EP2 receptor mediated cAMP release is augmented by PGF 2 alpha activation of the FP receptor via the calcium-calmodulin pathway

Abstract

Prostaglandins exert their effects on target cells by coupling to specific G protein-coupled receptors (GPCRs) that are often co-expressed in the same cells and use alternate and in some cases opposing intracellular signaling pathways. This study investigated the cross-talk that influences intracellular signaling and gene expression profiling in response to co-activation of the EP2 and FP prostanoid receptors in Ishikawa cells stably expressing both receptors (FPEP2 cells). In this study we show that in FPEP2 cells, PGF alone does not alter adenosine 3',5'-cyclic monophosphate (cAMP) production, but in combination with Butaprost enhances EP2 receptor mediated cAMP release compared to treatment with Butaprost alone. PGF-mediated potentiation of cAMP release was abolished by antagonism of the FP receptor, inhibition of phospholipase C (PLC) and inositol phosphate receptor (IP3R) whereas inhibition of protein kinase C (PKC) had no effect. Moreover, inhibition of calcium effectors using calmodulin antagonist (W7) or Ca(2+)/calmodulin-dependent kinase II (CaMK-II) inhibitor (KN-93) abolished PGF potentiation of Butaprost-mediated cAMP release. Using siRNA molecules targeted against the adenylyl cyclase 3 (AC3) isoform, we show that AC3 is responsible for the cross-talk between the FP and EP2 receptors. Using gene array studies we have identified a candidate gene, Spermidine/N1-acetyltransferase (SAT1), which is regulated by this cAMP mediated cross-talk. In conclusion, this study demonstrates that co-activation of the FP and EP2 receptors results in enhanced release of cAMP via FP receptor-G alpha(q)-Ca(2+)-calmodulin pathway by activating calcium sensitive AC3 isoform.

Fig. 1

EP2 and FP receptor analysis in FPS32 and FPEP2 cells. Relative mRNA expression of (A) EP2 receptor (B) EP1, EP3, EP4 and FP receptors as determined by quantitative real-time RT-PCR analysis in FPS32 and FPEP2 cells. Expression levels in FPEP2 clones are expressed as fold increase above FPS32 cells (n = 4; ***, P < 0.0001). (C) Protein expression of FP and EP2 receptors normalized for loading against ß-actin and expressed as fold increase above FPS32 cells as determined by Western blot analysis. (D) Localization of the FP (FPR) and EP2 (EP2R) receptor in FPS32 and FPEP2 cells as determined by immunofluorescence microscopy. Control cells (C) were incubated with preadsorbed primary antibody using specific blocking peptides. (E) The effect of 5 and 10 min treatments with vehicle or Butaprost on cAMP release in FPS32 and FPEP2 cells as determined by cAMP ELISA analysis (n = 4; **, P < 0.001; ***, P < 0.0001). (F) The effect of 60 min PGF stimulation on IP response in FPS32 and FPEP2 cell lines. Data are expressed as fold increase above non-stimulated samples (n = 5).

Fig. 2

PGF enhances Butaprost-stimulated cAMP production via FP receptor-Gα_q coupling and PLC activation. (A) IP release in FPEP2 cells after treatment with PGF (10^− 10 to 10^− 6 M) or (5 μM) Butaprost alone or PGF (10^− 10 to 10^− 6 M) and Butaprost (5 μM) together for 60 min as determined by an IP assay. Data are expressed as fold increase above non-stimulated sample (n = 4). (B) cAMP accumulation in FPEP2 cells after 5 min treatment with vehicle, Butaprost and/or PGF (n = 4). (C) cAMP accumulation in FPEP2 cells treated with vehicle, Butaprost and/or PGF in the presence/absence of the EP2 receptor antagonist (AH6809), FP receptor antagonist (AL-8810) or Gα_q inhibitor (YM-254890) for 5 min (n = 4). (D) cAMP in cells treated with vehicle Butaprost and/or PGF or Butaprost and/or PGF together with the PLC inhibitor (10 µM U73122), IP3-R blocker (40 μm 2-APB) or PKC inhibitor (1 μm Ro-31-822) for 5 min (n = 4; **, P < 0.001).

Fig. 3

PGF potentiates Butaprost-stimulated cAMP through calmodulin-CaMK-II pathway by activating AC3 isoform in FPEP2 cells. (A) cAMP accumulation in FPEP2 cells after 5 min treatment with vehicle, Butaprost and/or PGF in the presence/absence of inhibitors for calmodulin (W7) or CaMK-II (KN-93) (n = 4). (B) Real-Time RT-PCR analysis of AC1 and AC3 in FPEP2 cells transfected with specific AC siRNA compared to the control siRNA (scrambled sequence) (n = 4). (C) cAMP accumulation in control, AC1 or AC3 knockdown FPEP2 cells after 5 min treatment with vehicle, Butaprost and/or PGF as determined by cAMP analysis (n = 4; **, P < 0.001).

Fig. 4

PGF potentiates Butaprost-regulated SAT1 gene expression in FPEP2 cells via the calcium sensitive AC3 isoform. (A) Relative expression of SAT1 in FPEP2 cells after 4, 6 and 8 h treatments with vehicle, Butaprost and/or PGF (n = 4). (B) Relative expression of SAT1 in FPEP2 cells after 6 h treatment with vehicle, Butaprost and/or PGF in the absence or presence of the FP receptor antagonist (AL8810) (n = 4). (C) Relative expression of SAT1 in FPEP2 cells transfected with siRNA and subsequently treated for 6 h with vehicle, Butaprost and/or PGF (n = 4); (**, P < 0.001).

Fig. 5

Gα_q-mediated potentiation of Butaprost-induced cAMP release. Butaprost activates Gα_s-coupled EP2 receptors resulting in a rapid increase in intracellular cAMP accumulation while PGF by itself does not alter cAMP production. However, co-treatment of the cells with both ligands leads to the Gα_q-mediated activation of PLC and release of IP3 from the plasma membrane. Subsequently, IP3 via the IP3 receptor (IP3R) mediates the release of Ca²⁺ from intracellular stores leading to calmodulin-CaMK-II dependent potentiation of cAMP release via the calcium sensitive AC3 isoform and modulation of gene transcription such as SAT1.