Novel eicosanoid pathways

This article reviews a lecture I was honored to present at the Leon Wolfe Symposium in Montreal on March 25, 2004. The lecture described my research career, which started with my interaction with Wolfe at the Montreal Neurological Institute as a postdoctoral fellow and research associate and was followed by additional research discoveries after I left Montreal for my first academic position at the Research Institute, The Hospital for Sick Children and University of Toronto. The article consists of two parts. The first part involves the discovery (in Wolfe’s laboratory) of a new pathway of arachidonic acid, in which a bicyclic prostanoid structure (later called prostacyclin by John Vane and his group) was described, and its further development in Toronto, which led to the discovery of the conversion of the bicyclic prostanoid into 6-keto prostaglandin F_1α. The second part deals with the hepoxilin pathway, a pathway I discovered during a sabbatical leave in Japan with Professor Shozo Yamamoto, which was followed by a stay of several months in the laboratory of Professor Bengt Samuelsson in Sweden. I deal with the historical aspects of both pathways and end with interesting novel aspects of hepoxilin stable antagonist analogs in the treatment of solid tumors in experimental animals.     

Prostaglandin F(2alpha) regulates cytokine responses of mast cells through the receptors for prostaglandin E

There is an increasing body of evidence that prostanoids modulate mast cell functions and contribute to the development of allergic inflammation. The present study aimed to identify an undetermined function of prostaglandin (PG) F_2α in mast cell activation and the signaling mechanism involved in it. Simultaneous quantification of prostanoids by liquid chromatography/tandem mass spectrometry revealed the constitutive release of PGF_2α, thromboxane B₂, and 6-keto-PGF_1α from bone marrow-derived mast cells (BMMCs). Upon activation of BMMCs by lipopolysaccharide, the cytokine production in BMMCs was enhanced when the culture was supplemented with PGF_2α. However, F prostanoid receptor—a selective receptor for PGF_2α—was not detected in BMMCs. Further investigations performed using prostanoid receptor antagonists revealed an alternative mechanism wherein the receptors for PGE species—E prostanoid receptors—mediated the PGF_2α signal in BMMCs. The present study provides an insight into a novel function of PGF_2α, i.e., an autocrine accelerator for mast cell activation.     

Synthesis of Ethyl Tfa- and Tfp- Amino Acid-p-Aminobenzoates and Their Juvenile Hormone Test

Adipocytes can function as endocrine cells secreting a variety of adipocytokines including tumor necrosis factor (TNF)-α. Treatment of cultured mouse 3T3-L1 preadipocytes with TNF-α induced apoptosis, as was evident from increases in nuclear condensation and caspase-3 activity, but differentiated adipocytes during the maturation phase showed resistance to apoptosis by TNF-α. Antioxidants effectively reduced TNF-α-induced apoptosis in preadipocytes, indicating the involvement of reactive oxygen species. Exposure of preadipocytes to calcium ionophore A23187 reduced TNF-α-induced apoptosis, which was accompanied by increased production of prostaglandins (PGs) E₂ and PGF_2α. TNF-αpreferentially promoted gene expression of cyclooxygenase (COX)-2 without affecting that of COX-1. Consistently, NS-398, a COX-2 inhibitor, stimulated TNF-α-induced apoptosis, which was reversed by exogenous PGE₂ and PGF_2α. These results indicate that endogenous PGE₂ and PGF_2α synthesized by preadipocytes through the induction of COX-2 can serve as anti-apoptotic factors against apoptosis by TNF-α.     

Coupling of COX-1 to mPGES1 for prostaglandin E2 biosynthesis in the murine mammary gland

The mammary gland, like most tissues, produces measurable amounts of prostaglandin E2 (PGE2), a metabolite of arachidonic acid produced by sequential actions of two cyclooxygenases (COX-1 and COX-2) and three terminal PGE synthases: microsomal prostaglandin E2 synthase-1 (mPGES1), mPGES2, and cytosolic prostaglandin E2 synthase (cPGES). High PGE2 levels and COX-2 overexpression are frequently detected in mammary tumors and cell lines. However, less is known about PGE2 metabolic enzymes in the context of normal mammary development. Additionally, the primary COX partnerships of terminal PGE synthases and their contribution to normal mammary PGE2 biosynthesis are poorly understood. We demonstrate that expression of COX-1, generally considered constitutive, increases dramatically with lactogenic differentiation of the murine mammary gland. Concordantly, total PGE2 levels increase throughout mammary development, with highest levels measured in lactating tissue and breast milk. In contrast, COX-2 expression is extremely low, with only a modest increase detected during mammary involution. Expression of the G(s)-coupled PGE2 receptors, EP2 and EP4, is also temporally regulated, with highest levels detected at stages of maximal proliferation. PGE2 production is dependent on COX-1, as PGE2 levels are nearly undetectable in COX-1-deficient mammary glands. Interestingly, PGE2 levels are similarly reduced in lactating glands of mPGES1-deficient mice, indicating that PGE2 biosynthesis results from the coordinated activity of COX-1 and mPGES1. We thus provide evidence for the first time of functional coupling between COX-1 and mPGES1 in the murine mammary gland in vivo.     

