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Cloning and expression of a prostaglandin E receptor EP3 subtype from human erythroleukaemia cells.

ABSTRACT: Prostaglandins inhibit platelet activation by stimulating intracellular cyclic AMP formation. We have postulated that intracellular cyclic AMP levels in platelets are buffered by a distinct prostaglandin receptor that mediates inhibition of cyclic AMP formation. In order to provide evidence for the model, we have cloned the cDNA coding for a prostaglandin receptor EP3 subtype, which is coupled to inhibition of adenylate cyclase, from the megakaryocytic cell line human erythroleukaemia (HEL) cells. A PCR-generated hybridization probe, produced using primers based on the sequence of the mouse prostaglandin EP3 receptor published by Sugimoto, Namba, Honda, Hayashi, Negishi, Ichikawa and Narumiya [(1992) J. Biol. Chem. 267, 6463-6466], was used to screen a lambda gt11 HEL cell cDNA library. The composite full-length cDNA clone HEP3, generated from the two partial clones pHEP3-7 and pHEP3-5, is 1.6 kb long with an open reading frame coding for 390 amino acids. This clone is 83% identical to the alpha subtype of the mouse EP3 receptor. The full-length construct was transfected into COS-1 cells. The cloned receptor exhibited the properties of a prostaglandin EP3 subtype, inhibiting forskolin-stimulated cyclic AMP formation in response to prostaglandin E2 (PGE2) and binding PGE2 with high specificity and a Kd of 3.2 nM. Radiolabelled PGE2 could be displaced by prostaglandins in the order PGE2 = PGE1 > iloprost = PGD2. Northern blot analysis revealed that the receptor is also present in human kidney.

SUBMITTER: Kunapuli SP

PROVIDER: S-EPMC1137934 | biostudies-other | 1994 Mar

REPOSITORIES: biostudies-other

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Project description:Background and aimsInvolvement of prostaglandin E(2) (PGE(2)) receptors EP(1), EP(2), and EP(4) in the formation of aberrant crypt foci (ACF) and/or intestinal polyps has been suggested. In contrast, EP(3) appears to have no influence on the early stages of colon carcinogenesis. In the present study, we examined expression of PGE(2) receptor subtypes EP(1), EP(2), EP(3), and EP(4) in normal colon mucosa and colon cancers, and assessed the contribution of EP(3) to colon cancer development.MethodsmRNA expression of PGE(2) receptor subtypes EP(1), EP(2), EP(3), and EP(4) in normal colon mucosa and colon cancers in azoxymethane (AOM) treated mice and rats, and in humans, were examined by reverse transcription-polymerase chain reaction (RT-PCR), quantitative real time RT-PCR, and immunohistochemical analyses. Evaluation of the role of EP(3) was performed by intraperitoneal injection of AOM, using EP(3) receptor knockout mice. Effects of EP(3) receptor activation on cell growth of human colon cancer cell lines were examined using ONO-AE-248, an EP(3) selective agonist. Moreover, EP(3) expression in colon cancer cell lines was analysed with or without 5-aza-2'-deoxycytidine (5-aza-dC) treatment.ResultsExpression levels of EP(1) and EP(2) mRNA were increased in cancer tissues. EP(4) mRNA was constantly expressed in normal mucosa and cancers. In contrast, expression of EP(3) mRNA was markedly decreased in colon cancer tissues, being 5% in mice, 9% in rats, and 28% in humans compared with normal colon mucosa, analysed by quantitative real time RT-PCR. Immunohistochemical staining demonstrated the rat EP(3) receptor protein to be expressed in epithelial cells of normal mucosa and some parts of small carcinomas but hardly detectable in large carcinomas of the colon. Colon cancer development induced by AOM in EP(3) receptor knockout mice was enhanced compared with wild-type mice, with a higher incidence of colon tumours (78% v 57%) and mean number of tumours per mouse (2.17 (0.51) v 0.75 (0.15); p<0.05). Expression of EP(3) mRNA was detected in only one of 11 human colon cancer cell lines tested. Treatment with 5 microM of an EP(3) selective agonist, ONO-AE-248, resulted in a 30% decrease in viable cell numbers in the HCA-7 human colon cancer cell line in which EP(3) was expressed. Treatment with 5-aza-dC restored EP(3) expression in CACO-2, CW-2, and DLD-1 cells but not in WiDr cells, suggesting involvement of hypermethylation in the downregulation of EP(3) to some extent.ConclusionThe PGE(2) receptor subtype EP(3) plays an important role in suppression of cell growth and its downregulation enhances colon carcinogenesis at a later stage. Hypermethylation of the EP(3) receptor gene could occur and may contribute towards downregulating EP(3) expression to some extent in colon cancers.

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Cloning and expression of a prostaglandin E receptor EP3 subtype from human erythroleukaemia cells.

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