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International audience; Background. Mature B lymphocytes alter the recovery of cardiac function after acute myocardial infarction (MI) in mice. Follicular B cells and marginal zone B (MZB) cells are spatially distinct mature B cell populations in the spleen and exert specific functional properties. miR21/Hypoxia-inducible factor (HIF)α-related pathways have been shown to govern B cell functions. Objectives. We aimed to unravel the distinct role of MZB cells and that of endogenous activation of miR21/ HIFα signalling in MZB cells during post-ischemic injury. Methods. Acute MI was induced by permanent ligation of the left anterior descending coronary artery in mice. Cardiac function and remodeling were assessed using echocardiography and immunohistochemistry. To determine the specific role of MZB cells, we used mice with B cell lineage-specific conditional deletion of Notch signaling, which leads to selection deficiency of MZB cells. To evaluate the role of HIF-1α isoform, we generated mice with MZB cell lineage specific conditional deletion of Hif1a. Results. Acute MI prompted miR21-dependent increase of HIF-1α, particularly in splenic MZB cells. MZB cell deficiency and MZB cell-specific deletion of miR21 or Hif1a improved cardiac function after acute MI. miR21/HIF-1α signaling in MZB cells was required for Tolllike receptor dependent expression of the monocyte chemo-attractant protein CCL7, leading to increased mobilization of inflammatory monocytes to the ischemic myocardium and to adverse post-ischemic cardiac remodeling. Conclusions. This work reveals a novel function for miR21/HIF-1α pathway in splenic MZB cells with potential major implications for the modulation of cardiac function after acute MI.

High-density lipoproteins (HDLs) represent a family of particles characterized by the presence of apolipoprotein A-I (apoA-I) and by their ability to transport cholesterol from peripheral tissues back to the liver. In addition to this function, HDLs display pleiotropic effects including antioxidant, anti-apoptotic, anti-inflammatory, anti-thrombotic or anti-proteolytic properties that account for their protective action on endothelial cells. Vasodilatation via production of nitric oxide is also a hallmark of HDL action on endothelial cells. Endothelial cells express receptors for apoA-I and HDLs that mediate intracellular signalling and potentially participate in the internalization of these particles. In this review, we will detail the different effects of HDLs on the endothelium in normal and pathological conditions with a particular focus on the potential use of HDL therapy to restore endothelial function and integrity.

Background and Purpose— Low levels of low-density lipoprotein-cholesterol (LDL-C) are suspected to be associated with a risk of hemorrhagic transformation after ischemic stroke. We assessed the risk of hemorrhagic transformation after cerebral ischemia/reperfusion in mice with low levels of LDL-C resulting from proprotein convertase subtilisin kexin 9 (PCSK9) deficiency. Methods— PCSK9 −/− and PCSK9 +/+ mice were fed with a high-fat/high-cholesterol (21%/0.15%) diet for 1 month. Plasma lipids were measured using colorimetric assays. PCSK9 −/− and PCSK9 +/+ mice (n=15 per group) were subjected to a 4-hour intraluminal occlusion of the middle cerebral artery followed by 20 hours of reperfusion. Spontaneous hemorrhagic transformation was assessed by quantification of hemoglobin in ischemic tissue. In vitro, a cell model of blood–brain barrier was used to test endothelial barrier integrity in response to decreasing concentrations of LDL-C from 1 to 0.25g/L in ischemia/reperfusion conditions. Results— PCSK9 −/− mice had lower LDL-C, high-density lipoprotein-cholesterol, and total cholesterol levels than PCSK9 +/+ mice before and after 1 month on the high-fat/high-cholesterol diet. Hemoglobin concentration in ischemic cerebral tissue was not different between PCSK9 −/− and PCSK9 +/+ mice (31.5 [18.9–60.1] and 32.8 [14.7–69.9] ng/mg protein, respectively; P =0.81). Infarct volume was also similar in both groups ( P =0.66). Incubation of human cerebral endothelial cells with decreasing concentrations of LDL-C under ischemia/reperfusion conditions did not alter blood–brain barrier permeability. Conclusions— Low levels of LDL-C did not increase the risk of hemorrhagic transformation after cerebral ischemia/reperfusion in mice. Our observations suggest that PCSK9 inhibition, leading to LDL-C lowering, should not increase hemorrhagic complications after acute ischemic stroke.

