Regulation of prostaglandin EP₁ and EP₄ receptor signaling by carrier-mediated ligand reuptake

Abstract

After synthesis and release from cells, prostaglandin E₂ (PGE₂) undergoes reuptake by the prostaglandin transporter (PGT), followed by cytoplasmic oxidation. Although genetic inactivation of PGT in mice and humans results in distinctive phenotypes, and although experiments in localized environments show that manipulating PGT alters downstream cellular events, a direct mechanistic link between PGT activity and PGE₂ (EP) receptor activation has not been made. Toward this end, we created two reconstituted systems to examine the effect of PGT expression on PGE₂ signaling via two of its receptors (EP₁ and EP₄). In human embryonic kidney cells engineered to express the EP₁ receptor, exogenous PGE₂ induced a dose-dependent increase in cytoplasmic Ca²⁺. When PGT was expressed at the plasma membrane, the PGE₂ dose–response curve was right-shifted, consistent with reduction in cell surface PGE₂ availability; a potent PGT inhibitor acutely reversed this shift. When bradykinin was used to induce endogenous PGE₂ release, PGT expression similarly induced a reduction in Ca²⁺ responses. In separate experiments using Madin–Darby Canine Kidney cells engineered to express the PGE₂ receptor EP₄, bradykinin again induced autocrine PGE₂ signaling, as judged by an abrupt increase in intracellular cAMP. As in the EP₁ experiments, expression of PGT at the plasma membrane caused a reduction in bradykinin-induced cAMP accumulation. Pharmacological concentrations of exogenous PGE₂ induced EP₄ receptor desensitization, an effect that was mitigated by PGT. Thus, at an autocrine/paracrine level, plasma membrane PGT regulates PGE₂ signaling by decreasing ligand availability at cell surface receptors.

Introduction

In recent years, mechanisms mediating prostaglandin E₂ (PGE₂) signal termination have become increasingly clear. Following synthesis, release, and binding to its cognate receptors (EP_1-4), PGE₂ is metabolically inactivated by a two-step process, consisting first of energetically active uptake across the plasma membrane by the prostaglandin transporter (PGT) (SLCO2A1) (Kanai et al. 1995), and then cytoplasmic oxidative inactivation by 15-OH prostaglandin dehydrogenase (HPGD) (Tai et al. 2002). PGE₂ influx across the plasma membrane by PGT is rate limiting for this inactivation, because heterologous expression of HPGD without PGT is insufficient to result in PGE₂ oxidation (Nomura et al. 2004).

Based on this model, our laboratory has advanced the hypothesis that PGE₂ signaling is akin to synaptic signaling (Nomura et al. 2005). Notably, both neurotransmitters and prostanoids are synthesized by inducible enzymes (Saadat et al. 1989; Smith et al. 2000; Stichtenoth et al. 2001; Murakami and Kudo 2004); both systems involve triggered release of ligand into the extracellular compartment (Greengard 2001; Kudo and Murakami 2002); both sets of G-protein coupled receptors (GPCRs) utilize similar molecular signaling and regulatory mechanisms (Lefkowitz 1993; Neuschafer-Rube et al. 2004); and both sets of ligands undergo reuptake by plasma membrane carriers that are located on the ligand-releasing cell (Kanai et al. 1995; Bao et al. 2002; Nomura et al. 2005; Gether et al. 2006; Kristensen et al. 2011).

Despite the attractiveness of this hypothesis, it has not been shown definitively that PGE₂ reuptake by PGT can control signaling at a local level. Although genetic inactivation of PGT in mice results in patent ductus arteriosus (Chang et al. 2010), and humans who are homozygous null at the PGT locus have pachydermoperiostosis (Busch et al. 2012; Diggle et al. 2012; Sasaki et al. 2012; Seifert et al. 2012; Zhang et al. 2012), in both cases systemic levels of PGE₂ are increased, and ancillary studies suggest that these phenotypes might result from elevated circulating PGE₂ acting as a hormone (Ueda et al. 1980; Ringel et al. 1982; Drvaric et al. 1989; Cattral et al. 1994; Letts et al. 1994; Reese et al. 2000). Moreover, although experiments using localized (autocrine/paracrine) model systems have been interpreted to indicate that PGT alters PGE₂ signaling, the biological events measured in these studies were considerably distal to the EP receptors (Henry et al. 2005; Gordon et al. 2008; Subbaramaiah et al. 2011). Thus, a direct mechanistic link between cell surface PGT activity and EP receptor activation has not been made.

