Prostaglandin E2 facilitates subcellular translocation of the EP4 receptor in neuroectodermal NE-4C stem cells

Prostaglandin E2 (PGE₂) is a lipid mediator released from the phospholipid membranes that mediates important physiological functions in the nervous system via activation of four EP receptors (EP1-4). There is growing evidence for the important role of the PGE₂/EP4 signaling in the nervous system. Previous studies in our lab show that the expression of the EP4 receptor is significantly higher during the neurogenesis period in the mouse. We also showed that in mouse neuroblastoma cells, the PGE₂/EP4 receptor signaling pathway plays a role in regulation of intracellular calcium via a phosphoinositide 3-kinase (PI3K)-dependent mechanism. Recent research indicates that the functional importance of the EP4 receptor depends on its subcellular localization. PGE₂-induced EP4 externalization to the plasma membrane of primary sensory neurons has been shown to play a role in the pain pathway. In the present study, we detected a novel PGE₂–dependent subcellular trafficking of the EP4 receptor in neuroectodermal (NE-4C) stem cells and differentiated NE-4C neuronal cells. We show that PGE₂ induces EP4 externalization from the Golgi apparatus to the plasma membrane in NE-4C stem cells. We also show that the EP4 receptors translocate to growth cones of differentiating NE-4C neuronal cells and that a higher level of PGE₂ enhances its growth cone localization. These results demonstrate that the EP4 receptor relocation to the plasma membrane and growth cones in NE-4C cells is PGE₂ dependent. Thus, the functional role of the PGE₂/EP4 pathway in the developing nervous system may depend on the subcellular localization of the EP4 receptor.     

Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation

We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E₂ (PGE₂), a bioactive lipid mediator in various physiological or pathological conditions. PGE₂ is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE₂ signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE₂ or specific agonists. All four PGE₂ receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE₂ and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE₂ (17-PT PGE₂) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE₂ signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts.     

Prostaglandin E2 produced by inducible COX-2 and mPGES-1 promoting cancer cell proliferation in vitro and in vivo

Aim

Many cancers originate and flourish in a prolonged inflammatory environment. Our aim is to understand the mechanisms of how the pathway of prostaglandin E₂ (PGE₂) biosynthesis and signaling can promote cancer growth in inflammatory environment at cellular and animal model levels.

Main methods

In this study, a chronic inflammation pathway was mimicked with a stable cell line that over-expressed a novel human enzyme consisting of cyclooxygenase isoform-2 (COX-2) linked to microsomal (PGE₂ synthase-1 (mPGES-1)) for the overproduction of pathogenic PGE₂. This PGE₂-producing cell line was co-cultured and co-implanted with three human cancer cell lines including prostate, lung, and colon cancers in vitro and in vivo, respectively.

Key findings

Increases in cell doubling rates for the three cancer cell types in the presence of the PGE₂-producing cell line were clearly observed. In addition, one of the four human PGE₂ subtype receptors, EP₁, was used as a model to identify PGE₂-signaling involved in promoting the cancer cell growth. This finding was further proven in vivo by co-implanting the PGE₂-producing cells line and the EP₁-positive cancer cells into the immune deficient mice, after that, it was observed that the PGE₂-producing cells promoted all three types of cancer formation in the mice.

Significance

This study clearly demonstrated that the human COX-2 linked to mPGES-1 is a pathway that, when mediated by the EP, is linked to promoting cancer growth in a chronic inflammatory environment. The identified pathway could be used as a novel target for developing and advancing anti-inflammation and anti-cancer interventions.     

Expression of prostaglandin E2 receptor subtypes, EP2 and EP4, in the rat hippocampus after cerebral ischemia and ischemic tolerance

