Regulation of Receptor for Advanced Glycation End Products (RAGE) Ectodomain Shedding and Its Role in Cell Function

Abstract

The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor that can undergo proteolysis at the cell surface to release a soluble ectodomain. Here we observed that ectodomain shedding of RAGE is critical for its role in regulating signaling and cellular function. Ectodomain shedding of both human and mouse RAGE was dependent on ADAM10 activity and induced with chemical activators of shedding (ionomycin, phorbol 12-myristate 13-acetate, and 4-aminophenylmercuric acetate) and endogenous stimuli (serum and RAGE ligands). Ectopic expression of the splice variant of RAGE (RAGE splice variant 4), which is resistant to ectodomain shedding, inhibited RAGE ligand dependent cell signaling, actin cytoskeleton reorganization, cell spreading, and cell migration. We found that blockade of RAGE ligand signaling with soluble RAGE or inhibitors of MAPK or PI3K blocked RAGE-dependent cell migration but did not affect RAGE splice variant 4 cell migration. We finally demonstrated that RAGE function is dependent on secretase activity as ADAM10 and γ-secretase inhibitors blocked RAGE ligand-mediated cell migration. Together, our data suggest that proteolysis of RAGE is critical to mediate signaling and cell function and may therefore emerge as a novel therapeutic target for RAGE-dependent disease states.

Keywords: ADAM; S100 proteins; cell migration; cell signaling; ectodomain shedding; matrix metalloproteinase (MMP); receptor for advanced glycation end products (RAGE).

FIGURE 1.

RAGE undergoes constitutive and inducible ectodomain shedding in HEK-293 cells. A, schematic of RAGE showing the major protein domains (ECD, transmembrane region (TM), and ICD). Protein sizes for full-length and cleaved ECD human or mouse RAGE are shown in kDa. B, stably transfected HEK-293 cells expressing empty vector control, hRAGE, or mRAGE were incubated for 24 h in serum-free medium to allow constitutive shedding to occur. Blotting was performed on concentrated conditioned medium and total cell lysate with anti-RAGE antibodies. Cell lysates were probed with anti-β actin as a loading control. Images are representative of three independent experiments. C and D, stably transfected HEK-293 cells expressing empty vector, hRAGE, or mRAGE were serum-starved overnight, and fresh medium was added for experiments. RAGE shedding was induced with PMA (200 nm), ionomycin (Iono) (1 μm), or APMA (25 μm) with non-stimulated (NS) control treated with vehicle (DMSO) for 1 h. Medium and lysate were collected as above, and Western blotting (WB) was performed for RAGE. Images are representative of three independent experiments. E and F, human and mouse RAGE shedding was measured by ELISA using conditioned medium collected as above. In addition to PMA, ionomycin, and APMA stimulation, stimulation of shedding by 1% FBS was measured by ELISA. Data are means ± S.E. from three independent experiments. Statistical difference between groups was assessed by one-way ANOVA compared with non-stimulated where * denotes significant differences (p < 0.05) between groups. Error bars represent S.E. G, RAGE shedding was measured by ELISA after incubation of HEK-293 hRAGE-expressing cells with RAGE ligands (S100B and CML-human serum albumin; 1 μg/ml) or RAGE inhibitor (FPS-ZM1; 1 μm). Data are means ± S.E. from three independent experiments (n = 3). Statistical difference between groups was assessed by one-way ANOVA compared with non-stimulated where * denotes significant differences (p < 0.05) between groups. Error bars represent S.E. H, shedding of RAGE was induced with the phosphatase inhibitors cantharidin, calyculin A, and sodium pervanadate. Stably transfected HEK-293 cells expressing hRAGE were serum-starved overnight, and fresh medium was added for experiments. Shedding was induced for 1 h with cantharidin (500 μm), calyculin A (50 nm), and sodium pervanadate (10 μm) with non-stimulated (NS) control treated with vehicle (DMSO). Data are means ± S.E. from three independent experiments (n = 3). Statistical difference between groups was assessed by one-way ANOVA compared with non-stimulated where * denotes significant differences (p < 0.05) between groups. Error bars represent S.E.

