Prostaglandin E2 Receptor EP2 Subtype
EP2 is a G-protein-coupled seven-transmembrane receptor. Its N-terminal region is located in the extracellular, while the C-terminal regions in the cell cytoplasm. The middle section is constituted by the seven-transmembrane helix structure, three extracellular loops, and three intracellular loops. The activated G-proteins (Gs) are generally coupled with the third ring of cytoplasmic surface. Prostanoids, including prostaglandins (PGs) and thromboxanes (TXs), are metabolites of arachidonic acid and called 20 carbonate compounds. They are synthesized and released to couple with specific receptor aiming to exert their actions. Prostanoids exert their effects with the change of second messenger levels. In the mid-1960s, prostanoids had been noticed to cause changes in cyclic adenosine monophosphate (cAMP) levels and phosphatidylinositol (PI) turnover and free Ca2+ concentrations in the cell. In 1972, Kuehl et al. reported that there are many specific high-affinity binding sites in tissues and cells for the prostanoids (Kuehl and Humes 1972). These binding sites were later named prostaglandin receptors by Coleman et al. They integrated the previous results and proposed prostaglandins have several subtypes: TXs, prostaglandin I (PGI), prostaglandin E (PGE), prostaglandin F (PGF), and prostaglandin D (PGD). Their corresponding specific receptors are called TP, IP, EP, FP, and DP receptors. Like the other EP receptors, EP2 receptor has the seven putative transmembrane domains characteristic of G-protein-coupled receptors (Coleman et al. 1990). In addition, in 1994, the pharmacologically defined human EP2 subtype had been originally cloned and confirmed that the EP2 receptor is involved in a variety of biochemical reactions (Regan et al. 1994). Furthermore, to date, several articles reported that EP2 receptor activation in the human systems plays a role in both positive and negative effects.
PGE2 is most widely characterized in animal species and mediate various pathophysiological reactions, such as inflammation, pain, immunoregulation, mitogenesis, plasticity, and cell injury. When tissues and organs are exposed to stimuli, as the inducible COX isoform, COX-2 is rapidly induced by cell injury or excessive neuronal activity with simultaneous induction of membrane-associated PGE synthase-1 (mPGES-1 or PTGES), which produces PGE2 from COX-2-derived prostaglandin H2 (PGH2). As a stimulatory G-protein (Gs), EP2 receptor activation by PGE2 provokes adenylate cyclase (AC) synthesis and secretion, resulting in an increase of cytosolic cAMP production, which in turn initiates multiple downstream events via activation of protein kinase A (PKA) (Jiang and Dingledine 2013a). PKA directly induces phosphorylation and activation of transcription factors, such as the cAMP-responsive element-binding (CREB) protein, which upregulates Bcl-2 gene and brain-derived neurotrophic factor gene expression and mediates neuronal plasticity, long-term memory formation, neuronal survival, and neurogenesis in the brain (Jiang and Dingledine 2013b). Exchange protein directly activated by cAMP (Epac), another target of cAMP, has been identified to exist as two isoforms: Epac1 and Epac2. Epac is known as Rap guanine nucleotide exchange factor 3 (RAPGEF3) and Rap guanine nucleotide exchange factor 4 (RAPGEF4), respectively. They only differ in that Epac2 has an extra cAMP binding site and a ras-association domain for subcellular localization (Bos 2006). Epac activation by cAMP contributes to increase of downstream effectors Rap1/Rap2, which mediate a wide range of biological processes including the control of cell adhesion and cell-cell junction formation, insulin secretion, neurotransmitter release, etc. (Jiang and Dingledine 2013a). It is worth mentioning that the AC50 for Epac is higher than the AC50 for PKA, meaning that PKA is activated at a lower concentration of cAMP than Epac.
2-Physiological in Ovulation and Fertilization
The EP2 receptor plays an important role in ovulation and fertilization. Previous study showed that female EP2 receptor knockout (EP2−/−) mice have significantly reduced pregnancy rates, and when these animals do become pregnant, they deliver smaller litters than EP2+/+ and wild-type mice. The follow-up tests indicated that EP2−/− females are infertile secondary to failure of the released ovum to become fertilized in vivo. Since the EP2−/− ova could be fertilized in vitro, this suggest that the EP2 receptor may contribute to the microenvironment in which fertilization takes place (Tilley et al. 1999). Hizaki et al. proposed that EP2−/− mice reduced fertility is partly attributable to reduction in ovulation and predominantly attributable to severe failure of fertilization. In addition, compared with wild-type cumulus cells, the EP2−/− cumulus cells of the complexes in oviducts just before fertilization were less expanded, indicating that EP2 is involved in cumulus expansion required for successful fertilization (Hizaki et al. 1999).
