David Communi

Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1

Therapeutic angiogenesis is likely to require the administration of factors that complement each other. Activation of the receptor tyrosine kinase (RTK) Flk1 by vascular endothelial growth factor (VEGF) is crucial, but molecular interactions of other factors with VEGF and Flk1 have been studied to a limited extent. Here we report that placental growth factor (PGF, also known as PlGF) regulates inter- and intramolecular cross talk between the VEGF RTKs Flt1 and Flk1. Activation of Flt1 by PGF resulted in intermolecular transphosphorylation of Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1. Even though VEGF and PGF both bind Flt1, PGF uniquely stimulated the phosphorylation of specific Flt1 tyrosine residues and the expression of distinct downstream target genes. Furthermore, the VEGF/PGF heterodimer activated intramolecular VEGF receptor cross talk through formation of Flk1/Flt1 heterodimers. The inter- and intramolecular VEGF receptor cross talk is likely to have therapeutic implications, as treatment with VEGF/PGF heterodimer or a combination of VEGF plus PGF increased ischemic myocardial angiogenesis in a mouse model that was refractory to VEGF alone.

Specific Recruitment of Antigen-presenting Cells by Chemerin, a Novel Processed Ligand from Human Inflammatory Fluids

Dendritic cells (DCs) and macrophages are professional antigen-presenting cells (APCs) that play key roles in both innate and adaptive immunity. ChemR23 is an orphan G protein–coupled receptor related to chemokine receptors, which is expressed specifically in these cell types. Here we present the characterization of chemerin, a novel chemoattractant protein, which acts through ChemR23 and is abundant in a diverse set of human inflammatory fluids. Chemerin is secreted as a precursor of low biological activity, which upon proteolytic cleavage of its COOH-terminal domain, is converted into a potent and highly specific agonist of ChemR23, the chemerin receptor. Activation of chemerin receptor results in intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of p42–p44 MAP kinases, through the Gi class of heterotrimeric G proteins. Chemerin is structurally and evolutionary related to the cathelicidin precursors (antibacterial peptides), cystatins (cysteine protease inhibitors), and kininogens. Chemerin was shown to promote calcium mobilization and chemotaxis of immature DCs and macrophages in a ChemR23-dependent manner. Therefore, chemerin appears as a potent chemoattractant protein of a novel class, which requires proteolytic activation and is specific for APCs.

Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid organs and inflamed skin

Chemerin is a chemotactic agent that was recently identified as the ligand of ChemR23, a serpentine receptor expressed by activated macrophages and monocyte-derived dendritic cells (DCs). This paper shows that blood plasmacytoid and myeloid DCs express functional ChemR23. Recombinant chemerin induced the transmigration of plasmacytoid and myeloid DCs across an endothelial cell monolayer. In secondary lymphoid organs (lymph nodes and tonsils), ChemR23 is expressed by CD123+ plasmacytoid DCs and by CD1a+ DC-SIGN+ DCs in the interfollicular T cell area. ChemR23+ DCs were also observed in dermis from normal skin, whereas Langerhans cells were negative. Chemerin expression was selectively detected on the luminal side of high endothelial venules in secondary lymphoid organs and in dermal endothelial vessels of lupus erythematosus skin lesions. Chemerin+ endothelial cells were surrounded by ChemR23+ plasmacytoid DCs. Thus, ChemR23 is expressed and functional in plasmacytoid DCs, a property shared only by CXCR4 among chemotactic receptors. This finding, together with the selective expression of the cognate ligand on the luminal side of high endothelial venules and inflamed endothelium, suggests a key role of the ChemR23/chemerin axis in directing plasmacytoid DC trafficking.

Neutrophil-mediated maturation of chemerin : A link between innate and adaptive immunity

Dendritic cells and macrophages are professional APCs that play a central role in initiating immune responses, linking innate and adaptive immunity. Chemerin is a novel chemoattractant factor that specifically attracts APCs through its receptor ChemR23. Interestingly, chemerin is secreted as a precursor of low biological activity, prochemerin, which upon proteolytic removal of a C-terminal peptide, is converted into a potent and highly specific agonist of its receptor. Given the fact that APCs are often preceded by polymorphonuclear cells (PMN) in inflammatory infiltrates, we hypothesized that PMN could mediate chemerin generation. We demonstrate here that human degranulated PMNs release proteases that efficiently convert prochemerin into active chemerin. The use of specific protease inhibitors allowed us to identify the neutrophil serine proteases cathepsin G and elastase as responsible for this process. Mass spectrometry analysis of processed prochemerin showed that each protease generates specifically a distinct form of active chemerin, differing in their C terminus and initially identified in human inflammatory fluids. These findings strongly suggest that bioactive chemerin generation takes place during the early stages of inflammation, underscoring the functional contribution of chemerin as a bridge between innate and adaptive immunity.

