Evaluation Of Free-fatty Acid Receptor-4 (ffa4) On Modulation Of Ros Generation And Cox-2 Expression Via The C-terminal β-arrestin Phosphosensor

Abstract

Agonism of the G-protein coupled free-fatty acid receptor-4 (FFA4) has been shown to promote numerous anti-inflammatory effects in macrophages that arise due to interaction with β-arrestin partner proteins upon the receptor phosphorylations. Humans express functionally distinct short and long FFA4 splice variants, such that FFA4-S signals through both Gαq/11 and β-arrestin, while FFA4-L is intrinsically biased solely towards β-arrestin signaling. Recently, we (and others) have shown that phosphorylation of the FFA4-S and FFA4-L C-terminal tail is responsible for β-arrestin intractability and signaling. However, there is no apparent phosphorylation or arrestin recruitment in the C-terminal truncated mutant FFA4-L-Δ356, indicating that the C-terminal of FFA4-L plays a critical role in β-arrestin recruitment. Given the significance of β-arrestin in FFA4 anti-inflammatory function, the first objective of this study was to examine the role of the C-terminal β-arrestin phosphosensor in FFA4-S and FFA4-L signaling. To reach this objective, we employed PMA induced COX-2 expression, LPS induced NF-κB activity, and ERK1/2 phosphorylation in murine Raw 264.7 macrophages.

The second objective was to assess the role of dietary omega-3 fatty acid supplementation on FFA4 anti-inflammatory effect. To reach this objective, we supplemented rat diets with fish oil and flaxseed oil, and studied the proinflammatory cytokine TNF-α release in the rat colon. Our data reveal for the first time that both FFA4 isoforms modulate PMA-induced ROS generation, and that abolishment of the FFA4-S but not FFA4-L C-terminal phosphosensor, is detrimental to this effect. Furthermore, we show that while both isoforms reduce PMA-induced expression of COX-2, removal of the FFA4-S phosphosensor significantly decreases this response, suggesting that these effects of FFA4-S are β-arrestin mediated. On the contrary, FFA4-S, as well as the truncated C-terminal congener lacking the β-arrestin phosphosensor were both able to reduce LPS-induced NF-κB activity and ERK1/2 phosphorylation. However, FFA4-L and its corresponding mutant were incapable of modulating either, suggesting that these responses are mediated by G-protein coupling. In this thesis, it has been shown that the agonism of FFA4-S and FFA4-L with DHA and PKC activator lead to phosphorylation. However, there is no apparent phosphorylation or β-arrestin recruitment in the C-terminal truncated mutant FFA4-L-Δ356, indicating that the C-terminal of FFA4-L plays a critical role in β-arrestin recruitment. We also showed that supplementation of diets with 10 % fish oil or flaxseed oil for a period of 7 weeks in rats facilitates up regulation of FFA4, and decreases proinflammatory cytokine TNF-α in the rat colon. Taken together, our data reveals important structure-function and signaling differences between the two FFA4 isoforms, and for the first time links FFA4 to modulation of ROS in macrophages. The anti-inflammatory effect of FFA4 agonism in rat colon is also discussed.