Here, we coexpressed either of two representative PGE₂ (“EP”) receptors, along with the prostaglandin reuptake carrier PGT, in kidney cell lines. We then acutely manipulated cell surface PGT function and directly measured either signaling through the receptors or ligand-induced receptor desensitization. Our results demonstrate that plasma membrane PGT modulates the concentration of cell surface PGE₂ in an autocrine/paracrine signaling mode. As a result, PGT directly modulates ligand access to, and activation of, EP₁ and EP₄ receptors.

Materials and Methods

Cell lines

Wild-type human embryonic kidney-293 cells (WT-HEK), wild-type Madin–Darby Canine Kidney (WT-MDCK) cells, and MDCK cells expressing green fluorescence protein-tagged PGT (PGT-GFP-MDCK) (Endo et al. 2002) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. HEK cells stably expressing the EP₁ receptor (EP₁-HEK) (Peti-Peterdi et al. 2003) were cultured in F12 medium supplemented with 10% FBS, 1% P/S, 1.2 g/L NaHCO₃, and 0.05 mg/mL G418. We generated a derivative of the EP₁-HEK line stably expressing PGT (PGT-EP₁-HEK) by transfecting EP₁-HEK cells with human PGT cloned into the pcDNA3.1/Hygro (+) vector (Invitrogen, Grand Island, NY), followed by selection with hygromycin (0.25 mg/mL) in F12 media as described for EP₁-HEK.

mRNA quantification

EP₁, PGT, and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNAs in EP₁-HEK cells and in PGT-EP₁-HEK cells were quantified by qRT-PCR using the SYBR system (Invitrogen). The EP₁ (mouse) primers used were: 5′-CATCCGCTAGGCTCAGGTTA-3′ (forward) and 5′-AGCAGGAGCCAAGTTCCAG-3′ (backward). The PGT (human) primers used were: 5′-TGTACAGGAGTTGGCAGAGC-3′ (forward) and 5′-AGCGACACCTCTACTAGCCG-3′ (backward). The GAPDH (human) primers used were: 5′-AATGAAGGGGTCATTGATGG-3′ (forward) 5′-AAGGTGAAGGTCGGAGTCAA-3′ (backward).

Measurement of PGT function

PGT-mediated ³H-PGE₂ uptake was measured using methods previously described (Kanai et al. 1995; Chi et al. 2006).

Protein quantification by Western blotting

Protein extraction and quantification by Western blotting were conducted using methods previously described (Syeda et al. 2012). EP₁ was probed with an anti-EP₁ polyclonal antibody (Cayman Chemical, Ann Arbor, MI) overnight at 4°C, with a goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody as secondary antibody.

Measurements of intracellular Ca²⁺ transients

Changes in cytosolic Ca²⁺ levels were measured in cells loaded with the ratiometric Ca²⁺ indicator Fura-2 AM (Molecular Probes, Grand Island, NY) and imaged on an epifluorescence microscope as previously described (Suadicani et al. 2004, 2006). Experiments were performed with cells bathed in Tyrode solution ([in mmol/L] 137.0 NaCl, 2.7 KCl, 0.5 MgCl₂, 1.8 CaCl₂, 12.0 NaHCO₃, 0.5 NaH₂PO₄, 5.5 glucose, and 5 HEPES; pH 7.4). Values of intracellular Ca²⁺ levels were obtained from Fura-2 ratio images using an in vitro calibration curve.

