Expression Of Prostaglandin Transporter Research Articles

Elevated PGT promotes proliferation and inhibits cell apoptosis in preeclampsia by Erk signaling pathway

Prostaglandins participate in maternal recognition of pregnancy, implantation and maintenance of gestation. Prostaglandin transporter (PGT), as a candidate molecule of prostaglandin carriers, might be involved in the pathogenesis of preeclampsia. In preeclampsia (PE) patients’ placental tissue, we identified PGT by RNA sequencing, measured its expression pattern by quantitative real-time PCR and Western blot. PGT was found to be upregulated in preeclamptic placental tissue. The expression pattern of PGT in PE was double confirmed by eight Gene Expression Omnibus (GEO) databases. In abortion tissues at 6–8 weeks, we then observed the cellular location of PGT by Immunofluorescence technique (IF) and found PGT located in trophoblast cell of the placenta of early pregnancy. In vitro studies revealed that forced expression of PGT in HTR8/Sveno cell inhibited its apoptosis, but promoted its proliferation by activating Erk signaling. In vivo study, we used reduced uterine perfusion pressure (RUPP) rat model and L-NAME-induced preeclampsia-like rats to study the possible role of PGT in preeclampsia. And PGT was found to be upregulated in both preeclampsia rat models by Immunohistochemical (IHC) staining. Newly identified PGT plays an important role in trophoblast proliferation via Erk signaling, providing new insights for understanding the pathogenesis of PE.

Quyu Shengji Formula Facilitates Diabetic Wound Healing via Inhibiting the Expression of Prostaglandin Transporter.

Background Quyu Shengji Formula (QSF), a Chinese medicine formula widely used in the clinic, has proven therapeutic effects on diabetic ulcers. Nevertheless, the potential mechanism of how QSF cures diabetic ulcer remains elusive. Objective To assess the mechanism of QSF against wound healing defects in diabetes. Methods Db/db mice were adopted to determine the therapeutic potential of QSF. Further histology analysis was performed by hematoxylin and eosin (H&E) staining. Moreover, the expression patterns of prostaglandin transporter (PGT), prostaglandin E2 (PGE2), and angiogenesis factor vascular endothelial growth factor (VEGF) were evaluated by immunostaining (IHC) analysis, ELISA assay, real-time quantitative polymerase chain reaction (RT-qPCR), and western blot analysis in vivo. Human dermal microvascular endothelial cells (HDMECs) and the shRNA interference technique were used to explore the effects of QSF on cell migration, PGT, PGE2, and angiogenesis factor VEGF in vitro. Results Applied QSF on the wound of db/db mice significantly accelerated wound closure. Reductions of PGT and elevations of PGE2 and increased angiogenesis factor VEGF levels were shown after QSF treatment in vivo and in vitro. Furthermore, QSF promoted HDMEC migration. Inhibition of the expression of PGT by shRNA reversed phenotypes of QSF treatment in vitro. Conclusion Taken together, our findings reveal that QSF ameliorates diabetes-associated wound healing defects by abolishing the expression of PGT.

Reduction of prostaglandin transporter predicts poor prognosis associated with angiogenesis in gastric adenocarcinoma.

Prostaglandin (PG) E2 promotes gastrointestinal carcinogenesis and tumor progression. The total amount of biologically active PGE2 in tissues is determined by a balance of PG biosynthesis and degradation pathways, which involve the PG transporter (PGT). We investigated PGT in gastric adenocarcinoma by determining its expression pattern and examining associations of PGT with prognosis and tumor angiogenesis. PGT expression was determined by immunohistochemistry in advanced gastric adenocarcinoma specimens obtained from 96 patients who underwent surgical resection. Correlations between PGT expression level and clinicopathological factors were statistically analyzed. Angiogenesis in the tumor tissue was evaluated by counting the number of microvessels. The role of PGT in mRNA and protein expression of vascular endothelial growth factor (VEGF) was examined in gastric cancer cells stimulated by PGE2 . Based on multivariate and Kaplan-Meier analyses, negativity for PGT expression was an independent poor prognostic factor. There were more microvessels in PGT-negative tumors than in PGT-positive tumors. Transfection of AGS and MKN7 gastric cancer cells with PGT-specific siRNA led to increased VEGF mRNA and protein expression accompanied by increased PGE2 in the culture media. PGT expression is an independent predictor of poor survival and is associated with tumor angiogenesis in gastric adenocarcinoma.

