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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.

Guo TC, Gamil AA, Koenig M, Evensen Ø - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Norwegian University of Life Sciences, Faculty of Veterinary Medicine and Biosciences, Sea Lice Research Centre, P.O. Box 8146 Dep., 0033 Oslo, Norway.

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.

No MeSH data available.


Related in: MedlinePlus

Modulation of responses of defined genes after PGE2 treatment and LPS stimulation in TO cells.Immune response to PGE2 stimulation in TO cells. A) CXCL-10; B) CCL4; C) IL8; D) IL-1β and F) IL6. Different timepoints post LPS stimulation is are indicated. * = p<0.05; ** = p<0.001. Average ± SEM is shown (n = 3).
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pone.0120483.g006: Modulation of responses of defined genes after PGE2 treatment and LPS stimulation in TO cells.Immune response to PGE2 stimulation in TO cells. A) CXCL-10; B) CCL4; C) IL8; D) IL-1β and F) IL6. Different timepoints post LPS stimulation is are indicated. * = p<0.05; ** = p<0.001. Average ± SEM is shown (n = 3).

Mentions: To study whether PGE2 can modulate immune response in TO cells, TO cells were treated with LPS or pretreated with 1 or 10 μM PGE2) for 1h and subsequently stimulated with LPS. The cells were then incubated for 2, 6, and 12h and sampled for analysis. Stimulation of TO cells with LPS alone resulted in up-regulation of all the examined inflammatory genes (Fig. 6 A-F; control). The up-regulation was most significant at 6 hours post stimulation for all genes, except IL-6. PGE2 pre-incubation lowered the induction of CXCL-10 (Fig. 6A), CCL-4 (Fig. 6B), IL-8 (Fig. 6C) and IL-1β (Fig. 6D) gene expression but had no clear effect on TNF-α (Fig. 6 E). For IL-6 the expression level was lower at 2h but increased beyond control at 12h, seemingly delaying the response at early time while inducing a second wave at later time (Fig. 6F). The induced inhibition was found to be time dependent, with the most pronounced effect being seen at 6 hours post stimulation. Interestingly, CCL-4 (Fig. 6B) was significantly down-regulated by PGE2 at all the examined time points.


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.

Guo TC, Gamil AA, Koenig M, Evensen Ø - PLoS ONE (2015)

Modulation of responses of defined genes after PGE2 treatment and LPS stimulation in TO cells.Immune response to PGE2 stimulation in TO cells. A) CXCL-10; B) CCL4; C) IL8; D) IL-1β and F) IL6. Different timepoints post LPS stimulation is are indicated. * = p<0.05; ** = p<0.001. Average ± SEM is shown (n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383505&req=5

pone.0120483.g006: Modulation of responses of defined genes after PGE2 treatment and LPS stimulation in TO cells.Immune response to PGE2 stimulation in TO cells. A) CXCL-10; B) CCL4; C) IL8; D) IL-1β and F) IL6. Different timepoints post LPS stimulation is are indicated. * = p<0.05; ** = p<0.001. Average ± SEM is shown (n = 3).
Mentions: To study whether PGE2 can modulate immune response in TO cells, TO cells were treated with LPS or pretreated with 1 or 10 μM PGE2) for 1h and subsequently stimulated with LPS. The cells were then incubated for 2, 6, and 12h and sampled for analysis. Stimulation of TO cells with LPS alone resulted in up-regulation of all the examined inflammatory genes (Fig. 6 A-F; control). The up-regulation was most significant at 6 hours post stimulation for all genes, except IL-6. PGE2 pre-incubation lowered the induction of CXCL-10 (Fig. 6A), CCL-4 (Fig. 6B), IL-8 (Fig. 6C) and IL-1β (Fig. 6D) gene expression but had no clear effect on TNF-α (Fig. 6 E). For IL-6 the expression level was lower at 2h but increased beyond control at 12h, seemingly delaying the response at early time while inducing a second wave at later time (Fig. 6F). The induced inhibition was found to be time dependent, with the most pronounced effect being seen at 6 hours post stimulation. Interestingly, CCL-4 (Fig. 6B) was significantly down-regulated by PGE2 at all the examined time points.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Norwegian University of Life Sciences, Faculty of Veterinary Medicine and Biosciences, Sea Lice Research Centre, P.O. Box 8146 Dep., 0033 Oslo, Norway.

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.

No MeSH data available.


Related in: MedlinePlus