A distinct basic fibroblast growth factor (FGF-2)/FGF receptor interaction distinguishes urokinase-type plasminogen activator induction from mitogenicity in endothelial cells.

Abstract

Basic fibroblast growth factor (FGF-2) induces cell proliferation and urokinase-type plasminogen activator (uPA) production in fetal bovine aortic endothelial GM 7373 cells. In the present paper we investigated the role of the interaction of FGF-2 with tyrosinekinase (TK) FGF receptors (FGFRs) in mediating uPA up-regulation in these cells. The results show that FGF-2 antagonists suramin, protamine, heparin, the synthetic peptide FGF-2(112-155), and a soluble form of FGFR-1 do not inhibit FGF-2-mediated uPA up-regulation at concentrations that affect growth factor binding to cell surface receptors and mitogenic activity. In contrast, tyrosine phosphorylation inhibitors and overexpression of a dominant negative TK-mutant of FGFR-1 abolish the uPA-inducing activity of FGF-2, indicating that FGFR and its TK activity are essential in mediating uPA induction. Accordingly, FGF-2 induces uPA up-regulation in Chinese hamster ovary cells transfected with wild-type FGFR-1, -2, -3, or -4 but not with TK-FGFR-1 mutant. Small unilamellar phosphatidyl choline:cholesterol vesicles loaded with FGF-2 increased uPA production in GM 7373 cells in the absence of a mitogenic response. Liposome-encapsulated FGF-2 showed a limited but significant capacity, relative to free FGF-2, to interact with FGFR both at 4°C and 37°C and to be internalized within the cell. uPA up-regulation by liposome-encapsulated FGF-2 was quenched by neutralizing anti-FGF-2 antibodies, indicating that the activity of liposome-delivered FGF-2 is mediated by an extracellular action of the growth factor. Taken together, the data indicate that a distinct interaction of FGF-2 with FGFR, quantitatively and/or qualitatively different from the one that leads to mitogenicity, is responsible for the uPA-inducing activity of the growth factor.

Blocking the FGF/FGFR system as a ⿿two-compartment⿿ antiangiogenic/antitumor approach in cancer therapy

“…As an example, the peptide comprising the amino acid sequence FGF2 impaired the interaction of FGF2 with FGFR1 [125] and similar peptides were successfully employed to specifically deliver antiblastic drugs to FGFR-overexpressing tumor cells [126]. In addition, vaccination against FGFR1 showed antitumor activity in vivo [127] and anti-FGFR neutralizing antibodies blocked FGF2-mediated angiogenesis in vivo [113,[128][129][130].…”

Biological activity of substrate-bound basic fibroblast growth factor (FGF2): recruitment of FGF receptor-1 in endothelial cell adhesion contacts

“…After 6 h, adherent cell monolayers were incubated for 2 h at 48C with 125 I-FGF2 (10 ng/ml) and its binding to high anity FGFRs was evaluated (Rusnati et al, 1996). FGF2-adherent cells showed a signi®cant reduction in the capacity to bind 125 I-FGF2 at the apical side of the cell monolayer (5+2 c.p.m./well) when compared to cells adherent to FN (151+20 c.p.m./well) or to tissue culture plastic (225+30 c.p.m./well) (n=3).…”

“…GM7373 cells were transfected with 91023b-¯g in association (10 : 1, wt:wt) with pCB7 according to a calcium phosphate precipitation protocol as described (Rusnati et al, 1996) and selected with 250 mg/ml of G418 (Sigma, St. Louis, MO, USA). Parallel cultures were transfected with pCEP4-¯g 1.2 and selected with 200 mg/ml of hygromycin B (Boehringer Mannheim GmbH, Mannheim).…”

“…Parallel cultures were transfected with pCEP4-¯g 1.2 and selected with 200 mg/ml of hygromycin B (Boehringer Mannheim GmbH, Mannheim). For each transfection, resistant clones were tested for 125 I-FGF2 binding capacity (Rusnati et al, 1996) and by Western blotting using anti-FGFR1 antibody (Upstate Biotechnology, Lake Placid, NY, USA).…”

Identification and Characterization of a Fibroblast Growth Factor (FGF) Binding Domain in the Cysteine-rich FGF Receptor

“…The majority of FGF-mediated signaling events appear to require FGFRs, but additional requirements involving internalization and intracellular transport of the FGF ligand have been proposed (28 -30). A role for other FGF-binding proteins in mediating FGF signaling is thus expected, since these recent reports have identified FGF receptor tyrosine kinase-independent activities of FGFs that include the stimulation of DNA synthesis (29) (29 -31) and the regulation of plasminogen activator activity (32,33). In addition, mutants of both FGF-1 and FGF-2 have revealed that the biological activities of these factors do not entirely correlate with their ability to stimulate tyrosine phosphorylation of the FGFRs (34).…”