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J Biol Chem, Vol. 274, Issue 28, 19919-19924, July 9, 1999


Receptor for Advanced Glycation End Products (RAGE)-mediated Neurite Outgrowth and Activation of NF-kappa B Require the Cytoplasmic Domain of the Receptor but Different Downstream Signaling Pathways*

Henri J. HuttunenDagger §, Carole Fages§, and Heikki Rauvala

From the Laboratory of Molecular Neurobiology, Institute of Biotechnology, and Department of Biosciences, Division of Biochemistry, University of Helsinki, P. O. Box 56 (Viikinkaari 5), University of Helsinki, Helsinki FIN-00014, Finland

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Receptor for advanced glycation end products (RAGE) mediates neurite outgrowth in vitro on amphoterin-coated substrates. Ligation of RAGE by two other ligands, advanced glycation end products or amyloid beta -peptide, is suggested to play a role in cell injury mechanisms involving cellular oxidant stress and activation of the transcription factor NF-kappa B. However, the RAGE signaling pathways in neurite outgrowth and cell injury are largely unknown. Here we show that transfection of RAGE to neuroblastoma cells induces extension of filopodia and neurites on amphoterin-coated substrates. Furthermore, ligation of RAGE in transfected cells enhances NF-kappa B-dependent transcription. Both the RAGE-mediated neurite outgrowth and activation of NF-kappa B are blocked by deletion of the cytoplasmic domain of RAGE. Moreover, dominant negative Rac and Cdc42 but not dominant negative Ras inhibit the extension of neurites induced by RAGE-amphoterin interaction. In contrast, the activation of NF-kappa B is inhibited by dominant negative Ras but not Rac or Cdc42. These data suggest that distinct signaling pathways are used by RAGE to induce neurite outgrowth and regulate gene expression through NF-kappa B.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The receptor for advanced glycation end products (RAGE)1 is a member of the immunoglobulin superfamily of cell-surface molecules and shares closest homology with the neural cell adhesion molecule NCAM (1, 2). RAGE exhibits a wide tissue distribution (3) and interacts with a range of ligands.

Advanced glycation end products (AGE) resulting from nonenzymatic glycation of proteins and lipids accumulate during normal aging and at an accelerated rate in diabetes (4-6). In diabetes, hyperglycemia-driven accumulation of AGEs has been suggested to be involved in the pathogenesis of diabetic vascular disease. The interaction of AGEs with RAGE, one of their cell-surface receptors, results in perturbation of a variety of vascular homeostatic functions and has been shown to play a major role in the development of diabetic vasculopathy (7-9).

In Alzheimer's disease, deposition of amyloid beta -peptide containing plaques in the brain correlates with progressive neuronal dysfunction leading to dementia (10, 11). Extracellular amyloid beta -peptide induces neuronal cell death presumably by interaction with cell-surface receptors. Neuronal RAGE has been shown to interact with amyloid beta -peptide resulting in cellular oxidant stress and activation of NF-kappa B (12). Moreover, ligation of neuronal RAGE by amyloid beta -peptide has been shown to trigger a proinflammatory pathway leading to activation of microglial cells in Alzheimer's disease (13).

Amphoterin is a heparin-binding, neurite outgrowth-promoting protein isolated from neonatal rat brain (14-17). Amphoterin is abundantly expressed in the central and peripheral nervous systems during the later phases of embryonic development (14, 18, 19). RAGE binds amphoterin in a saturable and dose-dependent manner (20). Furthermore, anti-RAGE F(ab')2 or soluble ectodomain of RAGE blocks neurite outgrowth of cortical neurons on amphoterin-coated substrates. Amphoterin and RAGE are coexpressed in the developing rat nervous system. Since AGEs and amyloid beta -peptide are not expected to be present under non-pathophysiological conditions, amphoterin has been suggested to be the physiological ligand for RAGE (20).