We investigated the distribution and time course of expression of two subtypes of prostaglandin E₂ (PGE₂) receptors, EP2 and EP4, in a rat model of cerebral ischemia and ischemic tolerance. Adult male Sprague-Dawley rats were subjected to either lethal global ischemia (10 min) with or without sublethal ischemic preconditioning (3 min), or ischemia only (3 min). A short 3-min cerebral ischemia and a 3-min ischemia followed by a second lethal ischemia enhanced the expression of EP2 and EP4 receptors in CA1 pyramidal neurons of the hippocampus. In tolerance-acquired CA1 neurons, the immunoreactivities of EP2 and EP4 were upregulated after 4 h and 12 h, respectively. The immunoreactivities were most prominent at 3 days and were sustained for at least 14 days, consistent with results of immunoblotting experiments. However, immunoreactivities for these PGE₂ receptors increased in reactive glial cells in the vulnerable CA1 and hilar regions of rats subjected to lethal ischemia without ischemic preconditioning. Most of the EP2 immunoreactivity occurred in microglial cells and some astrocytes, whereas increased immunoreactivity for EP4 was found only in astrocytes. These data suggest that ischemia and the induction of ischemia tolerance have different regulatory effects on the expression of EP2 and EP4 receptors. Moreover, PGE₂ may exert its unique pathophysiological functions in relation to delayed neuronal death and ischemic tolerance induction in the rat hippocampus via specific PGE₂ receptors.This research was supported by a grant (M103KV010019 04K2201 01930) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of the Republic of Korea.     

Prostaglandin E2 receptors in asthma and in chronic rhinosinusitis/nasal polyps with and without aspirin hypersensitivity

Chronic rhinosinusitis with nasal polyps (CRSwNP) and asthma frequently coexist and are always present in patients with aspirin exacerbated respiratory disease (AERD). Although the pathogenic mechanisms of this condition are still unknown, AERD may be due, at least in part, to an imbalance in eicosanoid metabolism (increased production of cysteinyl leukotrienes (CysLTs) and reduced biosynthesis of prostaglandin (PG) E₂), possibly increasing and perpetuating the process of inflammation. PGE₂ results from the metabolism of arachidonic acid (AA) by cyclooxygenase (COX) enzymes, and seems to play a central role in homeostasis maintenance and inflammatory response modulation in airways. Therefore, the abnormal regulation of PGE₂ could contribute to the exacerbated processes observed in AERD. PGE₂ exerts its actions through four G-protein-coupled receptors designated E-prostanoid (EP) receptors EP1, EP2, EP3, and EP4. Altered PGE₂ production as well as differential EP receptor expression has been reported in both upper and lower airways of patients with AERD. Since the heterogeneity of these receptors is the key for the multiple biological effects of PGE₂ this review focuses on the studies available to elucidate the importance of these receptors in inflammatory airway diseases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0100-7) contains supplementary material, which is available to authorized users.     

Prostaglandin E2 suppresses β1-integrin expression via E-prostanoid receptor in human monocytes/macrophages

β₁-Integrins mediate cell attachment to different extracellular matrix proteins, intracellular proteins, and intercellular adhesions. Recently, it has been reported that prostaglandin E₂ (PGE₂) has anti-inflammatory properties such as inhibition of the expression of adhesion molecules or production of chemokines. However, the effect of PGE₂ on the expression of β₁-integrin remains unknown. In this study, we investigated the effects of PGE₂ on the expression of β₁-integrin in the human monocytic cell line THP-1 and in CD14⁺ monocytes/macrophages in human peripheral blood. For this, we examined the role of four subtypes of PGE₂ receptors and E-prostanoid (EP) receptors on PGE₂-mediated inhibition. We found that PGE₂ significantly inhibited the expression of β₁-integrin, mainly through EP₄ receptors in THP-1 cells and CD14⁺ monocytes/macrophages in human peripheral blood. We suggest that PGE₂ has anti-inflammatory effects, leading to the inhibited expression of β₁-integrin in human monocytes/macrophages, and that the EP₄ receptor may play an important role in PGE₂-mediated inhibition.     

Role of the prostaglandin E2/E-prostanoid 2 receptor signalling pathway in TGFβ-induced mice mesangial cell damage

The prostaglandin E2 receptor, EP2 (E-prostanoid 2), plays an important role in mice glomerular MCs (mesangial cells) damage induced by TGFβ1 (transforming growth factor-β1); however, the molecular mechanisms for this remain unknown. The present study examined the role of the EP2 signalling pathway in TGFβ1-induced MCs proliferation, ECM (extracellular matrix) accumulation and expression of PGES (prostaglandin E₂ synthase). We generated primary mice MCs. Results showed MCs proliferation promoted by TGFβ1 were increased; however, the production of cAMP and PGE₂ (prostaglandin E₂) was decreased. EP2 deficiency in these MCs augmented FN (fibronectin), Col I (collagen type I), COX2 (cyclooxygenase-2), mPGES-1 (membrane-associated prostaglandin E1), CTGF (connective tissue growth factor) and CyclinD1 expression stimulated by TGFβ1. Silencing of EP2 also strengthened TGFβ1-induced p38MAPK (mitogen-activated protein kinase), ERK1/2 (extracellular-signal-regulated kinase 1/2) and CREB1 (cAMP responsive element-binding protein 1) phosphorylation. In contrast, Adenovirus-mediated EP2 overexpression reversed the effects of EP2-siRNA (small interfering RNA). Collectively, the investigation indicates that EP2 may block p38MAPK, ERK1/2 and CREB1 phosphorylation via activation of cAMP production and stimulation of PGE₂ through EP2 receptors which prevent TGFβ1-induced MCs damage. Our findings also suggest that pharmacological targeting of EP2 receptors may provide new inroads to antagonize the damage induced by TGFβ1.     

