EP300 Protects from Light-Induced Retinopathy in Zebrafish.

Exposure of rhodopsin to bright white light can induce photoreceptor cell damage and degeneration. However, a comprehensive understanding of the mechanisms underlying light-induced retinopathy remains elusive. In this study, we performed comparative transcriptome analysis of three rodent models of light-induced retinopathy, and we identified 37 genes that are dysregulated in all three models. Gene ontology analysis revealed that this gene set is significantly associated with a cytokine signaling axis composed of signal transducer and activator of transcription 1 and 3 (STAT1/3), interleukin 6 signal transducer (IL6ST), and oncostatin M receptor (OSMR). Furthermore, the analysis suggested that the histone acetyltransferase EP300 may be a key upstream regulator of the STAT1/3-IL6ST/OSMR axis. To examine the role of EP300 directly, we developed a larval zebrafish model of light-induced retinopathy. Using this model, we demonstrated that pharmacological inhibition of EP300 significantly increased retinal cell apoptosis, decreased photoreceptor cell outer segments, and increased proliferation of putative Müller cells upon exposure to intense light. These results suggest that EP300 may protect photoreceptor cells from light-induced damage and that activation of EP300 may be a novel therapeutic approach for the treatment of retinal degenerative diseases.

Structural optimization and structure-functional selectivity relationship studies of G protein-biased EP2 receptor agonists.

The modification of the novel G protein-biased EP2 agonist 1 has been investigated to improve its G protein activity and develop a better understanding of its structure-functional selectivity relationship (SFSR). The optimization of the substituents on the phenyl ring of 1, followed by the inversion of the hydroxyl group on the cyclopentane moiety led to compound 9, which showed a 100-fold increase in its G protein activity compared with 1 without any increase in ß-arrestin recruitment. Furthermore, SFSR studies revealed that the combination of meta and para substituents on the phenyl moiety was crucial to the functional selectivity.

Discovery of G Protein-Biased EP2 Receptor Agonists.

To identify G protein-biased and highly subtype-selective EP2 receptor agonists, a series of bicyclic prostaglandin analogues were designed and synthesized. Structural hybridization of EP2/4 dual agonist 5 and prostacyclin analogue 6, followed by simplification of the ω chain enabled us to discover novel EP2 agonists with a unique prostacyclin-like scaffold. Further optimization of the ω chain was performed to improve EP2 agonist activity and subtype selectivity. Phenoxy derivative 18a showed potent agonist activity and excellent subtype selectivity. Furthermore, a series of compounds were identified as G protein-biased EP2 receptor agonists. These are the first examples of biased ligands of prostanoid receptors.

IOP-Lowering Effect of ONO-9054, A Novel Dual Agonist of Prostanoid EP3 and FP Receptors, in Monkeys.

PURPOSE: The purpose of this study was to determine whether a better IOP reduction can be observed in conscious, normotensive monkeys treated with ONO-9054, a novel dual EP3 and FP receptor agonist, compared with prostaglandin F2α analogs. METHODS: The binding affinities and agonistic activities of ONO-AG-367, a carboxylic acid of ONO-9054, to prostanoid receptors were assessed. The IOP-lowering effect of ONO-9054 in monkeys was analyzed after a single (0.3, 3, or 30 µg/mL) or 7-day repeated (30 µg/mL, every day) topical ocular administration. Ophthalmologic and histopathologic evaluations of the eye were performed after 4-week ocular administration of ONO-9054 (30 µg/mL, twice a day) in monkeys. RESULTS: The ONO-AG-367 exhibited high affinity for both EP3 and FP receptors and potent agonist activity, with EC50 values of 28.6 nM for the EP3 receptor and 22.3 nM for the FP receptor. Single and repeated topical ocular administration of ONO-9054 caused IOP reductions in normotensive monkeys. The maximum IOP reductions on day 7 observed with ONO-9054 (7.3 ± 0.8 mm Hg) were significantly greater than those observed with latanoprost (50 µg/mL, 4.9 ± 0.4 mm Hg) or travoprost (40 µg/mL, 5.1 ± 0.6 mm Hg). In ophthalmologic and histopathologic evaluations, slight and transient mydriasis was occasionally observed and no histopathologic lesions attributable to ONO-9054 were noted. CONCLUSIONS: A more profound and longer-lasting reduction in IOP in normotensive monkeys can be observed with ONO-9054, which simultaneously stimulates both EP3 and FP receptors, compared with prostaglandin analogs.

Curcumin Inhibits the proteinase-activated receptor-2-triggered prostaglandin E2 production by suppressing cyclooxygenase-2 upregulation and Akt-dependent activation of nuclear factor-κB in human lung epithelial cells.

We performed this study to determine if curcumin affects pro-inflammatory responses to activation of proteinase-activated receptor-2 (PAR2) in human pulmonary adenocarcinoma A549 cells. Curcumin completely inhibited the PAR2-triggered prostaglandin E(2) (PGE(2)) production, but notably not interleukin-8 release. Cyclooxygenase-2 (COX-2) upregulation, but not its upstream activation of mitogen-activated protein kinases, caused by PAR2 stimulation was partially inhibited by curcumin. Curcumin inhibited the PAR2-triggered phosphorylation of I-κB, an indicator for nuclear factor-κB (NF-κB) activation, and also its upstream signal Akt, which is known to contribute to PAR2-triggered PGE(2) formation, but not COX-2 upregulation. Collectively, curcumin inhibits the PAR2-triggered PGE(2) production by suppressing COX-2 upregulation and Akt/NF-κB signals in A549 cells.

