Abstract
Naturally occurring prostaglandin E2 (PGE2) plays a role in inflammatory responses through eicosanoid signaling pathways. PGE2 is impermeable to cell membranes at physiological pH and needs solute carrier across the membranes; however, it remains unclear how intercellular concentrations of PGE2 are regulated under the condition of inflammation. We aimed to clarify a role of organic anion-transporting polypeptide 2A1 (OATP2A1/SLCO2A1), also known as prostaglandin transporter (PGT), in PGE2 release from cells. Human bronchial epithelial BEAS-2B cells were treated with lipopolysaccharide (LPS), and PGT inhibitors were tested to evaluate contribution of PGT to PGE2 release by assessing its extracellular concentration and characterizing PGT-mediated PGE2 efflux in Xenopus laevis oocytes. As a result, LPS elevated mRNA expression of a pro-inflammatory cytokine IL6 and extracellular concentration of PGE2 in human bronchial epithelial BEAS-2B cells. PGT inhibitors tested (e.g. bromocresol green (BCG), bromosulfophthalein (BSP), and PGB1) significantly inhibited efflux of PGE2 from oocytes expressing PGT. Similarly, the amount of released PGE2 from the BEAS-2B cells decreased in the presence of BCG and BSP by 45 and 44% respectively while TGBz increased the concentration by 71%, suggesting that PGT mediates the release. In conclusion, these results imply a role of PGT in regulating intra- and extracellular concentrations of PGE2 in response to cells under inflammatory conditions.
Introduction
Prostaglandin E2 (PGE2) plays a major role in eicosanoid signaling pathways, which are well preserved among vertebrates. Naturally occurring PGE2 exerts diverse physiological actions such as inflammatory responses, uterine contraction, bone resorption, and induction of hyperthermia (Smith 1992). PGE2 is synthesized via cyclooxygenase (COX) 1 and 2 from arachidonic acid (Helliwell et al. 2004), and the precursor is further converted to PGE2 by cytosolic or microsomal PGE2 synthase (c/m PGES). PGE2 signals in either autocrine or paracrine manners through G-protein-coupled PGE receptors (e.g. EP1, EP2, EP3, and EP4), which differ in tissue distribution; therefore, PGE2 activates different Gproteins depending on the subtype of receptor that is bound to and then transduces diverse physiological actions (Bos et al. 2004). The downstream events have been studied extensively; however, current evidence is still missing the fundamental basis of how release of PGE2 is regulated.
Prostaglandins (PGs) including PGE2 are organic anions with pKa values of around 5 and exist in charged form at physiological pH (Roseman & Yalkowsky 1973). Hence, PGs are impermeable to cell membranes (Bito & Baroody 1975) and have been suggested not to cross plasma membranes without an aid of transporter systems. There is substantial evidence that organic anion-transporting polypeptides (OATPs) are involved in translocation of PGE2 across the plasma membranes (Abe et al. 1999, Tamai et al. 2000, Kullak-Ublick et al. 2001). Among those, OATP2A1/SLCO2A1 has been characterized as a solute carrier for PG transporter (PGT), particularly PGE2, as it was isolated from adult human kidneys (Lu et al. 1996). PGT mediates PGE2 cellular uptake, which can be driven by an exchange of organic anions such as lactate (Chan et al. 2002). PGT-mediated PGE2 uptake from the extracellular environment is an initial step for degradation of PGE2 by 15-hydroxy PG dehydrogenase (PGDH) in the cytoplasm (Nomura et al. 2004). Expression of PGT may be coordinately regulated with COXs (Bao et al. 2002). Furthermore, previous reports have shown that inflammatory effect of stannous chloride, by which heme oxygenase 1 is induced, is attenuated by a resultant elevation of PGE2 cellular absorption mediated by augmented activity of PGT. Therefore, PGT expression may be associated with inflammatory responses.
