PGH1, the Precursor for the Anti-Inflammatory Prostaglandins of the 1-series, Is a Potent Activator of the Pro-Inflammatory Receptor CRTH2/DP2

Prostaglandin H1 (PGH1) is the cyclo-oxygenase metabolite of dihomo-γ-linolenic acid (DGLA) and the precursor for the 1-series of prostaglandins which are often viewed as “anti-inflammatory”. Herein we present evidence that PGH1 is a potent activator of the pro-inflammatory PGD2 receptor CRTH2, an attractive therapeutic target to treat allergic diseases such as asthma and atopic dermatitis. Non-invasive, real time dynamic mass redistribution analysis of living human CRTH2 transfectants and Ca2+ flux studies reveal that PGH1 activates CRTH2 as PGH2, PGD2 or PGD1 do. The PGH1 precursor DGLA and the other PGH1 metabolites did not display such effect. PGH1 specifically internalizes CRTH2 in stable CRTH2 transfectants as assessed by antibody feeding assays. Physiological relevance of CRTH2 ligation by PGH1 is demonstrated in several primary human hematopoietic lineages, which endogenously express CRTH2: PGH1 mediates migration of and Ca2+ flux in Th2 lymphocytes, shape change of eosinophils, and their adhesion to human pulmonary microvascular endothelial cells under physiological flow conditions. All these effects are abrogated in the presence of the CRTH2 specific antagonist TM30089. Together, our results identify PGH1 as an important lipid intermediate and novel CRTH2 agonist which may trigger CRTH2 activation in vivo in the absence of functional prostaglandin D synthase

The role of prostaglandin E2-EP4 receptor in the regulation of endothelial barrier function

Endothelzellen kleiden die inneren Gefäßwände aus, die das gesamte Kreislaufsystem unseres Körpers durchziehen. Sie fungieren als selektive Schranke zwischen dem Gefäßlumen und den umliegenden Geweben. Prostaglandin E2 (PGE2) besitzt eine wichtige Funktion bei Entzündungsreaktionen wie etwa Fieber, Schmerz oder Tumorentstehung. Während einer Entzündungsreaktion stellen Immunzellen, Fibroblasten und das Epithelium die Hauptquelle von PGE2 dar. Das Ziel meiner Studie war es, den Einfluss von PGE2, insbesondere von seinem Rezeptor EP4 auf die endotheliale Barrierenfunktion zu untersuchen. Dazu wurde zunächst der EP4-Rezeptor mittels einer RNA-Interferenz-Methode stillgelegt und das Ergebnis konnte erfolgreich auf der mRNA- und der Protein-Ebene gezeigt werden. Außerdem konnte ein funktioneller Einfluss gezeigt werden. Ein Ausschalten des Rezeptors führte zu einem verringerten elektrischen Widerstand nach einer Aktivierung der EP4-Rezeptoren mittels PGE2 bzw. einem selektiven Agonisten. Zusätzlich wurden Änderungen in Zell-Zell-Kontakten durch Mikroskopie untersucht. Hier konnte eine Störung der Endothelzellen mittels Thrombin durch die Vorbehandlung mit PGE2 und dem selektiven EP4 Agonisten verhindert werden. Ferner wurde der Einfluss auf Zellzyklus und die Apoptose von Endothelzellen untersucht. Während Aktivierung der EP4 Rezeptoren keinen Einfluss auf den Zellzyklus zeigte, konnte eine Vorbehandlung mit PGE2 und dem EP4 Agonisten vor einer Staurosporin-induzierten Apoptose schützen. Zusammenfassend ist zu vermerken, dass diese Daten den EP4 Rezeptor als einen potentiellen Angriffspunkt für eine Therapie betreffend Krankheiten mit erhöhtem Aufkommen vaskulärer Fehlfunktionen darstellen. Die Aktivierung dieser EP4 Rezeptoren führt in weiterer Folge zur Aktivierung von Signaltransduktionsketten, die die Barrierefunktion verstärken. Daher offenbaren diese Daten eine gute Grundlage für weitere Untersuchungen, um die betreffenden EP4-Rezeptor-Signalwege zu erforschen.Endothelial cells form a thin cell layer at the inner wall of vessels that line the entire circulatory system. They act as a selective barrier between vessel lumen and the surrounding tissue. Dysfunction of this barrier is involved in various diseases such as inflammation or atherosclerosis. Prostaglandin E2 (PGE2) plays a crucial role during immune responses including fever, pain and tumorigenesis. During an immune response, inflammatory cells, fibroblasts and the epithelium are the main source of PGE2.The aim of my study was to investigate the influence of PGE2 and its receptor, EP4, on the endothelial barrier function. To this end, EP4 receptor of human microvascular endothelial cells of the lung was silenced via RNA interference and shown on mRNA and protein levels. Additionally, the functional impact of EP4 receptor silencing was observed by measuring electrical impedance of endothelial monolayers. Here, silencing of EP4 receptor resulted in a decreased barrier enhancement in response to PGE2 and the EP4 selective agonist ONO AE1-329. The EP4 receptor-induced morphological changes of cell-cell junctions were visualized by fluorescence microscopy. Thrombin-induced disruption of the endothelial monolayer resulting in disorganization could be prevented by pretreatment with PGE2 and the selective EP4 agonist. Furthermore, the impact of the EP4 receptor on endothelial cell cycle and apoptosis was considered. While activation of EP4 receptor did not promote the cell cycle, pretreatment with PGE2 and the EP4 agonist showed protective effects upon staurosporine-induced apoptosis.Taken together, these data suggest the EP4 receptor as a potential target for treating diseases with increased vascular permeability such as acute lung injury. Activation of this receptor stimulates so far not yet fully understood downstream signaling that enhances the endothelial barrier. Therefore, this finding provides a good basis for further investigation of possible signaling pathways.submitted by Nora KampitschZsfassung in dt. und engl. SpracheGraz, Univ., Masterarb., 2012(VLID)22429

Prostaglandin H₁ (PGH₁) stimulates Ca²⁺ mobilization from intracellular stores in CRTH2 transfectants and primary human eosinophils.