G protein-coupled receptors (GPCRs) are well-known drug targets. However, a question mark remains for the more than 100 orphan GPCRs as current deorphanisation strategies failed to identify specific ligands for these receptors. Recent advances have shown that orphan GPCRs may have important functions that are ligand-independent. Orphan GPCRs can modulate the function of well-defined drug targets such as GPCRs with identified ligands and neurotransmitter transporters though physical association with those molecules. Thus, compounds that bind to orphan GPCRs and allosterically regulate the function of the interacting partner or even disrupt the interaction with the latter could become new drugs.

Les recepteurs couples aux proteines G (RCPG) ou recepteurs a 7 domaines transmembranaires (7TM) representent la plus grande famille de proteines de la surface cellulaire. Cette famille de proteines controle de nombreux processus physiologiques. Son implication dans de nombreuses pathologies fait de cette classe de proteines la cible d’environ 50 % des medicaments couramment prescrits aujourd’hui. Cependant, malgre les efforts realises par la recherche academique et l’industrie, une centaine de RCPG sont encore orphelins. Ces derniers presentent donc un interet particulier puisqu’ils constituent autant de cibles potentielles pour la decouverte de nouvelles drogues. Les strategies de recherche concernant les RCPG orphelins ont consiste essentiellement en l’identification de leurs ligands naturels. Tout comme dans le cas de certains recepteurs orphelins nucleaires, des donnees recentes ont montre que des RCPG orphelins peuvent reguler, par heterodimerisation et independamment d’un ligand, la fonction d’autres recepteurs ayant un ligand connu. Cette caracteristique permettrait, d’une part, de mieux comprendre la diversite extraordinaire offerte par les RCPG et, d’autre part, d’ouvrir de nouvelles perspectives concernant l’identification de la fonction de ces RCPG orphelins, quels que soient les domaines therapeutiques consideres.

G-protein-coupled receptors (GPCRs) are important drug targets and are involved in virtually every biological process. However, there are still more than 140 orphan GPCRs, and deciphering their function remains a priority for fundamental and clinical research. Research on orphan GPCRs has concentrated mainly on the identification of their natural ligands, whereas recent data suggest additional ligand-independent functions for these receptors. This emerging concept is connected with the observation that orphan GPCRs can heterodimerize with GPCRs that have identified ligands, and by so doing regulate the function of the latter. Pairing orphan GPCRs with their potential heterodimerization partners will have a major impact on our understanding of the extraordinary diversity offered by GPCR heterodimerization and, in addition, will constitute a novel strategy to elucidate the function of orphan receptors that needs to be added to the repertoire of 'deorphanization' strategies.

One-third of the approximately 400 nonodorant G protein-coupled receptors (GPCRs) are still orphans. Although a considerable number of these receptors are likely to transduce cellular signals in response to ligands that remain to be identified, they may also have ligand-independent functions. Several members of the GPCR family have been shown to modulate the function of other receptors through heterodimerization. We show that GPR50, an orphan GPCR, heterodimerizes constitutively and specifically with MT(1) and MT(2) melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT(2) did not modify MT(2) function, GPR50 abolished high-affinity agonist binding and G protein coupling to the MT(1) protomer engaged in the heterodimer. Deletion of the large C-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT(1) without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT(1). Pairing orphan GPCRs to potential heterodimerization partners might be of clinical importance and may become a general strategy to better understand the function of orphan GPCRs.