EP₄ cell surface expression

WT-MDCK and PGT-GFP-MDCK cells were transiently transfected with a human EP₄-expressing cDNA (UMR cDNA Resource Center, http://www.cdna.org) using lipofectamine reagent (Invitrogen) 48 h after being seeded. At 24 h after transfection, the cells were treated with exogenous PGE₂ so as to obtain immediate medium PGE₂ concentrations of 0, 10, 20, 30, or 50 nmol/L. These experiments were performed in the absence or presence of the PGT inhibitor T26A (Chi et al. 2011) (5 μmol/L) at 37°C for 10 min. Immediately before harvesting the cells, media were collected to determine the result of contact with the cell monolayers on extracellular PGE₂ concentrations. Cells were then collected and lysed with buffer containing 10 mmol/L Tris-HCl, pH 7.4, 1 mmol/L EDTA, 10 mmol/L MgCl₂, 0.1 mmol/L PSF, and 20 μmol/L indomethacin to inhibit any further PGE₂ synthesis. Cell surface EP₄ receptor expression was quantified by [³H]-PGE₂ binding as previously described (Nishigaki et al. 1996). Briefly, the harvested cells were homogenized using a Potter–Elvehjem homogenizer in an ice-cold lysing buffer. The homogenate was centrifuged at 800 rpm for 5 min, and the supernatant was further centrifuged at 19,500 rpm for 45 min at 4°C. The pellet was washed once and then resuspended in 100 μL buffer A (20 mmol/L HEPES-NaOH, pH 7.4, 1 mmol/L EDTA, 10 mmol/L MgCl₂) containing 4 nmol/L [³H]-PGE₂ (170,000 dpm) and incubated at 30°C for 1 h. One milliliter cold buffer A was then added to stop binding. The mixture was filtered through a glass fiber which was then transferred to a scintillation vial. Ten milliliter scintillation solution was added to the vial and [³H]-PGE₂ was counted by scintillation counting. Nonspecific binding was determined using a 1000-fold excess of unlabeled PGE₂ in the incubation mixture. The specific binding was calculated by subtracting the nonspecific binding value from the total binding value.

PGE₂ measurement

Extracellular PGE₂ concentrations were quantified using an enzyme-linked immunoassay kit (Cayman Chemical) according to the manufacturer's protocol.

cAMP measurement

WT-MDCK and PGT-GFP-MDCK cells were transfected with the EP₄-expressing vector 24 h after being seeded onto six-well plates. Twenty-four hours after transfection, they were incubated with the cAMP/cGMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 250 μmol/L) for an additional 18 h. Cells were then stimulated either with 10 μmol/L bradykinin or with various concentrations of exogenous PGE₂ (0, 10, 20, 30, or 50 nmol/L), in the absence or presence of T26A (5 μmol/L), at 37°C for 30 and/or 10 min, respectively. Thereafter, cells were washed twice with phosphate-buffered saline (PBS) and were lysed with PBS containing 0.1 mol/L HCl and 0.1% triton-X 100 (250 μL/well) at room temperature for 15 min. Cell lysates were centrifuged (10,000g, 4°C for 10 min), and cAMP in the supernatants was measured using an enzyme-linked immunoassay (ELISA) kit from Cayman Chemical according to the manufacturer's protocol.

Results

Generation of a cell line coexpressing PGT and EP₁

To investigate functional interplay between the PGT transporter and EP₁ receptors, we began with a cell line that stably overexpresses the mouse EP₁ receptor, “EP₁-HEK” (Peti-Peterdi et al. 2003). From this, we generated a new cell line, “PGT-EP₁-HEK,” which stably overexpresses both mouse EP₁ and human PGT. Figure 1A shows that PGT mRNA expression in PGT-EP₁-HEK cells is about 140-fold that of EP₁-HEK cells. Figure 1B shows that PGT-EP₁-HEK cells exhibit significant tracer PGE₂ uptake, whereas EP₁-HEK cells exhibit almost no PGE₂ uptake. Figure 1C and D show that EP₁ mRNA and protein expression in PGT-EP₁-HEK cells were not different from that in the original EP₁-HEK cells.

Figure 1.