<p><b>A</b>–<b>D</b>: HEK293 cells stably expressing CRTH2 (CRTH2-HEK) were transiently transfected with a chimeric Gαqi5 protein to channel the Gi-sensitive CRTH2 receptor to mobilization of intracellular Ca²⁺. Cells were loaded with a Ca²⁺ fluorophore and CRTH2-specific Ca²⁺ traces were recorded over time upon challenge with the indicated agonists (<b>A</b>: PGD₂, <b>B</b>: PGH₁, <b>C</b>: PGH₂). <b>A</b>–<b>C</b>: representative data (mean + SEM of triplicate determinations. <b>D</b>: Maximum responses of all experiments were normalized to Ca²⁺ flux induced by 1 µM PGD₂ (mean + SEM, n = 3). <b>E</b>: PGH₁ induces Ca²⁺ mobilization in human eosinophils via CRTH2. Intracellular free Ca²⁺ levels were quantified by flow cytometry as described in the methods section. The level of Ca²⁺ mobilization in response to vehicle without agonist was set to 100%. Ca²⁺ mobilization upon addition of PGH₁, PGH₂, and PGD₂ is inhibited in the presence of 1 µM of the CRTH2 specific antagonist TM30089. Data are presented as the mean + SEM from 5 experiments conducted in triplicate, each experiment involving eosinophils from a separate donor.</p

PGH₁ activates human Th2 cells via CRTH2.

<p>Induction of Ca²⁺ mobilization (A–C) and cell migration (D–F) in human Th2 cells in response to the indicated concentrations of PGD₂, PGH₁, and PGH₂, respectively. The level of cell migration in response to medium without agonist was set to 1 fold. Both Ca²⁺ mobilization and cell migration are inhibited in the presence of 1 µM of the CRTH2 specific antagonist TM30089. Pooled data is expressed as the mean ± SEM from 3 experiments conducted in duplicate, each experiment involving Th2 cells from a separate donor. Statistical analysis was performed for vehicle vs. TM30089 treated cells and is indicated as (*) for p<0.05, as (**) for p<0.01 and as (***) for p<0.001.</p

PGH₁ induces eosinophil adhesion to human pulmonary microvascular endothelial cells under flow conditions.

<p>Eosinophils were pre-incubated with vehicle (<b>A</b>–<b>C</b>) or 10 µM CRTH2-specific antagonist TM30089 (<b>D</b>–<b>F</b>) for 10 min at room temperature followed by treatment with vehicle (<b>A</b>, <b>D</b>), 1 µM PGH₁ (<b>B</b>, <b>E</b>) or 30 nM PGD₂ (<b>C</b>, <b>F</b>) for 10 min at 37°C. Eosinophils were then superfused over human pulmonary microvascular endothelial cells grown on VenaEC biochips (Cellix, Dublin) for 5 min at 37°C. Representative images were taken 5 min after start of the superfusion (<b>A</b>–<b>F</b>). <b>G</b>: averaged data from <b>A</b>–<b>F</b>, quantified by computerized image analysis. Data are shown as mean + SEM of 4 experiments. *P<0.05 PGD₂ versus TM30089+PGD₂ and PGH₁ versus TM30089+PGH₁.</p

Prostaglandin H₁ (PGH₁) fully activates CRTH2 in living CRTH2-HEK cell transfectants.

<p><b>A</b>–<b>E</b>, abilities of PGH₁, selected prostaglandins, and the PGH₁ precursor dihomo-γ-linolenic acid (DGLA) to stimulate CRTH2 signaling using dynamic mass redistribution (DMR) technology. Cells were challenged with increasing concentrations of the indicated ligands and DMR was recorded as a measure of receptor activity (representative optical traces). <b>F</b>, transformation of optical traces (<b>A</b>–<b>E</b>) into concentration effect curves. Molar log EC₅₀ values were </p><p>PGH₁: −6.37±0.12; PGH₂: −7.09±0.08; PGD₁: −6.92±0.16; PGD₂: −7.95±0.09</p> (mean values ± SEM, n = 3).<p></p

PGH₁ activates human eosinophils via CRTH2.

<p>Human eosinophils were treated with the indicated concentrations of PGD₂, PGH₁, and PGH₂, respectively, and chemotactic activation was measured in eosinophil shape change assays. Eosinophil shape change is inhibited in the presence of 1 µM of the CRTH2-specific antagonist TM30089. Note: rank order of PG potency matches well with the results obtained in CRTH2-HEK transfectants using DMR assays (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033329#pone-0033329-g001" target="_blank"><b>Figure 1F</b></a>). Results are expressed as the mean ± SEM of 3 experiments conducted in triplicate with a separate donor used in each experiment. Statistical analysis was performed for vehicle vs. TM30089 treated cells and is indicated as (**) for p<0.01 and as (***) for p<0.001.</p