Heterodimerization has been documented for several members of the G protein-coupled receptor (GPCR) superfamily, including the closely related MT1 and MT2 melatonin receptors. However, the relative abundance of hetero- versus homodimers and the specific properties, which can be attributed to each form, are difficult to determine. Using a bioluminescence resonance energy transfer (BRET) donor saturation assay, we show that half-maximal MT1/MT2 heterodimer formation is reached for expression levels as low as 4000 receptors per cell. The relative propensity of MT1 homodimer and MT1/MT2 heterodimer formation are similar, whereas that for the MT2 homodimer formation is 3- to 4-fold lower. These data indicate that both the relative expression level of each receptor isoform and the affinities between monomers may determine the actual proportion of homo- and heterodimers. The specific interaction of ligands with the MT1/MT2 heterodimer was studied using a BRET-based assay as a readout for the conformational changes of the heterodimer. An MT1/MT2 heterodimer-specific profile and ligands selective for the MT1/MT2 heterodimer compared with the MT2 homodimer could be identified. Classic radioligand binding and BRET studies suggest that heterodimers contain two functional ligand binding sites that maintain their respective selectivity for MT1 and MT2 ligands. Occupation of either binding site is sufficient to induce a conformational change within the heterodimer. Taken together, these results show that the probability of GPCR heterodimer formation may be equal to or even higher than that of the corresponding homodimers and that specific properties of heterodimers can be revealed using a BRETbased ligand/receptor interaction assay. A growing number of observations suggest that G proteincoupled receptors (GPCR) form homodimers and heterodimerize with other members of the same receptor superfamily. Heterodimerization may have important consequences in terms of receptor function, because significant changes in ligand binding, signaling, or trafficking were observed for sev

Several studies report that high-density lipoproteins (HDLs) can carry α1-antitrypsin (AAT; an elastase inhibitor). We aimed to determine whether injection of exogenous HDL, enriched or not in AAT, may have protective effects against pulmonary emphysema. After tracheal instillation of saline or elastase, mice were randomly treated intravenously with saline, human plasma HDL (75 mg apolipoprotein A1/kg), HDL-AAT (75 mg apolipoprotein A1-3.75 mg AAT/kg), or AAT alone (3.75 mg/kg) at 2, 24, 48, and 72 hours. We have shown that HDL-AAT reached the lung and prevented the development of pulmonary emphysema by 59.3% at 3 weeks (alveoli mean chord length, 22.9 ± 2.8 μm versus 30.7 ± 4.5 μm; P0.001), whereas injection of HDL or AAT alone only showed a moderate, nonsignificant protective effect (28.2 ± 4.2 μm versus 30.7 ± 5 μm [P = 0.23] and 27.3 ± 5.66 μm versus 30.71 ± 4.96 μm [P = 0.18], respectively). Indeed, protection by HDL-AAT was significantly higher than that observed with HDL or AAT (P = 0.006 and P = 0.048, respectively). This protective effect was associated (at 6, 24, and 72 h) with: (1) a reduction in neutrophil and macrophage number in the bronchoalveolar lavage fluid; (2) decreased concentrations of IL-6, monocyte chemoattractant protein-1, and TNF-α in both bronchoalveolar lavage fluid and plasma; (3) a reduction in matrix metalloproteinase-2 and matrix metalloproteinase-9 activities; and (4) a reduction in the degradation of fibronectin, a marker of tissue damage. In addition, HDL-AAT reduced acute cigarette smoke-induced inflammatory response. Intravenous HDL-AAT treatment afforded a better protection against elastase-induced pulmonary emphysema than AAT alone, and may represent a significant development for the management of emphysema associated with AAT deficiency.

