Evidence for a Second Receptor for Prostacyclin on Human Airway Epithelial Cells That Mediates Inhibition of CXCL9 and CXCL10 Release

Abstract

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,18,19,] 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 (Ͼ10,000-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 iloprostinduced 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.

Molecular Mechanisms Regulating the Vascular Prostacyclin Pathways and Their Adaptation during Pregnancy and in the Newborn

“…The PR family comprises eight classic members: DP1, EP1-4, FP, IP, and TP, which correlate with their primary endogenous ligands PGD 2 , PGE 2 , PGF 2␣ , PGI 2 , and TXA 2 , respectively (Funk, 2001) (Table 2). Although cloning studies have not provided data indicative of IP receptor subtypes (Wilson et al, 2011), binding studies using radiolabeled PGI 2 analogs have suggested the presence of two IP receptor subtypes: the classic IP1 receptor, mainly identified in peripheral tissues, and IP2 receptor, largely expressed in the central nervous system (CNS), mainly in the thalamus, cerebral cortex, and striatum (Takechi et al, 1996). However, recent binding and functional studies have suggested coexpression of functionally distinct IP1 and IP2 receptors in the human airway epithelial cell line BEAS-2B (Wilson et al, 2011).…”

“…Although cloning studies have not provided data indicative of IP receptor subtypes (Wilson et al, 2011), binding studies using radiolabeled PGI 2 analogs have suggested the presence of two IP receptor subtypes: the classic IP1 receptor, mainly identified in peripheral tissues, and IP2 receptor, largely expressed in the central nervous system (CNS), mainly in the thalamus, cerebral cortex, and striatum (Takechi et al, 1996). However, recent binding and functional studies have suggested coexpression of functionally distinct IP1 and IP2 receptors in the human airway epithelial cell line BEAS-2B (Wilson et al, 2011). Splice variants of EP3, FP, and TP receptors have also been described in human tissues (Norel, 2007).…”

“…IP antagonists can also have different affinities for IP receptor subtypes. For instance, in the human respiratory epithelial cell line BEAS-2B the affinity of RO3244794 is ϳ10-fold higher for IP2 than for IP1 receptor (Wilson et al, 2011).…”

“…Iloprost has equivalent binding affinity for IP and EP1, appreciable affinity for EP4, and only slightly lower affinity for EP3 receptor. In comparison, carbacyclin has comparable binding affinity for IP, DP, EP, and FP receptors, whereas isocarbacyclin has affinity for EP2 and EP3 receptors (Wise, 2003;Hata and Breyer, 2004;Wilson et al, 2011). Cicaprost and beraprost have higher affinity and are considered better pharmacological tools than iloprost to investigate IP receptormediated biological responses (Tanaka et al, 2004).…”

“…In the human airway epithelial cell line BEAS-2B, PGI 2 analogs act with different potencies for IP1 and IP2 receptors to independently regulate two distinct functions. For example, iloprost activates CRE and glucocorticoid receptor response element reporter through IP1 but not IP2 receptor, whereas 15-deoxy-TIC inhibits chemokine CXCL9 and CXCL10 release through IP2 with minimal activity at IP1 receptor (Wilson et al, 2011). …”