Pharmacological evidence from RA trials and preclinical models of arthritis points to the importance of COX metabolites in the pathogenesis of this disease (1
). Modulation of PGE2 levels in animal models of arthritis has demonstrated the importance of this prostanoid in arthritis progression (13
). In this study, we evaluated the role of individual prostaglandin EP receptors in an animal model of RA. The exact contribution of individual EP receptors is not evident from the literature. To address this question, four individual EP receptor–deficient mouse lines were generated by homologous recombination. Incidence and severity of arthritis were not significantly different between EP1–/–
, or EP3–/–
mice and their respective genetic wild-type controls. However, the EP4 receptor–deficient mice displayed reduced severity and incidence of arthritis than did their wild-type controls.
We considered several mechanisms to explain the resistance of EP4 receptor–deficient mice to developing CAIA. We examined the systemic and hepatic inflammatory responses in EP4+/+
animals. In CAIA, SAA levels in EP4+/+
, unlike EP4–/–
animals, were eightfold higher than the levels in their respective nondiseased counterparts. Both PGE2 exudate and IL-6 serum/exudate levels were significantly reduced in the diseased EP4 receptor–deficient animals compared with wild-type controls, suggesting a reduction in both local and systemic inflammation. Portanova and colleagues studied the role of PGE2 in rat antigen-induced arthritis using a PGE2-neutralizing monoclonal antibody. Animals receiving this antibody had decreased arthritis and serum IL-6 levels (13
). Interestingly, several investigators have shown that IL-6–deficient mice develop significantly less arthritis than do wild-type controls (41
). These experiments, in combination with our results, reveal an association between EP4 receptors, IL-6, and arthritis.
As described earlier, both EP2 and EP4 are coupled to Gαs
, stimulation of adenylyl cyclase, and increased concentration of intracellular cAMP. However, only EP4–/–
mice displayed reduced CAIA, suggesting that certain properties beyond proximal signal transduction are responsible for the phenotypic difference between EP2 receptor–deficient and EP4 receptor–deficient mice. These include differences in both the pattern and level of expression of these two receptors. In general, in wild-type mice, EP4 is expressed at higher levels than is EP2 in most tissues and cells examined in our studies. While both receptors can be detected on most cell types, the relative expression of the two receptors can vary dramatically. In addition, differences have been noted in PGE2-mediated receptor desensitization and internalization between EP2 and EP4 receptors expressed in mammalian cells. EP4 receptors underwent rapid agonist-induced desensitization and internalization within 10 minutes, whereas EP2 receptors displayed no such response, even after treatment with an agonist for 30 minutes (44
). Recent studies have also highlighted the divergence of EP2 and EP4 receptors in signaling pathways located downstream of adenylyl cyclase stimulation (46
). Furthermore, EP2 and EP4 receptors display different affinities for PGE2, PGE2 metabolites (e.g., 15-keto-PGE2), and synthetic analogues (e.g., butaprost free acid, 11-deoxy-PGE1), ranging from sixfold (PGE2) to greater than 200-fold (butaprost free acid) (14
). It is not surprising that EP2 receptor–deficient and EP4 receptor–deficient mice display distinct phenotypes based on the significant differences in biochemical properties and ligand binding profiles of the EP2 and EP4 receptors.
We demonstrate that genetic ablation of EP4 receptors normalizes SAA levels back to baseline, consistent with a complete reversal of the systemic and possibly hepatic inflammatory processes. The liver contributes to the inflammatory process by expressing and secreting acute-phase proteins such as SAA (47
). Livers isolated from diseased animals express EP4 receptor mRNA, consistent with previously published reports. In these in vitro studies, hepatocytes, following a period of incubation with IL-6, expressed EP4 mRNA (48
). In vivo, IL-1β and IL-6 administration induce acute-phase proteins (50
). Our data show that IL-1β mRNA levels were significantly reduced in diseased EP4–/–
livers compared with diseased wild-type controls. Collectively, our data is consistent with the participation of hepatic EP4 receptors in the acute phase response of our experimental model. However, we cannot rule out the possibility that SAA levels are indirectly related to the decrease in arthritis.
To further investigate the role of cytokines, we next examined the role of EP4 receptors on cytokine release from macrophages isolated from arthritic mice. Cytokines secreted by monocytic phagocytes are an important component of the inflammatory response in RA (52
). Peritoneal macrophages freshly isolated from diseased wild-type animals express EP4 mRNA and generate significantly more IL-6 than do EP4–/–
peritoneal macrophages also isolated from diseased animals. This was not due to a generalized signal transduction abnormality, as demonstrated by the similar responsiveness of EP4–/–
macrophages following treatment with LPS. Our observations are consistent with published reports describing a role for EP4 receptors in cytokine release and inflammation (21
). Furthermore, these studies suggest that while EP4 receptor stimulation may be important in dampening extracellular TNF-α release, within the context of our experimental model, the reduction in IL-6 is associated with a curtailment in inflammation.
EP2, EP3, and EP4 receptors are expressed in arthritic synovial cells (16
). EP4 receptor mRNA is increased during the course of synovitis in the joints of arthritic rats (16
) and in synovial fibroblasts isolated from patients with RA (19
). Matrix metalloproteases (MMPs) located in the synovial space (synovium and cartilage) likely contribute to cartilage degradation. Bone samples isolated from EP4–/–
mice express significantly less MMP2 and MMP13 than do EP4+/+
cells following PGE2 treatment (55
). Recent data (56
) suggest that MMP13 degrades type II collagen, resulting in the formation of a neoepitope detectable by the 9A4 monoclonal antibody described in our histopathological analyses. We demonstrate that type II collagen breakdown is significantly reduced in our histopathological analyses consistent with the proposed role of MMP activity.
In CAIA, genetic deletion of COX2 results in a phenotype similar to that of the EP4–/–
). The majority of COX2–/–
mice showed no clinical or histological signs of arthritis. COX2 is a key enzyme involved in the metabolism of arachidonic acid to prostaglandin H2, which is converted to biologically active prostaglandins such as PGE2. Pharmacological inhibition of COX2 enzymatic activity in animal models of arthritis (58
) and RA clinical trials leads to improved signs and symptoms of disease (1
). Our results suggest that the COX2-dependent PGE2 response in CAIA is mediated by EP4 receptors. We propose a model whereby EP4 receptors participate in both local and systemic inflammatory responses leading to the development of polyarthritis.
In summary, we characterized the role of EP receptors in an experimental model of arthritis. Targeted genetic disruption of EP1, EP2, or EP3 receptors had no impact on the development of arthritis. In contrast, EP4 receptor–deficient mice displayed little or no clinical sign of arthritis and deterioration of juxta-articular structures and bone. Inflammation biomarkers were also significantly diminished compared with EP4+/+ animals. Our results support the importance of both PGE2 and EP4 receptors in inflammation and suggest a novel point of pharmacological intervention in the treatment of RA.