Expression of angiopoietin-1 in human glioblastomas regulates tumor-induced angiogenesis: in vivo and in vitro studies.

To define a role for the angiopoietin/Tie2 system in astrocytoma angiogenesis, we examined the expression of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) in these tumors by immunohistochemistry and in situ hybridization. Furthermore, we studied in vitro the effects elicited by glioblastoma cell-secreted Ang1 or by recombinant Ang1 on functions of endothelial cells (ECs). Our observations of astrocytomas show that a stage-specific induction of angiopoietins occurs and is correlated with angiogenic phases of different intensity. Ang1 expression was found in a few astrocytes scattered in the tumor at all stages of astrocytoma progression. In blood vessels, Ang1 mRNA increased progressively in high-grade glioblastomas, in which the number of vessels was higher than in low-grade tumors. Ang2 was detected in tumor cells and in ECs in high-grade astrocytomas, whereas its expression was negligible in low-grade tumors. Coculture of glioblastoma cell lines producing Ang1 with endothelium demonstrated a key role of this ligand in the control of EC network organization. We found that recombinant Ang1 in vitro induces EC spreading and reorganization of the cell monolayer into cordlike structures. These results suggest that Ang1 directly acts on ECs by modulating cell-cell and cell-matrix associations and promoting the differentiation phase of angiogenesis.

Concomitant activation of pathways downstream of Grb2 and PI 3-kinase is required for MET-mediated metastasis.

The Met tyrosine kinase - the HGF receptor - induces cell transformation and metastasis when constitutively activated. Met signaling is mediated by phosphorylation of two carboxy-terminal tyrosines which act as docking sites for a number of SH2-containing molecules. These include Grb2 and p85 which couple the receptor, respectively, with Ras and PI 3-kinase. We previously showed that a Met mutant designed to obtain preferential coupling with Grb2 (Met2xGrb2) is permissive for motility, increases transformation, but - surprisingly - is impaired in causing invasion and metastasis. In this work we used Met mutants optimized for binding either p85 alone (Met2xPI3K) or p85 and Grb2 (MetPI3K/Grb2) to evaluate the relative importance of Ras and PI 3-kinase as downstream effectors of Met. Met2xPI3K was competent in eliciting motility, but not transformation, invasion, or metastasis. Conversely, MetP13K/Grb2 induced motility, transformation, invasion and metastasis as efficiently as wild type Met. Furthermore, the expression of constitutively active PI 3-kinase in cells transformed by the Met2xGrb2 mutant, fully rescued their ability to invade and metastasize. These data point to a central role for PI 3-kinase in Met-mediated invasiveness, and indicate that simultaneous activation of Ras and PI 3-kinase is required to unleash the Met metastatic potential.

Tumor necrosis factor-alpha regulates expression of vascular endothelial growth factor receptor-2 and of its co-receptor neuropilin-1 in human vascular endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-alpha does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-alpha up-regulates in a dose- and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As inferred by nuclear run-on assay and transient expression of VEGFR-2 promoter-based reporter gene construct, the cytokine increased the transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding of nuclear transcription factor Sp1 to the promoter consensus sequence, blocked activation of VEGFR-2, suggesting that the up-regulation of the receptor required Sp1 binding sites. TNF-alpha increased the cellular amounts of VEGFR-2 protein and tripled the high affinity 125I-VEGF-A165 capacity without affecting the Kd of ligand-receptor interaction. As a consequence, TNF-alpha enhanced the migration and the wound healing triggered by VEGF-A165. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-alpha is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-alpha-mediated pathological settings.

Uncoupling of Grb2 from the Met receptor in vivo reveals complex roles in muscle development.

Hepatocyte growth factor (HGF) and its receptor, the Met tyrosine kinase, are determinants of placenta, liver, and muscle development. Here, we show that Met function in vivo requires signaling via two carboxy-terminal tyrosines. Mutation of both residues in the mouse genome caused embryonal death, with placenta, liver, and limb muscle defects, mimicking the phenotype of met null mutants. In contrast, disrupting the consensus for Grb2 binding allowed development to proceed to term without affecting placenta and liver but caused a striking reduction in limb muscle coupled to a generalized deficit of secondary fibers. These data show that the requirements for Met signaling vary depending on the tissue and reveal a novel role for HGF/ Met in late myogenesis.

Specific uncoupling of GRB2 from the Met receptor. Differential effects on transformation and motility.

The biological effects of hepatocyte growth factor/scatter factor are mediated by autophosphorylation of its receptor, the Met tyrosine kinase, on two carboxyl-terminal tyrosines. These phosphotyrosines (Y1349VHVNATY1356VNV) are multifunctional docking sites for several effectors. Grb2, the adaptor for the Ras guanyl-nucleotide exchanger SOS, binds to Tyr1356 in the YVNV motif. By site-directed mutagenesis we either abrogated or duplicated the Grb2 consensus, without interfering with the other effectors. Loss of the link with Grb2 severely impaired transformation. The same mutation, however, had no effect on the "scattering" response, indicating that the level of signal which can be reached by Grb2-independent routes is permissive for motility. Duplication of the Grb2 binding site enhanced transformation and left motility unchanged. Thus, two Met-mediated biological responses, motility and growth, can be dissociated on the basis of their differential requirement for a direct link with Ras.