Although previous studies have identified RAGE as a potential therapeutic target both in diabetic vascular disease and in Alzheimer's disease, the basic cell biology of RAGE is not well understood. The ligation of RAGE with either of the pathophysiologically relevant ligands, AGE or amyloid beta -peptide, is suggested to result in generation of cellular oxidant stress and activation of the transcription factor NF-kappa B (12, 21). It is still not clear whether RAGE induces oxidant stress by tethering these oxidizing agents on the cell surface or by mechanisms of cell signaling. However, RAGE-mediated induction of oxidant stress has been shown to activate a Ras-MAP kinase pathway that may eventually lead to the nuclear translocation of NF-kappa B (22). It is not known whether this signaling pathway is responsible for RAGE-mediated neurite outgrowth on amphoterin-coated substrates. Moreover, the proximal components of the RAGE signaling pathway are unknown.

In this study, we demonstrate that the cytoplasmic domain of RAGE is required for both RAGE-mediated neurite outgrowth and activation of NF-kappa B-dependent transcription. We also show that not Ras but the Rho family small GTPases Rac and Cdc42 are involved in the neurite outgrowth induced by RAGE-amphoterin interaction. However, we were unable to find evidence for the involvement of Rac and Cdc42 in the RAGE-mediated activation of NF-kappa B suggesting that a signaling mechanism consisting of two parallel pathways can be activated by the cytoplasmic domain of RAGE.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- Human RAGE cDNA was a generous gift from Dr. David Stern, Columbia University, New York. The cytoplasmic domain mutant (Delta 367-404) was generated by polymerase chain reaction using the following primers: 5'-ATAGTCGACATGGCAGCCGG-3' and 5'-ATAGTCGACTTACCGCCTTTGCCA-3'. Full-length RAGE and the cytoplasmic deletion mutant were subcloned into pcDNA3 expression vector (Invitrogen) containing a neomycin selection cassette. The full-length cDNAs for C3 transferase, N17Rac, and N17Cdc42 were a generous gift from Dr. Alan Hall, MRC, London, UK. The cDNA for N17H-Ras was a generous gift from Dr. Johan Peränen, University of Helsinki. These cDNAs were subcloned into pRK5 expression vector containing a Myc epitope tag. The cis-reporter plasmid pNFKB-Luc (Stratagene) contained a luciferase cDNA under a regular TATA box and an enhancer element with five NF-kappa B-binding sites. pFC-MEKK (the catalytic domain of MEKK, 360-672; Stratagene) was used as a positive control in the NF-kappa B assays. The authenticity of all constructs was confirmed by sequencing.

Cell Culture and Transfections-- N18 mouse neuroblastoma cells and C6 rat glioma cells were cultured in Dulbecco's modified Eagle's medium supplemented with 100 units/ml penicillin G and 0.1 mg/ml streptomycin and 10% fetal calf serum. N18 cells were transfected with Tfx-50TM reagent (Promega) according to the manufacturer's instructions. G418 (600 µg/ml; Life Technologies, Inc.) was added to the medium 48 h after transfection to select the stably transfected clones. The expression of RAGE was verified by Northern blotting using the 1.2-kilobase RAGE cDNA as a probe. For further experiments the stable transfectants were cultured in a medium containing 100 µg/ml G418. C6 cells were transfected by electroporation. Briefly, 7.5 × 106 cells in the normal culture medium containing 5 mM BES (Sigma) were added to an electroporation cuvette (0.4-cm electrode gap; Bio-Rad) together with 5 µl of carrier DNA (10 mg/ml herring sperm DNA; Roche Molecular Biochemicals) and 10 µg of construct DNA. 1:1:2 ratio of RAGE:dominant negative mutant:cis-reporter plasmid was used. After 5 min on ice the cells were electroporated with a Gene-Pulser (Bio-Rad) at 340 V and 500 millifarads. After a 10-min incubation at 40 °C the cells were washed with medium and plated on regular tissue culture plates (Corning Glass).