Lysophosphatidic acid modulates prostaglandin signalling in bovine steroidogenic luteal cells

We examined whether lysophosphatidic acid affects prostaglandin biosynthesis, transport, and signalling in bovine steroidogenic luteal cells. The aim of the present study was to determine the influence of LPA on PGE2 and PGF2α synthesis and on the expression of enzymes involved in PG biosynthesis (PTGS2, mPGES-1, cPGES, mPGES-2, PGFS and 9-KPR), prostaglandin transporter (PGT), and prostaglandin receptors (EP1, EP2, EP3, EP4 and FP) in bovine steroidogenic luteal cells. We found that LPA inhibited PGF2α synthesis in steroidogenic luteal cells. Moreover, LPA increased mPGES1 and cPGES and decreased PGFS expression in cultured bovine steroidogenic luteal cells. Additionally, LPA stimulated EP2 and EP4 receptor and PGT expression. This study suggests that LPA activity in the bovine CL directs the physiological intraluteal balance between the two main prostanoids towards luteotropic PGE2.

Sa1993a Reduced Expression of Prostaglandin Transporter Promotes Angiogenesis in Gastric Cancer

Background & Aims: Colorectal cancer (CRC) cells (unlike normal colonic epithelial cells) secrete VEGF, which drives these cells proliferation and promotes tumor-related angiogenesis. The mechanism(s) of increased, aberrant VEGF expression in CRC cells is not known, but is likely related to transcriptional activation of VEGF gene.We hypothesized that: 1) upregulated importin-α in CRC cells (which facilitates nuclear transport of transcription factors: HIF1α and CREB, and is essential for VEGF gene promoter activation) is the underlying mechanism for increased VEGF expression in these cells and CRC cell proliferation, and, 2) inhibition and/or silencing of importin-α in CRC cells will inhibit VEGF gene activation and CRC cell proliferation. Ivermectin is a specific inhibitor of importin α nuclear transport (Biochem J. 2012: 1;443(3):851-6). We examined whether treatment of CRC cell lines with ivermectin could inhibit CRC cell growth. Methods: We used: (a) human CRC cell lines HCT116 & HT29 and (b) normal colonic epithelial cells NCM356 & NCM460. We treated cultured CRC cells with ivermectin (50 μM, 6 hr; specific inhibitor of importin-α nuclear transport), with importin α specific siRNA or control RNA (100nM, 48 hr). Add-back studies included treatment with exogenous VEGF (20ng/ml). Studies: 1) mRNA and protein expression of importin-α and VEGF by Real-Time RT-PCR, and/or Western blotting and immunostaining, respectively; 2) VEGF secretion into culture medium by ELISA; 3) translocation of importin to the nucleus in CRC vs. normal cells; and 4) cell proliferation by BrdU assay. Results: 1) CRC cell lines express high levels of VEGF and secrete large amounts of VEGF (up to 1530 pg/ml) into culture medium at 24 hours vs. normal epithelial cells, which only secrete minimal VEGF ( 40% (p< 0.05); 3) Selective inhibition of importin-α in CRC cells using ivermectin significantly reduced VEGF expression by ~3-fold (p<0.01) and proliferation of these cells by 2.4-fold (p<0.01). Treatment with exogenous VEGF partly reversed inhibited by ivermectin cell proliferation; 4) Importin-α silencing in CRC cells significantly reduced VEGF expression and proliferation of these cells by 2.2-fold and 2fold (both p<0.01), respectively; treatment with exogenous VEGF following importin-α silencing reversed inhibition of cell proliferation by 2-fold (p < 0.01). Conclusions: 1) VEGF gene activation in CRC cells requires functional importin-α; 2) importin-α mediates increased proliferation of CRC cells; downregulation of importin-α with specific siRNA or its inhibition with specific inhibitor ivermectin inhibits CRC cell proliferation; 3) These studies suggest a potential therapeutic role of ivermectin in inhibiting CRC cell proliferation.

