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    Fotsis-Murphy Laboratory

    Cell Signaling and Membrane Trafficking: the VEGF and TGFβ family cascades

    No cell lives in isolation. In multicellular organisms, an enormous variety of chemicals, including small molecules, peptides, and proteins are used in intercellular communication. The signaling molecule acts as a ligand, which binds to a structurally complementary site on the extracellular or membrane-spanning domains of the receptor. Binding of a ligand to its receptor causes a conformational change in the cytosolic domain or domains of the receptor that ultimately induces specific cellular responses. The output of a signaling process depends not only on activation of a particular set of signaling molecules but also on the endocytic route of the ligand/receptor complex, which determines where and for how long the signal is emitted. Traditionally, endocytosis has been recognized as a means to terminate signaling via degradation of activated receptor complexes after their internalization from the cell surface. Recent data, however, suggest that the signaling machinery exploits the compartmentalization and the functional specialization of the endocytic pathway, beyond its conventional role in cargo degradation.

    At least five entry routes from the plasma membrane have been recognized to internalize various types of cargo. These are: 1. the clathrin-mediated route, 2. macropinocytosis that shares mechanistic properties with the phagocytic pathway, 3. the APPL pathway, 4. the caveolar route, and 5. the non-clathrin, non-caveolar pathway that ferries lipid raft components and extracellular fluid into the GPI-anchored protein enriched early endosomal compartment (GEEC). Cargo entering via these different routes is transported to a series of intracellular compartments from where it is either recycled to the cell surface via recycling endosomes or directed to degradative compartments (late endosomes and lysosomes). These endocytic organelles constitute endocytosis ‘substations’ that function as signaling platforms where the ligand/receptor complex binds and activates specific effectors, before it moves to the next organelle-substation. In this respect, the spatio-temporal regulation of the activity of ligand/receptor complexes is critical for the final output of the respective signaling cascades.

    VEGF signaling:

    Vascular Endothelial Growth Factors (VEGFs) are the most important regulators of vessel morphogenesis. VEGFs participate in the regulation of vasculogenesis, angiogenesis and lymphangiogenesis processes that are important for regeneration of obstructed vessels. VEGFs are secreted, dimeric glycoproteins of approximately 40 kDa and in mammals the VEGF family consists of five members, VEGF-A, B, C, D and placental growth factor (PLGF). Moreover, alternative splicing of several of the VEGF family members gives rise to isoforms with different biological activities. The VEGF ligands bind in an overlapping pattern to three receptor tyrosine kinases (RTKs), known as VEGF receptor-1, -2 and -3 (VEGFR1–3), as well as to co-receptors such as heparan sulphate proteoglycans (HSPGs) and neuropilins. VEGFR2 promotes migration, proliferation and differentiation of endothelial cells (ECs) via a number of cascades that activate a multitude of second messengers including ERK1/2 and p38 MAPKs.

    Current work focuses on understanding the molecular mechanisms of VEGF signaling, which is critical for the development of therapeutic angiogenesis or the generation of vascularised tissue engineered constructs. Towards this purpose, we have carried out DNA microarray analysis on mRNA collected from early passage human umbilical vein cells (HUVECs) at several time points post VEGF-induction. From approximately 22,000 genes examined, VEGF increased the expression of 116 genes, whereas it decreased the expression of 30 genes. We are currently investigating the signaling cascades responsible for the increased or decreased expression of several of the identified VEGF-regulated genes and the contribution of these genes in the generation of new vessels by VEGF.

    Current work on VEGF signaling:

    1. Continuing the work on VEGF-regulated transcription by expanding the investigation of additional VEGF-regulated genes, thereby generating valuable knowledge about the signaling circuits that regulate VEGF-dependent transcription and the contribution of the regulated genes in the regulation of key angiogenic responses of endothelial cells, such as proliferation, migration and survival.
    2. Studying the role of membrane trafficking in the outcome of VEGF/VEGFR complex signaling (in collaboration with the Christoforidis team)
    3. Investigating the vasculogenic signaling of VEGF. Towards this purpose, we are developing a protocol for the in vitro differentiation of hES and hips cells to hemangioblasts. As further differentiation of hemangioblasts to endothelial cells depends solely on VEGF, this system will allow the elucidation of the signaling cascades and the transcriptional circuits of VEGF responsible for the differentiation of precursor cells to endothelium (screening small molecules and siRNA libraries, proteomics, gene expression by microarrays, etc).