Preparation of Recombinant and Glycated Proteins-- Baculovirus-derived recombinant amphoterin and HB-GAM were prepared and purified as described previously (17, 23). AGE-modified bovine serum albumin was prepared by preincubation of BSA (1 mM; fraction V, Sigma) with 1 M glucose at 37 °C for 10 weeks in PBS containing 0.1 µg/ml pepstatin, 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, 1.5 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and 1 mM NaN3. The unincorporated glucose was removed by dialysis against PBS (24 h, twice). The concentration of AGE-BSA was determined by the method of Bradford (Bio-Rad).

Neurite Outgrowth Assays and Immunocytochemistry-- Neurite outgrowth assays were performed essentially as described before (24). Subconfluent, serum-starved N18 cells were detached by incubation in PBS containing 0.5 mM EDTA for 10 min followed by vigorous pipeting. Nunc Lab-Tek chamber slides were coated by incubating with recombinant amphoterin or HB-GAM solution (10-20 µg/ml) at 37 °C for 1 h. Cells were plated on coated wells and grown in serum-free Dulbecco's modified Eagle's medium containing 10 mg/ml bovine serum albumin for 24 h and fixed with 4% paraformaldehyde for 20 min. After permeabilization with 0.2% Triton X-100 for 10 min, the cells were blocked with 2% BSA in PBS and incubated for 1 h at room temperature with anti-Myc tag antibodies (1:500; clone 9E10, Upstate Biotechnology). The primary antibodies were detected with fluorescein isothiocyanate-labeled goat anti-mouse antibodies (Jackson ImmunoResearch Laboratories, Inc.). F-actin was detected simultaneously with TRITC-labeled phalloidin (Sigma). Conventional fluorescence (Olympus Provis 70) and confocal laser fluorescence (Zeiss LSM 410 Invert Laser Scan) microscopes were used to analyze the results. The proportion of neurite-bearing cells (processes longer than one diameter of the cell) was counted on five random fields of three independent experiments. Statistically significant differences between control and experimental conditions (Student's t test) are indicated with asterisks in the bar graphs. PD98059, wortmannin (Calbiochem), and N-acetyl-L-cysteine (Sigma) were used in indicated concentrations and added to cells 30 min before plating on amphoterin matrix.

NF-kappa B-Luciferase Assay-- Serum-starved C6 cells were detached by incubation in PBS containing 0.5 mM EDTA for 10 min followed by vigorous pipeting. 60-mm non-tissue culture-treated plates were coated with recombinant amphoterin as described above. Equal amount of cells (~2 × 106 cells) in serum-free Dulbecco's modified Eagle's medium containing 10 mg/ml bovine serum albumin was plated on amphoterin plates and regular tissue culture plates to which AGE-BSA was added after letting the cells attach for 2 h. The cells were stimulated under these conditions for 20-24 h after which luciferase activity was determined (48 h post-transfection) using standard reagents (Stratagene) and measured in a luminometer (Bio-Orbit, 1254 Luminova).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RAGE-mediated Neurite Outgrowth on Amphoterin Matrix Requires the Cytoplasmic Domain of the Receptor-- In order to study further the role of RAGE as a neurite outgrowth receptor for amphoterin, we produced stable N18 neuroblastoma cell lines expressing either a full-length RAGE or a deletion mutant lacking the cytoplasmic domain (amino acids 367-404) of RAGE. These cells were serum-starved and grown overnight on glass slides coated with either 10 or 20 µg/ml of recombinant amphoterin. Filamentous actin was then stained with TRITC-phalloidin to study morphological differences between the cell lines. As shown in Fig. 1, cells expressing the full-length RAGE (designated as N18/RAGE) were capable of extending numerous filopodia on slides coated with 10 µg/ml amphoterin (Fig. 1A), whereas cells expressing the cytoplasmic deletion mutant (designated as N18/RAGEDelta cyto; Fig. 1B) or mock-transfected cells (Fig. 1C) did not display such morphology. When grown on 20 µg/ml amphoterin-coated slides a proportion of N18/RAGE cells was capable of extending neurites (Fig. 1D). In contrast, N18/RAGEDelta cyto or N18/mock cells grew few processes and hardly any neurites (Fig. 1, E and F, respectively). In comparison to the cells transfected with the cytoplasmic domain deletion mutant of RAGE (2.7 ± 2.1%) or empty vector (3.1 ± 1.7%), the cells expressing full-length RAGE (20.0 ± 5.6%) had nearly 10 times higher capacity of growing neurites on amphoterin matrix (Fig. 1G, black bars).