Revascularization of ischemic tissues by PDGF-CC via effects on endothelial cells and their progenitors

The angiogenic mechanism and therapeutic potential of PDGF-CC, a recently discovered member of the VEGF/PDGF superfamily, remain incompletely characterized. Here we report that PDGF-CC mobilized endothelial progenitor cells in ischemic conditions; induced differentiation of bone marrow cells into ECs; and stimulated migration of ECs. Furthermore, PDGF-CC induced the differentiation of bone marrow cells into smooth muscle cells and stimulated their growth during vessel sprouting. Moreover, delivery of PDGF-CC enhanced postischemic revascularization of the heart and limb. Modulating the activity of PDGF-CC may provide novel opportunities for treating ischemic diseases.

THE DIVERSITY AND POSSIBLE FUNCTIONS OF THE INOSITOL POLYPHOSPHATE 5-PHOSPHATASES

Distinct forms of inositol and phosphatidylinositol polyphosphate 5-phosphatases selectively remove the phosphate from the 5-position of the inositol ring from both soluble and lipid substrates, i.e., inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), inositol 1,3,4, 5-tetrakisphosphate (Ins(1,3,4,5)P4), phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) or phosphatidylinositol 3,4, 5-trisphosphate (PtdIns(3,4,5)P3). In mammalian cells, this family contains a series of distinct genes and splice variants. All inositol polyphosphate 5-phosphatases share a 5-phosphatase domain and various protein modules probably responsible for specific cell localisation or recruitment (SH2 domain, proline-rich sequences, prenylation sites, etc.). Type I Ins(1,4,5)P3 5-phosphatase also uses Ins(1,3,4,5)P4 but not the phosphoinositides as substrates. This enzyme is targeted to specific membranes by means of a prenylation site. Type II 5-phosphatases can use both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 as substrates. Five mammalian enzymes and multiple splice variants are known: INPP5P or inositol polyphosphate 5-phosphatase II, OCRL (a Golgi protein implicated in the Lowe oculocerebrorenal syndrome), synaptojanin (a protein involved in the recycling of synaptic vesicles), SHIP 1 and SHIP 2 (or SH2-containing inositol 5-phosphatases). As discussed in this review, the substrate specificity, regulatory mechanisms, subcellular localisation and tissue specificity indicate that the different 5-phosphatase isoforms may play specific roles. As known in the dephosphorylation of tyrosine containing substrates by the tyrosine protein phosphatases or in the metabolism of cyclic nucleotides by the cyclic nucleotide phosphodiesterases, inositol polyphosphate 5-phosphatases directly participate in the control of second messengers in response to both activation or inhibitory cell signalling.

Activation of Dual Oxidases Duox1 and Duox2 DIFFERENTIAL REGULATION MEDIATED BY cAMP-DEPENDENT PROTEIN KINASE AND PROTEIN KINASE C-DEPENDENT PHOSPHORYLATION

Dual oxidases were initially identified as NADPH oxidases producing H2O2 necessary for thyroid hormone biosynthesis. The crucial role of Duox2 has been demonstrated in patients suffering from partial iodide organification defect caused by bi-allelic mutations in the DUOX2 gene. However, the Duox1 function in thyroid remains elusive. We optimized a functional assay by co-expressing Duox1 or Duox2 with their respective maturation factors, DuoxA1 and DuoxA2, to compare their intrinsic enzymatic activities under stimulation of the major signaling pathways active in the thyroid in relation to their membrane expression. We showed that basal activity of both Duox isoenzymes depends on calcium and functional EF-hand motifs. However, the two oxidases are differentially regulated by activation of intracellular signaling cascades. Duox1 but not Duox2 activity is stimulated by forskolin (EC50 = 0.1 μm) via protein kinase A-mediated Duox1 phosphorylation on serine 955. In contrast, phorbol esters induce Duox2 phosphorylation via protein kinase C activation associated with high H2O2 generation (phorbol 12-myristate 13-acetate EC50 = 0.8 nm). These results were confirmed in human thyroid cells, suggesting that Duox1 is also involved in thyroid hormonogenesis. Our data provide, for the first time, detailed insights into the mechanisms controlling the activation of Duox1–2 proteins and reveal additional phosphorylation-mediated regulation.