    TGFβ family signaling:

    The transforming growth factor β (TGFβ) family of ligands consists of evolutionary conserved pleiotropic secreted cytokines, which include TGFβ1, Activins and bone morphogenetic proteins (BMPs). Individual members of this family play crucial roles in multiple processes throughout development and in the maintenance of tissue homeostasis in adult life. As a consequence, deranged signaling by TGFβ family members has been implicated in many human diseases, including cancer, fibrosis, autoimmune and vascular diseases.

    TGFβ ligands trigger heteromeric complex formation between specific transmembrane type I and type II Ser/Thr kinase receptors, in which the type II receptor transphosphorylates and activates the type I receptor. R-SMADs are phosphorylated by type I receptor, and in turn can form heteromeric complexes with SMAD4. These activated SMAD complexes accumulate in the nucleus, where they directly or indirectly bind to specific promoter region on target genes together with transcription factors and/or co-activators/repressors.

    TGFβ receptors localize to both raft and non-raft membrane domains and the internalization route dictates whether signaling or degradation will ensue. Internalization of TGFβ receptors, via the clathrin-coated pathway into an EEA1 and SARA positive endocytic compartment, where SARA recruits non-phosphorylated SMAD2/3 to the activated receptors for phosphorylation, promotes downstream signaling. However, internalization via the raft-caveolar pathway, where SMAD7 and SMURF2 are localized, promotes ubiquitin-dependent receptor degradation. Indeed, inhibition of this pathway leads to receptor stabilization, suggesting that trafficking of receptors to the SARA positive early endosomes functions to sequester receptors from the rafts and caveolae, thereby stabilizing the receptors. Thus, partitioning between these two internalization pathways appears to be a dynamic and balanced process influencing the signaling outcome of the activated TGFβ family receptors. There is sparse information about the role of internalization of TGFβ family receptors via the other routes or the internalization route of Activin A.

    Current work focuses on:

    1. Studying the interconnection between TGFβ signaling and membrane trafficking
      • Identification of SARA-interacting proteins
      • Elucidation of the internalisation route of Activin A
    2. Elucidating the regulation of Activin A signalling
      • Identification and functional characterization of ActRIIB and ALK4-interacting proteins
      • Identification of regulators of Activin A signaling using high throughput siRNA screens

    Current work extends the investigation of TGFβ family signaling in stem cell biology. More specifically we are:

    1. Investigating the interplay between Activin A/BMP4 signaling and Nanog fluctuations during self-renewal and differentiation of human ES and iPS cells. For this reason we have generated human induced pluripotent stem cells (hips) from human fibroblasts.
    2. Elucidating the role of Activin A/BMP4 signaling in the regulation cell division symmetry/asymmetry during self-renewal and differentiation of human ES and iPS cells.

    Centrosome cycle and the inter-connection with membrane trafficking

    The main functional roles attributed to the centrosome, the major microtubule organizing centre of metazoans, are related to cell locomotion, sensory perception and division. The role of vesicular trafficking in the regulation of the centrosome cycle has been largely unexplored. Recently, however, several studies have indicated the involvement of molecules and/or complexes of the trafficking routes in centrosome positioning, duplication and regulation. Functional screens have revealed communication between the outer nuclear envelope, the Golgi apparatus, the endosomal recycling compartment and centrosomes, while other studies underline the involvement of the ESCRT complex proteins in centrosome function. We have found an involvement of an endosomal Rho protein, namely RhoD, in centrosome duplication and possible links between the centrosome’s structural and functional integrity to vesicular trafficking. We are investigating the molecular mechanism of action of RhoD with an extension to asymmetric cell division.