Introduction La proproteine convertase subtilisine/kexine type 9 (PCSK9) est un inhibiteur naturel du recepteur aux LDL. L’inhibition de PCSK9 est une nouvelle therapie prometteuse pour abaisser le LDL cholesterol (LDL-C). Toutefois, quelques etudes suggerent que des taux tres bas de LDL-C pourraient etre associes a une augmentation du risque de transformation hemorragique apres une ischemie/reperfusion cerebrale. Nous avons etudie ce risque de transformation hemorragique a partir d’un modele de souris avec des taux tres abaisses de LDL-C par deletion du gene PCSK9 et soumis a une ischemie/reperfusion cerebrale. Materiel et methodes Des souris deletees pour le gene PCSK9 (PCSK9−/−) et leurs controles (PCSK9++) ont suivi un regime riche en cholesterol pendant un mois, puis le profil lipidique a ete analyse. Le modele d’ischemie/reperfusion cerebrale a ete realise par 4 heures d’occlusion intraluminale de l’artere cerebrale moyenne puis 20 heures de reperfusion. La concentration en hemoglobine dans le tissu cerebral ischemique a ete mesuree par une methode Elisa en ng/mg de proteines totales. Le volume d’infarctus cerebral a ete mesure par le logiciel Image J apres coloration vitale mitochondriale au TTC. Les donnees ont ete representees en mediane [min–max]. Le test statistique non parametrique utilise etait le Mann-Whitney avec un p Resultats Apres regime riche en cholesterol, les souris PCSK9−/− avaient un taux de LDL-C inferieur a celui des souris PCSK9++ (0,20 [0,09–0,35] et 0,33 [0,21–0,53] g/L ; p = 0,0005). Le modele d’ischemie/reperfusion cerebrale a ete realise chez 15 souris PCSK9−/− et 15 souris PCSK9++. Une souris dans chaque groupe est decedee apres l’ischemie cerebrale. Vingt-quatre heures apres l’ischemie/reperfusion cerebrale, la concentration en hemoglobine dans le tissu ischemique etait similaire chez les souris PCSK9−/− et PCSK9++ (4,5 [2,7–8,6] et 4,7 [2,1–10] ng/mg ; p = 0,81). Le volume d’infarctus cerebral n’etait pas different entre les deux groupes de souris (p = 0,66). Discussion Des taux tres abaisses de LDL-C apres deletion du gene PCSK9 n’augmentent pas le risque de transformation hemorragique apres une ischemie/reperfusion cerebrale. Ces resultats confortent la securite d’utilisation des inhibiteurs de PCSK9 chez les patients a risque d’accidents vasculaires cerebraux.

Leptin mediates its metabolic effects through several leptin receptor (LEP-R) isoforms. In humans, long (LEPRb) and short (LEPRa,c,d) isoforms are generated by alternative splicing. Most of leptin’s effects are believed to be mediated by the OB-Rb isoform. However, the role of short LEPR isoforms and the possible existence of heteromers between different isoforms are poorly understood. Using BRET1 and optimized co-immunoprecipitation, we observed LEPRa/b and LEPRb/c heteromers located at the plasma membrane and stabilized by leptin. Given the widespread coexpression of LEPRa and LEPRb, our results suggest that LEPRa/b heteromers may represent a major receptor species in most tissues.Structured summaryMINT-7714817: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRb (uniprotkb:P48357-1) by anti tag co-immunoprecipitation (MI:0007)MINT-7714785: LEPRc (uniprotkb:P48357-2) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357-2) by bioluminescence resonance energy transfer (MI:0012)MINT-7714951, MINT-7714744: LEPRa (uniprotkb:P48357-3) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by bioluminescence resonance energy transfer (MI:0012)MINT-7714859: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by anti tag co-immunoprecipitation (MI:0007)MINT-7714885, MINT-7714672: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRb (uniprotkb:P48357-1) by bioluminescence resonance energy transfer (MI:0012)MINT-7714835: LEPRa (uniprotkb:P48357-3) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by anti tag co-immunoprecipitation (MI:0007)MINT-7714914, MINT-7714723, MINT-7714759: LeprB (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRa (uniprotkb:P48357-3) by bioluminescence resonance energy transfer (MI:0012)MINT-7714703, MINT-7714936, MINT-7714772: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357-2) by bioluminescence resonance energy transfer (MI:0012)MINT-7714872: LEPRb (uniprotkb:P48357-1) physically interacts (MI:0915) with LEPRc (uniprotkb:P48357-2) by anti tag co-immunoprecipitation (MI:0007)