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  		Fig. 1.   Deletion of the cytoplasmic domain of RAGE blocks process growth on amphoterin matrix. N18 neuroblastoma cells were stably transfected either with full-length RAGE (A and D), the cytoplasmic domain deletion mutant RAGEDelta cyto (B and E), or empty vector (C and F). Serum-starved cells were grown on glasses coated with either 10 µg/ml amphoterin (A-C) or 20 µg/ml amphoterin (D-F) for 20 h and stained with TRITC-phalloidin to visualize F-actin. Proportions of neurite-bearing cells on amphoterin (black bars) or HB-GAM matrices (gray bars) were calculated (G) in five random fields in three independent experiments. The expression of RAGE was confirmed by Northern blotting (H). All values represent the mean ± S.D. (n = 3). *, p < 0.05; bars, 10 µm (A-C); 30 µm (D-F).

To demonstrate that RAGE-mediated neurite outgrowth was specific for amphoterin-coated substrates, parallel experiments were performed with the cells plated on HB-GAM-coated slides. HB-GAM is another neurite outgrowth-promoting protein with similar characteristics as amphoterin, such as high affinity binding to heparin and a sequence rich in basic amino acids (for a review see Ref. 25). N18/RAGE cells grown on HB-GAM matrix displayed no morphological difference in comparison to N18/RAGEDelta cyto or N18/mock cells (<5% neurite-bearing cells in each) (Fig. 1G, gray bars). Thus, the neurite outgrowth-promoting effect of RAGE is specific for amphoterin and requires the presence of the cytoplasmic domain of the receptor.

Rac and Cdc42 but Not Ras-MAP Kinase Pathway Are Required for RAGE-mediated Neurite Outgrowth-- The Rho family of small GTPases is known to regulate various aspects of the actin cytoskeleton. In fibroblasts, Rho regulates the formation of stress fibers and focal complexes, Rac regulates membrane ruffling and the formation of lamellipodia, and Cdc42 regulates the formation of filopodia (reviewed in Ref. 26). Recent evidence suggests that they may also be involved in neuritogenesis and growth cone signaling (27-29). Considering the filopodial morphology of N18/RAGE cells grown on amphoterin matrix, we wanted to determine whether the Rho family GTPases are involved in RAGE signaling. Dominant negative mutants (T17N) of Rac or Cdc42 or a Rho-inhibitory molecule C3 transferase were expressed in N18/RAGE cells. As shown in Fig. 2, transient overexpression of either N17Rac or N17Cdc42 in N18/RAGE cells completely abolished neurite outgrowth on amphoterin matrix, whereas expression of the C3 transferase did not have a significant effect on RAGE-mediated neurite outgrowth.


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  		Fig. 2.   Rac and Cdc42 but not Rho or Ras are required for RAGE-mediated neurite outgrowth on amphoterin matrix. N18 neuroblastoma cells stably transfected with full-length RAGE were transiently transfected with Myc-tagged C3 transferase (A and E) or Myc-tagged dominant negative mutants N17Rac (B and F), N17Cdc42 (C and G), or N17Ras (D and H). After the serum-starved cells were grown on amphoterin matrix (20 µg/ml) for 20 h, F-actin was visualized with TRITC-phalloidin (A-D) and expression of transfected plasmids with monoclonal anti-Myc tag antibody (9E10) (E-H). Proportions of Myc-stained cells bearing neurites were calculated (I). Control cells were transfected with beta -galactosidase and stained with monoclonal anti-beta -galactosidase antibody. All values represent the mean ± S.D. (n = 3). **, p < 0.01; bars, 20 µm.

