Murine and human autotaxin alpha, beta, and gamma isoforms: gene organization, tissue distribution, and biochemical characterization.

Autotaxin is a type II ectonucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that it also has a lysophospholipase D activity. This enzyme probably provides most of the extracellular lysophosphatidic acid from lysophosphatidylcholine. The cloning and tissue distribution of the three isoforms (imaginatively called alpha, beta, and gamma) from human and mouse are reported in this study, as well as their tissue distribution by PCR in the human and mouse. The fate of the alpha isoform from human was also studied after purification and using mass spectrometry. Indeed, this particular isoform expresses the intron 12 in which a cleavage site is present, leading to a rapid catabolism of the isoform. For the human isoform gamma and the total autotaxin mRNA expression, quantitative PCR is presented in 21 tissues. The isoforms were expressed in two different hosts, insect cells and Chinese hamster ovary cells, and were highly purified. The characteristics of the six purified isoforms (pH and temperature dependence, K(m) and V(max) values, and their dependence on metal ions) are presented in this study. Their sensitivity to a small molecule inhibitor, hypericin, is also shown. Finally, the specificity of the isoforms toward a large family of lysophosphatidylcholines is reported. This study is the first complete description of the reported autotaxin isoforms.

Cellular knock-down of quinone reductase 2: a laborious road to successful inhibition by RNA interference.

NRH:quinone oxidoreductase 2 (QR2) is a long forgotten oxidoreductive enzyme that metabolizes quinones and binds melatonin. We used the potency of the RNA interference (RNAi)-mediated gene silencing to build a cellular model in which the role of QR2 could be studied. Because standard approaches were poorly successful, we successively used: (1) two chemically synthesized fluorescent small interfering RNA (siRNA) duplexes designed and tested for their gene silencing capacity leading to a maximal 40% QR2 gene silencing 48h post-transfection; (2) double transfection and cell-sorting of high fluorescent siRNA-transfected HT22 cells further enhancing QR2 RNAi silencing to 88%; (3) stable QR2 knock-down HT22 cell lines established with H1and U6 promoter driven QR2 short hairpin RNA (shRNA) encoding vectors, resulting in a 71-80% reduction of QR2 enzymatic activity in both QR2 shRNA HT22 cells. Finally, as a first step in the study of this cellular model, we observed a 42-48% reduction of menadione/BNAH-mediated toxicity in QR2 shRNA cells compared to the wild-type HT22 cells. Although becoming widespread and in some cases effective, siRNA-mediated cellular knock-down proves in the present work to be of marginal efficiency. Much development is required for this technique to be of general application.

Novel 1,3-dicarbonyl compounds having 2(3H)-benzazolonic heterocycles as PPARgamma agonists.

A series of 1,3-dicarbonyl compounds having 2(3H)-benzazolonic heterocycles has been synthesized and tested for PPARgamma agonist activity. SAR were developed and revealed that 6-acyl-2(3H)-benzothiazolone derivatives with 1,3-dicarbonyl group were the most potent. IP administration of compound 22 exhibited comparable levels of glucose and triglyceride correction to PO administration of rosiglitazone in the ob/ob mouse studies.

Characterization of the melatoninergic MT3 binding site on the NRH:quinone oxidoreductase 2 enzyme.

Melatonin acts through a series of molecular targets: the G-protein coupled receptors, MT1 and MT2, and a third binding site, MT3, recently identified as the enzyme NRH:quinone oxydoreductase 2 (QR2). The relationship between the multiple physiological functions of melatonin and this enzyme remains unclear. Because of the relationship of QR2 with the redox status of cells, these studies could bring the first tools for a molecular rationale of the antioxidant effects of melatonin. In the present paper, we used a QR2-stably expressing cell line and hamster kidneys to compare the 2-[125I]-iodomelatonin and 2-[125I]-iodo-5-methoxycarbonylamino-N-acetyltryptamine binding data, and to characterize the MT3 binding site. We designed and tested compounds from two distinct chemicals series in a displacement assay of the two MT3 ligands, 2-[125I]-iodomelatonin and 2-[125I]-iodo-5-methoxycarbonylamino-N-acetyltryptamine from their cloned target. We also tested their ability to inhibit QR2 catalytic activity. These compounds were separated into two classes: those that bind within the catalytic site (and being inhibitors) and those that bind outside it (and therefore not being inhibitors). Compounds range from potent ligands (K(i) = 1 nM) to potent inhibitors (14 nM), and include one compound [NMDPEF: N-[2-(2-methoxy-6H-dipyrido[2,3-a:3,2-e]pyrrolizin-11-yl)ethyl]-2-furamide] active on both parameters in the low nanomolar range. To dissect the physio-pathological pathways in which QR2, MT3 and melatonin meet, one needs more compounds binding to MT3 and/or inhibitors of QR2 enzymatic activity. The compounds described in the present paper are new tools for such a task.

Molecular evidence that melatonin is enzymatically oxidized in a different manner than tryptophan: investigations with both indoleamine 2,3-dioxygenase and myeloperoxidase.