International audience; Leukocytes from individuals with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a rare immunodeficiency, and bearing a wild-type CXCR4 ORF (WHIM(WT)) display impaired CXCR4 internalization and desensitization upon exposure to CXCL12. The resulting enhanced CXCR4-dependent responses, including chemotaxis, probably impair leukocyte trafficking and account for the immunohematologic clinical manifestations of WHIM syndrome. We provided here evidence that GPCR kinase-3 (GRK3) specifically regulates CXCL12-promoted internalization and desensitization of CXCR4. GRK3-silenced control cells displayed altered CXCR4 attenuation and enhanced chemotaxis, as did WHIM(WT) cells. These findings identified GRK3 as a negative regulator of CXCL12-induced chemotaxis and as a candidate responsible for CXCR4 dysfunction in WHIM(WT) leukocytes. Consistent with this, we showed that GRK3 overexpression in both leukocytes and skin fibroblasts from 2 unrelated WHIM(WT) patients restored CXCL12-induced internalization and desensitization of CXCR4 and normalized chemotaxis. Moreover, we found in cells derived from one patient a profound and selective decrease in GRK3 products that probably resulted from defective mRNA synthesis. Taken together, these results have revealed a pivotal role for GRK3 in regulating CXCR4 attenuation and have provided a mechanistic link between the GRK3 pathway and the CXCR4-related WHIM(WT) disorder.

In mammals, the circadian hormone melatonin targets two seven-transmembrane-spanning receptors, MT1 and MT2, of the G protein-coupled receptor (GPCR) super-family. Evidence accumulated over the last 15 yrs convincingly demonstrates that GPCRs, classically considered to function as monomers, are actually organized as homodimers and heterodimerize with other GPCR family members. These dimers are formed early in the biosynthetic pathway and remain stable throughout the entire life cycle. A growing number of observations demonstrate that GPCR oligomerization may occur in native tissues and may have important consequences on receptor function. The formation of MT1 and MT2 homodimers and MT1/MT2 heterodimers has been shown in heterologous expression systems at physiological expression levels. Formation of MT1/MT2 heterodimers remains to be shown in native tissues but is suggested by the documented co-expression of MT1 and MT2 in many melatonin-sensitive tissues, such as the hypothalamic suprachiasmatic nuclei, retina, arteries, and adipose tissue. Considering that multiple GPCRs are expressed simultaneously in most cells, the possible engagement into heterodimeric complexes has to be considered and taken into account for the interpretation of experimental data obtained from native tissues and knockout animals.

Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase that plays a major role in blood homoeostasis and reproduction in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two homologous active sites, have been characterized. So far, several ACEs from invertebrates have been cloned, but only in insects. They are soluble and display a single active site. Using biochemical procedures, an ACE-like activity was detected in our model, the leech, Theromyzon tessulatum. Annelida is the most distant phylum in which an ACE activity has been observed. To gain more insight into the leech enzyme, we have developed a PCR approach to characterize its mRNA. The approx. 2 kb cDNA has been predicted to encode a 616-amino-acid soluble enzyme containing a single active site, named TtACE (T. tessulatum ACE). Surprisingly, its primary sequence shows greater similarity to vertebrates than to invertebrates. Stable in vitro expression of TtACE in transfected Chinese-hamster ovary cells revealed that the leech enzyme is a functional metalloprotease. As in mammals, this 79 kDa glycosylated enzyme functions as a dipeptidyl carboxypeptidase capable of hydrolysing angiotensin I to angiotensin II. However, a weak chloride inhibitory effect and acetylated N-acetyl-SDKP (Ac SDAcKP) hydrolysis reveal that TtACE activity resembles that of the N-domain of mammalian ACE. In situ hybridization shows that its cellular distribution is restricted to epithelial midgut cells. Although the precise roles and endogenous substrates of TtACE remain to be identified, characterization of this ancestral peptidase will help to clarify its physiological roles in non-insect invertebrate species.