On the other hand, several groups have implied that PGT is involved in release of PGE2 from cells (Chan et al. 1998, 2002, Banu et al. 2003); however, contribution of PGT to PGE2 release from cells remains elusive under physiological conditions. Therefore, we hypothesize that PGT secretes cytoplasmic PGE2 from cells in response to extracellular inflammatory stimuli, which may accelerate PGE2-mediated signaling or enhance inflammatory events in an autocrine manner. Previously, COX and EP receptors have been studied extensively as inflammatory regulators and established as pharmacological targets to control inflammatory reactions. EP receptor antagonists are currently in clinical trials as anti-inflammatory drugs. Compared with those two major factors, limited information is available regarding trans-cellular transport of PGE2 by PGT. In this study, in order to understand the roleof PGT in regulating extracellular concentrations ofPGE2 in response to inflammation, we studied theeffectofPGT inhibitors on PGE2 release and pro-inflammatorycytokine IL6 mRNA expression in human bronchial epithelial cells treated with an inducing agent, lipopolysaccharide (LPS).
Materials and methods
Reagents and antibodies
[5,6,8,11,12,14,15-3H]PGE2 ([3H]PGE2; 7400 GBq/mmol) was obtained from Perkin Elmer Life Science (Boston, MA, USA). Bromosulfophthalein (BSP), bromocresol green (BCG), and PGE2, dibutylhydroxytoluene (BHT) and d4-PGE2 were purchased from Sigma–Aldrich Company, Wako Pure Chemical Industries, Ltd. (Tokyo, Japan), and Cayman Chemicals & Co. (Ann Arbor, MI, USA) respectively. TGBz-T34 (TGBz) and PGB1 (PGB1) were provided from Ono Pharmaceutical Co., Ltd. (Osaka, Japan). Anti-human PGT rabbit polyclonal antibody was kindly obtained from DrMichel A Fortier (Université Laval, Quebec, Canada). All other compounds and reagents were obtained from Sigma–Aldrich Company, Invitrogen, Wako Pure Chemical Industries, Ltd., or Nacalai Tesque, Inc. (Kyoto, Japan).
Cell culture
Human type 2 pulmonary epithelial A549, airway epithelial Calu-3, and mouse embryonic fibroblast 3T3-L1 cells were obtained from American Type Cell Collection (Manassas, VA, USA), and human bronchial epithelial BEAS-2B cells were purchased from DS Parma Biomedical Co., Ltd. (Osaka, Japan). All cells were grown in DMEM (Life Technology) supplemented with 10% (v/v) fetal bovine serum (Invitrogen, Life Technology), 100 units/ml penicillin, and 100 μg/ml streptomycin, at 37 °C in an atmosphere of 5% CO2. BEAS-2B cells were incubated with different concentrations of LPS for 24 h and then mRNA expression of human PGT and IL6 was determined by quantitative real-time RT-PCR.
Enzyme immunoassay for PGE2 determination
BEAS-2B cells were plated in 60 mm tissue culture plate, and the culture medium was replaced with medium not containing fetal bovine serum when the cells completely attached to the plate. An aliquot of extracellular medium was collected 24 h after LPS was added at the indicated concentration and diluted with enzyme immunoassay (EIA) buffer. The diluted samples were subjected to PGE2 EIA kit (Cayman Chemicals & Co.). According to the manufacturer's protocol, PGE2 concentration was quantified by measuring optical density at 420 nm using a micro-plate reader, ARVO MX (Perkin Elmer Life Science).
Uptake and efflux of PGE2 from Xenopus laevis oocytes
X. laevis oocytes were prepared and injected with in vitro copied RNA (cRNA) of PGT and then subjected to transport assays using radiolabeled PGE2. In brief, oocytes were micro-injected with 50 nl of either deionized water or PGT cRNA solution (1 μg/μl) and placed in modified Barth's solution (MBS; 96 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4) containing 50 μg/ml gentamicin for 3 days at 18 °C. PGT uptake was initiated by placing water- orcRNA-injected oocytes into uptake medium (ND96; 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH7.4) containing [3H]PGE2 (1.0 nM) at room temperature for the indicated time. At the end of the uptake, the oocytes were washed with ice-cold MBS and intracellular accumulation of [3H]PGE2 was measured with a liquid scintillation counter (LSC 5100, Aloka, Tokyo, Japan). For measuring efflux of PGE2 from oocytes, water- or cRNA-injected oocytes were micro-injected with a 50 nl aliquot of concentrated [3H]PGE2 (80 nM) solution and then the oocytes were placed with uptake medium (pH 7.4) containing unlabeled PGE2 (10 μM) as described previously (Chan et al. 1998). Efflux of [3H]PGE2 was measured for 120 min. For the inhibition study, the assay was performed in the absence (control) or the presence of inhibitor, e.g. BCG, BSP, TGBz, or PGB1 at the indicated concentrations. The effect of these inhibitors on uptake and efflux was assessed at 60 and 90 min respectively. The kinetic analysis was conducted as described previously (Shirasaka et al. 2010).
