which were treated for 6 h with nystatin (Fig. 6C). Exposure of MelJuSo cells to 25 ␮g/mL nystatin did not reduce sRAGE in the cell supernatant but rather caused an increase, suggesting that the integrity of cholesterol-rich/lipid raft membranes is not critical for RAGE cleavage. However, these membranes could modulate sRAGE level by excluding the factors involved in the shedding of the receptor. This result could be significant, since modulation of the other secreted form of the receptor, the endogenous soluble splice variant of RAGE, was shown to inhibit tumorigenesis, both by directly changing tumor cell survival, migration, and invasion, and by suppresing the angiogenesis (Kalea et al. 2010). While the current manuscript was under revision, Quade-Lyssy et al. have shown that cholesterol lowering compounds, statins, could promote RAGE shedding, increasing the sRAGE production of HEK cells overexpressing hRAGE or of mouse alveolar epithelial cells at endogenous level (Quade-Lyssy et al. 2013). In conclusion, in the present study we found that melanoma cells with different degrees of malignancy express RAGE at similar levels, although there can be a fine tuning of the receptor function in these cells by the isoform type produced and posttranslational events. We found differences in RAGE oligomerization, such as the requirement for higher orderly oligomeric species formation in primary melanoma cells MelJuSo, compared with the metastatic SK-Mel28 cell line. We obtained results suggesting a higher degree of N-glycosylation of the receptor, oligomerization, and targeting to specialized membrane domains (cholesterol-rich/ lipid rafts) in MelJuSo cells compared with SK-Mel28 cells. Moreover, we show that metastatic SK-Mel28 cells have a much reduced capacity of generating sRAGE, both endogenously and by ectodomain shedding. The functional relevance of these differences requires further investigations.

Acknowledgements We would like to thank Dr. Simona Ghenea for help with the cloning of pGEX-6P-1-sRAGE and Dr. Viorica Ivan for critically reading the manuscript. This work was supported from European Social Fund POSDRU/89/1.5/S/60746, the Romanian Academy project 3, Institute of Biochemistry of the Romanian Academy, and the reintegration grant 14/1.10.2007 to I.P., from the National Council of Scientific Research in Higher Education (CNCSIS).

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