Properties of EP₁-HEK and PGT-EP₁-HEK cell lines. (A and C) PGT and EP₁ mRNA expression levels in EP₁-HEK and PGT-EP₁-HEK cell lines. Cells were seeded onto six-well plates and harvested 72 h after seeding. Total RNA was extracted and mRNA was determined by qRT-PCR. PGT-EP₁-HEK cells expressed ∼140-fold more PGT mRNA than EP₁-HEK cells, whereas EP₁-HEK and PGT-EP₁-HEK cells expressed comparable levels of EP₁ mRNA. (B) ³H-PGE₂ uptake in EP₁-HEK and PGT-EP₁-HEK cell lines. Cells were seeded onto 24-well plates and ³H-PGE₂ uptake was measured 72 h after seeding. PGT-expressing cells exhibited 60- to 70-fold more ³H-PGE₂ uptake than EP₁-HEK control cells. (D) EP₁ protein expression levels in EP₁-HEK and PGT-EP₁-HEK cell lines. Cells were seeded onto 10-cm plates and harvested 72 h after seeding. Protein was extracted and quantified by Western blot. Upper panel shows a representative immunoblot; lower panel shows quantitation of three blots. EP₁-HEK and PGT-EP₁-HEK cells exhibited no difference in EP₁ protein expression. Values are mean ± SEM (n = 3 for each experiment). ***P < 0.0001 by t-test.

PGT modulates EP₁-mediated Ca²⁺ release in response to exogenously applied PGE₂

EP₁ receptor activation triggers intracellular Ca²⁺ release (Sugimoto and Narumiya 2007). Indeed, in both EP₁-HEK and PGT-EP₁-HEK cells, activation of EP₁ by exogenous PGE₂ (0.3 nmol/L to 3 μmol/L) induced a concentration-dependent rise in intracellular Ca²⁺ levels (Fig. 2A). However, in PGT-EP₁-HEK cells, which express the PGT reuptake carrier, the dose–response curve for PGE₂ was significantly right-shifted (Fig. 2A). The EC₅₀ (half-maximal effective concentration) value for PGE₂ in EP₁-HEK cells was 12.8 nmol/L (95% CI = 7.2–22.8 nmol/L, n = 37 cells), whereas in PGT-EP₁-HEK cells it was 86.9 nmol/L (95% CI = 51.7–146.0 nmol/L, n = 211 cells) (P < 0.01). Of note, this rightward shift of the PGE₂ dose–response curve cannot be attributed to differing levels of EP₁ expression (see Fig. 1C and D). The maximal response to PGE₂ was slightly but not significantly lower in PGT-EP₁-HEK compared to EP₁-HEK cells (Fig. 2A).

Figure 2.

PGT modulates PGE₂-induced Ca²⁺ responses and medium PGE₂ levels. (A) Dose-dependent responses of the increase in intracellular Ca²⁺ concentration induced by PGE₂ stimulation of EP₁-HEK (n = 37 cells), and of PGT-EP₁-HEK cells in the absence (n = 211 cells) and presence of the PGT blocker T26A (5 μmol/L, n = 340 cells). PGT shifted the PGE₂/Ca²⁺ dose–response curve to the right; PGT inhibition by T26A reversed this effect (see text for statistical analyses). (B) Final PGE₂ concentration in media following 10 min exposure to EP₁-HEK or PGT-EP₁-HEK monolayers in the presence and absence of T26A (5 μmol/L). PGT expression lowered the medium PGE₂ concentration. Values are mean ± SEM (n = 3 for each experiment). *P < 0.05.

Acute inhibition of PGT transport in PGT-EP₁-HEK cells with T26A (5 μmol/L) resulted in significantly higher maximal Ca²⁺ responses to PGE₂ (P < 0.05) compared to nontreated PGT-EP₁-HEK cells (Fig. 2A). T26A shifted the PGE₂ dose–response curve directionally, albeit not statistically significantly, back toward that of EP1-HEK cells (EC₅₀ = 51.8 nmol/L; 95% CI = 36.3–73.9 nmol/L, n = 324). Given the known effect of PGT to mediate PGE₂ uptake from the extracellular compartment (Nomura et al. 2004), the data of Figure 2A suggest that, at any given concentration of PGE₂ added to the culture medium, plasma membrane PGT lowers the PGE₂ concentration at the cell surface, as revealed by a reduction in EP₁ receptor activation.