RT-PCR study
Total RNA was prepared from BEAS-2B cells treated or untreated with LPS at the indicated concentration and then reverse-transcribed to cDNA. mRNA expression of PGT, IL6, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was measured by quantitative RT-PCR, using gene-specific primers as follows: sense 5′-CTGTGGAGACAATGGAATCGAG-3′ and antisense 5′-CACGATCCTGTCTTTGCTGAAG-3′ for PGT, sense 5′-ATGTAGCCGCCCCACACAGA-3′ and antisense 5′-CATCCATCTTTTTCAGCCAT-3′ for mIL6, and sense 5′-GGTGAAGGTCGGAGTCAACGGA-3′ and antisense 5′-GAGGGATCTCGCTCCTGGAAGA-3′ for G3PDH. The fold change in these genes, normalized to G3PGH dehydrogenase (Gapdh) and calculated relative to the expression in wild-type mouse cells, was analyzed by 2−ΔΔCT methods (Livak & Schmittgen 2001).
Western blot
To determine protein expression of PGT, cells were lysed in RIPA buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris at pH 8.0) by sonication. A 50 or 100 μg aliquot of the total cell lysate was separated in SDS polyacrylamide gel and then electrotransferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The blots were probed with anti-human PGT rabbit polyclonal antibody at 1:1000 dilution or with anti-G3PDH rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) at 1:2000 dilution, followed by appropriate secondary antibodies conjugated to HRP (Invitrogen, Life Technology). Corresponding expression was detected with electrochemical luminescence assay (Wako).
Immunocytochemistry
BEAS-2B cells were plated in eight-well culture slides (BDFalcon, Franklin Lakes, NJ, USA) at a density of 1×105 cells/well and then fixed in 4% paraformaldehyde. Immunostaining was performed by incubating the slides with 1:20 dilution of anti-human PGT rabbit polyclonal antibody for 1 h, followed by staining with 1:400 dilution of AlexaFluor 594-conjugated anti-rabbit IgG (secondary antibody) for 1 h at room temperature. The slides were counterstained with Hoechst 33342 and then mounted with Vectashield (Vector Laboratories Burlingame, CA, USA). Antigenic peptide consisting of amino acids 622–643 of human PGT (CFISWRVKKNKEYNVQKAAGLI) was synthesized in Medical and Biological Laboratories (Nagoya, Japan). The primary antibody to PGT was incubated with the peptide at a molar ratio of 1:100 for 1 h at room temperature and then used to stain cells as a negative control. Fluorescence was examined with a confocal laser microscope LSM710 (Carl Zeiss, Hertfordshire, UK).
PGT knockdown study
BEAS-2B cells were plated in 12-well plates, and 12 h later, they were transfected with 5 nM control siRNA (Silencer Select Negative Control #1 siRNA) or siRNA mixture against SLCO2A1 gene (Silencer Select Validated siRNAs of s13097 and s13098) with lipofectamine RNAi Max (Invitrogen) according to the manufacturer's protocol. The cells were further cultured for 48 h and then culture medium was changed to fresh medium not containing fetal bovine serum. At the same time, mRNA expression of PGT was detected according to the method described earlier. For PGE2 assay, the medium was collected from each well (450 μl) and PGE2 was extracted with hexane/ethyl acetate/2% formic acid solution (19:19:1, v/v/v) containing d4-PGE2 (10 ng/ml) as internal standard and BHT (500 ng/ml) to stabilize PGE2 during sample preparation. The resultant organic phase was evaporated completely and reconstituted with 100 μl mobile phase 0.1% formic acid/acetonitrile (65:35, v/v) before LC/MS/MS analysis. The concentrations of PGE2 were quantified with a LC/MS/MS system consisting a AB Sciex API 3200TM triple quadrupole mass spectrometer (AB Sciex, Foster City, CA, USA) coupled with an LC-20AD ultra-fast liquid chromatography system (Shimadzu Co., Kyoto, Japan). The mobile phase composed of the mobile phase at flow rate of 0.2 ml/min. Mercury MS (C18, 20×4.0 mm, Luna 3 μm, Phenomenex, Torrance, CA, USA) was used as the analytical column. Injection volume was 30 μl per sample. Samples were kept at 4 °C during the analysis. The analytes were detected using electrospray-negative ionization and monitoring the mass transitions m/z 351.1→271 for PGE2 and m/z 355.1→275.2 for d4-PGE2. Analyst software version 1.4 was used for data manipulation.