The binding of RAGE to the two other ligands, AGE and amyloid beta -peptide, has been shown to result in induction of cellular oxidant stress (12, 21). Recently RAGE-mediated induction of cellular oxidant stress has been shown to trigger a Ras-dependent MAP kinase pathway (22). Moreover, phosphatidylinositol 3-kinase has been shown to be recruited to Ras by oxidant stress (30). We therefore determined whether components of this Ras-MAP kinase pathway are involved in RAGE-mediated neurite outgrowth. Expression of a dominant negative mutant (T17N) of Ha-Ras in N18/RAGE cells did not affect RAGE-mediated neurite outgrowth (Fig. 2, D, H, and I). To demonstrate also that the other components of the known RAGE signaling pathway are dispensable for RAGE-mediated neurite outgrowth, N18/RAGE cells were grown on amphoterin matrix in the presence of either an antioxidant N-acetyl-L-cysteine, a MEK (MAP kinase kinase) inhibitor PD98059, or a PI 3-kinase inhibitor wortmannin. None of these compounds had a significant effect on RAGE-mediated neurite outgrowth (Fig. 3). These data indicate that both Rac and Cdc42 but not Rho or Ras-MAP kinase pathway are involved in RAGE-mediated neurite outgrowth.


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  		Fig. 3.   Inhibition of redox stress, MEK, or PI 3-kinase has no effect on RAGE-mediated neurite outgrowth on amphoterin matrix. Serum-starved N18 neuroblastoma cells stably transfected with full-length RAGE were grown overnight in the presence of either an antioxidant N-acetyl-L-cysteine (NAC), a MEK inhibitor PD98059, or a PI 3-kinase inhibitor wortmannin. The cells were stained with TRITC-phalloidin, and the proportions of neurite-bearing cells were calculated in five random fields in three independent experiments. The highest concentration of Me2SO (0.5%) used in experimental conditions was added to the control cells. All values represent the mean ± S.D. (n = 3).

The Cytoplasmic Domain of RAGE Is Required for Activation of NF-kappa B by RAGE Ligands-- To determine whether the cytoplasmic domain of RAGE is also required for RAGE-mediated activation of nuclear factor-kappa B, we expressed the full-length RAGE or the cytoplasmic domain deletion mutant of RAGE together with an NF-kappa B-responsive reporter gene (luciferase) construct in C6 rat glioma cells. This system was used because high transient expression levels could be obtained together with high efficiency transfection. Cells were serum-starved and stimulated overnight either on amphoterin matrix (20 µg/ml) or with AGE-modified BSA in solution (500 µg/ml). The enhancement of NF-kappa B-dependent transcription was then quantitated from the cell lysates by measuring the luciferase activity. Non-stimulated mock-transfected cells were used as a control of basal transcriptional activity. As shown in Fig. 4A, both amphoterin (black bars) and AGE (gray bars) were capable of activating NF-kappa B-dependent transcription in the cells transfected with full-length RAGE. With both amphoterin (337 ± 86%) and AGE (377 ± 104%) the activation of NF-kappa B-dependent transcription was significantly higher than in control cells. The level of NF-kappa B-dependent transcription in the cells transfected with RAGEDelta cyto was not remarkably higher than in similarly treated mock-transfected cells. This indicates that the cytoplasmic domain of RAGE is also needed for activation of NF-kappa B, and in addition to AGE and amyloid beta , amphoterin is also capable of activating NF-kappa B.


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  		Fig. 4.   Activation of NF-kappa B by RAGE ligation requires both the cytoplasmic domain of RAGE and functional Ras but not Rho family GTPases. A, C6 glioma cells were transiently transfected with an NF-kappa B-responsive cis-reporter gene construct together with either full-length RAGE, the cytoplasmic domain deletion mutant RAGEDelta cyto, or empty vector. Serum-starved cells were grown overnight either on amphoterin matrix (20 µg/ml; black bars) or in the presence of AGE (500 µg/ml; gray bars). Non-stimulated mock-transfected cells were used as a control of basal transcriptional activity (hatched bar). Relative luciferase activity is shown. B, C6 glioma cells were transiently transfected with an NF-kappa B-responsive cis-reporter gene construct together with the full-length RAGE and one of the dominant negative constructs N17Rac, N17Cdc42, N17Ras, or C3 transferase. Serum-starved cells were stimulated as in panel A. Relative luciferase activity is shown. All values represent the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01.

