Fig. 4

Impact of galectins on FGFR1 activity. a and b Impact of extracellular galectin-1 and -3 on the activation of FGFR1 and downstream signaling. Serum starved NIH3T3 cells were incubated for 15 min with FGF1 (50 ng/ml) and heparin (10 U/ml) or galectin-1 and -3 (0.5–10 μg/ml) either in the absence (a) or presence (b) of FGFR kinase inhibitor PD173074 (100 nM). Cells were lysed and activation of cellular signaling cascades was assessed with western blotting. c FGFR1 dependence of galectins signaling. Serum starved U2OS-R1 and U2OS-R1-K514R cells were incubated for 15 min with FGF1 (50 ng/ml) and heparin (10 U/ml) or galectin-1 and galectin-3 (10 μg/ml). Cellular signaling was studied with western blotting. d Anti-apoptotic activity of galectin-1 assessed with annexin assay. NIH3T3 cells were serum starved to induce apoptosis and treated with FGF1 (200 ng/ml) and heparin (10 U/ml), galectin-1 or galectin-3 (10 μg/ml) in the presence or absence of FGFR inhibitor (100 nM PD173074) for 24 h. Average values from three experiments +/− SD are shown. Student t-test was applied for statistical analysis (** p < 0.005; n.s. – not significant). e, f Anti-apoptotic activity of galectin-1 fully depends on FGFR1 kinase activity. U2OS-R1 or U2OS-R1-K514R cells were subjected to serum starvation for 24 h to induce apoptosis. Cells were treated with FGF1 (200 ng/ml) and heparin (10 U/ml) or galectin-1 (10 μg/ml) for 16 h. Next, caspase-3/7 activity was determined, normalized to cells untreated with FGF1 and galectins and denoted as relative caspase-3/7 activity. All experiments were performed three times. Average values +/− SD are shown. Student t-test was applied for statistical analysis (*p < 0.05; ** p < 0.005; n.s. – not significant). g Galectins induce cell proliferation. Serum starved NiH3T3 cells were treated with galectin-1 and galectin-3 (1–20 μg/ml) or FGF1 (1 ng/ml) and heparin (10 U/ml) in the presence or absence of 100 nM PD173074. Cells were incubated at 37 °C, 5% CO2 for 48 h and cell proliferation was determined with Alamar Blue. Average values from six experiments +/− SEM are shown. h Model of galectin-1 and galectin-3 impact on FGFR1. By binding to the sugar chains on the extracellular region of FGFR1 galectins differentially modulate membrane distribution of the receptor and its function. Dimeric galectin-1 induces formation of FGFR1 dimers/clusters that are tyrosine-phosphorylated and initiate downstream signaling resulting in cell proliferation and avoidance of apoptosis. In contrast, larger pentameric galectin-3 triggers extensive FGFR1 cross-linking on the cell surface. In these clusters FGFR1 molecules are separated from each other in orientation that does not permit receptor activation. FGFR1 lattice induced by galectin-3 retains receptor on the cell surface, downregulating constitutive internalization of the receptor in the absence of the growth factor