Expression of prostaglandin transporter in colorectal cancer tissues and its relationship with clinicopathological features

To investigate the expression of prostaglandin transporter (PGT) in colorectal cancer (CRC) tissues and its relationship with clinicopathological features. The mRNA and protein levels of PGT were determined by real-time PCR, Western blot and immunohistochemical methods in cancer tissues and adjacent normal tissue from 80 patients with colorectal cancer and their relationship with clinicopathological features was analyzed. Compared with the adjacent normal tissue of colorectal cancer, the PGT mRNA relative expression (0.57 ± 0.33 vs. 2.33 ± 1.20) and the PGT protein expression in cancer tissues decreased significantly [PGT/GAPDH 0.45 ± 0.16 vs. 0.78 ± 0.23, integral A 718.7 ± 359.4 vs. 10412.0 ± 6423.3, average A 0.03 ± 0.01 vs. 0.12 ± 0.09, all P<0.01]. Lower mRNA and protein expressions of PGT in colorectal cancer were associated with depth of invasion T3 to T4 and TNM stage III( to IIII( (P<0.01), while not associated with gender, age, tumor location and differentiation degree (all P>0.05). Expression levels of PGT mRNA and protein in colorectal cancer tissue are significantly down-regulation. PGT expression is associated with invasion depth and late stages.

Canine placental prostaglandin E2 synthase: expression, localization, and biological functions in providing substrates for prepartum PGF2alpha synthesis.

The prepartum output of PGF2alpha in the bitch is associated with increased placental PGE2-synthase (PTGES) mRNA levels. Contrasting with this is a decreased expression of PGF2alpha-synthase (PGFS/AKR1C3) in uteroplacental compartments during prepartum luteolysis, suggesting an involvement of alternative synthetic pathways in PGF2alpha synthesis, for example, conversion of PGE2 to PGF2alpha. However, because the expression and possible functions of the respective PTGES proteins remained unknown, no further conclusion could be drawn. Therefore, a canine-specific PTGES antibody was generated and used to investigate the expression, cellular localization, and biochemical activities of canine uteroplacental PTGES throughout pregnancy and at prepartum luteolysis. Additionally, the biochemical activities of these tissues involved in the conversion of PGE2 to PGF2alpha were investigated. The endometrial PTGES was localized in the uterine surface epithelium at preimplantation and in superficial and deep uterine glands, endothelial cells, and myometrium throughout pregnancy and at parturition. Placental signals were mostly in the trophoblast. The biochemical properties of recombinant PTGES protein were confirmed. Additionally, expression of two PGE2-receptors, PTGER2/EP2 and PTGER4/EP4, revealed their decreasing expression during luteolysis. In contrast, the uteroplacental expression of prostaglandin transporter (PGT) was strongly elevated prior to parturition. These localization patterns resembled that of PTGES. The increased expression of PTGES and PGT at parturition, together with the accompanying decreased levels of PGE2-receptors and the capability of canine uterine and placental homogenates to take part in the conversion of PGE2 to PGF2alpha, as found in this study, suggest that PGE2 could be used locally as a substrate for prepartum PGF2alpha synthesis in the dog.

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

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.

Su1982 Reduced Expression of Prostaglandin Transporter is an Independent Predictor of Poor Prognosis in Gastric Adenocarcinoma Associated With Tumor Angiogenesis

Su1982 Reduced Expression of Prostaglandin Transporter is an Independent Predictor of Poor Prognosis in Gastric Adenocarcinoma Associated With Tumor Angiogenesis

Abstract 524: Cyclooxygenase pathway in ovarian cancer.