FIGURE 2.

Regulation of RAGE ectodomain shedding in HEK-293 cells. A and B, stably transfected HEK-293 cells expressing hRAGE were serum-starved overnight and treated for 1 h with the PKC inhibitors Gö6976 (2 μm) and GFX109203X (2.5 μm) or metalloproteinase inhibitor BB94 (10 μm). Fresh medium was added, and shedding was induced for 1 h with either PMA (200 nm) (A) or ionomycin (1 μm) (B) with inhibitors re-added at the time of the experiment. The calcium chelator EGTA (10 mm) was added at the time of stimulation for 1 h. RAGE shedding was quantified in conditioned medium by human ELISA. Statistical difference between groups was assessed by one-way ANOVA where * denotes significant differences (p < 0.05) between groups. Error bars represent S.E. C and D, stably transfected HEK-293 cells expressing hRAGE were serum-starved overnight and treated for 1 h with either the p38 inhibitor SB203580 (10 μm), SAPK/JNK inhibitor SP600125 (100 μm), MEK inhibitor U0126 (10 μm), or phosphoinositide 3-kinase inhibitor LY-294002 (50 μm). Fresh medium was then added, and shedding was induced for 1 h with either PMA (200 nm) or ionomycin (1 μm) with inhibitors re-added at the time of the experiment. RAGE shedding was quantified in conditioned medium by human ELISA. Data are means ± S.E. from three independent experiments (n = 3). Statistical difference between groups was assessed by one-way ANOVA where * denotes significant differences (p < 0.05) between groups. Error bars represent S.E. E and F, stably transfected HEK-293 cells expressing hRAGE were serum-starved overnight and treated for 1 h with either the ADAM10 inhibitor GI254023X (5–20 μm) or metalloproteinase inhibitor BB94 (10 μm). Fresh medium was then added, and shedding was induced for 1 h with either PMA (200 nm) or ionomycin (1 μm) with inhibitors re-added at the time of the experiment. RAGE shedding was quantified in conditioned medium by human ELISA. Data are means ± S.E. from three independent experiments (n = 3). Statistical difference between groups was assessed by one-way ANOVA where * denotes significant differences (p < 0.05) between groups. Error bars represent S.E. NS, not-stimulated; Iono, ionomycin.

FIGURE 3.

RAGE ectodomain shedding occurs within a single region of the extracellular domain. A, schematic of the His₆-2HA N-terminally tagged human RAGE construct and isoform used in this study. The RAGE ECD, transmembrane region (TM), and ICD are shown. The predicted cleavage site at the JM domain should result in a shed ECD RAGE product of ∼50 kDa as indicated by the arrow. B, HEK-293 cells were transfected with a His₆-2HA N-terminally tagged human RAGE construct, and 48 h later shedding was induced with PMA (200 nm), ionomycin (1 μm), or APMA (25 μm) with non-stimulated (NS) control treated with vehicle (DMSO) for 1 h. Blots were performed on conditioned medium and total cell lysate with anti-His and anti-RAGE antibodies. Cell lysates were probed with anti-β actin as a loading control. Images are representative of three independent experiments. WB, Western blotting; Iono, ionomycin.

FIGURE 4.