To test this hypothesis further, we added exogenous PGE₂ to EP₁-HEK and PGT-EP₁-HEK cell monolayers so as to achieve initial PGE₂ concentrations of 0, 10, 20, 30, and 50 nmol/L. We then measured PGE₂ concentrations in the bathing media after 10 min exposure to the monolayers. As shown in Figure 2B, in EP₁-HEK cell monolayers, the medium PGE₂ concentrations at 10 min were not different from the initial concentrations, indicating that, in the absence of PGT, the added PGE₂ remained in the extracellular compartment. Treatment with T26A (5 μmol/L) did not affect medium PGE₂ levels in EP₁-HEK cells. However, in PGT-EP₁-HEK cells, the medium PGE₂ concentration after 10 min exposure to the monolayer was significantly reduced compared to that of control cells and, in these cells, acute inhibition of PGT with T26A returned medium PGE₂ levels at 10 min to levels similar to those in EP₁-HEK cells (Fig. 2B). Taken together, the data of Figure 2 indicate that PGT expression at the cell membrane affects both PGE₂ availability for signaling at the cell surface as well as the bulk-fluid phase PGE₂ concentration.

PGT controls endogenous PGE₂ bioavailability and modulates autocrine/paracrine PGE₂ signaling via the EP₁ receptor

Cells synthesize and release endogenous PGE₂ in response to diverse stimuli, such as bradykinin (Miller 2006). We have shown previously that PGT modulates the net release of PGE₂ into the bulk cell culture medium in MDCK cells in response to bradykinin (Nomura et al. 2005). Here, we confirmed these results in HEK cells. We treated EP₁-HEK and PGT-EP₁-HEK cells with bradykinin (10 μmol/L) in the absence and presence of the PGT blocker T26A (5 μmol/L). As shown in Figure 3A, although bradykinin-induced PGE₂ release from both EP₁-HEK and PGT-EP₁-HEK cells, the PGE₂ concentration in the medium bathing PGT-EP₁-HEK cells was significantly lower than that in the medium bathing EP₁-HEK cells. Inhibiting PGT with T26A blocked the effect of PGT to lower PGE₂ in the medium (Fig. 3A). These results thus confirm those we reported previously using MDCK monolayers (Nomura et al. 2005).

Figure 3.

PGT modulates EP₁ Ca²⁺ responses induced by autocrine/paracrine PGE₂ signaling. (A) PGE₂ concentrations in media of EP₁-HEK and PGT-EP₁-HEK cells treated with 10 μmol/L bradykinin in the absence or presence of 5 μmol/L T26A for 30 min at 37°C. Medium [PGE₂] was lower in cells expressing PGT, but not when PGT was inhibited with T26A. Values are mean ± SEM (n = 3 for each experiment). *P < 0.05. (B) Bradykinin-induced EP₁ signaling (Ca²⁺ responses) in WT-HEK, EP₁-HEK, and PGT-EP₁-HEK cells. EP₁-expressing cells had a Ca²⁺ response to bradykinin above that of wild-type cells. In cells that were also expressing PGT, the PGT inhibitor T26A doubled the degree of signaling via EP₁. Data are mean ± SEM (n ≥ 4 for each experiment), with >100 cells analyzed per experiment and per experimental condition. *P < 0.05, ***P < 0.001.

Next, we examined the ramifications of this PGT effect on autocrine EP₁ signaling. Bradykinin induces intracellular Ca²⁺ release in HEK 293 cells by activating B2 receptors (Kramarenko et al. 2009). All three cell lines (WT-HEK, EP₁-HEK, and PGT-EP₁-HEK) responded to bradykinin with an increase in intracellular Ca²⁺ levels, although the amplitude of the response was about threefold higher (P < 0.05) in the two cell lines overexpressing the EP₁ receptor compared to wild-type cells (Fig. 3B). Thus, most of the bradykinin-induced Ca²⁺ response in EP₁-HEK and PGT-EP₁-HEK cells results from signaling through EP₁. The amplitude of the Ca²⁺ response to bradykinin in PGT-EP₁-HEK cells doubled when these cells were treated with the PGT blocker T26A (Fig. 3B). Taken together, the data of Figure 3 indicate that PGT controls the concentration of extracellular PGE₂ in this autocrine signaling system, and that the resulting cell surface PGE₂ concentration is reflected in the degree of signaling through EP₁ receptors.

