Figure 1.
Stimulation of RAGE shedding via ligand-induced activation of different G protein-coupled receptors in HEK cells.
The cell line co-expressing RAGE and the PAC1 receptor (PAC1/RAGE) was stimulated with PACAP-27, cells co-expressing RAGE and the V2 vasopressin receptor (V2R/RAGE) were treated with arginine-vasopressin (AVP) and cells co-expressing RAGE and the oxytocin receptor (OTR/RAGE) were stimulated with oxytocin (OT). As control, cell lines were mock-stimulated with comparable volumes of water. After stimulation with 300 nM of each hormone for 4 h the cell culture supernatant was collected and the proteins were precipitated. Secreted RAGE (sRAGE) was detected by Western blotting using the polyclonal antibody 3260, followed by an anti-rabbit antibody labeled with horseradish peroxidase and ECL. Full-length RAGE and actin were detected in cell lysates by Western blot analysis. For quantification the experiments were done in triplicates. (A) Western Blots for secreted RAGE (sRAGE) in cell culture supernatants, full-length RAGE (RAGE fl) and Actin in cell lysates. (B) Quantitative analysis: Shown are the mean effects ± S.D., significance was determined by the One-way ANOVA Bonferroni test (* = P<0.05; ** = 0.01; *** = P<0.001).
Figure 2.
Induction of RAGE shedding at the cell surface with PACAP-27.
Cell-surface proteins of full-length RAGE and PAC1 coexpressing cells (PAC1/RAGE cells) were biotinylated using a membrane-impermeable biotinylation reagent. PACAP-27 and control treatment was then performed for 2 h. The amount of secreted biotinylated (A) and cell-anchored biotinylated RAGE (B) was determined separately under constitutive or PACAP-stimulated conditions after isolation of biotinylated proteins. RAGE was detected by Western blotting using antibody Mab5328 and ECL; typical blots are shown. Left image, amount of secreted biotinylated RAGE; right images, cell surface-located biotinylated RAGE and actin in total cell lysates. For quantification the experiments were performed in triplicates. Shown are the mean effects ± S.D., significance was determined by the One-way ANOVA Bonferroni test (* = P<0.05; ** = P<0.01).
Figure 3.
Stimulation of RAGE shedding with different PACAP peptides in PAC1/RAGE cells.
(A) PAC1/RAGE cells were incubated for 4 h with either 300 nM acetylated PACAP-38, PACAP-38, PACAP-27 or VIP; H2O: mock-treated cells (negative control). Then sRAGE was detected and quantified by Western blotting as described in Figure 1. Shown are the mean effects ± S.D., significance was determined by the One-way ANOVA Bonferroni test (ns = P>0.05; *** = P<0.001). (B) RAGE expressing cells were incubated for 4 h with PMA (1 µM), PACAP-27 (300 nM) or DMSO as control. Detection and quantification of secreted RAGE as well as significance was determined as described in figure part A; (ns = P>0.05; ** = P<0.01).
Figure 4.
PACAP-induced dose-dependent shedding of RAGE.
PAC1/RAGE cells were treated for 4 h with increasing PACAP-27 concentrations (ranging from 10 nM up to 3000 nM). Afterwards the medium was collected and sRAGE was identified with the Ab3260 as described in Figure 1. For quantification, the experiments were done in triplicates. Shown are the mean effects ± S.D. Full-length RAGE and actin were detected in total cell lysates by Western blot analysis.
Figure 5.
Time dependence of RAGE shedding induced by either PACAP (A) or PMA (B).
Upper figure part: representative Western blots for detection of sRAGE, full-length RAGE (RAGE fl) and actin; middle figure part: Quantitative analysis of sRAGE amounts; lower figure part: Quantification of full-length RAGE expression. PAC1/RAGE cells were incubated for different periods of time (15 min up to 360 min) using either 300 nM PACAP-27 or 1 µM PMA. sRAGE was detected and quantified as described in Figure 1. Full-length RAGE and actin were analyzed in cell lysates by Western blotting using either the monoclonal anti-RAGE antibody 5328 or anti-actin antibody A2066. Detection was performed with ECL. For quantification of sRAGE and full-length RAGE, the experiments were done in triplicates. Shown are the mean effects ± S.D.
