Prostaglandin E(2) couples through EP(4) prostanoid receptors to induce IL-8 production in human colonic epithelial cell lines.
OBJECTIVE
Prostaglandin (PG) E(2) and interleukin (IL)-8 are simultaneously increased during the inflammation that characterizes numerous pathologies such as inflammatory bowel disease. IL-8 is a potent neutrophil chemo-attractant and activator, and can initiate and/or exacerbate tissue injury. PGE(2) signals principally through prostanoid receptors of the EP(2) and/or EP(4) subtypes to promote cAMP-dependent cellular functions. The aim of this study was to identify the role of the EP(2) and EP(4) receptor subtype(s) on two human colonic epithelial cell lines (Caco-2 and T84), in regulating PGE(2)-induced IL-8 production.


METHODS
To identify the causative receptor, we knocked-down and over-expressed EP(2) and EP(4) receptor subtypes in colonic epithelial cells and studied the effect of several selective EP(2)/EP(4) receptor agonists and antagonists. The inductions of IL-8 and EP receptor mRNA and protein expression were determined by real-time PCR and western blot analysis. The affinity of PGE(2) and Bmax values for the EP(2) and EP(4) receptor on colonic epithelial cells were determined by radioligand-binding assays with [(3)H]PGE(2).


RESULTS
PGE(2) had the highest affinity for the EP(4) receptor subtype and promoted a robust stimulation of cAMP-dependent IL-8 synthesis. This effect was mimicked by a selective EP(4) receptor agonist, ONO-AE1-329, and abolished by silencing the EP(4) receptor gene by using siRNA techniques, a selective EP(4) receptor antagonist (ONO-AE3-208) and a selective inhibitor (Rp-cAMP) of cAMP-dependent protein kinase.


CONCLUSIONS
These findings suggest that initiation and progression of colonic inflammation induced by IL-8 could be mediated, at least in part, by PGE(2) acting via the EP(4) receptor subtype.
Dey
I
I
Departments of Microbiology and Infectious Diseases, Health Sciences Centre, University of Calgary, AB, Canada.
Giembycz
M A
MA
Chadee
K
K
D015153:Blotting, Western; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism; D015232:Dinoprostone / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis*; D008024:Ligands; D011869:Radioligand Assay; D018078:Receptors, Prostaglandin E / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D058309:Receptors, Prostaglandin E, EP4 Subtype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D015854:Up-Regulation
D016209:Interleukin-8; D008024:Ligands; C545650:PTGER4 protein, human; D018078:Receptors, Prostaglandin E; D058309:Receptors, Prostaglandin E, EP4 Subtype; D015232:Dinoprostone