Cellular senescence involves an intracrine prostaglandin E2 pathway in human fibroblasts

Cyclooxygenase 2 and release of prostaglandin E₂ are involved in many responses including inflammation and are upregulated during cellular senescence. However, little is known about the role of lipid inflammatory mediators in senescence. Here, we investigated the mechanism by which the COX-2/PGE₂ axis induces senescence. Using the NS398 specific inhibitor of COX-2, we provide evidence that reactive oxygen species by-produced by the COX-2 enzymatic activity are negligible in front of the total senescence-associated oxidative stress. We therefore investigated the role of PGE₂ by invalidating the PGE₂ synthases downstream of COX-2, or the specific PGE₂ receptors, or by applying PGE₂ or specific agonists or antagonists. We evaluated the effect on senescence by evaluating the senescence-associated proliferation arrest, the percentage of senescence-associated β-galactosidase-positive cells, and the expression of senescent molecular markers such as IL-6 and MCP1. We show that PGE₂ acting on its EP specific receptors is able to induce both the onset of senescence and the maintenance of the phenotype. It did so only when the PGE₂/lactate transporter activity was enhanced, indicating that PGE₂ acts on senescence more via the pool of intracellular EP receptors than via those localized at the cell surface. Treatment with agonists, antagonists and silencing of the EP receptors by siRNA revealed that EP3 was the most involved in transducing the intracrine effects of PGE₂. Immunofluorescence experiments confirmed that EP3 was more localized in the cytoplasm than at the cell surface. Taken together, these results suggest that COX-2 contributes to the establishment and maintenance of senescence of normal human fibroblasts via an independent-ROS and a dependent-PGE₂/EPs intracrine pathway.     

Prostaglandin E2 modulates components of the Wnt signaling system in bone and prostate cancer cells

Both Wnt signaling and prostaglandin E₂ (PGE₂) play pivotal roles in bone development, remodeling, osteoporosis and prostate cancer (PCa) bone metastases. We investigated the effects of PGE₂ on Wnt signaling in osteoblast-lineage cells and Wnt-inhibitor expression in PCa cells. We demonstrate that low dose PGE₂ (0.1 μM) promotes Wnt signaling while higher doses of PGE₂ (1.0-10 μM) inhibit these same parameters in osteoblast-lineage cells. The differential effects of low vs high-dose PGE₂ on pre-osteoblasts may be attributed to dose-dependent modulation of prostaglandin receptor (EP) subtype expression; with lower doses increasing the expression the cAMP-stimulatory EP4 receptor subtype and higher doses increasing the expression of the cAMP-inhibitory EP3 receptor subtype. Moreover, we demonstrate that high expression levels of COX-2 and PGE₂ promote the secretion of Wnt inhibitors from prostate cancer cells. These data demonstrate that there are dose-dependent effects of PGE₂ on Wnt activation in osteoblast-lineage cells and Wnt-inhibitor expression in PCa cells which may have clinical implications in the management.     

HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A2α dependent PGE2via both PKA and PKB pathways

Cytosolic phospholipase A₂α (cPLA₂α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA₂α which coincided with a significant increase in cell proliferation. The inhibition of cPLA₂α activity by pyrrophenone or by antisense oligonucleotide against cPLA₂α (AS) or inhibition of prostaglandin E₂ (PGE₂) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE₂. The secreted PGE₂ activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE₂. But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE₂. AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA₂α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA₂α-dependent PGE₂ production. PGE₂via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.     