Activation of NF-kappa B by RAGE Depends on Ras but Not the Rho Family GTPases-- In addition to the central role of Rho family small GTPases in the regulation of the actin cytoskeleton, they have also been shown to function as regulators of specific transcription factors such as NF-kappa B (31). As Rac and Cdc42 are clearly involved in RAGE-mediated neurite outgrowth, we wanted to see whether, in addition to Ras, these Rho family small GTPases are also involved in RAGE-mediated activation of NF-kappa B. C3 transferase or one of the dominant negative mutants N17Rac, N17Cdc42, or N17Ras were transfected into C6 cells together with full-length RAGE and the NF-kappa B-responsive reporter gene construct. The relative luciferase activity in proportion to similarly stimulated RAGE-transfected cells was measured from cell lysates after an overnight stimulation with either amphoterin (20 µg/ml; black bars) or AGE (500 µg/ml; gray bars) and is shown in Fig. 4B. The NF-kappa B-dependent transcriptional activity was only inhibited in cells co-transfected with N17Ras. The dominant negative Ras was able to inhibit significantly both amphoterin-induced (67 ± 4.3%) and AGE-induced (77 ± 7.5%) activation of NF-kappa B-dependent transcription, whereas inhibition of Rho, Rac, or Cdc42 had no significant effect. These data indicate that Ras but not the Rho family members are involved in the RAGE-mediated activation and apparent nuclear translocation of NF-kappa B.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

During the last few years, the signal transduction pathways responsible for neuronal differentiation and neurite outgrowth have been a subject for intense research. We have been interested in a particular receptor, namely RAGE, recently shown to be capable of mediating neurite outgrowth on amphoterin-coated substrates (20). Amphoterin is a member of the high mobility group proteins (17) and has been shown, in addition to its putative function in the nucleus, to have extracellular functions in the developing nervous and hematopoietic systems (14-19, 32-35). Hori et al. (20) showed that RAGE binds amphoterin in a dose-dependent manner, mediates neurite extension on amphoterin-coated substrates, and that amphoterin and RAGE have a spatially and temporally similar expression pattern in the developing nervous system. The effect of the two other ligands of RAGE, AGE and amyloid beta -peptide (1, 12), on cells is dramatically different. Both can generate significant cellular oxidant stress and activate transcription factor NF-kappa B in a RAGE-dependent manner (12, 21). Despite intensive investigation it is still unknown whether RAGE induces oxidant stress by bringing AGE and amyloid beta -peptide, both sources of reactive oxygen species themselves (21, 36), close to the cell surface or by mechanisms of cell signaling. Thus, the signaling mechanism of RAGE presents several interesting questions. In this study our aim was to determine whether RAGE acts as a signaling receptor in a manner that requires the cytoplasmic domain of the receptor.

Transfection of full-length RAGE to N18 neuroblastoma cells is sufficient to induce neurite outgrowth on amphoterin matrix further confirming the role of RAGE as a neurite outgrowth receptor of amphoterin. However, when a cytoplasmic domain deletion mutant is transfected to N18 cells neurite outgrowth is reduced to the level of mock-transfected cells. This implies that the cytoplasmic domain of RAGE interacts with a molecule or perhaps a signaling complex necessary to initiate neurite extension. When RAGE-transfected N18 cells are grown on a lower concentration of amphoterin, extension of numerous filopodia rather than neurites is observed. This suggests a dose dependence of amphoterin-induced morphological changes.