Prostanoids receptors signaling in different diseases/cancers progression

Abstract

Prostanoids, that is, prostaglandins (PGs) PGE₂, PGF_2α, PGI₂, PGD₂ and thromboxane A₂(TXA₂), are the oldest members of the eicosanoid family. The PGs are a family of lipid mediators formed in response to various stimuli. They are transported into the extracellular microenvironment by specific multidrug resistance-associated proteins (MRPs) after synthesis. Once exported to the microenvironment, prostanoids bind to G-protein coupled receptors that contain seven transmembrane spanning domains. There are eight types of the prostanoid receptors conserved in mammals from mouse to human. They are the PGD receptor (DP), four subtypes of the PGE receptor (EP₁, EP₂, EP₃ and EP₄), the PGF receptor (FP), PGI receptor (IP) and TXA receptor (TP). Recently, several studies have revealed the roles of PG receptor signaling in various pathological conditions, and suggest that selective manipulation of the prostanoid receptors may be beneficial in treatment of the pathological conditions. Here we review these recent findings of roles of prostanoid receptor signaling and their therapeutic implications.     

Understanding the local actions of lipids in bone physiology

The adult skeleton is a metabolically active organ system that undergoes continuous remodeling to remove old and/or stressed bone (resorption) and replace it with new bone (formation) in order to maintain a constant bone mass and preserve bone strength from micro-damage accumulation. In that remodeling process, cellular balances – adipocytogenesis/osteoblastogenesis and osteoblastogenesis/osteoclastogenesis – are critical and tightly controlled by many factors, including lipids as discussed in the present review.Interest in the bone lipid area has increased as a result of in vivo evidences indicating a reciprocal relationship between bone mass and marrow adiposity. Lipids in bones are usually assumed to be present only in the bone marrow. However, the mineralized bone tissue itself also contains small amounts of lipids which might play an important role in bone physiology. Fatty acids, cholesterol, phospholipids and several endogenous metabolites (i.e., prostaglandins, oxysterols) have been purported to act on bone cell survival and functions, the bone mineralization process, and critical signaling pathways. Thus, they can be regarded as regulatory molecules important in bone health. Recently, several specific lipids derived from membrane phospholipids (i.e., sphingosine-1-phosphate, lysophosphatidic acid and different fatty acid amides) have emerged as important mediators in bone physiology and the number of such molecules will probably increase in the near future. The present paper reviews the current knowledge about: (1°) bone lipid composition in both bone marrow and mineralized tissue compartments, and (2°) local actions of lipids on bone physiology in relation to their metabolism. Understanding the roles of lipids in bone is essential to knowing how an imbalance in their signaling pathways might contribute to bone pathologies, such as osteoporosis.     

Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

The prothrombotic mediator thromboxane A₂ is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.     

Gamahonolides A,B, and Gamahorin,Novel Antifungal Compounds from Stromata of Epichloe typhina on Phleum pratense

Four novel antifungal compounds, gamahonolides A and B, gamahorin, and 5-hydroxyl-4-phenyl-2(5H)-furanone, were isolated from stromata of Epichloe typhina on Phleum pratense. Their structures were determined by spectroscopic methods. The absolute configuration of gamahonolide A was determined by its ORD spectrum and ¹H-NMR shift difference between the diastereomeric pair of its O-methylmandelates. The stereochemistry of gamahorin was determined by NOE difference spectra and its CD spectrum.     

Coupling between cyclooxygenases and terminal prostanoid synthases

Biosynthesis of prostanoids is regulated by three sequential enzymatic steps, namely phospholipase A2, cyclooxygenase (COX), and terminal prostanoid synthase. Recent evidence suggests that lineage-specific terminal prostanoid synthases, including prostaglandin (PG) E2, PGD2, PGF2alpha, PGI2, and thromboxane synthases, show distinct functional coupling with upstream COX isozymes, COX-1 and COX-2. This can account, at least in part, for segregated utilization of the two COX isozymes in distinct phases of PG-biosynthetic responses. In terms of their localization and COX preference, terminal prostanoid synthases are classified into three categories: (i) the perinuclear enzymes that prefer COX-2, (ii) the cytosolic enzyme that prefers COX-1, and (iii) the translocating enzyme that utilizes both COXs depending on the stimulus. Additionally, altered supply of arachidonic acid by phospholipase A2s significantly affects the efficiency of COX-terminal prostanoid synthase coupling. In this review, we summarize our recent understanding of the coupling profiles between the two COXs and various terminal prostanoid synthases.