Identification and characterization of an endogenous chemotactic ligand specific for FPRL2

Chemotaxis of dendritic cells (DCs) and monocytes is a key step in the initiation of an adequate immune response. Formyl peptide receptor (FPR) and FPR-like receptor (FPRL)1, two G protein–coupled receptors belonging to the FPR family, play an essential role in host defense mechanisms against bacterial infection and in the regulation of inflammatory reactions. FPRL2, the third member of this structural family of chemoattractant receptors, is characterized by its specific expression on monocytes and DCs. Here, we present the isolation from a spleen extract and the functional characterization of F2L, a novel chemoattractant peptide acting specifically through FPRL2. F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein, an intracellular tetrapyrolle-binding protein. The peptide binds and activates FPRL2 in the low nanomolar range, which triggers intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of extracellular signal–regulated kinase 1/2 mitogen-activated protein kinases through the Gi class of heterotrimeric G proteins. When tested on monocytes and monocyte-derived DCs, F2L promotes calcium mobilization and chemotaxis. Therefore, F2L appears as a new natural chemoattractant peptide for DCs and monocytes, and the first potent and specific agonist of FPRL2.

Role of neutrophil proteinase 3 and mast cell chymase in chemerin proteolytic regulation

Chemerin is a potent chemotactic factor that was identified recently as the ligand of ChemR23, a G protein‐coupled receptor expressed by mononuclear phagocytes, dendritic cells (DCs), and NK cells. Chemerin is synthesized as a secreted precursor, prochemerin, which is poorly active on ChemR23. However, prochemerin can be converted rapidly into a full ChemR23 agonist by proteolytic removal of a carboxy‐terminal peptide. This maturation step is mediated by the neutrophil‐derived serine proteases elastase and cathepsin G. In the present work, we have investigated proteolytic events that negatively control chemerin activity. We demonstrate here that neutrophil‐derived proteinase 3 (PR3) and mast cell (MC) chymase are involved in the generation of specific chemerin variants, which are inactive, as they do not induce calcium release or DC chemotaxis. Mass spectrometry analysis showed that PR3 specifically converts prochemerin into a chemerin form, lacking the last eight carboxy‐terminal amino acids, and is inactive on ChemR23. Whereas PR3 had no effect on bioactive chemerin, MC chymase was shown to abolish chemerin activity by the removal of additional amino acids from its C‐terminus. This effect was shown to be specific to bioactive chemerin (chemerin‐157 and to a lesser extent, chemerin‐156), as MC chymase does not use prochemerin as a substrate. These mechanisms, leading to the production of inactive variants of chemerin, starting from the precursor or the active variants, highlight the complex interplay of proteases regulating the bioactivity of this novel mediator during early innate immune responses.

Angiotensin II Inhibits Insulin-Stimulated GLUT4 Translocation and Akt Activation through Tyrosine Nitration-Dependent Mechanisms

Angiotensin II (Ang II) plays a major role in the pathogenesis of insulin resistance and diabetes by inhibiting insulins metabolic and potentiating its trophic effects. Whereas the precise mechanisms involved remain ill-defined, they appear to be associated with and dependent upon increased oxidative stress. We found Ang II to block insulin-dependent GLUT4 translocation in L6 myotubes in an NO- and O2 .−-dependent fashion suggesting the involvement of peroxynitrite. This hypothesis was confirmed by the ability of Ang II to induce tyrosine nitration of the MAP kinases ERK1/2 and of protein kinase B/Akt (Akt). Tyrosine nitration of ERK1/2 was required for their phosphorylation on Thr and Tyr and their subsequent activation, whereas it completely inhibited Akt phosphorylation on Ser473 and Thr308 as well as its activity. The inhibitory effect of nitration on Akt activity was confirmed by the ability of SIN-1 to completely block GSK3α phosphorylation in vitro. Inhibition of nitric oxide synthase and NAD(P)Hoxidase and scavenging of free radicals with myricetin restored insulin-stimulated Akt phosphorylation and GLUT4 translocation in the presence of Ang II. Similar restoration was obtained by inhibiting the ERK activating kinase MEK, indicating that these kinases regulate Akt activation. We found a conserved nitration site of ERK1/2 to be located in their kinase domain on Tyr156/139, close to their active site Asp166/149, in agreement with a permissive function of nitration for their activation. Taken together, our data show that Ang II inhibits insulin-mediated GLUT4 translocation in this skeletal muscle model through at least two pathways: first through the transient activation of ERK1/2 which inhibit IRS-1/2 and second through a direct inhibitory nitration of Akt. These observations indicate that not only oxidative but also nitrative stress play a key role in the pathogenesis of insulin resistance. They underline the role of protein nitration as a major mechanism in the regulation of Ang II and insulin signaling pathways and more particularly as a key regulator of protein kinase activity.