Abstract Objectives: To examine the role of the cyclooxygenase (COX) pathway in ovarian cancer. Cyclooxygenases, specifically COX1 and COX2, are frequently overexpressed in malignancies and lead to the synthesis of prostaglandins. A particularly important prostaglandin, PGE2 is exported from the cell into the extracellular milieu by the multi-drug resistance protein 4 (MRP4) transporter where it acts in a paracrine and autocrine manner by activating a family of G-protein coupled receptors (EP1-4). PGE2 is transported into the cell by the prostaglandin transporter (PGT) and catabolized by 15-prostaglandin dehydrogenase (15-PGDH). It has been previously shown that COX1, rather than COX2, is overexpressed in ovarian cancer. Little is known about the role of other members of this pathway, including EP receptors, PGT, MRP4, and 15-PGDH, in this disease. Methods: HOSE (benign immortalized ovarian epithelial cells) and two ovarian cancer cell lines, SKOV3 and OVCAR3, were grown under standard conditions. Cells were harvested and RNA and protein were isolated. Quantitative RT-PCR was done using the SYBR Green protocol to examine the expression of members of the cyclooxygenase pathway and expressed relative to HOSE cells. Results: In both ovarian cancer cell lines relative to HOSE, expression of COX2, PGT, EP4, and EP1 was decreased. In SKOV3 cells, COX1 and 15-PGDH expression was decreased but MRP4 levels were comparable to levels detected in HOSE cells. In OVCAR3 cells, COX1 and 15-PGDH expression levels were increased and MRP4 was decreased relative to HOSE cells. PGE2 levels in media will be measured to determine the net effect of these changes. Protein levels of the members of the cyclooxygenase pathway will be determined by western blot. Conclusions: It is clear that several members of the cyclooxygenase pathway are expressed differently in malignant versus normal ovarian cells. Studies in progress will determine the functional significance of these differences. Citation Format: Gautam G. Rao, Tyler Kochel, Namita Kundu, Jocelyn Reader, Amy Fulton. Cyclooxygenase pathway in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 524. doi:10.1158/1538-7445.AM2013-524

Prostaglandin E2 functions as a luteotrophic factor in the dog

The luteal phase in dogs is governed by many poorly understood regulatory mechanisms. Functioning of the corpus luteum (CL) is unaffected by hysterectomy. Recently, the role of prostaglandins in regulating canine CL function was addressed suggesting a luteotrophic effect of prostaglandin E2 (PGE2) during the early luteal phase. However, compelling functional evidence was lacking. The potential of PGE2 to stimulate steroidogenesis was tested in canine primary luteal cells isolated from developing CL of non-pregnant dogs. In addition, the luteal expression of prostaglandin transporter (PGT) and steroidogenic acute regulatory protein (STAR) was demonstrated and characterized in CL from non-pregnant bitches during the course of dioestrus as well as from pregnant animals during the pre-implantation, post-implantation and mid-gestation periods of pregnancy and during luteolysis; the luteal expression of PGE2 receptors (EP2 and EP4) has been investigated at the protein level throughout pregnancy. Our findings show that PGE2 is an activator of STAR expression in canine luteal cells from early luteal phase, significantly up-regulating STAR promoter activity and protein expression resulting in increased steroidogenesis. The 3βHSD (HSD3B2) and P450scc (CYP11A1) expression remained unaffected by PGE2 treatment. The expression of PGT was confirmed in CL during both pregnancy and dioestrus and generally localized to the luteal cells. After initial up-regulation during the earlier stages of the CL phase, its expression declined towards the luteal regression. Together with the demonstration of EP2 and EP4 throughout pregnancy, and the decline in EP2 at prepartum, our findings further support our hypothesis that intra-luteal PGE2 may play an important role in regulating progesterone secretion in the canine CL.

Prostaglandin Transporter Modulates Wound Healing in Diabetes by Regulating Prostaglandin-Induced Angiogenesis

Prostaglandin transporter (PGT) mediates prostaglandin (PG) catabolism and PG signal termination. The prostanoid PGE(2), which induces angiogenesis and vasodilation, is diminished in diabetic skin, suggesting that PGT up-regulation could be important in wound healing deficiency, typified by diabetic foot ulcer. We hypothesized that up-regulation of PGT in hyperglycemia could contribute to weakened PGE(2) signaling, leading to impaired angiogenesis and wound healing. In human dermal microvascular endothelial cells (HDMECs), exposure to hyperglycemia increased PGT expression and activity up to threefold, accompanied by reduced levels of PGE(2). Hyperglycemia reduced HDMEC migration by 50% and abolished tube formation. Deficits in PGE(2) expression, HDMEC migration, and tube formation could be corrected by treatment with the PGT inhibitor T26A, consistent with the idea that PGT hyperactivity is responsible for impairments in angiogenesis mediated by PG signaling. In vivo, PGT expression was profoundly induced in diabetes and by wounding, correlating with diminished levels of proangiogenic factors PGE(2) and VEGF in cutaneous wounds of diabetic mice. Pharmacological inhibition of PGT corrected these deficits. PGT inhibition shortened cutaneous wound closure time in diabetic mice from 22 to 16 days. This effect was associated with increased proliferation, re-epithelialization, neovascularization, and blood flow. These data provide evidence that hyperglycemia enhances PGT expression and activity, leading to diminished angiogenic signaling, a possible key mechanism underlying defective wound healing in diabetes.