Effects of hypothalamic microinjection of PGE2 on body temperature and sympathetic nervous activities in the rabbit

The febrile response and sympathetic nervous response to hypothalamic microinjections of prostaglandin E₂ (PGE₂) were investigated in anesthetized rabbits. Microninjection of PGE₂ (500–1000 ng) caused an increase in rectal temperature of more than 0.3°C in 13 of 50 loci in the preoptic and anterior hypothalamic area (PO/AH). At 8 of these 13 loci, PGE₂ elicited response patterns in the sympathetic nervous system, such as an increase in cutaneous sympathetic nervous activity and decrease in renal sympathetic nervous activity. This pattern of sympathetic nervous responses was induced with a simultaneous increase in rectal temperature of more than 0.5°C. The 8 loci were distributed in the preoptic area, especially in the vicinity of the supraoptic nucleus. Electrolytic lesions of this region were made bilaterally, and intracerebroventricular injection of PGE₂ (8 µg/kg) was found to inhibit fever and sympathetic activity. The results demonstrate that the action of PGE₂ is responsible for the response patterns of sympathetic twigs during fever. The preoptic area, especially in the vicinity of the supraoptic nucleus, is most sensitive to PGE₂ for the patternized response of sympathetic neurons and fever.     

Corn Silk Induced Cyclooxygenase-2 in Murine Macrophages

Stimulation of murine macrophages with corn silk induced cyclooxygenase (COX)-2 with secretion of PGE₂. Expression of COX-2 was inhibited by pyrolidine dithiocarbamate (PDTC), and increased DNA binding by nuclear factor kappa B (NF-κB), indicating that COX-2 induction proceeds also via the NF-κB signaling pathway. A specific inhibitor of COX-2 decreased the expression level of inducible nitric oxide synthase (iNOS) stimulated by corn silk. PGE₂ elevated the expression level of iNOS, probably via EP2 and EP4 receptors on the surface of the macrophages.     

Autocrine prostaglandin E2 signaling promotes tumor cell survival and proliferation in childhood neuroblastoma

Background

Prostaglandin E₂ (PGE₂) is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2) has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE₂ production, the expression pattern and localization of PGE₂ receptors and intracellular signal transduction pathways activated by PGE₂.

Principal Findings

A high expression of the PGE₂ receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE₂ and stimulation of serum-starved neuroblastoma cells with PGE₂ increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE₂ (dmPGE₂) increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE₂. Similarly, PGE₂ receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner.

Conclusions

These findings demonstrate that PGE₂ acts as an autocrine and/or paracrine survival factor for neuroblastoma cells. Hence, specific targeting of PGE₂ signaling provides a novel strategy for the treatment of childhood neuroblastoma through the inhibition of important mediators of tumor-promoting inflammation.     

Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ – prostaglandin E2 dependent proliferation of growth plate chondrocytes

Prostaglandin E₂ (PGE₂) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE₂ pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE₂ receptors) is essential. We therefore examined the production of PGE₂ in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE₂ on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE₂ receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE₂ synthesis was determined by mass spectrometry, cell proliferation by DNA [³H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE₂ into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE₂-dependent proliferation. Exogenously added PGE₂ stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10^-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE₂ was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE₂ release, which stimulates cell proliferation via the EP1 receptor.     

Prostaglandin E<Subscript>2</Subscript> (PGE<Subscript>2</Subscript>) suppresses natural killer cell function primarily through the PGE<Subscript>2</Subscript> receptor EP4

The COX-2 product prostaglandin E₂ (PGE₂) contributes to the high metastatic capacity of breast tumors. Our published data indicate that inhibiting either PGE₂ production or PGE₂-mediated signaling through the PGE₂ receptor EP4 reduces metastasis by a mechanism that requires natural killer (NK) cells. It is known that NK cell function is compromised by PGE₂, but very little is known about the mechanism by which PGE₂ affects NK effector activity. We now report the direct effects of PGE₂ on the NK cell. Endogenous murine splenic NK cells express all four PGE₂ receptors (EP1-4). We examined the role of EP receptors in three NK cell functions: migration, cytotoxicity, and cytokine release. Like PGE₂, the EP4 agonist PGE₁-OH blocked NK cell migration to FBS and to four chemokines (ITAC, MIP-1α, SDF-1α, and CCL21). The EP2 agonist, Butaprost, inhibited migration to specific chemokines but not in response to FBS. In contrast to the inhibitory actions of PGE₂, the EP1/EP3 agonist Sulprostone increased migration. Unlike the opposing effects of EP4 vs. EP1/EP3 on migration, agonists of each EP receptor were uniformly inhibiting to NK-mediated cytotoxicity. The EP4 agonist, PGE₁-OH, inhibited IFNγ production from NK cells. Agonists for EP1, EP2, and EP3 were not as effective at inhibiting IFNγ. Agonists of EP1, EP2, and EP4 all inhibited TNFα; EP4 agonists were the most potent. Thus, the EP4 receptor consistently contributed to loss of function. These results, taken together, support a mechanism whereby inhibiting PGE₂ production or preventing signaling through the EP4 receptor may prevent suppression of NK functions that are critical to the control of breast cancer metastasis.     