Proteinase-activated receptor-2-triggered prostaglandin E(2) release, but not cyclooxygenase-2 upregulation, requires activation of the phosphatidylinositol 3-kinase/Akt / nuclear factor-kappaB pathway in human alveolar epithelial cells.

Proteinase-activated receptor-2 (PAR2) triggers upregulation of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)) formation in human alveolar epithelial A549 cells. This COX-2 upregulation appears to involve the Src / epidermal growth factor (EGF) receptor / p38 MAP kinase (p38MAPK) pathway and also the cAMP-response element-binding protein (CREB) pathway. Here, we investigated the roles of nuclear factor-kappaB (NF-kappaB)-related signals in the PAR2-triggered PGE(2) release / COX-2 upregulation in A549 cells. The PAR2-triggered PGE(2) release was clearly blocked by an inhibitor of the NF-kappaB pathway. Stimulation of PAR2 actually caused phosphorylation of inhibitor-kappaB, an indicator of NF-kappaB activation, an effect being blocked by inhibitors of MEK, phosphatidylinositol 3-kinase (PI3-kinase), and Akt, but little or not by inhibitors of p38MAPK and JNK. Stimulation of PAR2 also caused phosphorylation of Akt, an effect suppressed by inhibitors of PI3-kinase and MEK. Nonetheless, the PAR2-triggered upregulation of COX-2 was resistant to inhibitors of NF-kappaB, PI3-kinase, and Akt, but was attenuated by inhibitors of MEK and JNK. Stimulation of PAR2 induced phosphorylation of CREB, an effect abolished by an inhibitor of MEK but not inhibitors of p38MAPK and EGF receptor. These findings demonstrate that the MEK / ERK / PI3-kinase / Akt / NF-kappaB pathway is involved in PAR2-triggered PGE(2) formation, but not upregulation of COX-2 that is dependent on activation of ERK/CREB and JNK in addition to p38MAPK.

Evidence that PAR2-triggered prostaglandin E2 (PGE2) formation involves the ERK-cytosolic phospholipase A2-COX-1-microsomal PGE synthase-1 cascade in human lung epithelial cells.

We investigated possible involvement of three isozymes of prostaglandin E synthase (PGES), microsomal PGES-1 (mPGES-1), mPGES-2 and cytosolic PGES (cPGES) in COX-2-dependent prostaglandin E(2) (PGE(2)) formation following proteinase-activated receptor-2 (PAR2) stimulation in human lung epithelial cells. PAR2 stimulation up-regulated mPGES-1 as well as COX-2, but not mPGES-2 or cPGES, leading to PGE(2) formation. The PAR2-triggered up-regulation of mPGES-1 was suppressed by inhibitors of COX-1, cytosolic phospholipase A(2) (cPLA(2)) and MEK, but not COX-2. Finally, a selective inhibitor of mPGES-1 strongly suppressed the PAR2-evoked PGE(2) formation. PAR2 thus appears to trigger specific up-regulation of mPGES-1 that is dependent on prostanoids formed via the MEK/ERK/cPLA(2)/COX-1 pathway, being critical for PGE(2) formation.

Signal transduction for formation/release of interleukin-8 caused by a PAR2-activating peptide in human lung epithelial cells.

Proteinase-activated receptor-2 (PAR2) plays a dual role in the respiratory system, being pro- and anti-inflammatory. In human lung epithelial cells (A549), PAR2 activation causes release of interleukin-8 (IL-8) in addition to prostaglandin E(2) (PGE(2)). In the present study, we thus investigated PAR2-triggered signal transduction pathways causing IL-8 formation in A549 cells. SLIGRL-NH(2), a PAR2-activating peptide, but not LSIGRL-NH(2), a scrambled peptide, elicited release of IL-8 from A549 cells for 18 h, as measured by the ELISA method, an effect being suppressed by inhibitors of MEK, JNK, EGF receptor-tyrosine kinase (EGFR-TK), Src, pan-tyrosine kinases and protein kinase C, but not p38 MAP kinase or cyclooxygenase. SLIGRL-NH(2) also up-regulated IL-8 at protein and mRNA levels, as determined by Western blotting and RT-PCR, respectively. The PAR2-triggered up-regulation of IL-8 protein and mRNA was blocked by an inhibitor of MEK, but not clearly by inhibitors of JNK and EGFR-TK. SLIGRL-NH(2) actually phosphorylated JNK as well as ERK, the JNK activation being resistant to inhibitors of Src, pan-tyrosine kinases, protein kinase C and EGFR-TK. Our data suggest that PAR2-triggered IL-8 formation involves transcriptional up-regulation of IL-8 via the MEK-ERK pathway, while the JNK and EGF receptor pathways might rather contribute to a post-transcriptional process for the release of IL-8.