Abstract 1472: MRP4 and PGT, members of the COX-2 pathway, may contribute to breast cancer progression

Abstract Prostaglandin E2 (PGE2) is the major product of the cyclooxygenase 2 (COX-2) enzyme. PGE2 is a substrate of multiple drug resistance-associated protein 4 (MRP4) which exports it from the cell. PGE2 can then signal through any of its four GPCR-family receptors EP1-4. Each receptor is connected to a different cell function including adenylate cyclase activation, calcium flux, and inositol trisphosphate mobilization. Alternatively, extracellular PGE2 can be imported through prostaglandin transporter (PGT) and inactivated by 15-hydroxy prostaglandin dehydrogenase (15-PGDH). PGE2 plays a role in tumor progression as evidenced by the anti-tumor activity of COX-2 inhibitors. Adverse effects of these inhibitors have forced a search to identify other potential therapeutic targets in the COX-2 pathway (such as the receptors, transporters, and associated metabolic enzymes of PGE2). We hypothesize that disregulation of the COX-2 pathway can lead to increased metastatic potential in breast cancer. High extracellular PGE2 in the tumor microenvironment can stem from high COX-2 expression or elevated eicosanoid export activity. Continued prostaglandin signaling is thought to occur due to the interplay between increased expression of the exporting protein (MRP4) shuttling PGE2 into the pericellular space and decreased expression of the importing protein (PGT) removing PGE2 from the pericellular space. Higher concentrations of PGE2 in the pericellular space allow for increased signaling through PGE2 receptors EP1-4. Using a panel of breast cancer cell lines, we demonstrate that highly metastatic tumor cells can express low levels of COX-2, but still actively signal through PGE2 receptors. MRP4 mRNA and protein expression is increased in tumor cell lines with high metastatic potential while expression of PGT mRNA and protein is decreased in these cells when compared to cells with lower metastatic potential. This should lead to higher concentrations of extracellular PGE2 in the tumor microenvironment. Additionally, we have examined MCF7 cells stably transfected with aromatase and an anastrozole-resistant derivative of this cell line for expression of COX-2, EP4, EP1, MRP4 and PGT. At the mRNA level, COX-2, EP1, MRP4 and PGT decrease in the aromatase inhibitor-resistant cells. EP4 mRNA levels increase in the resistant cells. Examining the relative expression levels of all key members in the COX-2 pathway as they relate to tumor progression could highlight new therapeutic targets under this classification of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1472. doi:10.1158/1538-7445.AM2011-1472

Differential expression and regulation of prostaglandin transporter and metabolic enzymes in mouse uterus during blastocyst implantation

To examine the spatiotemporal expression and regulation of prostaglandin transporter (PGT), 15-hydroxy-PG dehydrogenase (15-PGDH), and carbonyl reductase 1 (CBR1) messenger RNA (mRNA) and protein in the mouse uterus during embryo implantation and in related models. Experimental animal study. University research laboratory. Sexually mature female Kunming strain white mice. Delayed and activated implantation, artificial decidualization, and subcutaneous injection of progesterone (P) and E(2) in ovariectomized mouse. The expression of mRNA and protein were detected by in situ hybridization and immunohistochemistry in mouse uterus. Prostaglandin transporter mRNA and protein were expressed in the subluminal stroma at implantation site on day 5 of pregnancy and then in decidua but were not detected at the interimplantation sites and in preimplantation or pseudopregnant uterus. The presence of an active blastocyst was required for PGT expression at the implantation site. Both 15-PGDH and CBR1 mRNA were detected in glandular epithelium on day 4 of pregnancies. The expression of 15-PGDH and CBR1 mRNA was also detected in postimplantation embryos. These data suggest that differentially expressed PGT and 15-PGDH may participate in PG signaling in mouse uterus during implantation and decidualization.