Rho family small GTPases are now widely accepted to be key regulators of the actin cytoskeleton (reviewed in Ref. 26). It is becoming apparent that these signaling molecules are also critical components of the cytoskeletally driven neurite outgrowth (27-29). The striking resemblance of the RAGE-transfected N18 cells grown on lower concentrations of amphoterin to the typical Cdc42-induced filopodial morphology (37) suggested that the Rho family members might be involved in RAGE signaling. Indeed, dominant negative constructs of both Rac and Cdc42 completely blocked the RAGE-mediated neurite outgrowth on amphoterin matrix, whereas inhibition of Rho by C3 transferase had no effect. This is reasonable since Rho has been shown to be the mediator of the lysophosphatidic acid-induced neurite retraction rather than the extension of neurites (28). Since inhibition of either Rac or Cdc42 alone is able to block RAGE-mediated neurite outgrowth, it seems that they lie on the same signaling pathway. In fact it was recently shown that Cdc42 can activate Rac through PAK, a downstream effector of both Cdc42 and Rac (38). However, considering the filopodial morphology of N18/RAGE cells on the lower concentration of amphoterin, it is possible that Cdc42 and Rac mediate their effects on the cytoskeleton independently, perhaps in different phases of neuritogenesis.

The p21-activated kinase (PAK) family of serine/threonine kinases has been identified as targets for active Rac and Cdc42 (39). PAK family members are considered as main candidate effectors mediating the downstream effect of Rac and Cdc42 on the actin cytoskeleton (40, 41). Recently, Daniels et al. (42) reported that membrane targeting of PAK1 via a C-terminal isoprenylation sequence is sufficient to induce neurite outgrowth in PC12 cells independently of PAK1 kinase activity. Quite interestingly, by using truncated mutants they found that an acidic glutamate/aspartate-rich region of PAK1 is necessary for neurite outgrowth. They speculated that this region might act to recruit signaling proteins to the plasma membrane, which is necessary to initiate neurite extension. The cytoplasmic domain of RAGE contains a similar highly acidic region. It will be interesting to see whether identification of molecules capable of binding to RAGE cytoplasmic domain will give further insight into the role of Rac and Cdc42 in RAGE-mediated neurite outgrowth. The involvement of PAK or other effectors of Rac and/or Cdc42 in RAGE signaling awaits further investigation.

One of the best characterized transcription factors, NF-kappa B, is classically linked to inflammation and stress responses. Recently, evidence has begun to accumulate that NF-kappa B is also involved in brain function, particularly following injury and in neurodegenerative conditions but also in neuronal development (reviewed in Ref. 43). Activation of NF-kappa B is a hallmark of RAGE ligation with either AGEs or amyloid beta -peptide (12, 21). In the vascular system RAGE-mediated activation of NF-kappa B has been shown to induce expression of genes such as vascular cell adhesion molecule-1 (VCAM-1), which might contribute to the development of diabetic vascular disease (44). In neurons RAGE-mediated activation of NF-kappa B in response to amyloid beta -peptide has been shown to induce expression of macrophage-colony-stimulating factor strengthening the inflammatory response in Alzheimer's brain (13). In this study we demonstrate that amphoterin is also capable of activating NF-kappa B through RAGE. However, it is likely that amphoterin-induced changes in gene expression contribute to other than inflammatory processes. Recently, nerve growth factor-dependent activation of NF-kappa B was shown to contribute to the survival of sympathetic neurons (45). It is possible that amphoterin could induce such a survival effect through RAGE-mediated activation of NF-kappa B. Interestingly, the analysis of the promoter region of RAGE gene revealed the presence of two functional NF-kappa B-binding sites (46) suggesting a possible autoregulatory loop in the regulation of RAGE expression. However, when this manuscript was under preparation a finding was published (47) showing that amphoterin-induced increase in RAGE expression is mediated by binding of amphoterin to RAGE resulting in Sp1 activation rather than activation of NF-kappa B. Soluble amphoterin was used in their study, whereas we have used matrix-bound amphoterin. In our NF-kappa B assay soluble amphoterin had a weaker but still significant effect on the activation NF-kappa B when compared with matrix-bound amphoterin (data not shown). Thus, the differences in their study and our results may lie somewhere else, for example in the use of different cell types or the length of stimulation. Also it should be noted that our results have been obtained not by measuring the nuclear localization of NF-kappa B but by measuring the activation of NF-kappa B-dependent transcription. Further studies will be required to establish the molecular mechanism responsible for RAGE-mediated activation of NF-kappa B both in vitro and in vivo.