Statistical analysis
Data are given as the mean of values obtained in at least three individual experiments with s.e.m. Statistical analyses were performed with the unpaired Student's t-test, and a probability of <0.05 (P<0.05) was considered to be statistically significant.
Results
PGT expression and PGE2 uptake activity in various cell lines derived from respiratory organ
To determine whether PGT is involved in cellular release of PGE2 from cells, several cell lines derived from human and rodent respiratory organs were tested for PGT mRNA expression and PGE2 uptake activity. As summarized in Table 1, significant mRNA expression of PGT was detected in 3T3-L1 and BEAS-2B cells, as these two cell lines exhibited cellular uptake of PGE2 at a significant level; therefore, in the following experiments, release of PGE2 was studied from BEAS-2B cells because it is derived from human bronchial epithelial cells. PGT protein expression in BEAS-2B cells was further studied by means of western blotting and immunocytochemistry. Western blotting showed that three bands between the sizes of 63 and 75 kDa, which were identical to those in HEK293 cells stably transfected with PGT cells, increased in a manner of the loaded amount of cell lysate based on densitometric analysis (Fig. 1A and B). Further fluorescence microscopic immunocytochemistry confirmed immunolocalization of PGT in the plasma membranes as well as cytoplasm in BEAS-2B cells because cells stained with pre-absorbed anti-PGT antibody with the synthesized antigenic peptide did not detect such red fluorescence (Fig. 1C and D).
PGT expression and uptake activity in various cell lines derived from respiratory organ. Each point represents the mean±s.e.m. (n=3)
| Cell lines | Origin | PGT mRNAa | PGE2 uptakeb |
|---|---|---|---|
| 3T3-L1 | Mouse fibroblast | + | + |
| A549 | Human lung carcinoma | − | − |
| BEAS-2B | Human bronchial epithelium | + | + |
| Calu-3 | Human lung carcinoma | − | ND |
| Macrophage | Rat alveolar macrophage | − | ND |
ND, not determined.
mRNA levels of PGT (normalized to G3PDH expression) in 3T3-L1, A549, BEAS-2B, Calu-3, and rat alveolar macrophage were analyzed by real-time RT-PCR.
Uptake of [3H]PGE2 (0.5 nM) was measured for 5 min by each cells at 37 °C transport buffer (pH 7.4). The criterion is whether or not inhibited uptake of PGE2 by PGT inhibitors.
PGT protein expression in BEAS-2B cells. (A) PGT expression in BEAS-2B cells was determined by western blotting. HEK293 cells transfected empty vector and PGT cDNA were used as negative and positive control respectively. (B) Band intensity for PGT protein was subject to densitometric analysis and the normalized values with the intensity of GAPDH are shown in the bar graph. Fluorescence microscopic analysis of PGT expressed in BEAS-2B cells was performed as described in the Materials and methods section by staining cells with anti-PGT (C) or pre-absorbed anti-PGT antibody (D). The nucleus was counterstained with Hoechst 33344 (2 μg/ml). Fluorescence was observed at 63× magnification under confocal laser microscopy. Scale bar represents 20 μm.
