Neuroprotective role of prostaglandin PGE2 EP2 receptor in hemin-mediated toxicity.

Heme (Fe(2+) protoporphyrin IX) and hemin (Fe(3+)), the prosthetic group of hemoprotein, are cytotoxic due to their ability to contribute to the production of reactive oxygen species, increased intracellular calcium levels, and stimulate glutamate-mediated excitotoxicity. Previous work by our group showed that blockade of the prostaglandin E2 (PGE2)-EP1 receptor reduced hemin-induced cytotoxicity in primary cortical neuronal cultures. However, the role of the prostaglandin E2 (PGE2)-EP2 receptor in hemin neurotoxicity remains unclear. Activation of the EP2 receptor in neurons results in increased cyclic AMP (cAMP) and protein kinase A signaling; therefore, we hypothesized that the activation of the EP2 receptor decreases hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wildtype-control (WT) and EP2(-/-) mice, we investigated the role of the EP2 receptor in hemin neurotoxicity by monitoring cell survival with the Calcein-AM live-cell and lactate dehydrogenase assays. MitoTracker staining was also performed to determine how mitochondria were affected by hemin. Hemin neurotoxicity in EP2(-/-) neurons was 37.2 ± 17.0% greater compared to WT neurons. Of interest, cotreatment with the EP2 receptor agonist, butaprost (1 and 10 µM), significantly attenuated hemin neurotoxicity by 55.7 ± 21.1% and 60.1 ± 14.8%, respectively. To further investigate signaling mechanisms related to EP2 receptor mediating cytoprotection, neurons were cotreated with hemin and activators/inhibitors of both the cAMP-protein kinase A/exchange protein directly activated by cAMP (Epac) pathways. Forskolin, a cAMP activator, and 8-pCPT-cAMP, an Epac activator, both attenuated hemin neurotoxicity by 78.8 ± 22.2% and 58.4 ± 9.8%, respectively, as measured using the lactate dehydrogenase assay. Together, the results reveal that activation of the EP2 receptor is protective against hemin neurotoxicity in vitro and these findings suggest that neuroprotection occurs through the cAMP-Epac pathway in neuronal cultures. Therefore, activation of the EP2 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin.

Prostaglandin E receptor subtype EP3 in conjunctival epithelium regulates late-phase reaction of experimental allergic conjunctivitis.

BACKGROUND: We previously demonstrated that the prostaglandin E(2) (PGE(2))-EP3 pathway negatively regulates allergic reactions in a murine allergic asthma model. OBJECTIVES: We investigated whether the PGE(2)-EP3 pathway also regulates the development of murine experimental allergic conjunctivitis (EAC). METHODS: The expression of EP3 was examined by means of RT-PCR and immunohistochemistry in wild-type mice, as well as by means of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining in mice deficient in EP3 (Ptger3(-/-) mice) carrying the beta-galactosidase gene at the EP3 gene locus. EAC was induced by immunization of mice with short ragweed pollen (RW), followed by challenge with eye drops of RW, and eosinophil infiltration and eotaxin-1 mRNA expression in the conjunctiva were examined. Mice were also treated with a topical application of an EP3-selective agonist during the elicitation phase. Quantitative RT-PCR was used to detect expression of COXs and prostaglandin E synthases, and ELISA was used to measure PGE(2) production in the eyelid. RESULTS: EP3 was constitutively expressed in conjunctival epithelium on the ocular surface. Ptger3(-/-) mice demonstrated significantly increased eosinophil infiltration in conjunctiva after RW challenge compared with wild-type mice. Consistently, significantly higher expression of eotaxin-1 mRNA was observed in Ptger3(-/-) mice. Conversely, treatment of wild-type mice with an EP3-selective agonist resulted in a significant decrease in eosinophil infiltration, which was blunted in Ptger3(-/-) mice. Expression of COX-2 and prostaglandin E synthases was upregulated and PGE(2) content was increased in the eyelids after RW challenge. CONCLUSION: These data suggest that PGE(2) acts on EP3 in conjunctival epithelium and downregulates the progression of EAC.

Prostacyclin prevents murine lung cancer independent of the membrane receptor by activation of peroxisomal proliferator--activated receptor gamma.

Overexpression of prostacyclin synthase (PGIS) decreases lung tumor multiplicity in chemical- and cigarette-smoke-induced murine lung cancer models. Prostacyclin signals through a single G-protein-coupled receptor (IP), which signals through cyclic AMP. To determine the role of this receptor in lung cancer chemoprevention by prostacyclin, PGIS-overexpressing mice were crossed to mice that lack the IP receptor [IP(-/-)]. Carcinogen-induced lung tumor incidence was similar in IP(+/+), IP(+/-), and IP(-/-) mice, and overexpression of PGIS gave equal protection in all three groups, indicating that the protective effects of prostacyclin are not mediated through activation of IP. Because prostacyclin can activate members of the peroxisomal proliferator-activated receptor (PPAR) family of nuclear receptors, we examined the role of PPARgamma in the protection of prostacyclin against lung tumorigenesis. Iloprost, a stable prostacyclin analogue, activated PPARgamma in nontransformed bronchial epithelial cells and in a subset of human non-small-cell lung cancer cell lines. Iloprost-impregnated chow fed to wild-type mice resulted in elevated lung macrophages and decreased lung tumor formation. Transgenic animals with lung-specific PPARgamma overexpression also developed fewer lung tumors. This reduction was not enhanced by administration of supplemental iloprost. These studies indicate that PPARgamma is a critical target for prostacyclin-mediated lung cancer chemoprevention and may also have therapeutic activity.

Molecular cloning and characterization of CLICK-III/CaMKIgamma, a novel membrane-anchored neuronal Ca2+/calmodulin-dependent protein kinase (CaMK).

During a screen for novel putative Ca(2+)/calmodulin-dependent protein kinase (CaMK)-like CREB kinases (CLICKs), we have cloned a full-length cDNA for CLICK-III/CaMKIgamma, an isoform of the CaMKI family with an extended C-terminal domain ending with CAAX motif (where AA is aliphatic acid). As expected from the similarity of its kinase domain with the other CaMKI isoforms, full activation of CLICK-III/CaMKIgamma required both Ca(2+)/CaM and phosphorylation by CaMKK. We also found that Ca(2+)/cAMP-response element-binding protein (CREB) was a good substrate for CLICK-III/CaMKIgamma, at least in vitro. Interestingly enough, CLICK-III/CaMKIgamma transcripts were most abundant in neurons, with the highest levels in limited nuclei such as the central nucleus of the amygdala (CeA) and the ventromedial hypothalamus. Consistent with the presence of the CAAX motif, CLICK-III/CaMKIgamma was found to be anchored to various membrane compartments, especially to Golgi and plasma membranes. Both point mutation in the CAAX motif and treatment with compactin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, disrupted such membrane localization, suggesting that membrane localization of CLICK-III/CaMKIgamma occurred in a prenylation-dependent way. These findings provide a novel mechanism by which neuronal CaMK activity could be targeted to specific membrane compartments.