Multiple signal transduction pathways through two prostaglandin E receptor EP₃ subtype isoforms expressed in human uterus

PGE₂ is known to induce uterine contraction by increasing intra-cellular Ca²⁺. In the present study, to investigate other functions of PGE₂ in human uterus, two EP₃ isoforms were isolated by the RT-PCR method using human uterus polyadenylated ribonucleic acid (RNA). These EP₃ isoforms, named EP_3-V and EP_3-VI, are composed of 402 and 393 amino acid residues, respectively, which are unique compared with EP₃ isoforms of other species. Their N-terminal 359 amino acid residues are identical to those of previously reported human EP₃ isoforms, whereas the two isoforms contained a novel amino acid sequence in their C-terminal tails. The dissociation constant values of EP_3-V and EP_3-VI for PGE₂ were 3.9 and 1.4 nmol/L, respectively, which were consistent with those of previously reported EPa isoforms. Signaling experiments revealed that M&B28767, an EP₃ agonist, not only inhibited forskolin-induced cAMP concentrations, but also activated mitogen-activated protein kinase in Chinese hamster ovary cells stably expressing EP_3-V and EP_3-VI. These responses were abolished by treatment with pertussis toxin. In addition, M&B28767 increased cAMP concentrations in EP_3-VI-expressing cells, whereas it did not in EP_3-V-expressing cells. M&B28767 did not stimulate phosphoinositide turnover in EP_3-V- or EP_3-VI-expressing cells. EP_3-V and EP_3-VI messenger RNAs (mRNAs) were detected abundantly in human uterus, whereas weak, but substantial, bands were detected in the lung and kidney in RT-PCR specific for each mRNA. In situ hybridization revealed EP_3-V and EP_3-VI mRNAs in the human myometrium, but not in the endometrium. The present study suggests that EP_3-V and EP_3-VI are possibly involved in the proliferation of cells in human myometrium.