Prostaglandin E2 increases the calcium concentration in rat brown adipocytes and their consumption of oxygen

Effects of prostaglandin E₂ (PGE₁) were examined on the oxygen consumption and intracellular calcium concentration of rat brown adipose tissue (BAT). PGE₂ 0.1 nM-1 μM increased oxygen consumption of the tissue blocks of BAT, with a maximum 2–13 min after PGE2 administration. PGE₂ was most effective at 1 and 10 nM, and the oxygen consumption was elevated for over 40 min. Pretreatment of BAT with indomethacin, a prostaglandin synthesis inhibitor, did not affect the increase in oxygen consumption induced by noradrenaline. PGE₂ at 1–10 nM gradually increased the intracellular calcium concentration of freshly dispersed single brown adipocytes by 3–4 times in 30 min. PGE₂ also increased the intracellular calcium concentration of brown adipocytes in calcium-free medium. These results raise the possibility that PGE₂ and noradrenaline affect heat genesis and metabolism of BAT independently.     

Analysing phosphorylation-based signalling networks by phospho flow cytometry

Analysis of signalling events by classical biochemical approaches is limited as the outcome is an averaged readout for protein activation of a single protein within a cell population. This is a clear restriction when addressing signalling events in mixed populations or subpopulations of cells. By combining flow cytometry with a panel of phosphospecific antibodies against several signal molecules simultaneously in a multi-parameter phospho flow cytometry analysis it is possible to obtain a higher level of understanding of the signal transduction dynamics at a single cell level. In addition, analysis of mixed cell populations makes it possible to study cells ex vivo in a state more closely resembling the in vivo situation. The multimeric analysis yields information on combinations of signals turned on and off in specific settings such as disease (signal nodes) that can be used for biomarker analysis and for development of drug screening strategies. Prostaglandin E₂ (PGE₂) is known to signal through four G-protein coupled transmembrane receptors, EP1-4, activating a multitude of potential signalling pathways. The analysis of the PGE₂ signalling network elicited by activation of the four EP receptors in lymphoid cells revealing several signalling nodes is reviewed as an example.     

Role of intracellular prostaglandin E2 in cancer-related phenotypes in PC3 cells

Prostaglandin E₂ (PGE₂) and hypoxia-inducible factor-1α (HIF-1α) affect many mechanisms that have been shown to play a role in prostate cancer. In PGE₂-treated LNCaP cells, up-regulation of HIF-1α requires the internalization of PGE₂, which is in sharp contrast with the generally accepted view that PGE₂ acts through EP receptors located at the cell membrane. Here we aimed to study in androgen-independent PC3 cells the role of intracellular PGE₂ in several events linked to prostate cancer progression. To this end, we used bromocresol green, an inhibitor of prostaglandin uptake that blocked the immediate rise in intracellular immunoreactive PGE₂ following treatment with 16,16-dimethyl-PGE₂. Bromocresol green prevented the stimulatory effect of 16,16-dimethyl-PGE on cell proliferation, adhesion, migration and invasion and on HIF-1α expression and activity, the latter assessed as the HIF-dependent activation of (i) a hypoxia response element-luciferase plasmid construct, (ii) production of angiogenic factor vascular endothelial growth factor-A and (iii) in vitro angiogenesis. The basal phenotype of PC3 cells was also affected by bromocresol green, that substantially lowered expression of HIF-1α, production of vascular endothelial growth factor-A and cell proliferation. These results, and the fact that we found functional intracellular EP receptors in PC3 cells, suggest that PGE₂-dependent intracrine mechanisms play a role in prostate cancer Therefore, inhibition of the prostaglandin uptake transporter might be a novel therapeutic approach for the treatment of prostate cancer.