Prostaglandin transporter expression in mouse brain during development and in response to hypoxia

Prostaglandins (PGs) are bioactive lipid mediators released following brain hypoxic–ischemic injury. Clearance and re-uptake of these prostaglandins occur via a transmembrane prostaglandin transporter (PGT), which exchanges PG for lactate. We used Western blot analyses to examine the PGT developmental profile and its regional distribution as well as changes in transporter expression during chronic hypoxia in the neonatal mouse brain. Microsomal preparations from four brain regions (cortex, hippocampus, cerebellum and brainstem/diencephalon) showed gradual increases in prostaglandin transporter expression in all brain regions examined from postnatal day 1 till day 30. There was a significant regional heterogeneity in the prostaglandin transporter expression with highest expression in the cortex, followed by cerebellum and hippocampus, and least expressed in the brainstem/diencephalon. To further delineate the pattern of prostaglandin transporter expression, separate astrocytic and neuronal microsomal preparations were also examined. In contrast to neurons, which had a robust expression of prostaglandin transporters, astrocytes had very little PGT expression under basal conditions. In response to chronic hypoxia, there was a significant decline in PGT expression in vivo and in neurons in vitro, whereas cultured astrocytes increased their PGT expression. This is the first report on PGT expression in the CNS and our studies suggest that PGTs have 1) a widespread distribution in the CNS; 2) a gradual increase and a differential expression in various regions during brain development; and 3) striking contrast in expression between glia and neurons, especially in response to hypoxia. Since PGTs play a role as prostaglandin–lactate exchangers, we hypothesize that PGTs are important in the CNS during stress such as hypoxia.

Effects of LPS stimulation on the expression of prostaglandin carriers in the cells of the blood‐brain and blood‐cerebrospinal fluid barriers

Prostaglandins produced in cerebral endothelial cells (CECs) are the final signal transduction mediators from the periphery to the brain during fever response. However, prostaglandins are organic anions at physiological pH, and they enter cells poorly using simple diffusion. Several transporters have been described that specifically transport prostaglandins across cell membranes. We examined the expression of the two principal prostaglandin carriers, prostaglandin transporter (PGT), and multidrug resistance-associated protein 4 (MRP4) in cells of the blood-brain barrier and in choroid epithelial cells in vitro as well as in vivo in rat brain in control conditions and after lipopolysaccharide (LPS) challenge. We detected PGT in primary cultures of rat CECs, astrocytes, pericytes, and choroid epithelial cells. LPS stimulation had no effect on the expression level of PGT in these cells; however, after LPS stimulation the polarized, dominantly luminal, expression pattern of PGT significantly changed. MRP4 is also expressed in CECs, and its level was not influenced by LPS treatment. In rat brain, PGT was highly expressed in the supraoptic and paraventricular nuclei of the hypothalamus, in the ependymal cell layer of the third ventricle, and in the choroid plexus. LPS treatment increased the expression of PGT in the supraoptic and paraventricular nuclei. Our results suggest that PGT and MRP4 likely play a role in transporting prostaglandins through the blood-brain and blood-cerebrospinal fluid barriers and may be involved in the maintenance of prostaglandin homeostasis in the brain and in the initiation of fever response.

Functional characterization of prostaglandin transporter and terminal prostaglandin synthases during decidualization of human endometrial stromal cells

Decidualization of endometrial stromal cells is essential for successful implantation and pregnancy. Prostaglandins (PG) have been shown to be required for the initiation and maintenance of decidualization in animal models. The transport of PG across the plasma membrane is mediated by carriers such as prostaglandin transporter (PGT). Our recent data have shown the expression of human PGT (hPGT) in the endometrium during the menstrual cycle. The objective of the present study was to characterize hPGT in decidualized stromal cells. Human endometrial stromal cells were treated with a combination of cAMP and medroxyprogesterone acetate to induce decidualization. Decidualization was confirmed by morphological differentiation and increased secretion of prolactin. A large increase in hPGT mRNA level, as measured by real-time PCR analysis, was observed in decidual cells compared with control. Similarly, a 2-fold up-regulation of hPGT and 3-12-fold increase in PG biosynthetic enzymes were obtained at the protein level. Decidual cells exhibited a higher isotopic PGE2 uptake and greater intracellular PG levels than control. The higher uptake of PG by decidual cells is highly likely to be mediated via hPGT. PGT is a newly identified regulator of PG action at the cellular level and likely contributes to the regulation of PG action in female reproductive processes.

