Showing papers by "Luigi Naldini published in 1992"

Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth

Abstract: Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit HGF binding to endothelial cells identifies two sites with different affinities The higher affinity binding site (Kd = 035 nM) corresponds to the p190MET receptor Sub-nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility HGF induces repairs of a wound in endothelial cell monolayer HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate

Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor.

Abstract: The extracellular protease urokinase is known to be crucially involved in morphogenesis, tissue repair and tumor invasion by mediating matrix degradation and cell migration. Hepatocyte growth factor/scatter factor (HGF/SF) is a secretory product of stromal fibroblasts, sharing structural motifs with enzymes of the blood clotting cascade, including a zymogen cleavage site. HGF/SF promotes motility, invasion and growth of epithelial and endothelial cells. Here we show that HGF/SF is secreted as a single-chain biologically inactive precursor (pro-HGF/SF), mostly found in a matrix-associated form. Maturation of the precursor into the active alpha beta heterodimer takes place in the extracellular environment and results from a serum-dependent proteolytic cleavage. In vitro, pro-HGF/SF was cleaved at a single site by nanomolar concentrations of pure urokinase, generating the active mature HGF/SF heterodimer. This cleavage was prevented by specific urokinase inhibitors, such as plasminogen activator inhibitor type-1 and protease nexin-1, and by antibodies directed against the urokinase catalytic domain. Addition of these inhibitors to HGF/SF responsive cells prevented activation of the HGF/SF precursor. These data show that urokinase acts as a pro-HGF/SF convertase, and suggest that some of the growth and invasive cellular responses mediated by this enzyme may involve activation of HGF/SF.

A functional domain in the heavy chain of scatter factor/hepatocyte growth factor binds the c-Met receptor and induces cell dissociation but not mitogenesis.

Abstract: We recently found that scatter factor (SF), a cell motility factor with a multimodular structure, is identical to hepatocyte growth factor (HGF), a potent mitogen of various cell types. SF/HGF is the ligand of the c-Met receptor tyrosine kinase. Here we used transient expression of naturally occurring and in vitro mutagenized cDNAs of SF/HGF to delineate the protein domains necessary for biological activity and binding to the c-Met receptor. (i) A single-chain SF/HGF resulting from the destruction of the protease cleavage site between heavy and light chain (Arg-494--> Gln) was largely inactive, indicating that proteolytic cleavage is essential for acquisition of the biologically active conformation. (ii) A SF/HGF splice variant encoding a protein with a 5-amino acid deletion in the first kringle domain was as highly active as the wild-type molecule. (iii) The separately expressed light chain (with serine protease homology) was inactive in all assays tested. (iv) The separate heavy chain as well as a naturally occurring splice variant consisting of the N terminus and the first two kringle domains bound the c-Met receptor, stimulated tyrosine auto-phosphorylation, and induced scattering of epithelial cells but not mitogenesis. These data indicate that a functional domain in the N terminus/first two kringle regions of SF/HGF is sufficient for binding to the Met receptor and that this leads to the activation of the downstream signal cascade involved in the motility response. However, the complete SF/HGF protein seems to be required for mitogenic activity.

The Receptor for the Hepatocyte Growth Factor-Scatter Factor: Ligand-Dependent and Phosphorylation-Dependent Regulation of Kinase Activity

Abstract: Hepatocyte Growth Factor (HGF) and Scatter Factor (SF) are identical molecules produced by the stromal fibroblats and non-parenchimal cells of many organs and also present in serum. HGF exerts an array of activities on epitelial cells, i.e. mitogenesis, dissociation of epithelial sheets, stimulation of cell motility, and promotion of matrix invasion. The receptor for HGF is the tyrosine kinase encoded by the MET proto-oncogene. It is widely expressed in normal epithelial tissues as a 190 kDa heterodimer of two disulfide-linked protein subunits. HGF binding triggers tyrosine autophosphorylation of the receptor β subunit in intact cultured cells. Autophosphorylation upregulates the kinase activity of the receptor, increasing the Vmax of the phosphotransfer reaction. The major phosphorylation site has been mapped to Tyr 1235. Negative regulation of the receptor kinase activity occurs through distinguishable pathways involving protein kinase C activation or increase in the intracellular Ca2+ concentration. Both lead to the phosphorylation of serine residue(s) in a unique tryptic phosphopeptide of the receptor and to a decrease in its tyrosine phosphorylation and kinase activity. Receptor autophosphorylation also triggers the signal transduction pathways inside the target cells. The phosphorylated receptor associates phosphatidylinositol 3-kinase, indicating that the generation of the D-3 phosphorylated inositol lipids is involved in effecting the motility and/or growth response to HGF.