Citation: Journal of Endocrinology 217, 3; 10.1530/JOE-12-0339
Effect of LPS on extracellular PGE2 concentration and IL6 expression in BEAS-2B cells
Effect of LPS on extracellular PGE2 concentration and IL6 expression was examined in BEAS-2B cells (Fig. 2). When the cells were treated with LPS, extracellular concentration of PGE2 and mRNA expression of IL6 were elevated in a dose-dependent manner of LPS. A somewhat sigmoidal relationship was observed between extracellular concentration of PGE2 and expression level of IL6 mRNA. When cells were treated with 0.3 μg/ml LPS, extracellular concentration of PGE2 reached a plateau. Therefore, PGE2 release was investigated in BEAS-2B cells treated with 0.1 μg/ml LPS in the following experiments. Under this condition, mRNA expression of PGT was lower in LPS-treated BEAS-2B cells than that in the untreated cells, and the expression level was unchanged in another 24 h in the treated cells (Fig. 3A). When mRNA expression of IL6 was also measured in the same manner, the expression reached the peak at 6 h in the treated cells and the level gradually decreased after 24-h exposure (Fig. 3B).
Concentration-dependent effect of LPS on PGE2 and IL6 expression in BEAS-2B cells. BEAS-2B cells were incubated in the absence or the presence of LPS (0.01, 0.03, 0.1, 0.3, and 1 μg/ml) at 37 °C. After 12 h of LPS incubation, extracellular PGE2 levels were determined by EIA, as described in the Materials and methods section. And after 24 h of LPS incubation, mRNA levels of IL6 were analyzed by real-time RT-PCR. The results were normalized to G3PDH expression. Each point represents the mean±s.e.m. (n=3).
Citation: Journal of Endocrinology 217, 3; 10.1530/JOE-12-0339
Effect of LPS on expression level of PGT and IL6 in BEAS-2B cells. BEAS-2B cells were incubated in the absence (open) or the presence (closed) of LPS (0.1 μg/ml) for various incubation periods. mRNA levels of PGT (A) and IL6 (B) were analyzed by real-time RT-PCR. The results were normalized to G3PDH expression. *P<0.05, significantly different from expression without LPS (control). Each point represents the mean±s.e.m. (n=3–4).
Citation: Journal of Endocrinology 217, 3; 10.1530/JOE-12-0339
We further examined the effects of BCG, BSP, TGBz, and PGB1, which have been characterized as inhibitors of PGT, on PGE2 release from BEAS-2B cells for 24 h. Among them, TGBz was reported as an inhibitor with higher affinity to PGT (Ki value of 3.7 μM). As shown in Fig. 4A, BCG (100 μM) and BSP (25 μM) reduced the extracellular concentration of PGE2 by 45.4 and 44.4% respectively while TGBz (25 μM) and PGB1 (25 μM) increased the concentration by 71 and 19% respectively. Similar observations were made for the effect of BCG and BSP on IL6 expression in the BEAS-2B cells. IL6 mRNA expression was attenuated in the presence of BCG and BSP by 43.9 and 94.6% respectively. By contrast, TGBz and PGB1 stimulated IL6 expression by 218 and 342% respectively (Fig. 4B).
Inhibitory effects of BCG, BSP, TGBz, and PGB1 on extracellular PGE2 and IL6 expression induced by LPS in BEAS-2B cells. BEAS-2B cells were incubated with LPS (0.1 μg/ml) in the absence (control) or the presence of each PGT inhibitor (100 μM BCG, 25 μM BSP, 25 μM TGBz, or 25 μM PGB1) for 24 h at 37 °C. (A) Extracellular PGE2 levels were determined by EIA, as described in the Materials and methods section. (B) mRNA levels of IL6 were analyzed by real-time RT-PCR. The results were normalized to G3PDH expression. *P<0.05, significantly different from expression without inhibitors (control). Each point represents the mean±s.e.m. (n=3).
Citation: Journal of Endocrinology 217, 3; 10.1530/JOE-12-0339
Effects of inhibitors on PGT-mediated PGE2 transport
Xenopus oocytes injected with PGT cRNA were employed in order to study the effect of these four inhibitors on PGT-mediated PGE2 transport across the plasma membranes. A significant increase in intracellular accumulation of PGE2 was observed in PGT cRNA-injected oocytes (Fig. 5A). All the four PGT inhibitors used in this study significantly reduced PGE2 uptake at 60 min (Fig. 5B). BCG, BSP, or PGB1 almost abolished the uptake, while TGBz had the least effect and inhibited as much as 40% of control uptake. Inhibitory potential of these four inhibitors was also verified by their 50% inhibitory concentration (IC50) to PGE2 uptake, which of BCG, BSP, TGBz, and PGB1 was estimated as 2.88±0.38, 1.46±0.20, 14.6±2.6, and 0.890±0.086 μM respectively (Fig. 6A, B, C and D).
