8-iso-PGE₂ Stimulates Anion Efflux from Airway Epithelial Cells via the EP₄ Prostanoid Receptor

Abstract

Isoprostanes are biologically active molecules, produced when reactive oxygen species mediate the peroxidation of membrane polyunsaturated fatty acids. Previous work has demonstrated that the isoprostane 8-iso-prostaglandin E(2) (PGE(2)) stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial anion secretion across the human airway epithelial cell line, Calu-3. Since isoprostanes predominantly achieve their effects via binding to prostanoid receptors, we hypothesized that this 8-iso-PGE(2) stimulation of CFTR activity was the result of the isoprostane binding to a prostanoid receptor. Using RT-PCR, immunoblotting, and immunofluorescence, we here demonstrate that Calu-3 cells express the EP(1-4) and FP receptors, and localize these proteins in polarized cell monolayers. Using iodide efflux as a marker for CFTR-mediated Cl(-) efflux, we investigate whether prostanoid receptor agonists elicit a functional response from Calu-3 cells. Application of the agonists PGE(2), misoprostol (EP(2), EP(3), and EP(4)) and PGE(1)-OH (EP(3) and EP(4)) stimulate iodide efflux; however, iloprost, butaprost, sulprostone, and fluoprostenol (agonists of the EP(1), EP(2), EP(3), and FP receptors, respectively) have no effect. The iodide efflux seen with 8-iso-PGE(2) is abolished by the EP(4) receptor antagonist AH23848, the CFTR inhibitor 172, and inhibition of PKA and the PI3K pathway. In conclusion, we demonstrate that although Calu-3 cells possess numerous prostanoid receptors, only the EP(4) subtype appears capable of eliciting a functional iodide efflux response, which is mediated via the EP(4) receptor. We propose that 8-iso-PGE(2), acting via EP(4) receptor, could play an important role in the CFTR-mediated response to oxidant stress, and which would be compromised in the CF airways.

Prostaglandin E2 Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Activity and Airway Surface Liquid Volume Requires Gap Junctional Communication

“…Palmer and colleagues showed in Calu-3 cells that PGE 2 activated CFTR via EP2-Rs and EP4-Rs (12). Joy and Cowley, in contrast, reported that Calu-3 cells express all four known EP-R subtypes, but point to EP4-Rs in the regulation of CFTR-dependent iodide efflux (28). Via RT-PCR, we only detected EP4-Rs in our Calu-3 cells, and EP4 inhibition was sufficient to prevent CFTR activation, confirming the results of Clayton and colleagues (29).…”

“…The production of PGE 2 via PLA 2 and COX activation and its paracrine stimulation of EP-Rs were proposed as a mechanism to amplify not only the engagement of A2B-Rs, but also of PARs (7,8,11,12,27,28). Controversy continues regarding which PGE 2 receptor subtype is involved in this signaling pathway.…”

H2O2 Stimulates Cystic Fibrosis Transmembrane Conductance Regulator through an Autocrine Prostaglandin Pathway, Using Multidrug-Resistant Protein–4

“…Previous studies showed that isoprostanoids, formed by nonenzymatic peroxidation, activated the Cl 2 secretion response in bovine tracheal epithelial cultures and Calu-3 cells (19,20). In contrast, we found that H 2 O 2 stimulated COX activity, resulting in prostanoid formation, as previously described in other cells (27).…”

“…However, hydroxyl radicals did not appear to play a role in the effects of H 2 O 2 on NHBE cells. Furthermore, isoprostanes have been reported to stimulate CFTR activity in cell lines (19), but the inhibition of H 2 O 2 -induced CFTR currents by COX inhibitors in NHBE cells suggests that isoprostanes, which are directly generated by H 2 O 2 exposure, were not responsible for the observed effects in fully differentiated airway epithelial cells.…”

“…Yet the mechanisms by which H 2 O 2 leads to the activation of the cAMP pathway are not understood. Although both Calu-3 cells (19) and bovine tracheal epithelia (20) have been shown to respond to isoprostanes through the activation of prostanoid receptors, suggesting that the oxidative stress-induced formation of isoprostanes may be a mechanism by which H 2 O 2 activates CFTR, findings in epithelial cell lines may not adequately reflect the processes relevant to the native human airway epithelium.…”