Identification of a protease-resistant RAGE splice variant. A, RAGE protein sequences around the proposed cleavage site were obtained from UniProt (72) and aligned using Jalview (61). These included mouse (Q62151_MOUSE), pig (A5A8Y1_PIG), horse (F6W335_HORSE), rhesus monkey (F1ABQ1_MACMU), human (Q15109_HUMAN), and chimpanzee (H2R7G0_PANTR) obtained from UniProt (72). Conserved amino acids are shown below the alignments; a “+” is shown if two or more residues are equally conserved. Amino acid residues are colored according to the ClustalX color scheme where a color is only applied if the amino acid meets the criteria specific for the residue type at the alignment position (64). The proposed cleavage site is shown above, and the exon coding for each region is shown below. B, schematic of the RAGE ectodomain shedding site. The RAGE ECD, transmembrane region (TM), and ICD are shown as a schematic with the sequence alignment of the JM region and transmembrane region shown below. Protein alignment of mRAGE with mRAGEv4 shows the 9 amino acids missing in mRAGEv4 as a result of alternative splicing of exon 9 of RAGE with the proposed cleavage site indicate by an arrow. C, stably transfected HEK-293 cells expressing mRAGE or mRAGEv4 were assessed for constitutive shedding. RAGE was detected by Western blotting using polyclonal antibodies raised against the total ECD of RAGE. Cells were incubated for 24 h in serum-free medium to allow constitutive shedding to occur, and blotting was performed on conditioned medium and total cell lysate with anti-RAGE antibodies. Cell lysate were probed with anti-β actin as a loading control. Images are representative of three independent experiments. D, cell surface RAGE expression of HEK-293 cells (vector control, mRAGE, or mRAGEv4) was assessed by flow cytometry. Cells were incubated sequentially with anti-RAGE and Alexa Fluor 647-labeled secondary antibodies, and cell surface RAGE expression was detected using a FACSCalibur flow cytometer. Images are representative of three independent experiments. E, stably transfected HEK-293 cells expressing mRAGE or mRAGEv4 were serum-starved overnight, and fresh medium was added for experiments. RAGE shedding was induced with PMA (200 nm), ionomycin (1 μm), or APMA (25 μm) with non-stimulated (NS) control treated with vehicle (DMSO) for 1 h. Medium and lysate were collected as above, and Western blotting was performed for RAGE. Images are representative of three independent experiments. F, RAGE shedding of HEK-293 cells (vector control, mRAGE, or mRAGEv4) was quantified by ELISA using total unconcentrated conditioned medium collected in D. In addition to PMA, ionomycin (Iono), and APMA stimulation, 1% FBS-stimulated shedding was measured by ELISA. Data are means ± S.E. from three independent experiments (n = 3). Error bars represent S.E.

FIGURE 5.

RAGE ectodomain shedding affects cell migration of C6 glioma cells. A, stably transfected C6 cells (vector control, mRAGE, or mRAGEv4) were generated and assessed for RAGE expression and shedding. Shedding of mRAGE from independently generated stable transfectants (denoted as mRAGE-A or -B and mRAGEv4-A or -B) was assessed by Western blotting (WB) using polyclonal antibodies raised against the total ECD of RAGE. Cells were incubated for 24 h in serum-free medium to allow constitutive shedding to occur. Blotting was performed on conditioned medium and total cell lysate with anti-RAGE antibodies. Cell lysates were probed with anti-β actin as a loading control. Images are representative of three independent experiments. B, C6 glioma cells expressing mRAGE, mRAGEv4, or empty vector (mock) were assessed for ligand-induced migration toward collagen I in transwell migration assays. Independently generated stable transfectants (denoted as mRAGE-A or -B and mRAGEv4-A or -B) were tested. Following 24h of migration, cells were fixed in crystal violet solution, and dye was extracted and quantified using spectrophotometry. Data are means ± S.E. from three independent experiments (n = 3). Statistical difference between groups was assessed by one-way ANOVA where * denotes significant differences (p < 0.05) between groups. Error bars represent S.E. C and D, C6 glioma cells stably expressing mRAGE, mRAGEv4, or empty vector (mock) were assessed for ligand-induced migration in transwell migration assays with S100B (C) or FBS (D). Cells were seeded into the upper chamber of a Boyden transwell filter and allowed to migrate toward 5 μg/ml S100B or 1% FBS stimulant for 24 h. Cells were fixed in crystal violet solution, and dye was extracted and quantified using spectrophotometry. Data are means ± S.E. from three independent experiments (n = 3). Statistical difference between groups was assessed by one-way ANOVA where * denotes significant differences (p < 0.05) between groups. Error bars represent S.E.

