P2Y12, a G protein-coupled receptor that plays a central role in platelet activation has been recently identified as the receptor targeted by the antithrombotic drug, clopidogrel. In this study, we further deciphered the mechanism of action of clopidogrel and of its active metabolite (Act-Met) on P2Y12 receptors. Using biochemical approaches, we demonstrated the existence of homooligomeric complexes of P2Y12 receptors at the surface of mammalian cells and in freshly isolated platelets. In vitro treatment with Act-Met or in vivo oral administration to rats with clopidogrel induced the breakdown of these oligomers into dimeric and monomeric entities in P2Y12 expressing HEK293 and platelets respectively. In addition, we showed the predominant association of P2Y12 oligomers to cell membrane lipid rafts and the partitioning of P2Y12 out of rafts in response to clopidogrel and Act-Met. The raft-associated P2Y12 oligomers represented the functional form of the receptor, as demonstrated by binding and signal transduction studies. Finally, using a series of receptors individually mutated at each cysteine residue and a chimeric P2Y12͞P2Y13 receptor, we pointed out the involvement of cysteine 97 within the first extracellular loop of P2Y12 in the mechanism of action of Act-Met. mechanism of action ͉ platelet ͉ antiaggregant M any G protein-coupled receptors (GPCRs) have been shown to assemble as homodimers, heterodimers, as well as larger oligomers (1, 2). The existence of such oligomeric entities raises questions as to their functional consequences as well as their physiological relevance. Heterologous expression systems have provided a variety of answers concerning ligand-dependent regulation of GPCR oligomeric states. Ligand binding, depending on the GPCR studied, can promote (3-10) or inhibit (11-13) dimer formation, as well as having no effect on preexisting constitutive homo-or heterodimers (14-25). The fact that heterodimerization may alter the pharmacological properties of a GPCR along with its internalization and signal transduction behavior is of critical importance (26, 27).Clustering, even for nonheptahelical receptors, now appears as a common feature of cell signaling. Specialized structures such as clathrin-coated pits, caveolae, and lipid rafts contain high concentrations of signaling molecules. Rafts represent dynamic assemblies of proteins and lipids, mostly sphingolipids and cholesterol (28,29). Proteins such as glycophosphatidylinositol-anchored proteins, nonreceptor tyrosine kinases, G␣ subunits of heterotrimeric G proteins, and palmitoylated proteins appear to localize to these microdomains (30). In addition, recent studies have shown that partitioning of proteins in and out of rafts can depend on their state of activation or dimerization (31-33). A variety of GPCR have also been identified in caveolae or rafts. These include ␣ and ␤-adrenergic receptors (34, 35), adenosine A1 receptor (36), angiotensin II type 1 receptor (37), muscarinic receptor (38), EDG1 receptor (39), bradykinin B1 and B2 receptor...