In recent years, some molecular basis of the impaired fertilization in EP2-/- mice was reported. Cumulus cells begin to exert immune cell-like functions upon ovulation with expression of a set of genes such as complement components, cytokine receptors, and CC chemokines. Such chemokine signaling stimulates integrin engagement to the extracellular matrix (ECM) proteins via the RhoA-ROCK-actomyosin pathway, and CCL7-CCR signaling increases the viscosity of the cumulus ECM so that it is strong enough to endure mechanical stress, but this chemokine action leads to the suppression of fertilization. However, the chemokine gene expression can be suppressed by PGE2-EP2 signaling in cumulus cells through the cAMP pathway (Sugimoto et al. 2015). In addition, the chemokine actions should be downregulated and the cumulus ECM should be disassembled, so that sperm can penetrate the cumulus ECM layer; the PGE2-EP2 system contributes to this process, which is required for successful fertilization. Although there are many detailed mechanics of EP2 receptor influence on ovulation and fertilization that are far from clear, EP2 receptor antagonist may be a suitable candidate as a contraceptive for women.
3-Physiological in Tumorigenesis and Progression
EP2 receptor participates in multiple processes of tumorigenesis and progression, including tumor cell proliferation, migration, angiogenesis, and immunosuppression (Jiang and Dingledine 2013a). Recent studies suggest that genetic ablation of EP2 reduces the number and size of intestinal polyps in the mouse model, and activation of EP2 can in turn boost expression of COX-2 and vascular endothelial growth factor (VEGF) in polyp tissues (Sonoshita et al. 2001). In addition, the EP2 agonist butaprost promotes growth and invasion of prostate tumor cells, while this effect can be blocked by the EP2 antagonist TG4-155 (Jiang and Dingledine 2013b). The effects of EP2 receptor on tumor are associated with activation of PI3K-Akt and Ras-ERK pathways, which promote tumor cell activities and can be blocked by the EP2 antagonist. EP2 activation can upregulate pro-inflammatory cytokines, including IL-1β and IL-6 in cancer cells, while downregulating antitumor cytokines such as IFN-γ and TNF-α in immune cells. IL-1β has been proved that its capability of promoting tumor growth, invasiveness, and angiogenesis and the elevated levels of IL-6 are related to progression of many types of cancer, including prostate, colorectal, breast, and ovarian cancers. In the future, the study of EP2 receptor may contribute to the management of tumor.
4-Physiological in Blood Pressure (BP) Regulation
The EP2 receptor is the most predominant PGE2 receptor in vascular smooth muscle cells and is associated with several pathological and physiological changes, such as arterial dilatation, spontaneous hypertension, atherosclerosis, etc. EP2 receptors via the cAMP pathway have the potential to control vascular tone and BP, which had been demonstrated via animal experiments in the past years. EP2−/− mice showed slightly elevated baseline systolic blood pressure and developed profound systolic hypertension when fed a high-salt diet, whereas the blood pressure did not change in wild-type mice. The hypertension can be quickly reversed by switching to normal salt diet and is reestablished by resumption of the high-salt diet, establishing salt sensitivity (Kennedy et al. 1999). Furthermore, intramedullary PGE2 infusion to anesthetized, uninephrectomized mice produced natriuresis and diuresis, an effect that was recapitulated by intramedullary infusion of the EP2 agonist butaprost. And the natriuresis was absent in EP2−/− mice. The results suggest an important role of the EP2 receptor in mediating the renal natriuretic action of PGE2, which is related to the blood pressure regulation (Chen et al. 2008).