FIGURE 6.

RAGE ectodomain shedding affects cell spreading and actin cytoskeleton rearrangement. A and B, cell adhesion/spreading assays were performed by seeding C6 glioma cells expressing mRAGE, mRAGEv4, or empty vector (mock) onto cell culture dishes coated with either PBS (control) or collagen I for 2 h. Independently generated stable transfectants (denoted as mRAGE-A or -B and mRAGEv4-A or -B) were tested. Adhered cells were fixed and imaged using Olympus CK2 (400× magnification shown in A) with surface area estimated using ImageJ. Surface area was calculated as a percentage of control PBS cells. Data are means ± S.E. from three independent experiments (n = 3). Statistical difference between groups was assessed by one-way ANOVA where * denotes significant differences (p < 0.05) between groups. Error bars represent S.E. C, C6 glioma cells expressing mRAGE, mRAGEv4, or empty vector (mock) were seeded on collagen-coated cell culture dishes for 2 h, fixed, and stained with rhodamine-conjugated phalloidin (actin)/anti-vinculin with GFP secondary antibody/DAPI. Cells were examined and photographed using an Olympus FV1000 microscope. Cells were examined and photographed using fluorescence microscopy. Images are representative of three independent experiments.

FIGURE 7.

RAGE ectodomain shedding affects cell signaling in C6 glioma cells. C6 glioma cells expressing mRAGE, mRAGEv4, or empty vector (mock) were seeded on collagen-coated cell culture dishes for 2 h. Cell lysates were then subject to immunoblotting with antibodies to phospho- (p) and total Src (B), Akt (C), ERK1/2 (D), and p38 (E). Samples were also subjected to Western blotting for RAGE and actin controls (A). Densitometry for three independent experiments was performed using ImageJ. Activation was calculated by normalization to total proteins levels, with percentage of induction calculated relative to control cells. Data are means ± S.E. from three independent experiments (n = 3). Statistical difference between groups was assessed by one-way ANOVA where * denotes significant differences (p < 0.05) between groups. Images are representative of three independent experiments. Error bars represent S.E.

FIGURE 8.

Cell migration induced by RAGE ectodomain shedding is dependent on signaling through ADAM10 and γ-secretase. C6 glioma cells expressing mRAGE, mRAGEv4, or empty vector (mock) were assessed for collagen I-induced migration in the presence of inhibitors of MEK (10 μm U0126) or PI3K (10 μm LY294002) (A), sRAGE (5 μg/ml) (B), metalloproteinases (10 μm BB94) (C), ADAM10 (5 μm GI254023X) (D), and γ-secretase (10 μm DAPT) (E). Following 24 h of migration, cells were fixed in crystal violet solution, and dye was extracted and quantified using spectrophotometry. Data are means ± S.E. from three independent experiments (n = 3). Statistical difference between groups was assessed by one-way ANOVA where * denotes significant differences (p < 0.05) between groups. Error bars represent S.E.

FIGURE 9.

Schematic model of the proposed mechanisms of RAGE ectodomain shedding and its regulation of cell function. A, following cell exposure to stimuli or calcium influx, PKC and PI3K signaling is activated (1). This in turn activates ADAM10 (2), which cleaves RAGE at the juxtamembrane region, releasing the RAGE ECD (3). A membrane-tethered C-terminal fragment (CTF) remains (4), and it can be cleaved by a γ-secretase to release the RAGE ICD (5). B, inhibition of pathways up- and downstream of RAGE ectodomain shedding affect cell migration. These include the inhibition of RAGE ligand binding (sRAGE), activation of proteases including ADAM10 (GI254023X) and γ-secretase (DAPT), and downstream signaling pathways (MEK and PI3K inhibition).