PGT modulates autocrine/paracrine signaling through the EP₄ receptor

In addition to Ca²⁺, another important second messenger for PGE₂ is intracellular cAMP, resulting from activation of either the EP₂ or EP₄ receptor by PGE₂. We tested the hypothesis that, as with the EP₁ receptor, PGT regulates EP₄-mediated autocrine/paracrine signaling. We constructed a cell system consisting of wild-type MDCK cells (“WT-MDCK”) or MDCK cells that were stably transfected with PGT tagged with GFP (green fluorescent protein) at the carboxyl terminus (“PGT-GFP-MDCK”) (Endo et al. 2002). These cell lines were then transiently transfected with an EP₄ receptor-expressing cDNA. As above, we stimulated both cell lines with bradykinin so as to induce acute, autocrine/paracrine signaling of EP₄ receptors by endogenous PGE₂.

As shown in Figure 4A, although bradykinin-induced the net release of PGE₂ from both cell lines, expression of PGT significantly reduced the extracellular PGE₂ concentration compared to control, whereas blocking PGT activity with T26A (5 μmol/L) reversed this reduction. As shown in Figure 4B, bradykinin-induced intracellular cAMP production was significantly lower in PGT-GFP-MDCK cells compared to controls, and T26A treatment reversed this effect. These findings demonstrate that, as with EP₁ signaling via intracellular Ca²⁺, PGT regulates autocrine/paracrine signaling by the EP₄ receptor via cAMP.

Figure 4.

PGT regulates cAMP accumulation in response to autocrine/paracrine EP₄-mediated PGE₂ signaling. WT-MDCK and PGT-GFP-MDCK cells were transiently transfected with the EP₄ receptor cDNA for 24 h, then incubated for 18 h with 250 μmol/L IBMX, and afterward treated with 10 μmol/L bradykinin in the absence or presence of 5 μmol/L T26A for 30 min. (A) PGE₂ concentrations in the media. (B) Intracellular cAMP accumulation. PGT expression reduced both the concentration of PGE₂ in the medium and the accumulation of intracellular cAMP in response to 30 min exposure to bradykinin. Both of these PGT effects were reversed by the PGT inhibitor T26A. Values are mean ± SEM (n = 3 for each experiment). *P < 0.05.

PGT modulates homologous desensitization of the EP₄ receptor in response to exogenous PGE₂

The EP₂, EP₃, and EP₄ receptors undergo homologous desensitization in response to pharmacological addition of extracellular PGE₂ (Negishi et al. 1993; Nishigaki et al. 1996; Ashby 1998). To test the hypothesis that PGT modulates agonist-induced EP₄ desensitization, we transiently transfected WT-MDCK cells and PGT-GFP-MDCK cells with the human EP₄ receptor cDNA and determined cell surface EP₄ expression by a ligand-binding assay in response to exogenous PGE₂ (0–50 nmol/L).

As shown in Figure 5A, in WT-MDCK cells (“WT”), exogenous PGE₂ caused dose-dependent EP₄ desensitization, such that exposure to 50 nmol/L PGE₂ induced desensitization of about 50% of cell surface EP₄ receptors. In contrast, in cells expressing PGT (“PGT-MDCK”), PGE₂-induced EP₄ desensitization was significantly abrogated. Although, T26A (5 μmol/L) had no effect on PGE₂-induced EP₄ desensitization in WT-MDCK cells, it inhibited PGT-mediated EP₄ retention on the cell surface.

Figure 5.

