EP3 prostanoid receptor isoforms utilize distinct mechanisms to regulate ERK 1/2 activation

Abstract

Summary Prostaglandin-E2 (PGE2) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE2 mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP1, EP2, EP3 and EP4. The EP3 prostanoid receptor is unique in that it has multiple isoforms generated by alternative mRNA splicing. These splice variants display differences in tissue expression, constitutive activity and regulation of signaling molecules. To date there are few reports identifying differential activities of EP3 receptor isoforms and their effects on gene regulation. We generated HEK cell lines expressing either the human EP3-Ia, EP3-II or EP3-III isoforms. Using immunoblot analysis we found that nM concentrations of PGE2 strongly stimulated the phosphorylation of ERK 1/2 by the EP3-II and EP3-III isoforms; whereas, ERK 1/2 phosphorylation by the EP3-Ia isoform was minimal and only occurred at μM concentrations of PGE2. Furthermore, the mechanisms of the PGE2 mediated phosphorylation of ERK 1/2 by the EP3-II and EP3-III isoforms were different. Thus, PGE2 stimulation of ERK 1/2 phosphorylation by the EP3-III isoform involves activation of a Gαi/PI3K/PKC/Src and EGFR-dependent pathway; while for the EP3-II isoform it involves activation of a Gαi/Src and EGFR-dependent pathway. These differences result in unique differences in the regulation of reporter plasmid activity for the downstream effectors ELK1 and AP-1 by the EP3-II and EP3-III prostanoid receptor isoforms.

EP3 receptor isoforms are differentially expressed in subpopulations of primate granulosa cells and couple to unique G-proteins

“…Stimulation of monkey EP3-5 with sulprostone decreased cAMP, likely via Gαi. Elevated cAMP with sulprostone and PTX is similar to previous reports of elevated cAMP with agonist and PTX in other cells, but the specific G proteins involved remain to be identified (Israel & Regan 2009, Talasila et al 2009). Sulprostone stimulation of EP3-9 augmented cAMP concentrations, likely through Gαs.…”

Epidermal growth factor receptor reactivation induced by E-prostanoid-3 receptor- and tumor necrosis factor-alpha-converting enzyme-dependent feedback exaggerates interleukin-8 production in airway cancer (NCI-H292) cells

“…In addition, pretreatment with the Gi-protein inhibitor pertussis toxin or with a cAMP analog decreased the second EGFR-P and IL-8 production induced by TGF-alpha, implicating a Gi-protein-coupled receptor such as the EP3 receptor in these responses. Recently, Gi-proteincoupled isoforms of the EP3 receptor have been reported to activate ERK1,2 EGFR-dependently in human embryonic kidney cells [35], identifying the EP3 receptor as a GPCR capable of activating EGFR. The present findings show that PGE2/EP3-induced EGFR feedback exaggerates IL-8 production in NCI-H292 cancer cells but not in NHBE (normal) cells, suggesting that positive feedback between the EGFR and PGE2/EP3 receptor pathways could increase IL-8 production in some airway cancers.…”

Activation of PGE2/EP2 and PGE2/EP4 signaling pathways positively regulate the level of PD‑1 in infiltrating CD8+ T cells in patients with lung cancer

“…A previous study revealed that EP4 is able to mediate PGE2-induced migration of A549 lung cancer cells (31). EP3 is distinct as it has multiple isoforms generated by alternative mRNA splicing, and EP3 has also been found to be crucial for tumor stroma formation and tumor growth (32,33). The functions of various PGE2 receptors in regulating the level of PD-1 expression were distinguished.…”