Involvement of Prostaglandin E2 in Production of Amyloid-β Peptides Both in Vitro and in Vivo

Takashi Homan,Tohru Mizushima,Shuh Narumiya,Yukihiko Sugimoto,Ken-ichiro Tanaka,Wataru Araki,Toshiharu Suzuki,Masami Narita,Tatsuya Hoshino,Tadashi Nakaya

doi:10.1074/jbc.m703087200

Abstract

Amyloid-beta peptides (Abeta), generated by proteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease (AD). Inflammation is also believed to be integral to the pathogenesis of AD. Here we show that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta in cultured human embryonic kidney (HEK) 293 or human neuroblastoma (SH-SY5Y) cells, both of which express a mutant type of APP. We have demonstrated using subtype-specific agonists that, of the four main subtypes of PGE(2) receptors (EP(1-4)), EP(4) receptors alone or EP(2) and EP(4) receptors together are responsible for this PGE(2)-stimulated production of Abeta in HEK293 or SH-SY5Y cells, respectively. An EP(4) receptor antagonist suppressed the PGE(2)-stimulated production of Abeta in HEK293 cells. This stimulation was accompanied by an increase in cellular cAMP levels, and an analogue of cAMP stimulated the production of Abeta, demonstrating that increases in the cellular level of cAMP are responsible for the PGE(2)-stimulated production of Abeta. Immunoblotting experiments and direct measurement of gamma-secretase activity suggested that PGE(2)-stimulated production of Abeta is mediated by activation ofgamma-secretase but not of beta-secretase. Transgenic mice expressing the mutant type of APP showed lower levels of Abeta in the brain, when they were crossed with mice lacking either EP(2) or EP(4) receptors, suggesting that PGE(2)-mediated activation of EP(2) and EP(4) receptors is involved in the production of Abeta in vivo and in the pathogenesis of AD.

Highlights

It has been repeatedly suggested that inflammation is important in the pathogenesis of Alzheimer disease (AD)
COX-1 is expressed constitutively in most cell types, whereas expression of COX-2 is induced by various factors including inflammatory cytokines and is responsible for the progression immunoassay; Exchange protein directly activated by cAMP (Epac), exchange protein directly activated by cAMP; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide; HEK, human embryonic kidney; NF-␬B, nuclear factor-␬B; NSAIDs, non-steroidal anti-inflammatory drugs; pCPT-cAMP, 8-(4-chlorophenylthio)-cAMP; pCPT-O-Me-cAMP, 8-(4-chloro-phenylthio)-2Ј-O-methyladenosine-3Ј-5Јcyclic monophosphate; PI3K, phosphatidylinositol 3-kinase; PGE2, prostaglandin E2; PGs, prostaglandins; Rock, Rho kinase; PKA, protein kinase A; RT, reverse transcriptase; sELISA, sandwich enzyme-linked immunosorbent; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CHAPSO, 3-[(3cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid
Firmed that this occurs in HEK293 cells and have shown In most of the intracellular signal transduction pathways that that PGE2-mediated activation of the EP4 receptor and the are downstream of the increase in the level of cellular cAMP, resulting increase in the cellular level of cAMP are respon- both PKA and PI3K play important roles [46]

Summary

EXPERIMENTAL PROCEDURES

HEK293 and SH-SY5Y cells expressing APPsw were from our laboratory stocks [34]. Sandwich Enzyme-linked Immunosorbent Assay (sELISA) for A␤ and EIA for cAMP—Cells were cultured for 24 h and the conditioned medium was subjected to sELISA using three types of specific monoclonal antibodies, as described previously [29, 34]. The supernatants were dried, re-suspended in the assay buffer, and applied to the EIA kit for measurement of cAMP, according to the manufacturer’s instructions. RT-PCR Analysis—Total RNA was extracted from cells using an RNeasy kit according to the manufacturer’s protocols. Samples (10 ␮g of RNA) were reverse transcribed using a firststrand cDNA synthesis kit according to the manufacturer’s instructions. Differences were considered to be significant for values of p Ͻ 0.05

RESULTS

PKA inhibitor

DISCUSSION