The growth inhibitory effect of PGE has been
The growth inhibitory effect of PGE2 has been linked to the ability of Gs coupled PGE2 receptors EP2 and EP4 to mediate elevation of cAMP , . Evidence suggests that this mechanism may not be the key cause of growth inhibition . In various cell types, EP4 receptor has been shown to utilize additional “nonclassical” cAMP transduction pathways, leading to phosphatidylinositol 3-kinase (PI3K) activation and to augmentation of NF-κB activity , . Using immunoblot analysis we showed that treatment with an EP4 receptor selective agonist caused decreased phosphorylation of NF-κB inhibitory IκBα, which was associated with increased amounts of unphosphorylated IκBα. The unphosphorylated proteins preserve NF-κB as dimers in the cytoplasm, thus preventing their translocation to the nucleus and transcription of NF-κB-dependent genes.
Several deficits have been identified in immature B cells, regarding the engagement of BCR-induced positive signals, including impaired activation of PKC, Akt, and Rel/NF-κB , , , . Both Akt and Rel/NF-κB synergistically regulate the expression and activation of cell survival and of cell cycle proteins such as BclX, Bfl-1, Mcl-1, Cyclin E, and E2F3 , , thus promoting proliferation of B cells. Inhibition of this positive signaling axis in pacap via NF-κB deficiency enhances B cell apoptosis and growth arrest following BCR stimulation , , . In the present study we show that pretreatment of the WEHI 231 cells with EP4 receptor agonist markedly reduces BCR triggered phosphorylation of IκBα, which results in the decreased transcription of NF-κB target genes such as TNF-α and IL-10. Although stimulation of EP4 receptor leads to activation of ERK1/2 and AKT in different cell types , , , , no such effects have been observed in immature B cell line WEHI 231. It appears that EP4 receptor stimulation at least in part enhances apoptosis of immature B cells, not by enhancing apoptotic pathways but by inhibiting pro-survival signals such as activation of NF-κB. Elucidation of chemokine suppression by PGE2 in macrophages resulted in the discovery of a novel PKA-independent signal transduction pathway mediated by EPRAP, the EP4 receptor-associated protein. EPRAP, a human analog of murine FEM1A, was found to interact directly with NF-κB1 protein p105, thus preventing its phosphorylation . Further investigations are required to investigate the precise mechanism by which EP4 receptor signaling modulates phosphorylation of IκBα. EP4 receptor activation could also lead to recruitment of β-arrestin 1 and 2 . It was reported that the interaction of β-arrestin 2 with IκBα prevents phosphorylation and degradation of IκBα, and thus attenuates activation of NF-κB and transcription of NF-κB target genes , .
Acknowledgements We thank Ono Pharmaceutical for supplying EP4 receptor antagonist ONO-AE3-208. We also thank Prof. Roger H. Pain for proof reading the manuscript. This work was supported by the Slovenian Research Agency grants BI-FR/08-10-003 and 1000-07-310183.
Introduction The vascular endothelium forms the barrier between blood circulation and the interstitial space and regulates the exchange of plasma components, adhesion and extravasation of leukocytes, and haemostasis . During inflammatory processes endothelial leakage occurs resulting in plasma extravasation and edema formation . The integrity of the endothelial barrier is tightly regulated by cell-to-cell contacts like adherent and tight junctions between adjacent cells and connection to the actin cytoskeleton , , . Prostanoids and phospholipids such as sphingosin-1-phosphate are involved in the regulation of endothelial barrier , . Prostaglandin (PG)E2 is the most abundant prostanoid in humans  and exerts a variety of biological functions through four different receptors (EP1–4), which differ in tissue specific gene expression . These receptors activate different signaling pathways: EP1 receptor binding leads to an increase of intracellular Ca levels, assumed of being coupled to a Gαq-protein. EP2 and EP4 receptors induce cyclic AMP (cAMP) production, whereas EP3 receptors couple to a Gαi-protein and inhibit cAMP synthesis . PGE2 is mainly regarded as a potent pro-inflammatory mediator due to its effects on vasodilation, vascular permeability and nociception . However, the role of PGE2 in the regulation of immune responses is more complex. Notably the lung represents a privileged organ with regard to PGE2 actions . In the airways, PGE2 shows an anti-inflammatory mode of action as it was demonstrated to inhibit the release of a number of cytokines and chemokines via activation of the EP4 receptor , ,  and inhibition of mast cell-induced bronchoconstriction via the EP2 receptors . However, EP1 and EP3 receptors play a minor role in regulation of inflammatory processes in the lung . PGE2 was shown to enhance the endothelial barrier function of human pulmonary artery endothelial cells via PKA and Epac/Rap activation leading to Rac activation and cytoskeletal remodeling . We recently revealed that PGE2 promotes barrier function in human pulmonary microvascular endothelial cells (HMVEC-L) via EP4 receptor-induced strengthening of the junction and reduces endothelial trafficking of neutrophils . Moreover, we recently demonstrated that PGE2, via activation of the EP4 receptor, also shows barrier promoting effects in vivo in a mouse model of lipopolysaccharide (LPS)-induced acute lung inflammation .