The catabolism of melatonin, whether naturally occurring or ingested, takes place via two pathways: approximately 70% can be accounted for by conjugation (sulpho- and glucurono-conjugation), and approximately 30% by oxidation. It is commonly thought that the interferon-induced enzyme indoleamine 2,3-dioxygenase (EC 1.13.11.42), which oxidizes tryptophan, is also responsible for the oxidation of 5-hydroxytryptamine (serotonin) and its derivative, melatonin. Using the recombinant enzyme expressed in Escherichia coli, we show in the present work that indoleamine 2,3-dioxygenase indeed cleaves tryptophan; however, under the same conditions, it is incapable of cleaving the two other indoleamines. By contrast, myeloperoxidase (EC 1.11.1.7) is capable of cleaving the indole moiety of melatonin. However, when using the peroxidase conditions of assay -- with H2O2 as co-substrate -- indoleamine 2,3-dioxygenase is able to cleave melatonin into its main metabolite, a kynurenine derivative. The present work establishes that the oxidative metabolism of melatonin is due, in the presence of H2O2, to the activities of both myeloperoxidase and indoleamine 2,3-dioxygenase (with lower potency), since both enzymes have Km values for melatonin in the micromolar range. Under these conditions, several indolic compounds can be cleaved by both enzymes, such as tryptamine and 5-hydroxytryptamine. Furthermore, melatonin metabolism results in a kynurenine derivative, the pharmacological action of which remains to be studied, and could amplify the mechanisms of action of melatonin.

Functional characterization of human neuropeptide Y receptor subtype five specific antagonists using a luciferase reporter gene assay.

Neuropeptide Y (NPY) has several receptors; one of them, the neuropeptide Y5 receptor (NPY5) seems involved in feeding behavior in mammals. Although this particular receptor has been extensively studied in the literature, the difficulties encountered to obtain a stable cell line expressing this recombinant receptor have impaired the development of tools necessary to establish its molecular pharmacology. We thus established a method for the functional study of new ligands. It is based upon the cotransfection in human melatonin receptor 1 (MT1)-overexpressing HEK293 cells of three plasmids encoding melanocortin receptor (MC5), neuropeptide Y5 receptor (NPY5) and a cyclic AMP response element-controlled luciferase. Once challenged with alphaMSH, the MC5 receptor activates the cyclic AMP response, through the coupling protein subunit G(s). In contrast, NPY5 agonists, through the NPY5 receptor which is negatively coupled to the same pathway, counteract the alphaMSH-mediated effect on cyclic AMP level. Using appropriate controls, this method can pinpoint compounds with antagonistic activity. Simple and straightforward, this system permits reproducible measurements of agonist or antagonist effects in the presence of neuropeptide Y, the natural agonist. This method has the advantage over already existing methods and beyond its apparent complexity, to enhance the cyclic AMP concentration at a 'physiological' level, by opposition to a forskolin-induced adenylate cyclase activation. Finally, to further validate this assay, we showed results from (1) a series of natural peptidic agonists that permitted the standardization and (2) a series of potent nonpeptidic antagonists (affinity >10(-9) M) that form a new class of active NPY5 receptor antagonists.

TNF skews monocyte differentiation from macrophages to dendritic cells.

Monocytes represent a large pool of circulating precursors of APCs, both macrophages and dendritic cells (DCs). It is thus important to identify the mechanisms by which microenvironment regulates monocyte differentiation. We have previously shown that, upon contact with resting stromal cells such as fibroblasts, monocytes differentiate into macrophages in an IL-6/M-CSF-dependent fashion. Yet, in the inflamed tissue, monocytes need to yield DCs for the adaptive immunity to be induced. Inasmuch as TNF and IL-1 are present at the site of inflammation, we tested their capacity to modulate monocyte differentiation into either macrophages or DCs. TNF, but not IL-1, induce monocytes to become DCs despite the presence of fibroblasts. TNF-induced DCs contain Langerin-positive cells and are able to induce allogenic T cell proliferation. Then, TNF was found to decrease the expression and internalization of the M-CSF receptor, thus overriding the IL-6/M-CSF pathway. Thus, TNF facilitates the induction of adaptive immunity by promoting DC differentiation not only from CD34+ progenitors but also from CD14+ blood precursors.

Autotaxin is released from adipocytes, catalyzes lysophosphatidic acid synthesis, and activates preadipocyte proliferation. Up-regulated expression with adipocyte differentiation and obesity.

Our group has recently demonstrated (Gesta, S., Simon, M., Rey, A., Sibrac, D., Girard, A., Lafontan, M., Valet, P., and Saulnier-Blache, J. S. (2002) J. Lipid Res. 43, 904-910) the presence, in adipocyte conditioned-medium, of a soluble lysophospholipase d-activity (LPLDact) involved in synthesis of the bioactive phospholipid lysophosphatidic acid (LPA). In the present report, LPLDact was purified from 3T3F442A adipocyte-conditioned medium and identified as the type II ecto-nucleotide pyrophosphatase phosphodiesterase, autotaxin (ATX). A unique ATX cDNA was cloned from 3T3F442A adipocytes, and its recombinant expression in COS-7 cells led to extracellular release of LPLDact. ATX mRNA expression was highly up-regulated during adipocyte differentiation of 3T3F442A-preadipocytes. This up-regulation was paralleled by the ability of newly differentiated adipocytes to release LPLDact and LPA. Differentiation-dependent up-regulation of ATX expression was also observed in a primary culture of mouse preadipocytes. Treatment of 3T3F442A-preadipocytes with concentrated conditioned medium from ATX-expressing COS-7 cells led to an increase in cell number as compared with concentrated conditioned medium from ATX non-expressing COS-7 cells. The specific effect of ATX on preadipocyte proliferation was completely suppressed by co-treatment with a LPA-hydrolyzing phospholipase, phospholipase B. Finally, ATX expression was found in mature adipocytes isolated from mouse adipose tissue and was substantially increased in genetically obese-diabetic db/db mice when compared with their lean siblings. In conclusion, the present work shows that ATX is responsible for the LPLDact released by adipocytes and exerts a paracrine control on preadipocyte growth via an LPA-dependent mechanism. Up-regulations of ATX expression with adipocyte differentiation and genetic obesity suggest a possible involvement of this released protein in the development of adipose tissue and obesity-associated pathologies.