Sequence Analysis and Identification of New Isoform of EP4 Receptors in Different Atlantic Salmon Tissues (Salmo salar L.) and Its Role in PGE2 Induced Immunomodulation In Vitro

Abstract

PGE2 plays an important role in a broad spectrum of physiological and pathological processes mediated through a membrane-bound G protein-coupled receptor (GPCR) called EP receptor. In mammals, four subtypes of EP receptor (EP 1-4) are identified and each of them functions through different signal transduction pathways. Orthologous EP receptors have also been identified in other non-mammalian species, such as chicken and zebrafish. EP4 is the only identified PGE2 receptor to date in Atlantic salmon but its tissue distribution and function have not been studied in any detail. In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5’ untranslated region was accompanied by silent mutation at nt 668. While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line), we failed to obtain the full-length product. Further investigation revealed different isoform of EP4 receptor in TO cells and we subsequently documented its presence in different Atlantic salmon tissues. These two isoforms of EP4 receptor share high homology in their first half of sequence but differ in the second half part with several deletion segments though the final length of coding sequence is the same for two isoforms. We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1β genes expression in a time dependent manner and without cAMP upregulation.

Molecular Characterization of E-Type Prostanoid Receptor 4 (EP4) from Ayu (Plecoglossus altivelis) and Its Functional Analysis in the Monocytes/Macrophages

“…In zebrafish, three EP4 genes showed unique expression patterns; and the EP4b transcripts were detected in all tested tissues except for the heart and testis and were highest in the gill [ 33 ]. In our study, we found that PaEP4L mRNA was expressed in all tested tissues with high expression in the intestine and gill, which was similar to that reported in other teleosts [ 19 , 20 ]. In mammals, emerging evidences had shown that chronic inflammation might increase EP4 expression in macrophages [ 34 , 35 ].…”

“…Previous studies in Atlantic salmon showed that EP4 mRNA expression was highest in the spleen followed by the head kidney, gill and intestine [ 19 , 20 ]. In zebrafish, three EP4 genes showed unique expression patterns; and the EP4b transcripts were detected in all tested tissues except for the heart and testis and were highest in the gill [ 33 ].…”

“…In Atlantic salmon, PGE2 pre-incubation lowered the induction of CXCL-10, CCL-4, IL-8, and IL-1β gene expression in MO/MΦ. However, whether this effect is mediated by EP4 remained unknown [ 20 ]. In this study, we found that PGE2 treatment significantly decreased the mRNA expression of IL-1β, IL-10, and TNF-α in ayu MO/MΦ, while anti-PaEP4L IgG neutralization could eliminate PGE2 effect on cytokine expression in MO/MΦ.…”

“…The procedure was performed following the manufacturer’s instructions. As this assay is based on competitive binding technique, the value of OD 450 is inversely proportional to the concentration of cAMP as described previously [ 20 ].…”

“…In recent years, EP4 has been attracting considerable attention due to its immunomodulatory activity in the mammalian immune response, and its homologs have been identified in various fish and shellfish, such as zebrafish ( Danio rerio ) [ 16 ], Atlantic salmon ( Salmo salar ) [ 17 ], and Hong Kong oyster ( Crassostrea hongkongensis ) [ 18 ]. The tissue mRNA expression profile of Atlantic salmon EP4 has been studied [ 19 , 20 ], and it is reported to be most abundantly expressed in the spleen followed by the head kidney, gill, and intestine, while the weakest expression is found in the heart and muscles [ 19 , 20 ]. However, the role of fish EP4 in modulating the immune response still remains unclear.…”