Figure 6.
Effect of metalloproteinase inhibitor GM6001 on PACAP-induced RAGE shedding.
PAC1/RAGE cells were pre-treated for 1 h with either 20 µM GM6001 or solvent, then 300 nM PACAP-27 was added for 2 h; DMSO: mock-treated cells (negative control). Secreted RAGE was detected and quantified as described in Figure 1. Full-length RAGE and actin were detected in total cell lysates by Western blot analysis. Shown are the mean effects ± S.D., significance was determined by the One-way ANOVA Bonferroni test (ns = P>0.05; ** = P<0.01). Full-length RAGE and actin were detected in total cell lysates by Western blot analysis.
Figure 7.
Effect of metalloproteinase inhibitor GI254023X on PACAP-induced RAGE shedding.
PAC1/RAGE cells were pre-treated for 1 h with either 25 µM (A), 1 µM (B) or 100 nM (C) GI254023X or solvent (DMSO), then 300 nM PACAP-27 was added for 2 h; DMSO: mock-treated cells (negative control). Soluble RAGE was detected and quantified as described in Figure 1. Full-length RAGE and actin were detected in total cell lysates by Western blot analysis. Shown are the mean effects ± S.D., significance was determined by the One-way ANOVA Bonferroni test (ns = P>0.05; *** = P<0.001).
Figure 8.
Effects of RNAi-mediated knock-down on the shedding of RAGE.
PAC1/RAGE cells were transfected with stealth RNAi oligonucleotide duplexes (Invitrogen) targeting either ADAM10 (AD10), ADAM17 (AD17), MMP9 or MMP2. As control (-) cells were transfected with a stealth RNAi control oligonucleotide duplex (Invitrogen). Experiments were performed 48 h after transfection. Secretion of RAGE was analyzed under unstimulated (H2O, n = 10) and PACAP-induced (300 nM, n = 5) conditions for 3 h. (A) Typical Western blots and gelatin zymography are shown, (B) Quantitative analysis of secreted RAGE. RAGE secreted into the cell culture supernatant and full-length RAGE in cell lysates were detected with antibody Mab5328. The enzymatic activity of MMP9 and MMP2 in cell culture supernatants was determined by gelatin zymography as described in Materials and Methods. In cell lysates full-length RAGE (fl-RAGE), ADAM10 (pro and mature (m) forms), and ADAM17 (pro and mature (m) forms) were detected with suitable antibodies (see “Materials and Methods”). As a loading control actin was detected in cell lysates by Western blotting. Shown in the quantitative analysis (B) are the mean effects ± S.D., significance was determined by the One-way ANOVA Bonferroni test (ns = P>0.05; * = P<0.05; ** = P<0.01; *** = P<0.001).
Figure 9.
Analysis of adenylate cyclase/protein kinase A (PKA) signaling on the PACAP-induced RAGE secretion.
(A) and (B), effect of protein kinase A inhibition; (C) effect of adenylate cyclase/protein kinase A activation. (A) and (B), PAC1/RAGE cells were pre-treated for 1 h with either PKA inhibitor H89 (5 µM, figure part A) or KT5720 (1 µM, figure part B) then 300 nM PACAP-27 was added for 2 h. The effects of PKA inhibitors were also analyzed in the absence of PACAP stimulation. Secreted RAGE was detected and quantified as described in Figure 1. Shown are the mean effects ± S.D., significance was determined by the One-way ANOVA (ns = P>0.05). For quantification the experiments were performed in triplicates. (C) Cells were incubated with either adenylate cyclase stimulator Forskolin (50 µM) or PKA activator 8-Br-cAMP (1 µM) for 2 h. Secreted RAGE in the cell culture supernatant was detected and quantified by Western Blot as described above. H2O, DMSO: mock-treated cells (negative controls).
Figure 10.
Effect of Ca2+ on the shedding of RAGE.