5-Pathophysiological Functions in Central Nervous System Diseases
In Intracerebral Hemorrhage (ICH)
PGE2-EP2 signaling pathway plays a complex role in the secondary injury following ICH. Several mechanisms, including cytotoxicity of blood, hypermetabolism, excitotoxicity, spreading depression, oxidative stress, inflammation, etc., are involved in these processes. After ICH onset, the inflammatory begins with the activation and accumulation of blood-derived leukocytes, astrocytes, and resident of microglia. Activation of inflammatory cells could facilitate the release of cytokines and chemokines and formation of reactive oxygen species, which had been reported to be modulated by the EP2 receptor (Wang and Doré 2007). The study by Leclerc et al. revealed that significantly less microgliosis and astrogliosis, smaller brain lesion volumes, and less ipsilateral hemispheric enlargement were seen in EP2−/− mice (Leclerc et al. 2015). In addition, the release of hemoglobin and its decomposition products, particularly the iron ion, would stimulate the glutamate receptors including NMDA, a-amino-3-hydroxy-5-methyl-4-isoxa-zolep-propionate (AMPA), kainate receptors, etc. and induce glutamate-mediated excitotoxicity (Sharp et al. 2008). Moreover, the acute excitotoxicity can be prevented by EP2 receptor activation with regulated cAMP levels, suggesting that EP2 receptor stimulation mediates neuroprotection against NMDA excitotoxicity (Ahmad et al. 2006). However, Takadera et al. had proposed that butaprost and forskolin (an activator of adenylate cyclase) are both showed to enhance NMDA-mediated neurotoxicity (Takadera and Ohyashiki 2006). These opposite results may be explained that under different doses of agonist, different downstream signaling pathways were activated. It is worth mentioning that iron, metabolite of RBC, has a detrimental effect on the secondary damage following ICH. And previous studies indicated that the EP2 receptor-induced signaling impairs the phagocytic capabilities of macrophages and microglia, both of which are responsible for clearance of iron in the brain (Leclerc et al. 2015). In conclusion, the EP2 receptor plays a complex role in ICH, and it may be a promising therapeutic target.
In Ischemic Stroke
Ischemic stroke results from critically reduced blood flow in one or more arteries of the brain or spinal cord. Ischemic stroke-induced secondary neurological deficits are closely associated with excitotoxic and anoxic injury, which can be protected by PGE2-EP2 receptor in a cAMP-dependent manner (McCullough et al. 2004). Previous studies showed that mice lacking EP2 exhibited significantly larger infarct size and larger stroke volumes compared to wild-type mice in models of both transient and permanent focal ischemia, with middle cerebral artery occlusion (McCullough et al. 2004; Liu et al. 2005). Ahmad et al. also obtained similar results, and they demonstrated that an injection of a selective EP2 agonist-ONO-AE1-259-01 into the lateral ventricle contributed to reduce neurological deficits and hemispheric infarct volumes (Ahmad et al. 2006). And blocking the activation of PKA could abolish this protective effect, suggesting that EP2-mediated neuroprotection is dependent on cAMP-PKA signaling. Taken together, activating the EP2-cAMP-PKA signaling may be an effective treatment strategy for ischemic stroke.
In Inflammatory Neurodegenerative Diseases
Activation of EP2 receptor in microglia might promote inflammation in several mouse models of neurodegenerative diseases including AD, PD, and ALS. Several mechanisms contribute to neuronal death in these diseases including neurotoxicity, glutamate receptor-mediated excitotoxicity, and innate immune activation (Minghetti 2004). And the deletion of EP2 receptors reduces the oxidative damage and amyloid burden in a model of AD, attenuates neurotoxicity by α-synuclein aggregation in a mouse model of PD, and improves motor strength while extending the survival of ALS mice. Moreover, these results indicate that cAMP-activated Epac, not PKA, appears to mediate the effect of EP2 receptors on inflammatory genes in classically activated microglia, which contribute to the delayed neurotoxicity. Compared with PKA, Epac has a lower affinity for cAMP: at the beginning of EP2 receptor activation, cAMP initially stimulates PKA signaling, whereas for sustained EP2 activation, the Epac pathway dominates as cytoplasmic cAMP levels continue to rise (Jiang and Dingledine 2013a). In general, EP2-cAMP-mediated Epac signaling pathway augments neurological deficits in chronic inflammatory diseases. This might provide a theoretical basis for the treatment of chronic neurodegenerative diseases.
EP2 receptors have been reported to be widely expressed in multiple tissues and are involved in regulating a series of pathology and physiology reactions. In these processes, since the complexity of its downstream signaling pathways, EP2 receptor may play a varied role in different environments. As has been described before, Ep2−/− mice showed significantly reduced pregnancy rates, and when these animals do become pregnant, they deliver smaller litters than Ep2+/+ and wild-type mice. EP2 receptor activation may play a positive role in this physiological process. However, when it comes to the tumorigenesis and progression, the EP2 receptor activation seems to promote the genesis of tumor and tumor cell growth. Even in the same disease, such as in the ICH, EP2 receptor showed opposite effects. Although there are still a lot of observed results that cannot be explained by the existing theories, further studies are needed to explore the mechanisms of EP2 receptor work in molecular levels; it is no denying that the EP2 receptor may be a promising therapeutic target when its selective agonists and antagonists are artificially synthesized.
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