ABSTRACT
Recombinant Fibroblast Growth Factor-4 (FGF4) and FGF2 induce extracellular signal-regulated kinase-1/2 activation and DNA synthesis in murine aortic endothelial (MAE) cells. These cells co-express the IIIc/Ig-3 loops and the novel glycosaminoglycan-modified IIIc/Ig-2 loops isoforms of FGF receptor-2 (FGFR2). The affinity of FGF4/FGFR2 interaction is 20-30 times lower than that of FGF2 and is enhanced by heparin. Overexpression of FGF2 or FGF4 cDNA in MAE cells results in a transformed phenotype and increased proliferative capacity, more evident for FGF2 than FGF4 transfectants. Both transfectants induce angiogenesis when applied on the top of the chick embryo chorioallantoic membrane. However, in contrast with FGF2-transfected cells, FGF4 transfectants show a limited capacity to growth under anchorage-independent conditions and lack the ability to invade 3D fibrin gel and to undergo morphogenesis in vitro. Also, they fail to induce hemangiomas when injected into the allantoic sac of the chick embryo. In conclusion, although exogenous FGF2 and FGF4 exert a similar response in MAE cells, significant differences are observed in the biological behavior of FGF4 versus FGF2 transfectants, indicating that the expression of the various members of the FGF family can differently affect the behavior of endothelial cells and, possibly, of other cell types, including tumor cells.
Subject(s)
Fibroblast Growth Factors/pharmacology , Fibroblast Growth Factors/physiology , Proto-Oncogene Proteins/pharmacology , Proto-Oncogene Proteins/physiology , 3T3 Cells/physiology , Allantois/blood supply , Animals , Cell Line , Chick Embryo , Chorion/blood supply , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/pharmacology , Fibroblast Growth Factor 4 , Fibroblast Growth Factors/biosynthesis , Fibroblast Growth Factors/genetics , Humans , Mice , Mice, Inbred BALB C , Neovascularization, Physiologic/physiology , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Recombinant Proteins/pharmacology , TransfectionABSTRACT
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 tyrosine-kinase (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 degrees C and 37 degrees 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.
