Rolf M. Nüsing

Cyclooxygenase-2 Controls Energy Homeostasis in Mice by de Novo Recruitment of Brown Adipocytes

Fat-Burning Fat In mammals, fat exists in two forms—the well-known white adipose tissue (WAT), which stores energy and is associated with obesity, and the lesser-known brown adipose tissue (BAT), which burns energy to generate heat. BATs role in human physiology was once thought to be restricted to newborns, but the recent discovery that adults also harbor functional BAT has re-ignited interest in the factors regulating BAT development and their potential as targets for anti-obesity therapies. Vegiopoulos et al. (p. 1158, published online 6 May; see the Perspective Ishibashi and Seale) now show that cyclooxygenase-2 (COX-2), an enzyme critical to prostaglandin synthesis, triggers fat progenitor cells in mice to differentiate into BAT rather than WAT. Mice overexpressing COX-2 displayed increased energy expenditure and were protected from diet-induced obesity. In mice, the development of energy-burning brown fat is regulated by an enzyme that is critical for prostaglandin synthesis. Obesity results from chronic energy surplus and excess lipid storage in white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) efficiently burns lipids through adaptive thermogenesis. Studying mouse models, we show that cyclooxygenase (COX)–2, a rate-limiting enzyme in prostaglandin (PG) synthesis, is a downstream effector of β-adrenergic signaling in WAT and is required for the induction of BAT in WAT depots. PG shifted the differentiation of defined mesenchymal progenitors toward a brown adipocyte phenotype. Overexpression of COX-2 in WAT induced de novo BAT recruitment in WAT, increased systemic energy expenditure, and protected mice against high-fat diet–induced obesity. Thus, COX-2 appears integral to de novo BAT recruitment, which suggests that the PG pathway regulates systemic energy homeostasis.

GPR109A (PUMA-G/HM74A) mediates nicotinic acid–induced flushing

Nicotinic acid (niacin) has long been used as an antidyslipidemic drug. Its special profile of actions, especially the rise in HDL-cholesterol levels induced by nicotinic acid, is unique among the currently available pharmacological tools to treat lipid disorders. Recently, a G-protein-coupled receptor, termed GPR109A (HM74A in humans, PUMA-G in mice), was described and shown to mediate the nicotinic acid-induced antilipolytic effects in adipocytes. One of the major problems of the pharmacotherapeutical use of nicotinic acid is a strong flushing response. This side effect, although harmless, strongly affects patient compliance. In the present study, we show that mice lacking PUMA-G did not show nicotinic acid-induced flushing. In addition, flushing in response to nicotinic acid was also abrogated in the absence of cyclooxygenase type 1, and mice lacking prostaglandin D(2) (PGD(2)) and prostaglandin E(2) (PGE(2)) receptors had reduced flushing responses. The mouse orthologue of GPR109A, PUMA-G, is highly expressed in macrophages and other immune cells, and transplantation of wild-type bone marrow into irradiated PUMA-G-deficient mice restored the nicotinic acid-induced flushing response. Our data clearly indicate that GPR109A mediates nicotinic acid-induced flushing and that this effect involves release of PGE(2) and PGD(2), most likely from immune cells of the skin.

Prostaglandin E2 Induces Vascular Relaxation by E-Prostanoid 4 Receptor-Mediated Activation of Endothelial Nitric Oxide Synthase

The present experiments were designed to test the hypothesis that prostaglandin (PG) E2 causes vasodilatation through activation of endothelial NO synthase (eNOS). Aortic rings from mice with targeted deletion of eNOS and E-prostanoid (EP) receptors were used for contraction studies. Blood pressure changes in response to PGE2 were measured in conscious mice. Single doses of PGE2 caused concentration-dependent relaxations during contractions to phenylephrine (EC50=5*10−8 mol/L). Relaxation after PGE2 was absent in rings without endothelium and in rings from eNOS−/− mice and was abolished by NG-nitro-l-arginine methyl ester and the soluble guanylate cyclase inhibitor 1H1,2,4-oxadiazolo-[4,3-a]quinoxalin-1-one. In PGE2-relaxed aortic rings, the cGMP content increased significantly. PGE2-induced relaxations were abolished by the EP4 receptor antagonist AE3–208 (10−8 mol/L) and mimicked by an EP4 agonist (AE1–329, 10−7 mol/L) in the presence of endothelium and eNOS only. Relaxations were attenuated significantly in rings from EP4−/− mice but normal in EP2−/−. Inhibitors of the cAMP-protein kinase A pathway attenuated, whereas the inhibitor of protein phosphatase 1C, calyculin (10−8 mol/L), abolished the PGE2-mediated relaxation. In aortic rings, PGE2 dephosphorylated eNOS at Thr495. Chronically catheterized eNOS−/− mice were hypertensive (137±3.6 mm Hg, n=13, versus 101±3.9 mm Hg, n=9) and exhibited a lower sensitivity of blood pressure reduction in response to PGE2 compared with wild-type mice. There was no difference in the blood pressure response to nifedipine. These findings show that PGE2 elicits EP4 receptor-mediated, endothelium-dependent stimulation of eNOS activity by dephosphorylation at Thr495 resulting in guanylyl cyclase–dependent vasorelaxation and accumulation of cGMP in aortic rings.

