Heparin-dependent fibroblast growth factor activities: effects of defined heparin oligosaccharides

Heparin and related molecules have been identified as important participants in fibroblast growth factor (FGF) signaling although the mechanisms of action remain unclear. We have used heparin oligosaccharides to examine steps in the signaling process which could be affected by the polysaccharide. Immobilized FGF-1 and FGF-2 bound all sizes of oligosaccharides tested, ranging from tetrasaccharide to decasaccharide, at physiological salt concentration. Each group of oligosaccharide was eluted from the FGF affinity columns in several peaks, and larger oligosaccharides showed higher apparent affinity for the immobilized growth factors compared to the shorter ones. Heparin hexasaccharides were the smallest fragments providing complete protection of FGF-1 and FGF-2 against trypsin digestion. Tetrasaccharides, however, were able to provide partial protection. The requirement of heparin for ligand-receptor interaction was evaluated in receptor binding assays using Sf9 insect cells engineered to overexpress different recombinant FGF receptor (FGFR) species including FGFR1 beta, FGFR1 alpha or FGFR4 at the cell surface. In these assays hexasaccharides were the smallest fragments capable of stimulating FGF-receptor interaction. Over the range of concentrations examined, neither hexasaccharides nor octasaccharides were able to stimulate receptor binding to the level attained by intact heparin. In fact, these oligosaccharides interfered with the ability of intact heparin in promoting FGF-receptor binding. The presence of both stimulatory and inhibitory activities in hexasaccharide and octasaccharide populations could be attributed to structural heterogeneity within the oligosaccharide preparations. However, similar observations were obtained with "highly-sulfated" structurally homogeneous preparations of hexasaccharide and octasaccharide, although these molecules generally had greater stimulatory and less inhibitory activity than their structurally heterogeneous counterparts. Hexasaccharides were found to be the smallest fragments able to potentiate the FGF-1-induced 3T3 cell proliferation while their effect on FGF-2 signaling was less clear. These observations suggest that heparin can modulate FGF-signaling at several stages with different end results.