The role of Gas6 in endothelial cell (EC) function remains incompletely characterized. Here we report that Gas6 amplifies EC activation in response to inflammatory stimuli in vitro. In vivo, Gas6 promotes and accelerates the sequestration of circulating platelets and leukocytes on activated endothelium as well as the formation and endothelial sequestration of circulating platelet-leukocyte conjugates. In addition, Gas6 promotes leukocyte extravasation, inflammation, and thrombosis in mouse models of inflammation (endotoxinemia, vasculitis, heart trans- IntroductionThe growth arrest-specific gene 6 (Gas6) binds to the receptor tyrosine kinases Axl, Tyro3, and Mer. 1 Gas6 is composed of a N-terminal gamma-carboxy-glutamic acid domain (Gla-domain), a loop region, 4 EGF-like repeats, and a C-terminal steroid hormone binding globulin-like (SHBG-like) domain). 1 Even though this molecule was discovered as a homologue of the anticoagulant protein S more than a decade ago, its role in vivo remains incompletely characterized. 2,3 Originally identified in fibroblasts, Gas6 is expressed in various cell types, including endothelial cells (ECs), 4 smooth muscle, 5 and bone marrow cells. 6 Gas6 and its receptors modify platelet activation and aggregation, 7-10 but the role of Gas6 in the interplay between platelets and other cell types, such as ECs and leukocytes, 11 during inflammatory conditions remains unclear.Several lines of evidence indeed suggest that Gas6 may affect ECs and leukocytes. ECs and leukocytes express Gas6 and its receptors, especially in conditions of inflammation and repair. 4,[12][13][14][15][16] Gas6 stimulates EC survival [17][18][19][20] and promotes angiogenesis by enforcing the adhesion of Axl-expressing ECs via homophilic interactions, 21-23 yet another study suggested that activation of Axl impairs tyrosine phosphorylation of vascular endothelial growth factor (VEGF) receptor-2. 24 The activity of Gas6 on leukocytes also remains incompletely understood. Indeed, genetic loss of Mer inhibits cytokine production by natural killer cells 25 while it stimulates tumor necrosis factor-␣ (TNF-␣) production by monocytes 14 and impairs clearance of apoptotic cells. 26 Loss of all 3 Gas6 receptors, on the other hand, induces lymphoproliferative disorders via hyperactivation of antigen-presenting cells, 27,28 but mice lacking Gas6 (Gas6 Ϫ/Ϫ ) do not develop autoimmune health problems (P.C., unpublished data, 2008). In humans, the plasma levels of Gas6 were found to be elevated during severe sepsis, a life-threatening condition involving increased interactions between ECs, leukocytes, and platelets. 29,30 However, exogenous Gas6 inhibits granulocyte adhesion to ECs, but only at very high doses. 31 Furthermore, the role of endogenous Gas6 in leukocyte extravasation in vivo was not studied. Here, by using our previously generated Gas6 Ϫ/Ϫ mice, 7 we studied whether Gas6 might play a role in EC activation and in the interactions between ECs, platelets, and leukocytes during inflammatory conditions. Methods MiceTh...

Adipose tissue is an active endocrine organ that produces a variety of secretory factors involved in the initiation of angiogenic processes. The bioactive peptide apelin is the endogenous ligand of the G protein-coupled receptor, APJ. Here we investigated the potential role of apelin and its receptor, APJ, in the angiogenic responses of human endothelial cells and the development of a functional vascular network in a model of adipose tissue development in mice. Treatment of human umbilical vein endothelial cells with apelin dose-dependently increased angiogenic responses, including endothelial cell migration, proliferation, and Matrigel(R) capillary tubelike structure formation. These endothelial effects of apelin were due to activation of APJ, because siRNA directed against APJ, which led to long-lasting down-regulation of APJ mRNA, abolished cell migration induced by apelin in contrast to control nonsilencing siRNA. Hypoxia up-regulated the expression of apelin in 3T3F442A adipocytes, and we therefore determined whether apelin could play a role in adipose tissue angiogenesis in vivo. Epididymal white adipose tissue (EWAT) transplantation was performed as a model of adipose tissue angiogenesis. Transplantation led to increased apelin mRNA levels 2 and 5 days after transplantation associated with tissue hypoxia, as evidenced by hydroxyprobe staining on tissue sections. Graft revascularization evolved in parallel, as the first functional vessels in EWAT grafts were observed 2 days after transplantation and a strong angiogenic response was apparent on day 14. This was confirmed by determination of graft hemoglobin levels, which are indicative of functional vascularization and were strongly increased 5 and 14 days after transplantation. The role of apelin in the graft neovascularization was then assessed by local delivery of stable complex apelin-targeting siRNA leading to dramatically reduced apelin mRNA levels and vascularization (quantified by hemogloblin content) in grafted EWAT on day 5 when compared with control siRNA. Taken together, our data provide the first evidence that apelin/APJ signaling pathways play a critical role in the development of the functional vascular network in adipose tissue. In addition, we have shown that adipocyte-derived apelin can be up-regulated by hypoxia. These findings provide novel insights into the complex relationship between adipose tissue and endothelial vascular function and may lead to new therapeutic strategies to modulate angiogenesis.

Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription through its sex-dependent temporal pattern of pituitary hormone secretion. CYP2C12 encodes a female-specific rat liver P450 steroid hydroxylase whose expression is activated by continuous GH stimulation of hepatocytes. Presently, we investigated the role of liver-enriched and GH-regulated transcription factors in the activation of CYP2C12 gene expression in GH-stimulated liver cells. Transcription of a CYP2C12 promoter-luciferase reporter gene in transfected HepG2 cells was activated 15-40-fold by the liverenriched hepatocyte nuclear factor (HNF) 3␣, HNF3␤, and HNF6. Synergistic interactions leading to an ϳ300-fold activation of the promoter by HNF3␤ in combination with HNF6 were observed. 5 -Deletion analysis localized the HNF6 response to a single 5 -proximal 96-nucleotide segment. By contrast, the stimulatory effects of HNF3␣ and HNF3␤ were attributable to five distinct regions within the 1.6-kilobase CYP2C12 proximal promoter. GH activation of the signal transducer and transcriptional activator STAT5b, which proceeds efficiently in male but not female rat liver, inhibited CYP2C12 promoter activation by HNF3␤ and HNF6, despite the absence of a classical STAT5-binding site. The female-specific pattern of CYP2C12 expression is thus proposed to reflect the positive synergistic action in female liver of liver-enriched and GH-regulated transcription factors, such as HNF3␤ and HNF6, coupled with a dominant inhibitory effect of GH-activated STAT5b that is manifest in males.GH 1 signals to hepatocytes and other target cells via its plasma membrane-bound receptor, which is a member of the cytokine/growth factor receptor superfamily (1). Binding of GH to the GH receptor induces receptor dimerization and activation of Janus kinase 2 (JAK2), a tyrosine kinase that interacts with the cytoplasmic domain of the GH receptor and phosphorylates both itself and the cytoplasmic domain of the GH receptor on multiple tyrosine residues. These phosphorylated tyrosines, in turn, serve as docking sites for downstream signaling molecules that contain Src homology 2 domains, including STAT transcription factors and insulin receptor substrates 1 and 2 (1, 2). These primary signaling molecules activate secondary messengers, such as diacylglycerol, calcium, and nitric oxide, and enzymes, such as mitogen-activated protein kinase, protein kinase C, phospholipase A 2 , and phosphatidylinositol 3Ј-kinase. These GH-stimulated signaling pathways regulate a variety of intracellular events, including gene transcription, metabolite transport, and enzymatic activity, and contribute to the overall regulation by GH of whole body growth and metabolism (3).The cytochrome P450 gene CYP2C12 encodes a steroid-disulfate 15␤-hydroxylase that is induced in the livers of female rats at puberty. At this same developmental stage, a related P450 gene, CYP2C11, which encodes a steroid 16␣-and 2␣-hydroxylase, is induced in the livers of male rats (4, 5). The differential expression of...

VEGFR-3 is essential for vascular development and maintenance of lymphatic vessel's integrity. Little is known about its cooperative effect with other receptors of the same family. Contrary to VEGFR-2, stimulation of VEGFR-3 by VEGF-C and -D failed to enhance its phosphorylation either in HEK293T or in PAE cells. These ligands were unable to induce angiogenesis of PAEC expressing VEGFR-3 alone. In the presence of VEGFR-2, VEGF-C and -D induced heterodimerization of VEGFR-3 with VEGFR-2. This heterodimerization was associated with enhanced VEGFR-3 phosphorylation and subsequent cellular responses as evidenced by the formation of capillary-like structures in PAE cells and proliferation of primary human endothelial cells expressing both receptors. Taken together, these results show for the first time that VEGFR-3 needs to be associated to VEGFR-2 to induce ligand-dependent cellular responses.