Release of sphingosine-1-phosphate from human platelets is dependent on thromboxane formation.

Summary.u2002 Background:u2002Platelets release the immune‐modulating lipid sphingosine‐1‐phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood. Objectives:u2002The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. Methods:u2002S1P was detected using thin‐layer chromatography in [3H]sphingosine‐labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. Results:u2002Release of S1P from platelets was stimulated with protease‐activated receptor‐1‐activating peptide (PAR‐1‐AP, 100u2003μm). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500‐mg single dose or 100u2003mg over 3u2003days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U‐46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC‐transport. Furthermore, thrombin‐induced release of S1P was attenuated in platelets from TP‐deficient mice. Supernatants from PAR‐1‐AP‐stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P‐dependent manner via S1P receptors‐1 and ‐3. These effects were inhibited by ASA‐pretreatment of platelets. Conclusions:u2002TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti‐inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.

Induction of prostanoid, nitric oxide, and cytokine formation in rat bone marrow derived macrophages by activin A

In this study we describe that activin A, a transforming growth factor (TGF) β‐like polypeptide affects the expression of inflammatory response genes and their products. In rat bone marrow derived macrophages 15u2003nM activin A caused the stimulation of prostaglandin (PG) E2 and thromboxane (TX) A2 formation, production of nitrite as a marker for nitric oxide (NO) and the release of the cytokines tumour necrosis factor (TNF) α and interleukin (IL) ‐1β. As shown by mRNA analysis induction of cyclo‐oxygenase‐2 and inducible nitric oxide synthase by activin A gave rise to the enhanced release of prostanoids and NO. Costimulation of bone marrow derived macrophages with 15u2003nM activin A and 100u2003nM 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA) potentiated the synthesis of prostanoids in a synergistic manner. With respect to NO formation the effect of activin A and TPA was additive. In contrast to the nitrite production activin A induced PGE2 synthesis was susceptible to tyrosine kinase inhibition by genistein and tyrphostin 46 (IC50 was 10 and 20u2003μM, respectively). This observed inhibition was caused by the selective suppression of activin A induced cyclo‐oxygenase‐2 mRNA expression. Further, the release of TNFα in the presence of activin A was potentiated by tyrosine kinase inhibition. In summary, we report that activin A exerts proinflammatory activity which results in the formation of prostanoids, NO and cytokines in rat bone marrow derived macrophages. Tyrosine kinase dependent and independent signalling pathways are involved leading to the increased synthesis of these metabolites. Based upon these results, we speculate that activin A may be considered as a possible component of inflammatory processes affecting at least the haematopoietic system.

Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors

Castor oil is one of the oldest drugs. When given orally, it has a laxative effect and induces labor in pregnant females. The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. Despite the wide-spread use of castor oil in conventional and folk medicine, the molecular mechanism by which ricinoleic acid acts remains unknown. Here we show that the EP3 prostanoid receptor is specifically activated by ricinoleic acid and that it mediates the pharmacological effects of castor oil. In mice lacking EP3 receptors, the laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP3 receptor gene in intestinal epithelial cells did not affect castor oil-induced diarrhea, mice lacking EP3 receptors only in smooth-muscle cells were unresponsive to this drug. Thus, the castor oil metabolite ricinoleic acid activates intestinal and uterine smooth-muscle cells via EP3 prostanoid receptors. These findings identify the cellular and molecular mechanism underlying the pharmacological effects of castor oil and indicate a role of the EP3 receptor as a target to induce laxative effects.

Dominant Role of Prostaglandin E2 EP4 Receptor in Furosemide-Induced Salt-Losing Tubulopathy: A Model for Hyperprostaglandin E Syndrome/Antenatal Bartter Syndrome

Increased formation of prostaglandin E2 (PGE2) is a key part of hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), a renal disease characterized by NaCl wasting, water loss, and hyperreninism. Inhibition of PGE2 formation by cyclo-oxygenase inhibitors significantly lowers patient mortality and morbidity. However, the pathogenic role of PGE2 in HPS/aBS awaits clarification. Chronic blockade of the Na-K-2Cl co-transporter NKCC2 by diuretics causes symptoms similar to HPS/aBS and provides a useful animal model. In wild-type (WT) mice and in mice lacking distinct PGE2 receptors (EP1-/-, EP2-/-, EP3-/-, and EP4-/-), the effect of chronic furosemide administration (7 d) on urine output, sodium and potassium excretion, and renin secretion was determined. Furthermore, furosemide-induced diuresis and renin activity were analyzed in mice with defective PGI2 receptors (IP-/-). In all animals studied, furosemide stimulated a rise in diuresis and electrolyte excretion. However, this effect was blunted in EP1-/-, EP3-/-, and EP4-/- mice. Compared with WT mice, no difference was observed in EP2-/- and IP-/- mice. The furosemide-induced increase in plasma renin concentration was significantly decreased in EP4-/- mice and to a lesser degree also in IP-/- mice. Pharmacologic inhibition of EP4 receptors in furosemide-treated WT mice with the specific antagonist ONO-AE3-208 mimicked the changes in renin mRNA expression, plasma renin concentration, diuresis, and sodium excretion seen in EP4-/- mice. The GFR in EP4-/- mice was not changed compared with that in WT mice, which indicated that blunted diuresis and salt loss seen in EP4-/- mice were not a consequence of lower GFR. In summary, these findings demonstrate that the EP4 receptor mediates PGE2-induced renin secretion and that EP1, EP3, and EP4 receptors all contribute to enhanced PGE2-mediated salt and water excretion in the HPS/aBS model.