PGT regulates EP₄ receptor desensitization in response to exogenous PGE₂. Wild-type MDCK (“WT”) and PGT-GFP-MDCK (“PGT-MDCK”) cells were transiently transfected with the EP₄-expressing cDNA, and 24 h later were exposed to pharmacological concentrations of exogenous PGE₂ (0–50 nmol/L, “applied medium [PGE₂]”) in the presence or absence of 5 μmol/L T26A. After 10 min, media were collected for determining residual extracellular PGE₂ concentrations by ELISA, and cells were harvested for quantifying the amount of cell surface EP₄ receptor using a binding assay. (A) Cell surface EP₄ receptor. Exogenous PGE₂ caused a reduction in cell surface EP₄ (i.e., desensitization) in wild-type MDCK cells (open circles); T26A had no effect on this desensitization (closed circles). When PGT was expressed, it abrogated the ability of PGE₂ to cause EP₄ desensitization (open triangles), an effect that was reversed by the PGT inhibitor T26A (closed triangles). (B) Media [PGE₂] at the conclusion of 10 min exposure to cell monolayers. PGT reduced the concentration of PGE₂ in the media (open triangles) compared to the lack of change in wild-type cells (open circles) or wild- type cells exposed to T26A (closed circles). T26A reversed the ability of PGT to lower media [PGE₂] (closed triangles). (C) Intracellular cAMP accumulation. PGT-GFP-MDCK cells (“PGT-MDCK”) were transfected with EP₄ as above, treated with 250 μmol/L IBMX for 18 h, and exposed to exogenous PGE₂ ± T26A for 10 min at various doses as above. Cells expressing uninhibited PGT (absent T26A, open triangles) exhibited a robust cAMP dose-response to PGE₂, whereas PGT-MDCK cells in which PGT was inhibited by T26A (open triangles) exhibited a significantly blunted cAMP response to exogenous PGE₂. Values are mean ± SEM (n = 3 for each experiment). *P < 0.05.

PGT-mediated modulation of EP₄ desensitization involves PGT-mediated removal of exogenous PGE₂, as shown in Figure 5B. The measured PGE₂ concentration in the medium 10 min after PGE₂ addition to the cell monolayer was significantly lower in PGT-GFP-MDCK cells compared to that in WT-MDCK cells. When PGT was acutely inhibited by T26A, extracellular PGE₂ levels measured in media exposed to PGT-GFP-MDCK cells were not different from those observed using WT-MDCK cells (Fig. 5B).

The downstream signaling consequences of these changes in EP₄ receptor desensitization are shown in Figure 5C. In PGT-GFP-MDCK cells, PGE₂ increased intracellular cAMP accumulation in a dose-dependent manner to a degree that was significantly greater when PGT was active than when it was inhibited (Fig. 5C).

Together, the data presented in Figure 5 suggest a model in which plasma membrane PGT determines the cell surface PGE₂ concentration which, in turn, determines the degree of short-term, agonist-induced desensitization. The degree of desensitization, in turn, determines the degree of intracellular cAMP accumulation in response to exogenous PGE₂.

Discussion

The present studies show that the PGT modulates PGE₂ signaling by altering ligand concentration at cell surface receptors. Our findings provide a molecular mechanism for several recent reports in which acute or chronic manipulation of PGT transporter activity altered downstream physiological events. These include relaxation of tracheal smooth muscle by stimulants of protease-activated receptor-2 (Henry et al. 2005), astrocyte-mediated cerebral vasodilation (Gordon et al. 2008), regulation of preadipocyte aromatase activity by PGE₂ (Subbaramaiah et al. 2011), and maintenance of a functional corpus luteum (Lee et al. 2013).

Our results may also provide insight into the mechanisms underlying failure of postnatal closure of the ductus arteriosus in mice (Chang et al. 2010), and of pachydermoperiostosis in humans, both of which result from homozygous null PGT alleles (Busch et al. 2012; Diggle et al. 2012; Sasaki et al. 2012; Seifert et al. 2012; Zhang et al. 2012). Although, as described above, an argument can be made that these phenotypes represent PGE₂ acting systemically as a hormone, the present results suggest that at least some of the vascular, skeletal, and/or dermal phenotypes resulting from PGT inactivation may represent abnormal autocrine/paracrine PGE₂ signaling.

The ability of the PGT transporter to compete with EP receptors for ligand is dependent on the respective abilities of the transporter and the receptors to bind, and turn over, the ligand. The binding constant of PGE₂ to PGT is about 90 nmol/L (Kanai et al. 1995), and that of PGE₂ for EP₁ and EP₄ is in the range of 2–20 nmol/L (Narumiya et al. 1999). Although PGT binds PGE₂ more weakly than does EP₁ or EP₄, it is apparently able to compete for the ligand even when EP receptors are overexpressed, suggesting that PGT likely turns over PGE₂ faster than do the receptors.