Uptake of PGE2 was determined by Xenopus oocytes expressing PGT. (A) Time course of PGE2 uptake was determined by Xenopus oocytes expressing PGT. Uptake of [3H]PGE2 (1 nM) by water-injected (open) and PGT cRNA-injected (closed) oocytes was measured over 120 min at 25 °C in MBS buffer (pH 7.4). (B) Effects of BCG, BSP, TGBz, and PGB1 on PGT-mediated uptake of PGE2. *P<0.05, significantly different from uptake without inhibitors (control). Each point represents the mean±s.e.m. (n=8–10).
Citation: Journal of Endocrinology 217, 3; 10.1530/JOE-12-0339
Inhibitory effects of BCG, BSP, TGBz, and PGB1 on PGE2 uptake by Xenopus oocytes expressing PGT. Uptake of [3H]PGE2 (1 nM) by water-injected or PGT cRNA-injected oocytes was measured in the absence (control) or the presence of BCG (A), TGBz (B), BSP (C), or PGB1 (D) at concentration ranges of 0.1–100 μM for 60 min at 25 °C in MBS buffer (pH 7.4). Each point represents the mean±s.e.m. (n=8–10).
Citation: Journal of Endocrinology 217, 3; 10.1530/JOE-12-0339
In order to determine whether these inhibitors have the same potential for efflux of PGE2, first PGT-mediated efflux of PGE2 was studied in Xenopus oocytes expressing PGT. The released amount of PGE2 from PGT-expressing oocytes was significantly greater than that from control oocytes (Fig. 7A). Furthermore, the cis-inhibitory effect on PGT-mediated PGE2 efflux was examined by injecting labeled PGE2 with or without inhibitors immediately before the assay and then PGE2 efflux was measured in the presence of 10 μM PGE2 in extracellular uptake buffer. BCG (100 μM), BSP (25 μM), and PGB1 (25 μM) reduced significantly PGT-mediated PGE2 efflux. TGBz unlikely affected the efflux, although the tested concentration was lower than other inhibitors because of its poor solubility (Fig. 7B).
Efflux of PGE2 by Xenopus oocytes expressing PGT. (A) Time course of PGE2 efflux was determined by Xenopus oocytes expressing PGT. Efflux of [3H]PGE2 (80 nM) by water-injected (open) and PGT cRNA-injected (closed) oocytes was measured over 120 min at 25 °C in MBS buffer (pH 7.4). An aliquot (50 nl) of [3H]PGE2 was injected into oocytes and then the five oocytes were transferred to 1 ml MBS buffer. *P<0.05, significantly different from efflux by water oocyte. (B) Effects of BCG, BSP, TGBz, and PGB1 on PGE2 efflux by Xenopus oocytes expression PGT. Efflux of [3H]PGE2 (80 nM) by water-injected or PGT cRNA-injected oocytes was measured for 90 min at 25 °C in MBS buffer (pH 7.4) in the absence (control) or presence of each inhibitor (100 μM BCG, 25 μM BSP, 3 μM TGBz or 25 μM PGB1). *P<0.05, significantly different from efflux without inhibitors (control). Each point represents the mean±s.e.m. (n=8–10).
Citation: Journal of Endocrinology 217, 3; 10.1530/JOE-12-0339
Effect of knocking down the PGT mRNA on release of PGE2 from BEAS-2B cells
To determine whether PGT is involved in PGE2 release, the PGT gene was knocked down in BEAS-2B cells. In cells transfected with two different siRNA mixtures, PGT mRNA expression was reduced to 30±2% of that of untransfected control cells (Fig. 8A). As arachidonic acid was added to the medium, the released amount of PGE2 from the transfected cells for 2 h significantly decreased to 1.05±0.06 ng/well from 1.55±0.07 ng/well for untransfected cells.
Effect of knocking down the PGT gene on release of PGE2 from BEAS-2B cells. (A) mRNA expression of human PGT was determined by real-time RT-PCR after 48 h of siRNA transfection. (B) Extracellular PGE2 amount was determined by LC/MS/MS, as described in the Materials and methods section. Control and siRNA-transfected BEAS-2B cells were incubated with 5 μM arachidonic acid for 2 h at 37 °C. *P<0.05, significantly different from control. Each column represents the mean±s.e.m. (n=5).