PAC1/RAGE cells were pre-incubated for 1 h with respective inhibitor and then 300 nM PACAP-27 was added for 2 h. Secreted RAGE in the cell culture supernatant was detected and quantified as described in Figure 1. Shown are the mean effects ± S.D., significance was determined by the One-way ANOVA test (ns = P>0.05; ** = P<0.01; *** = P<0.001). (A) Effect of protein kinase C inhibitors Gö6976 and Gö6983 (both 1 µM); (B) effect of the IP3 receptor modulator 2-APB (75 µM), and (C) effect of the calcium/calmodulin-dependent protein kinase II inhibitor CK59 (20 µM). In all experiments effects of inhibitors were also analyzed in the absence of PACAP stimulation.
Figure 11.
Measurement of intracellular calcium signaling.
PAC1/RAGE (A), V2R/RAGE and OTR/RAGE cells (B) as well as HEK cells (A/B) were incubated for 30 min with 1,5 µM fura 2-AM. Then the cells were suspended and collected by centrifugation (0.4 g), after suspending cells in calcium buffer, measurements were performed with a spectrofluorometer. The respective agonists were applied to the cells after 100 s. As a negative control, either PACAP-27 or OT and AVP were added to HEK cells. Rising intracellular Ca2+ levels are detected by an increase in the fluorescence ratio 340 nm/380 nm.
Figure 12.
Role of phosphatidylinositol 3-kinase in PACAP-induced RAGE shedding.
PAC1/RAGE cells were pre-incubated for 1 h with PI3K inhibitor LY294002 (50 µM) and then 300 nM PACAP-27 was added for 2 h; DMSO: mock-treated cells (negative control). Secreted RAGE in the cell culture supernatant was detected and quantified as described in Figure 1. Shown are the mean effects ± S.D., significance was determined by the Student’s unpaired t test (*** = P<0.001).
Figure 13.
Role of the MEK1 MAP kinase pathway in PACAP-induced RAGE shedding.
PAC1/RAGE cells were pre-incubated for 1 h with MEK1 Inhibitor PD98059 (50 µM) and then 300 nM PACAP-27 was added for 2 h. Secreted RAGE was detected and quantified as described in Figure 1. Shown are the mean effects ± S.D., significance was determined by the One-way ANOVA test (ns = P>0.05; ** = P<0.01).
Figure 14.
Influence of simultaneously added kinase inhibitors on the shedding of RAGE.
PAC1/RAGE cells were pre-incubated for 1 h with (A) MEK1 inhibitor PD98059 (50 µM) and PKC inhibitor Gö6983 (1 µM); (B) MEK1 inhibitor PD98059 (50 µM) and CaMKII inhibitor CK59 (20 µM) or (C) PI3K inhibitor LY294002 (50 µM) and PKC inhibitor Gö6983 (1 µM). Then 300 nM PACAP-27 was added for 2 h. Secreted RAGE was detected and quantified as described in Figure 1. Shown are the mean effects ± S.D., significance was determined by the One-way ANOVA test (ns = P>0.05; * = P<0.05).
Figure 15.
Model of the intracellular pathways involved in PACAP-induced RAGE shedding.
In this study we demonstrated an involvement of MAP kinase, PI3-kinase, PKC and CaMKII in PAC1 receptor-induced activation of metalloproteinase-mediated RAGE shedding. Molecular mechanisms linking signaling cascades with metalloproteinase activation are still unknown (dotted lines). CaMKII: calmodulin-dependent protein kinase II; DAG: diacylglycerol; IP3: inositol 1,4,5-triphosphate; MAP kinase: mitogen-activated protein kinase; PI3-kinase: phosphatidylinositol 3-kinase.
Figure 16.
Analysis of PACAP-induced RAGE shedding in vivo.
Mices (N = 6) were treated intranasally with PACAP-38 for 3 months. Afterwards mouse lungs were dissected and membrane proteins and soluble proteins were separated following tissue homogenization by ultracentrifugation. Secreted RAGE (sRAGE) and full-length RAGE (RAGE fl) were detected by Western blotting using an antibody directed against mouse RAGE (MAB1179) followed by an anti-rat antibody labeled with horse radish peroxidase, as loading control actin was detected. Shown are the mean effects ± S.D., significance was determined by the Student’s unpaired t test (ns = P>0.05; * = P<0.05).