Carcinogenesis Advance Access originally published online on July 8, 2009
Carcinogenesis 2009 30(9):1620-1627; doi:10.1093/carcin/bgp168
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Published by Oxford University Press 2009.
The prostaglandin receptor EP2 activates multiple signaling pathways and β-arrestin1 complex formation during mouse skin papilloma development
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
1 Present address: Drug Safety Evaluation Development Research Laboratories, Shionogi and Co., Ltd, Futaba-cho, Toyonaka, Osaka 561-0825, Japan
* To whom correspondence should be addressed. Tel: +1 919 541 7558; Fax: +1 919 541 0146; Email: langenb1{at}niehs.nih.gov
Prostaglandin E2 (PGE2) is elevated in many tumor types, but PGE2's contributions to tumor growth are largely unknown. To investigate PGE2's roles, the contributions of one of its receptors, EP2, were studied using the mouse skin initiation/promotion model. Initial studies indicated that protein kinase A (PKA), epidermal growth factor receptor (EGFR) and several effectors—cyclic adenosine 3',5'-monophosphate response element-binding protein (CREB), H-Ras, Src, protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)1/2—were activated in 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted papillomas and that PKA and EGFR inhibition (H89 and AG1478, respectively) decreased papilloma formation. EP2's contributions to the activation of these pathways and papilloma development were determined by inhibiting endogenous TPA-induced PGE2 production with indomethacin (Indo) and concomitantly treating with the EP2 agonist, CAY10399 (CAY). CAY treatment restored papilloma formation in TPA/Indo-treated mice and increased cyclic adenosine 3',5'-monophosphate and PKA activation as measured by p-CREB formation. CAY treatment also increased EGFR and Src activation and their inhibition by AG1478 and PP2 indicated that Src was upstream of EGFR. CAY also increased H-Ras, ERK1/2 and AKT activation, and AG1478 decreased their activation indicating EGFR being upstream. Supporting EP2's contribution, EP2–/– mice exhibited 65% fewer papillomas and reduced Src, EGFR, H-Ras, AKT and ERK1/2 activation. G protein-coupled receptor (GPCR) activation of EGFR has been reported to involve Src's activation via a GPCR–β-arrestin–Src complex. Indeed, immunoprecipitation of β-arrestin1 or p-Src indicated the presence of an EP2–β-arrestin1–p-Src complex in papillomas. The data indicated that EP2 contributed to tumor formation via activation of PKA and EGFR and that EP2 formed a complex with β-arrestin1 and Src that contributed to signaling and/or EP2 desensitization.
Abbreviations: AC, adenylate cyclase; AKT, protein kinase B; cAMP, cyclic adenosine 3',5'-monophosphate; CAY, CAY10399; COX, cyclooxygenase; CREB, cyclic adenosine 3',5'-monophosphate response element-binding protein; DMBA, 7,12-dimethylbenz(a)anthracene; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled receptor; Indo, indomethacin; NSAID, non-steroidal anti-inflammatory drug; PG, prostaglandin; PGE2, prostaglandin E2; PKA, protein kinase A; TPA, 12-O-tetradecanoylphorbol-13-acetate; WT, wild-type
These authors contributed equally to this work. Received January 29, 2009; revised June 30, 2009; accepted July 1, 2009.