FHF1 is a bona fide fibroblast growth factor that activates cellular signaling in FGFR-dependent manner

Abstract

Fibroblast growth factors (FGFs) via their receptors (FGFRs) transduce signals from the extracellular space to the cell interior, modulating pivotal cellular processes such as cell proliferation, motility, metabolism and death. FGF superfamily includes a group of fibroblast growth factor homologous factors (FHFs), proteins whose function is still largely unknown. Since FHFs lack the signal sequence for secretion and are unable to induce FGFR-dependent cell proliferation, these proteins were considered as intracellular proteins that are not involved in signal transduction via FGFRs. Here we demonstrate for the first time that FHF1 directly interacts with all four major FGFRs. FHF1 binding causes efficient FGFR activation and initiation of receptor-dependent signaling cascades. However, the biological effect of FHF1 differs from the one elicited by canonical FGFs, as extracellular FHF1 protects cells from apoptosis, but is unable to stimulate cell division. Our data define FHF1 as a FGFR ligand, emphasizing much greater similarity between FHFs and canonical FGFs than previously indicated. Video Abstract. (MP4 38460 kb).

Keywords: Apoptosis; Cell proliferation; FGF; FGFR; FHF; Glucose uptake; Signal transduction.

Fig. 1

Extracellular FHF1 activates FGFR1. a SDS-PAGE, b western blotting and c mass spectrometry analysis of recombinant FHF1 produced in bacterial expression system (calculated mass 24,302 Da). d CD spectrum of FHF1 between 205 and 260 nm. e Thermal denaturation curve of FHF1. f and g Activation of FGFR and downstream signaling by FHF1 and FGF1 in NIH3T3 (f) and U2OS-R1 (g) cells in the absence and presence of FGFR specific inhibitor (100 nM PD173074) assessed with western blotting. h Concentration-dependent activation of FGFR1 and ERKs cascades in NIH3T3 cells by FHF1 and FGF1 verified as in (f) and (g)

Fig. 2

FHF1 directly interacts with FGFRs. a ELISA analysis of binding of FHF1 and FGF1 to extracellular domains of FGFRs. Mean values of five independent experiments ± SEM are shown. Student t-test was applied for statistical analysis; ** p < 0.01, *** p < 0.001. b Scheme of SPR experiments using sensor chip with immobilized extracellular part of FGF receptors fused to the Fc fragment. c SPR analyses of FHF1 interaction with FGFRs immobilized at high density on CM5 sensor (9000 RU). d Kinetics of FHF1-FGFR1 interaction assessed with SPR. The FHF1 protein at the concentrations from 0.1 μM to 3.2 μM was injected on CM4 sensor surface with FGFR1-Fc immobilized at low density (1000 RU). Equilibrium dissociation constant (K_D) was calculated from saturation binding curve. e Soluble extracellular domains of all FGFRs block the activation of FGFR by FHF1 in NIH3T3 cells. Cells were pre-incubated with FGFR1–4 fused to Fc fragment and the ability of FHF1 and FGF1 to trigger cell response was assessed with western blotting

Fig. 3

Biological activities of FHF1. a Impact of FHF1 and FGF1 on cell proliferation in NIH3T3 and BaF3-R1c cells. The data shown are mean values ± SD of three independent experiments, presented as a percentage of maximal response. b Effect of FHF1 and FGF1 on glucose uptake in 3T3-L1 adipocytes. The data shown are mean values ± SD of three independent experiments expressed as a percentage of glucose uptake induced by serum. Student t-test was applied for statistical analysis; * p < 0.05, ** p < 0.01, n.s. – not significant. c Anti-apoptotic properties of FHF1 and FGF1 assessed by measurements of cell viability upon induction of apoptosis with 1 μM staurosporine in U2OS-R1 cells. The data shown are mean values ± SD of three independent experiments expressed as a percentage of viability of untreated cells; * p < 0.05, ** p < 0.01, *** p < 0.001. d Ratio of caspase 3/7 activity in serum-starved NIH3T3 cells stimulated with FHF1 and FGF1 to cell viability. The data shown are mean values ±SD of three independent experiments normalized toward untreated cells (relative caspase-3/7 activity). Student t-test was applied for statistical analysis; ** p < 0.01, *** p < 0.001. e PARP and caspase 3 cleavage in U2OS-R1 cells upon induction of apoptosis with 1 μM staurosporine and FHF1 or FGF1 treatment. f Long-term stability of FHF1 in cell conditioned media. FHF1 and FGF1 were incubated with NIH3T3 cells for 48 h, and then media containing recombinant proteins were aspirated and tested for their FGFR stimulatory activity with western blotting. Freshly prepared solution of recombinant FHF1 and FGF1 were used as controls