Anti-inflammatory Role of Microsomal Prostaglandin E Synthase-1 in a Model of Neuroinflammation

A major immunological response during neuroinflammation is the activation of microglia, which subsequently release proinflammatory mediators such as prostaglandin E2 (PGE2). Besides its proinflammatory properties, cyclooxygenase-2 (COX-2)-derived PGE2 has been shown to exhibit anti-inflammatory effects on innate immune responses. Here, we investigated the role of microsomal PGE2 synthase-1 (mPGES-1), which is functionally coupled to COX-2, in immune responses using a model of lipopolysaccharide (LPS)-induced spinal neuroinflammation. Interestingly, we found that activation of E-prostanoid (EP)2 and EP4 receptors, but not EP1, EP3, PGI2 receptor (IP), thromboxane A2 receptor (TP), PGD2 receptor (DP), and PGF2 receptor (FP), efficiently blocked LPS-induced tumor necrosis factor α (TNFα) synthesis and COX-2 and mPGES-1 induction as well as prostaglandin synthesis in spinal cultures. In vivo, spinal EP2 receptors were up-regulated in microglia in response to intrathecally injected LPS. Accordingly, LPS priming reduced spinal synthesis of TNFα, interleukin 1β (IL-1β), and prostaglandins in response to a second intrathecal LPS injection. Importantly, this reduction was only seen in wild-type but not in mPGES-1-deficient mice. Furthermore, intrathecal application of EP2 and EP4 agonists as well as genetic deletion of EP2 significantly reduced spinal TNFα and IL-1β synthesis in mPGES-1 knock-out mice after LPS priming. These data suggest that initial inflammation prepares the spinal cord for a negative feedback regulation by mPGES-1-derived PGE2 followed by EP2 activation, which limits the synthesis of inflammatory mediators during chronic inflammation. Thus, our data suggest a role of mPGES-1-derived PGE2 in resolution of neuroinflammation.

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.

Effects of selective COX-2 inhibition on prostanoids and platelet physiology in young healthy volunteers

Summary.u2002 Background:u2002Selective inhibitors of cyclooxygenase‐2 (COX‐2) called coxibs, are effective anti‐inflammatory and analgesic drugs. Recently, these drugs were associated with an increased risk for myocardial infarction and atherothrombotic events. The hypothesis of thromboxane‐prostacyclin imbalance has been preferred to explain these unwanted effects. Methods:u2002We studied the effects of 14u2003days intake of rofecoxib (25u2003mg q.d.), celecoxib (200u2003mg b.i.d.), naproxen (500u2003mg b.i.d.) and placebo in a randomized, blinded, placebo‐controlled study in young healthy volunteers (median age 25–30u2003years, each group nu2003=u200310). We assessed prostanoid metabolite excretion (PGE‐M, TXB2, 6‐keto‐PGF1α, 11‐dehydro‐TXB2, 2,3‐dinor‐TXB2, and dinor‐6‐keto‐PGF1α), the expression of platelet activation markers (CD62P, PAC‐1, fibrinogen), platelet‐leukocyte formation, the endogenous thrombin potential, platelet cAMP content and plasma thrombomodulin level. Results:u2002Naproxen suppressed biosynthesis of PGE‐M, prostacyclin metabolites and thromboxane metabolites and thrombomodulin levels. In contrast, both coxibs had an inhibitory effect only on PGE‐M, 6‐keto‐PGF1α, and on dinor‐6‐keto‐PGF1α, whereas TXB2, 2,3‐dinor‐TXB2 and 11‐dehydro‐TXB2 excretion were unaffected. None of the coxibs exerted significant effects on the expression of platelet activation markers, cAMP generation, platelet‐leukocyte formation, or on thrombomodulin plasma levels. Interestingly, platelet TXB2 release during aggregation was enhanced after coxib treatment following arachidonic acid or collagen stimulation. Conclusion:u2002In young healthy volunteers coxibs inhibit systemic PGE2 and PGI2 synthesis. Platelet function and expression of platelet aggregation markers are not affected; however, coxibs can stimulate TXB2 release from activated platelets. Combined decrease in vasodilatory PGE2 and PGI2 together with increased TXA2 in proaggregatory conditions may contribute to coxib side effects.