In this study, we show that the RAGE-mediated activation of NF-kappa B is dependent on the cytoplasmic domain of RAGE and functional Ras. Deletion of the cytoplasmic domain of RAGE blocked the activation of NF-kappa B-dependent transcription both with amphoterin and AGE. However, using the dominant negative approach again, we found that only Ras but not the Rho family GTPases is involved in the RAGE-mediated activation of NF-kappa B. The schematic model presented in Fig. 5 suggests that a similar, yet unknown, membrane-proximal signaling component is required for both amphoterin and AGE in RAGE signaling, but two parallel, independent pathways then lead to the activation of NF-kappa B or cytoskeletal reorganization. However, our experiments do not completely exclude the possibility of cross-talk between the Ras-MAP kinase and the Cdc42-Rac pathways in RAGE signaling. There is substantial evidence of such cross-talk between Ras and Rho pathways in other systems (37, 48). In addition, Rac has been shown to participate in the activation of NADPH complex in phagocytes (49, 50) but also in a redox-dependent pathway necessary for NF-kappa B activation in nonphagocytic cells (51). Thus an interesting possibility that Rac might regulate intracellular production of radicals required for RAGE-mediated activation of NF-kappa B still remains.


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  		Fig. 5.   Distinct signaling pathways are responsible for RAGE-mediated neurite outgrowth and activation of NF-kappa B. Activation of RAGE by AGEs induces generation of oxygen radicals by a yet unknown mechanism. Free radicals then activate a Ras-MAP kinase pathway eventually leading to the activation and nuclear translocation of NF-kappa B. RAGE-mediated neurite outgrowth on amphoterin-coated substrates requires both functional Rac and Cdc42, and inhibition of either one is sufficient to block neurite extension suggesting that Rac and Cdc42 lie on the same pathway. Inhibition of different components of the Ras-MAP kinase pathway does not affect RAGE-mediated neurite outgrowth, and on the other hand, inhibition of Cdc42-Rac pathway has no effect on the RAGE-mediated activation of NF-kappa B.

An intriguing scenario is appearing for the functions of RAGE and its three ligands: AGE, amyloid beta -peptide, and amphoterin. Detailed understanding of the RAGE signaling mechanisms is important because of the pathophysiological relevance of AGE and amyloid beta -peptide. On the other hand, amphoterin-induced neurite extension and the RAGE/Cdc42-Rac signaling pathway may be important in the formation of the neural connections during development and/or injury of the nervous system.

    ACKNOWLEDGEMENTS

We thank Dr. David Stern for generously providing us with the human RAGE cDNA; Dr. Alan Hall for providing us with the expression plasmids for C3 transferase and dominant negative Rac and Cdc42; and Dr. Johan Peränen for providing us with dominant negative Ras. The excellent technical assistance of Eeva-Liisa Saarikalle and Seija Lehto is gratefully acknowledged.

    FOOTNOTES

* This work was supported by grants from the Academy of Finland, the Sigrid Jusélius Foundation, and the Center for International Mobility Organization.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: +358-9-70859060; Fax: +358-9-70859068; E-mail: Henri.Huttunen@helsinki.fi.

§ Both authors are in the Helsinki Graduate School in Biotechnology and Molecular Biology.

    ABBREVIATIONS

The abbreviations used are: RAGE, receptor for advanced glycation end products; AGE, advanced glycation end products; HB-GAM, heparin-binding growth-associated molecule; PI 3-kinase, phosphatidylinositol 3-kinase; MAP kinase, mitogen activated protein kinase; MEK, MAP or extracellular signal-related kinase kinase; BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; PAK, p21-activated kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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