Prostaglandin E2 stimulates the production of amyloid-beta peptides through internalization of the EP4 receptor

Abstract

Amyloid-beta (Abeta) peptides, generated by the proteolysis of beta-amyloid precursor protein by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta through EP(2) and EP(4) receptors, and here we have examined the molecular mechanism. Activation of EP(2) and EP(4) receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP(2), but not EP(4), receptor-mediated stimulation of the Abeta production. In contrast, inhibitors of endocytosis suppressed EP(4), but not EP(2), receptor-mediated stimulation. Activation of gamma-secretase was observed with the activation of EP(4) receptors but not EP(2) receptors. PGE(2)-dependent internalization of the EP(4) receptor was observed, and cells expressing a mutant EP(4) receptor lacking the internalization activity did not exhibit PGE(2)-stimulated production of Abeta. A physical interaction between the EP(4) receptor and PS-1, a catalytic subunit of gamma-secretases, was revealed by immunoprecipitation assays. PGE(2)-induced internalization of PS-1 and co-localization of EP(4), PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP(4) receptor null mice. These results suggest that PGE(2)-stimulated production of Abeta involves EP(4) receptor-mediated endocytosis of PS-1 followed by activation of the gamma-secretase, as well as EP(2) receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease.

FIGURE 1.

Effect of inhibitors of endocytosis on PGE₂-stimulated production of Aβ in HEK293 cells. HEK293 cells expressing APPsw were preincubated for 1 h with 0.5 m sucrose (A and B) or 0.25 mg/ml concanavalin A (C and D) and further incubated for 24 h with or without 1 μm PGE₂ in the presence of the same concentration of each inhibitor as in the preincubation step. The amounts of Aβ40 and Aβ42 in the conditioned medium were determined by sandwich enzyme-linked immunosorbent assay and expressed relative to the control (without PGE₂) (A and C). After incubation with or without PGE₂ for 1 h, membrane fractions were prepared and subjected to γ-secretase-mediated peptide cleavage assay as described under “Experimental Procedures” (B and D). Values are given as means ± S.D. (n = 3). **, p < 0.01; *, p < 0.05.

FIGURE 2.

Effect of siRNA for clathrin on PGE₂-stimulated production of Aβ. HEK293 cells expressing APPsw were transiently transfected with siRNA for clathrin (siClathrin) (+) or non-silencing siRNA (−) (A–D). Cells were incubated with 1 μm PGE₂ (A–C) or 100 μm pCPT-cAMP (D) for 24 h (A, B, and D) or 1 h (C). Whole cell extracts were analyzed by immunoblotting with an antibody against clathrin or actin. The band intensity was determined and expressed relative to the control (A). The amounts of Aβ (B and D) and the γ-secretase activity (C) were determined and expressed as described in the legend for Fig. 1. Values are given as means ± S.D. (n = 3). **, p < 0.01; *, p < 0.05; n.s., not significant.

FIGURE 3.

PGE₂-dependent internalization of the EP₄ receptor and its contribution to PGE₂-stimulated production of Aβ. CHO-K1 cells expressing APPsw were transiently transfected with expression plasmid encoding EP₂ (A), EP₄ (A–C), or EP₄-t369 (A–C) or with control vector (B and C). Cells were preincubated for 1 h with antibody against HA and further cultured for 1 h (A and C) or 24 h (B) with or without 1 μm PGE₂. After incubation with secondary antibody, the cells were inspected using fluorescence microscopy. Pictures of both high (right) and low (left) magnification are shown (scale bar, 20 μm) in each of the three panels. (A) The amounts of Aβ (B) or γ-secretase activity (C) were determined and expressed as described in the legend for Fig. 1. Values are given as means ± S.D. (n = 3). **, p < 0.01; n.s., not significant.

FIGURE 4.

Effect of siRNA for Rab5 and Rab7 on PGE₂-stimulated production of Aβ. HEK293 cells expressing APPsw were transiently transfected with siRNA for Rab5 (siRab5) (A–C) or Rab7 (siRab7) (D–F), or with non-silencing siRNA (−) (A–F). Cells were incubated with 1 μm PGE₂ for 24 h (A, B, D, and E) or 1 h (C and F). Whole cell extracts were analyzed by immunoblotting as described in the legend for Fig. 2 (A and D). The amounts of Aβ (B and E) and γ-secretase activity (C and F) were determined and expressed as described in the legend for Fig. 1. Values are given as means ± S.D. (n = 3). **, p < 0.01; *, p < 0.05.

FIGURE 5.

Interaction between the EP₄ receptor and PS-1 and their PGE₂-dependent co-internalization. HEK293 cells expressing APPsw were transiently transfected with expression plasmid encoding the EP₄ receptor or EP₄-t369 or with control vector. Whole cell extracts were immunoprecipitated with antibody against HA. A, whole cell extracts (WCE) and the immunoprecipitates (IP) were analyzed by immunoblotting with antibody against HA or PS-1-NTF as described in the legend for Fig. 2 (n.d., not detectable). B, cells were surface-biotinylated and incubated with or without 1 μm PGE₂. Then, cells were treated with glutathione to cleave biotin from the surface proteins. Biotinylated proteins present in the cell lysates were precipitated with Neutravidin, and the precipitates were analyzed by immunoblotting with HA or PS-1-NTF as described in the legend for Fig. 2.

FIGURE 6.

Co-localization of EP₄ receptors, PS-1, and Rab7 in cells. HEK293 cells expressing APPsw were transiently transfected with an expression plasmid encoding the EP₄ receptor. Cells were preincubated with antibody against HA (A, B) for 1 h and further incubated for 1 h with or without (Cont, control) 1 μm PGE₂. After fixation, samples were incubated with antibody against PS-1-NTF (A, C) or Rab7 (B, C). After incubation with the respective secondary antibody, cells were inspected using fluorescence microscopy as described in the legend for Fig. 3.

FIGURE 7.

Mechanism for EP₄ receptor-mediated stimulation of production of Aβ in vivo. Membrane fractions (A and C) or whole cell extracts (B) were prepared from the brains of 3-month-old APPsw/EP₄^+/+ and APPsw/EP₄^−/− mice and subjected to a γ-secretase-mediated peptide cleavage assay (A) or immunoblotting with antibody against APP, PS-1-NTF or actin (B and C). Values are given as means ± S.D. (n = 6). **, p < 0.01 (A). The brain sections were prepared from the same mice and subjected to immunostaining as described in the legend for Fig. 6 (D). The ratio of cells with yellow spots (positive cells) to total cells in the brain sections (three sections/brain) was determined. Values are given as means ± S.D. (n = 8). **, p < 0.01 (E).