Citation: Journal of Endocrinology 217, 3; 10.1530/JOE-12-0339
Discussion
The intercellular concentration of PGE2 has to be strictly regulated because PGE2 is an inflammatory mediator. In this study, we aimed to clarify a role of PGT in PGE2 release from cells by analyzing PGT-mediated PGE2 efflux. Airway epithelial cells may function as both target and effector cells in response to external stimuli that cause inflammatory reactions because they can synthesize and release a variety of lipid mediators including PGE2, 15-hydroxyeicosatetraenoic acid, PGF2α, and platelet-activating factor (Churchill et al. 1989, Salari & Chan-Yeung 1989). Therefore, we investigated a contribution of PGT to a release of PGE2 from human bronchial epithelial BEAS-2B cells (Table 1).
Based on the two-step model of PG signals previously reported by Nomura et al., where PGE2 is first taken up by PGT and then broken down by intracellular PGDH, PGT has been widely accepted as an influx transporter for PGE2 such that it exerts an antiphlogistic action. By contrast, it remains unclear whether PGT contributes to the release of PGE2 produced by cells. In this study, we attempted to clarify a role of PGT in PGE2 secretion under inflammatory conditions. LPS is the microbial product that activates the innate immune system (Ulevitch & Tobias 1995) and upregulates various pro-inflammatory gene expressions including COX2 (PTGS2), CXCL2, IFIT2, CCL2, CCL7, and EDN1 (Aung et al. 2006). We, therefore, evaluated released PGE2 from cells treated with LPS and then observed the reduction of extracellular concentration in the presence of BCG or BSP (Fig. 4A). These two inhibitors were further demonstrated to block the efflux of PGE2 mediated by PGT when examined in heterologously PGT-expressing Xenopus oocytes (Fig. 7B). On the other hand, in the presence of TGBz, which exhibited inhibitory effects on uptake but not efflux of PGE2, the extracellular concentration of PGE2 was slightly increased in LPS-treated BEAS-2B cells. More conclusively, a knockdown assay showed a significant contribution of PGT to PGE2 release from BEAS-2B cells (Fig. 8). In addition, we learned that PGT protein may not be localized only at the plasma membrane but also in the cytoplasm (Fig. 1C and D). Because PGT expressed at the plasma membrane is considered to serve as an efflux transporter for PGE2, it was hard to judge the alteration in the expression of PGT protein responsible for PGE2 efflux in siRNA-treated cells by western blotting using their total lysates. In this study, therefore, we simply determined mRNA expression of PGT. The difference between decreases in mRNA expression of PGT and the amount of released PGE2 could be explained by contribution of other transporters to PGE2 efflux. PGE2 is a substrate of the multidrug resistance protein 4 (MRP4/ABCC4; Reid et al. 2003, Rius et al. 2005); however, Endter et al. reported that MRP4 mRNA was not detected in BEAS-2B cells. Therefore, we may rule out the contribution of MRP4 on PGE2 transport in this cell line (Endter et al. 2009). In addition to MRP4, other solute carriers may also be involved in the release of PGE2 as previous studies including ours reported that at least OATP1B1 (SLCO1B1), 2B1 (SLCO2B1), 1A2 (SLCO1A2), 3A1 (SLCO3A1), and 4A1 (SLCO4A1) mediate transport of PGE2 (Tamai et al. 2000, Kullak-Ublick et al. 2001, Adachi et al. 2003) and mRNA expression of OATP1A2, 1B3 (SLCO1B3), 3A1, 4A1, and 5A1 (SLCO5A1) was detected in BEAS-2B cells (Endter et al. 2009); however, their functional expression may be dependent on the cultural condition because of discrepancies of PGT expression between our study and their report. Hence, our siRNA study at least provides evidence of a significant contribution of PGT to PGE2 release from BEAS-2B cells, although the contribution of other PGE2 transporter needs to be clarified in response to LPS stimuli.