EP₁ signaling is mediated by intracellular Ca²⁺ release. Regardless of whether the source of PGE₂ was endogenous (i.e., postbradykinin) or exogenous, PGT lowered the amount of cell surface PGE₂ and reduced EP₁ signaling (Figs. 2, 3). Similarly, PGT reduced EP₄-mediated intracellular cAMP accumulation when the source of PGE₂ was endogenous (postbradykinin) (Fig. 4). However, when exogenous PGE₂ in pharmacological concentrations was introduced, EP₄ exhibited rapid desensitization in accord with prior studies (Nishigaki et al. 1996). In that case, PGT reduced cell surface PGE₂ and abrogated EP₄ desensitization, resulting in an increase in cAMP formation (Fig. 5). Thus, in the case of EP₄ signaling, the net result of competition for ligand between PGT and the EP₄ receptor depends on the source, timing, and concentration of the cell surface PGE₂.

As above, we have previously drawn an analogy between neurotransmitter release/reuptake at the synapse and PG release/reuptake by cells engaged in autocrine/paracrine PG signaling (Nomura et al. 2005). The present studies allow us to extend this analogy insofar as we now show that PGT, like neurotransmitter reuptake carriers, regulates availability of ligand for its GPCR. The analogy is of further interest in that both neurotransmitter and PG reuptake carriers are regulated by their respective ligands. Thus, dopamine activates the dopamine reuptake carrier through dopamine receptors (Zapata et al. 2007), and the PGT substrate PGF_2α inhibits PGT through the FP receptor (Vezza et al. 2001).

In the same way that inhibitors of neurotransmitter reuptake have been useful in the management of psychiatric diseases (Mann 2005), inhibitors of prostaglandin reuptake, by raising endogenous PG levels (Chi et al. 2011; Syeda et al. 2012), might be medicinally applicable for conditions in which exogenous prostaglandins are currently used, such as glaucoma (Stjernschantz 2004), pulmonary artery hypertension (Gomberg-Maitland and Olschewski 2008), and vascular insufficiency (Amendt 2005). Because we found that the PGT inhibitor T26A increased autocrine/paracrine signaling through EP₁ and EP₄ receptors only in the presence of PGT, it appears that T26A does not interact directly with these two PGE₂ receptors. Thus, T26A or its derivatives may offer a small-molecule approach to raising endogenous prostaglandin levels.

Finally, we note that there are currently approximately 120 orphan GPCRs (Civelli 2005; Oh et al. 2006) and 90 orphan SLC transporters (Dahlin et al. 2009; Schlessinger et al. 2010), all with unassigned ligands. The present findings show that regulatory competition between GPCRs and SLC carriers occurs outside of the synaptic cleft, and thus may represent a more general phenomenon than previously appreciated.

Glossary

DMEM

Dulbecco's modified Eagle's medium

EP₁-HEK

HEK cells stably expressing the PGE₂ receptor EP₁

EP

prostaglandin E₂ receptor

FBS

fetal bovine serum

Fura-2 AM

fura-2 acetoxymethyl ester

G418

geneticin

GAPDH

glyceraldehyde phosphate dehydrogenase

GFP

green fluorescence protein

GPCR

G-protein-coupled receptor

HEK

human embryonic kidney

HPGD

15-hydroxyprostaglandin dehydrogenase

HRP

horseradish peroxidase

MDCK

Madin–Darby Canine Kidney

P/S

penicillin/streptomycin

PBS

phosphate-buffered saline

PGE₂

prostaglandin E₂

PGF₂α

prostaglanding F₂α

PG

prostaglandin

PGT-EP₁-HEK

HEK cells stably expressing both the PGE₂ receptor EP₁ and the prostaglandin transporter PGT

PGT-GFP-MDCK

MDCK cells stably expressing GFP-tagged PGT

PGT

prostaglandin transporter

PSF

phenylmethyl sulfonyl fluoride

SLC

solute carrier

T26A

N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-4-((4-((2-(2-(2-benzamidoethoxy) ethoxy)ethyl)amino)-6-((4-hydroxyphenyl)amino)-1,3,5-triazin-2-yl)amino)benzamide

WT-HEK

wild-type HEK cell line

Disclosures

None declared.