ro3244794 and taprostene

Herein we provide evidence for the coexpression of two distinct prostacyclin (PGI(2)) receptors (IP) on BEAS-2B human airway epithelial cells. IP receptor heterogeneity initially was suggested by the finding that the rank orders of potency of PGI(2) and three structurally similar analogs [taprostene, iloprost, 15-deoxy-16-(m-tolyl)-17,18,19,20-tetranorisocarbacyclin (15-deoxy-TIC)] for the inhibition of chemokine (CXCL9 and CXCL10) release and for transcriptional activation/augmentation of cAMP response element and glucocorticoid response element luciferase reporters were distinct. Indeed, PGI(2), taprostene, and iloprost activated both reporters whereas 15-deoxy-TIC was inert. Conversely, 15-deoxy-TIC, PGI(2), and taprostene (but not iloprost) suppressed chemokine release. Further experiments established that iloprost did not antagonize the inhibitory effect taprostene or 15-deoxy-TIC on chemokine output. Likewise, 15-deoxy-TIC failed to antagonize taprostene- and iloprost-induced reporter transactivation. Thus, iloprost- and 15-deoxy-TIC-induced responses were apparently mediated via pharmacologically distinct receptors. In human embryonic kidney 293 cells overexpressing the human recombinant IP receptor receptor, 15-deoxy-TIC was considerably less potent (>10,000-fold) than iloprost and taprostene in promoting cAMP accumulation, yet in BEAS-2B cells, these analogs were equipotent. IP receptor heterogeneity was also supported by the finding that the affinity of the IP receptor antagonist R-3-(4-fluorophenyl)-2-[5-(4-fluorophenyl)-benzofuran-2-yl-methoxycarbonyl-amino] propionic acid (RO3244794) for the receptor mediating inhibition of chemokine release was approximately 10-fold lower than for the receptor mediating both transcriptional outputs. Finally, small interfering RNAs directed against the IP receptor gene, PTGIR, failed to block the suppression of chemokine output induced by taprostene and 15-deoxy-TIC, whereas taprostene- and iloprost-induced transcriptional responses were markedly attenuated. Collectively, these results indicate that PGI(2), taprostene and 15-deoxy-TIC suppress chemokine release from BEAS-2B cells by interacting with a novel IP receptor that we denote here as the "IP(2)" subtype.

Topics: Benzofurans; Cells, Cultured; Chemokine CXCL10; Chemokine CXCL9; Cyclic AMP; Dose-Response Relationship, Drug; Epoprostenol; HEK293 Cells; Humans; Iloprost; Propionates; Receptors, Epoprostenol; Receptors, Prostaglandin; Recombinant Proteins; Respiratory Mucosa

Prostacyclin receptor (IP-receptor) agonists display anti-inflammatory and antiviral activity in cell-based assays and in preclinical models of asthma and chronic obstructive pulmonary disease. In this study, we have extended these observations by demonstrating that IP-receptor activation also can enhance the ability of glucocorticoids to induce genes with anti-inflammatory activity. BEAS-2B bronchial epithelial cells stably transfected with a glucocorticoid response element (GRE) luciferase reporter were activated in a concentration-dependent manner by the glucocorticoid dexamethasone. An IP-receptor agonist, taprostene, increased cAMP in these cells and augmented luciferase expression at all concentrations of dexamethasone examined. Analysis of the concentration-response relationship that described this effect showed that taprostene increased the magnitude of transcription without affecting the potency of dexamethasone and was, thus, steroid-sparing in this simple system. RO3244794, an IP-receptor antagonist, and oligonucleotides that selectively silenced the IP-receptor gene, PTGIR, abolished these effects of taprostene. Infection of BEAS-2B GRE reporter cells with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA) also prevented taprostene from enhancing GRE-dependent transcription. In BEAS-2B cells and primary cultures of human airway epithelial cells, taprostene and dexamethasone interacted either additively or cooperatively in the expression of three glucocorticoid-inducible genes (GILZ, MKP-1, and p57(kip2)) that have anti-inflammatory potential. Collectively, these data show that IP-receptor agonists can augment the ability of glucocorticoids to induce anti-inflammatory genes in human airway epithelial cells by activating a cAMP/PKA-dependent mechanism. This observation may have clinical relevance in the treatment of airway inflammatory diseases that are either refractory or respond suboptimally to glucocorticoids.

Topics: Anilides; Anti-Inflammatory Agents, Non-Steroidal; Benzofurans; Bronchi; Cell Survival; Cells, Cultured; Colforsin; Cyclic AMP; Dexamethasone; Epoprostenol; Gene Expression; Genetic Vectors; Glucocorticoids; Humans; Intracellular Signaling Peptides and Proteins; Oligonucleotides; PPAR gamma; Propionates; Receptors, Epoprostenol; Respiratory Mucosa; Response Elements; RNA, Small Interfering; Transcriptional Activation