Effects of LPS stimulation on the expression of prostaglandin carriers in the cells of the blood-brain and blood-cerebrospinal fluid barriers

Prostaglandins produced in cerebral endothelial cells (CECs) are the final signal transduction mediators from the periphery to the brain during fever response. However, prostaglandins are organic anions at physiological pH, and they enter cells poorly using simple diffusion. Several transporters have been described that specifically transport prostaglandins across cell membranes. We examined the expression of the two principal prostaglandin carriers, prostaglandin transporter (PGT), and multidrug resistance-associated protein 4 (MRP4) in cells of the blood-brain barrier and in choroid epithelial cells in vitro as well as in vivo in rat brain in control conditions and after lipopolysaccharide (LPS) challenge. We detected PGT in primary cultures of rat CECs, astrocytes, pericytes, and choroid epithelial cells. LPS stimulation had no effect on the expression level of PGT in these cells; however, after LPS stimulation the polarized, dominantly luminal, expression pattern of PGT significantly changed. MRP4 is also expressed in CECs, and its level was not influenced by LPS treatment. In rat brain, PGT was highly expressed in the supraoptic and paraventricular nuclei of the hypothalamus, in the ependymal cell layer of the third ventricle, and in the choroid plexus. LPS treatment increased the expression of PGT in the supraoptic and paraventricular nuclei. Our results suggest that PGT and MRP4 likely play a role in transporting prostaglandins through the blood-brain and blood-cerebrospinal fluid barriers and may be involved in the maintenance of prostaglandin homeostasis in the brain and in the initiation of fever response.

Prostaglandin Signaling in the Renal Collecting Duct: RELEASE, REUPTAKE, AND OXIDATION IN THE SAME CELL

Prostaglandins mediate autacrine and paracrine signaling over short distances. We used the renal collecting duct as a model system to test the hypothesis that local control of prostaglandin signaling is achieved by expressing inactivation in the same cell as synthesis. Immunocytochemical studies demonstrated that renal collecting ducts in situ express the prostaglandin (PG) synthesis enzyme, cyclooxygenase-1 (COX-1), as well as both components of prostaglandin metabolic inactivation, i.e. the prostaglandin uptake carrier prostaglandin transporter (PGT) and the enzyme 15-hydroxyprostaglandin dehydrogenase. We characterized this system further using the collecting duct cell line Madin-Darby canine kidney (MDCK), which retains COX-2 and prostaglandin dehydrogenase expression but which has lost PGT expression. When we reintroduced PGT, it was correctly sorted to the apical membrane where it altered the sidedness of prostaglandin E2 (PGE2) release, a process we call "vectorial release via sided reuptake." Importantly, although COX-2 and prostaglandin dehydrogenase are expressed in the same MDCK cell, they must be compartmentalized because even in the presence of excess dehydrogenase newly synthesized PGE2 is released largely un-oxidized. However, when PGE2 undergoes first release and then PGT-mediated reuptake, significant oxidation takes place, suggesting that PGT imports PGE2 into the prostaglandin dehydrogenase compartment. Our data are consistent with a new model that offers significant new mechanisms for the fine control of eicosanoid signaling.

Expression of Prostaglandin Transporter in the Bovine Uterus and Fetal Membranes During Pregnancy1

Uteroplacental prostaglandins (PGs) play pivotal roles in the maintenance and termination of pregnancy in mammals. In the present study, we have characterized the expression of prostaglandin transporter (PGT) in placentome caruncles, intercaruncular tissues, fetal membranes, and utero-ovarian plexus during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. In caruncles and intercaruncular tissues, PGT mRNA (also known as SLC02A1) and PGT protein were highly expressed at the late stage of pregnancy compared to the early and mid stages, whereas the level of expression is constant and low in fetal membranes throughout pregnancy. PGT mRNA and PGT protein were expressed at a constant level in the utero-ovarian plexus both ipsilateral and contralateral to corpus luteum throughout the course of pregnancy. Overall, the relative expression of PGT mRNA and PGT protein were higher in caruncles than in intercaruncular tissue and fetal membranes, whereas no differences were detected between intercaruncular tissues and fetal membranes at any stage of gestation. Immunohistochemistry indicated that PGT was preferentially expressed in caruncular epithelial cells of placentomes and endometrial luminal epithelial and myometrial smooth muscle cells of the intercaruncular regions. The level of PGT expression was comparatively higher in maternal components than in fetal components. In conclusion, differential spatiotemporal tissue-specific expression of PGT in uterine and intrauterine tissues suggests a role for this transporter in the exchange of PGs between the maternal and the fetal compartments, as well as for intrauterine metabolism of PGs during pregnancy.