Differential Signaling and Gene Regulation Among Three Human EP3 Prostanoid Receptor Isoforms

Abstract

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 293 EBNA cell lines expressing the EP3-Ia, EP3-II, or EP3-III isoforms. After confirming the functional expression of each of these isoforms, we examined their activation of cellular signal transduction pathways.We found that each of these isoforms utilize distinct mechanisms to regulate ERK 1/2 phosphorylation and that these differences lead to unique regulation of the downstream effectors ELK-1 and AP-1. We also found MAPK dependent differences in regulation of cell proliferation. The EP3-III isoform increases cell proliferation in a MAPK dependent manner while the EP3-Ia dose dependently regulates cell proliferation via Gαi and not ERK 1/2. Activation of the EP3-II receptor had no effects on cell proliferation.To study differential gene regulation by these three EP3 receptor isoforms, we conducted microarray studies. Over 300 genes were differentially regulated by these isoforms. Quantitative real-time PCR analysis was used to validate 15 candidate genes. Five genes were chosen for further analysis of protein expression using immunoblotting, but only one of these, WT-1, was significantly increased following treatment with PGE2. WT-1, a transcription factor important for kidney and heart development, was strongly upregulated by PGE2 stimulation of the EP3-II receptor, but only weakly by the other isoforms.In conclusion, these studies show that the human EP3 prostanoid receptor isoforms are capable of distinct regulation of both signal transduction pathways and gene transcription. Elucidating the differential functions of EP3 receptor isoforms may allow for greater understanding of the diverse functions attributed to this receptor and their physiological functions.