Contribution of the two Gs-coupled PGE₂-receptors EP2-receptor and EP4-receptor to the inhibition by PGE₂ of the LPS-induced TNFα-formation in Kupffer cells from EP2-or EP4-receptor-deficient mice. Pivotal role for the EP4-receptor in wild type Kupffer cells

Abstract

Background/Aims: Prostaglandin E₂ (PGE₂) is known to inhibit the lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) formation in Kupffer cells via an increase in cAMP. Four receptor-subtypes have been cloned for PGE₂ so far. Two of them, the EP2-receptor and the EP4-receptor are linked to stimulatory Gs-proteins and could mediate the inhibition by PGE₂ of TNFα-formation.

Methods: The significance of both receptors for PGE₂-dependent inhibition of LPS-induced TNFα-formation was studied using Kupffer cells of mice in which either one of the two receptors had been eliminated by homologous recombination.

Results: The mRNAs of both receptors were expressed in wild type mouse Kupffer cells. Exogenous PGE₂ inhibited TNFα-formation in Kupffer cells lacking either EP2-receptor or EP4-receptor to a similar extent as in control cells, however, 10-fold higher PGE₂ concentrations were needed for half maximal inhibition in cells lacking the EP4-receptor than in control or EP2-receptor-deficient cells. The response to endogenous PGE₂ was blunted in EP4-receptor-deficient mice only and especially after prolonged incubation.

Conclusions: The data indicate, that PGE₂ can inhibit TNFα-formation via both the EP2- and the EP4-receptor and that, however, the EP4-receptor appears to be physiologically more relevant in Kupffer cells since it conferred a high affinity response to PGE₂.

Introduction

Prostanoids are involved in the regulation of hepatic functions during inflammation. They are released from non-parenchymal liver cells in response to a large array of inflammatory stimuli such as lipopolysaccharide (LPS) [1], zymosan [2], anaphylatoxins [3], [4] and proinflammatory cytokines [5]. Prostanoids modulate hepatocyte functions in a paracrine mode: they influence the glucogenic activity of the hepatocyte by regulation of enzyme activity [6], [7], [8], [9], control of the expression level of key glucogenic enzymes [10] and also indirectly via a modulation of sinusoidal blood flow [11]. Recently, they have been shown to attenuate the IL-6-induced acute phase response of the hepatocyte after an IL-6-dependent expression of previously absent Gs-coupled prostaglandin E₂-(PGE₂-) receptors [12]. Prostanoids also modulate non-parenchymal cell functions in paracrine and autocrine modes, e.g. thromboxane A₂ (TXA₂) or prostaglandin F_2α (PGF_2α) contract trans-differentiated hepatic stellate cells [13] while contraction is inhibited by PGE₁, PGE₂ or prostacyclin (PGI₂) [14], [15]. The LPS-induced release of NO [16], [17], reactive oxygen species, or cytokines [18] from Kupffer cells is inhibited by PGE₂. For the LPS-induced release of TNFα an autocrine feedback inhibition loop has been shown to exist [19], [20]: LPS and TNFα stimulate the release of PGE₂ from Kupffer cells. PGE₂ in turn attenuates the TNFα-production via an increase in intracellular cAMP [19]. It is not known so far, which prostanoid-receptor is involved in this signaling chain.

Prostanoids exert their effects on their target cells via heptahelical transmembrane receptors. For the five prostanoids prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α), prostaglandin D₂ (PGD₂), prostacyclin (PGI₂) and thromboxane A₂ (TXA₂) there exist eight types of G-protein-coupled receptors [21]. Four subtypes exist for PGE₂, that are coupled to different heterotrimeric G-proteins. The EP1-receptor (EP1-R) couples to Gq, the EP3-receptor (EP3-R) couples to Gi whereas the EP2-receptor (EP2-R) and the EP4-receptor (EP4-R) couple to Gs [22]. These receptors are expressed differentially in the four principal liver cells types, i.e. hepatocytes, Kupffer cells, sinusoidal endothelial cells and hepatic stellate cells [23]. Kupffer cells express both types of Gs-coupled PGE₂-R that might possibly confer the PGE₂-mediated cAMP-dependent inhibition of TNFα-production. Therefore, the question was addressed, which of the two receptors is part of the feedback inhibition loop. Using Kupffer cell cultures isolated from mice lacking either the EP2-R or the EP4-R it was shown, that both receptors can mediate the PGE₂-dependent inhibition of LPS-induced TNFα-formation and can at least partially substitute for each other in Kupffer cells of receptor deficient mice. However, the EP4-R appeared to be physiologically more relevant.