Amyloid-β (Aβ) peptides, generated by the proteolysis of β-amyloid precursor protein by β- and γ-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 Aβ 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 Aβ production. In contrast, inhibitors of endocytosis suppressed EP
4, but not EP
2, receptor-mediated stimulation. Activation of γ-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 Aβ. A physical interaction between the EP
4 receptor and PS-1, a catalytic subunit of γ-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 Aβ involves EP
4 receptor-mediated endocytosis of PS-1 followed by activation of the γ-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.Alzheimer disease (AD)
2 is the most common neurodegenerative disorder of the central nervous system and the leading cause of adult onset dementia. AD is characterized pathologically by the accumulation of tangles and senile plaques. Senile plaques are composed of the amyloid-β (Aβ) peptides Aβ40 and Aβ42 (
1,
2). To generate Aβ, β-amyloid precursor protein (APP) is first cleaved by β-secretase and then by γ-secretase. Cleavage of APP by α-secretase produces non-amyloidogenic peptides (
3,
4). The γ-secretase is an aspartyl protease complex composed of four core components, including presenilin (PS) 1 and PS2 (
5). Early onset familial AD is linked to three genes,
APP,
PS1, and
PS2 (
5,
6), strongly suggesting that γ-secretase-dependent production of Aβ is a key factor in the pathogenesis of AD. Therefore, cellular factors that affect the γ-secretase-dependent production of Aβ may be good targets for the development of drugs to prevent and treat AD.Both APP and PS-1 are transmembrane proteins, and their intracellular localization is controlled by secretory and endocytic pathways. These proteins are modified in the endoplasmic reticulum and trafficked to the cell surface through the
trans-Golgi network (TGN). Then, they are internalized again and trafficked to early endosomes. Next, they are trafficked to late endosomes and lysosomes (LEL), which are recycling endosomes that are targeted to the cell surface or the TGN (
7–
11). The production of Aβ seems to occur in a broad range of cellular compartments including the cell surface, TGN, and endosomes (
12). Abnormalities of secretory and endocytic pathways have been observed in the brains of AD patients (
9,
13). Importantly, factors that control these vesicle transport systems affect the production of Aβ. For example, overproduction of Rab5, a factor essential for traffic of vesicles to early endosomes, has been shown to stimulate the production of Aβ (
14), and SorL1 has been shown to reduce the production of Aβ by stimulating the traffic of APP in early endosomes to the TGN (
15,
16).It has been suggested that inflammation is important in the pathogenesis of AD; chronic inflammation has been observed in the brains of AD patients, and trauma to the brain and ischemia, both of which can activate inflammation, are major risk factors for AD (
17–
19). Cyclooxygenase (COX) is essential for the synthesis of prostaglandin E
2 (PGE
2), a potent inducer of inflammation and has two subtypes, COX-1 and COX-2. COX-1 is expressed constitutively, whereas expression of COX-2 is induced under inflammatory conditions and is responsible for the progression of inflammation (
20–
22). The following evidences of the involvement of PGE
2 (and COX-2) in the progression of AD suggest that they are good targets for the development of AD drugs: (i) Elevated levels of PGE
2 and overexpression of COX-2 have been observed in the brains of AD patients (
23–
25); (ii) the extent of COX-2 expression correlates with the amount of Aβ and the degree of progression of AD pathogenesis (
26); (iii) transgenic mice constitutively overexpressing COX-2 show aging-dependent neural apoptosis and memory dysfunction (
27); (iv) prolonged use of nonsteroidal anti-inflammatory drugs, inhibitors of COX, delays the onset and reduces the risk of AD (
28); (v) PGE
2 stimulates the production of reactive oxygen species in microglia cells, resulting in activation of β-secretase (
29).We recently reported that PGE
2 stimulates the production of Aβ in human embryonic kidney (HEK) 293 and human neuroblastoma (SH-SY5Y) cells that stably express a form of APP with two mutations (K651N/M652L) (APPsw) that elevate cellular and secreted levels of Aβ (
30). Similar results were reported by another group (
31). Using agonists and antagonists specific for each of the four PGE
2 receptors (EP
1, EP
2, EP
3, and EP
4), we found that EP
4 receptors alone and also both EP
2 and EP
4 receptors are involved in PGE
2-stimulated production of Aβ in HEK293 or SH-SY5Y cells, respectively (
30). Furthermore, experiments with transgenic mice suggest that EP
2 and EP
4 receptors are involved in the production of Aβ
in vivo (
30). Based on these results, we propose that antagonists of the EP
2 and/or EP
4 receptors may be therapeutically beneficial for the treatment of AD. Understanding the mechanism governing EP
2 and EP
4 receptor-mediated stimulation of production of Aβ by PGE
2 will be important for such drug development.Activation of EP
2 and EP
4 receptors causes activation of adenylate cyclase and an increase in the cellular level of cAMP (
32). We have shown that an EP
4 receptor agonist or both EP
2 and EP
4 receptor agonists increase the cellular level of cAMP in HEK293 or SH-SY5Y cells, respectively, and that a cAMP analogue, 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP), increases the level of Aβ in HEK293 cells (
30). These findings suggest that the cellular level of cAMP is important for PGE
2-stimulated production of Aβ. An increase in the cellular level of cAMP is known to activate protein kinase A (PKA), which is important for cAMP-regulated intracellular signal transduction (
33). However, an inhibitor of PKA, N-[2-(
p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), does not block PGE
2-stimulated production of Aβ in HEK293 cells (
30). Other cAMP-regulated signal transduction factors, such as phosphatidylinositol 3-kinase and Epac (exchange protein directly activated by cAMP), were also shown not to be involved in PGE
2-stimulated production of Aβ in HEK293 cells (
30). Thus, the mechanism whereby the activation of EP
2 and EP
4 receptors stimulates the production of Aβ has remained unknown. In this study, by using inhibitors of adenylate cyclase and PKA, we found that activation of the EP
2 receptor stimulates production of Aβ through activation of adenylate cyclase and PKA. We also propose that activation of the EP
4 receptor causes its co-internalization with PS-1 (γ-secretase) into endosomes and that this co-internalization is important for EP
4 receptor-mediated stimulation of Aβ production by PGE
2 through the activation of γ-secretase.
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