Another concern is alteration of the mechanism of PGT-mediated transport of PGE2. According to previous reports, PGE2 efflux was stimulated with trans-PGE2 with a K1/2 (inhibitory constant) of 290 nM and the effect was saturated at 10 μM when PGT was expressed in Xenopus oocytes (Chan et al. 1998). According to this report, we analyzed PGE2 secretion from Xenopus oocytes. In the case of PGE2 release from the mammalian cells, it has been reported that extracellular lactate stimulates PGT-mediated PGE2 release (Chan et al. 2002); however, the precise mechanism still remains to be determined. As LPS caused a significant release of PGE2, we decided not to use PGE2 as a trans-stimulator for PGT-mediated transport to avoid any physiological effect of PGE2 on the EP receptor, which may compromise the observed LPS-mediated PGE2 release. Biological events caused by LPS may optimize or regulate endogenous trans-stimulators as PGE2 is smoothly secreted. For a further knockdown assay, we employed LC/MS/MS to quantify PGE2 more consistently and accurately (Fig. 8). Unfortunately, sensitivity of detection was at most 30-fold decreased; therefore, we had to add arachidonic acid to detect PGE2 in extracellular medium at a significant level of PGE2. In this case, the stimulation effect of LPS was not clearly observed. This could be because PG synthesis induced by arachidonic acid may be much greater than that stimulated by LPS.
LPS may alter gene expression of PGT gene expression in BEAS-2B cells. In this study, we observed that LPS (0.1 μg/ml) had only a marginal decrease in PGT mRNA expression in BEAS-2B cells (Fig. 3A). Limited information is available for the effect of LPS on PGT expression. One report describes that LPS did not affect rat Pgt expression in both mRNA and protein forms in rat cerebral endothelial cells treated with LPS for up to 24 h at the same concentration we used (Tavakoli et al. 2001). This implies that PGT expression may not be sensitive to LPS stimuli. Comparing LPS-induced reduction of PGT mRNA expression for 48 h with the normal condition, this could be explained as a consequence of damage by LPS-induced production of pro-inflammatory cytokines; however, this should be addressed by future studies.
LPS is a component of the Gram-negative bacterial cell membrane and has been suggested to mediate inflammation via CD14/TLR4/MD2 receptor complexes. LPS signal is transduced through MAP kinase pathways and activates various transcription factors, resulting in induction of genes encoding pro-inflammatory cytokines, e.g. IL6 and TNF-a. Independently, it is reported that BEAS-2B cells express EP1, EP2, and EP4 receptors, and their exposure to PGE2 increases IL6 mRNA expression (Tavakoli et al. 2001). Interestingly, the effect of three PGT inhibitors (e.g. BCG, BSP, and TGBz) on PGT-mediated release (Fig.7B) similarly corresponded to their effect on IL6 mRNA expression in LPS-treated BEAS-2B cells (Fig. 4B). On the other hand, as shown in Figs 5B, 6D, and 7B, despite the inhibitory effect of PGB1 on PGT-mediated PGE2 uptake, PGB1 elevated extracellular PGE2 concentration and IL6 mRNA expression in LPS-treated BEAS-2B cells. This inconsistency may be possibly explained by several mechanisms; for instance, PGB1 may act directly on EP receptors as their agonist or antagonist, or extracellular PGB1 may not exert an inhibition of PGT-mediated release. These results suggest that extracellular PGE2, which may be adjustable by PGT, regulates signal through the EP receptor in an autocrine model. Accordingly, modulation of PGT-mediated release may provide an antiphlogistic benefit if further study defines such a role of PGT under inflammatory conditions.
In conclusion, our study implies a critical role of PGT in regulating the extracellular concentration of PGE2 by controlling its trans-cellular transport across the plasma membranes in cells under inflammatory conditions. Further studies should be warranted to clarify a transporter responsible for PGE2 and its alteration in gene expression and intracellular localization under inflammatory conditions. Better understanding of a precise mechanism that determines PGT-mediated PGE2 transport may provide a new rationale to efficiently control inflammatory responses induced by extracellular signaling.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.
Acknowledgements
The authors thank Mr Katsumasa Otake (former student of Tokyo University of Science), Mr Yuichi Yanagisawa, and Mr Hiroaki Kurashige (former students of Kanazawa University) for excellent technical assistance and kind advice.
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