Fibroblast growth factor blocks Sonic hedgehog signaling in neuronal precursors and tumor cells

Abstract

The Sonic hedgehog (Shh) and FGF signaling pathways regulate growth and differentiation in many regions of the nervous system, but interactions between these pathways have not been studied extensively. Here, we examine the relationship between Shh and FGF signaling in granule cell precursors (GCPs), which are the most abundant neural progenitors in the cerebellum and the putative cell of origin for the childhood brain tumor medulloblastoma. In these cells, Shh induces a potent proliferative response that is abolished by coincubation with basic FGF. FGF also inhibits transcription of Shh target genes and prevents activation of a Gli-responsive promoter in fibroblasts, which suggests that it blocks Shh signaling upstream of Gli-mediated transcription. FGF-mediated inhibition of Shh responses requires activation of FGF receptors and of ERK and JNK kinases, because it can be blocked by inhibitors of these enzymes. Finally, FGF promotes differentiation of GCPs in vitro and in vivo and halts proliferation of tumor cells from patched (ptc) mutant mice, a model for medulloblastoma. These findings suggest that FGF is a potent inhibitor of Shh signaling and may be a useful therapy for tumors involving activation of the hedgehog pathway.

Fig. 1.

FGF inhibits Shh-induced proliferation of purified GCPs. (A) GFP⁺ cells were sorted from Math1-GFP neonates and cultured for 48 h without (Control) or with recombinant Shh (3 μg/ml) and the indicated amounts of bFGF. Cells were pulsed with [³H]thymidine (3H-Td) and cultured overnight before being assayed for 3H-Td incorporation. (B) Effects of other receptor tyrosine kinase (RTK)-activating factors. GCPs were cultured with no stimuli (Con), Shh, or Shh plus bFGF (25 ng/ml), nerve growth factor (NGF) (100 ng/ml), PDGF-AA (10 ng/ml), insulin-like growth factor 1 (IGF-1) (25 ng/ml), or EGF (25 ng/ml). Con, control. Cells were assayed for 3H-Td incorporation as described above. Data represent means ± SEM of triplicate samples. ∗∗, P < 0.001 (one-way ANOVA).

Fig. 2.

FGF prevents induction of Shh target genes. (A) Inhibition of gli1 induction in GCPs. GCPs were cultured with no stimulus (Con), Shh, or Shh plus bFGF for the indicated times. RNA was analyzed by real-time RT-PCR with primers for gli1 or actin mRNA. gli1 expression was normalized to actin mRNA and divided by levels in control GCPs to calculate fold change. (B) Inhibition of Shh responses in Shh-Light2 cells. Cells were cultured for 48 h in the absence (Control) or presence of Shh plus the indicated concentrations of bFGF and assayed for luciferase activity. Data represent means ± SEM of four samples. ∗∗, P < 0.001 (one-way ANOVA).

Fig. 3.

FGF-mediated inhibition requires signaling through FGFRs. GCPs (A) or Shh-Light2 cells (B) were pretreated with vehicle (DMSO) or with the FGFR inhibitor PD173074 (0.05 μM for GCPs, 0.1 μM for Shh-Light2) for 1 h and then cultured for 48 h with no stimulus (Con) or Shh with or without bFGF. GCPs were assayed for 3H-Td incorporation, and Shh-Light2 cells were assayed for luciferase activity as described above. Data represent means ± SEM of three samples. ∗∗, P < 0.001 (one-way ANOVA).

Fig. 4.

FGF-mediated inhibition depends on MAPK activity. (A) ERK and JNK inhibitors block bFGF-mediated inhibition in GCPs. Cells were pretreated with DMSO, MEK inhibitor (MEKi) (U0126, 10 μM), JNK inhibitor (JNKi) (SP600125, 10 μM), or both for 1 h before addition of Shh in the presence or absence of bFGF. After 48 h, cells were pulsed with 3H-Td, cultured for 18 h, and assayed for thymidine incorporation. Data represent means ± SEM of six samples. ∗∗, P < 0.001 (one-way ANOVA). (B) FGF activates ERK and JNK in GCPs. GCPs were cultured with no stimulus (Con) or Shh in the presence or absence of bFGF for 30 min. Cells were lysed, and expression of phospho-ERK (P-ERK), ERK, phospho-c-jun (P-jun), c-jun, and actin were examined by using Western blotting. Note the increase in phospho-ERK and phospho-jun in FGF-treated cells. (C) FGF activates ERK and JNK in Shh-Light2 cells. Cells were pretreated with DMSO, FGFR inhibitor (FGFRi) (PD173074, 0.1 μM), or JNK inhibitor (JNKi) and then treated with Shh in the presence or absence of bFGF for 5 or 30 min. Levels of phospho-ERK, ERK, phospho-c-jun, c-jun, and actin were examined by using Western blotting. Note the increase in phospho-ERK and phospho-c-jun in FGF-treated cells. ERK phosphorylation is blocked by FGFR inhibitor, whereas c-jun phosphorylation is blocked by both FGFR inhibitor and JNK inhibitor.

Fig. 5.

bFGF promotes differentiation of GCPs in vitro. GCPs were cultured for 48–72 h with no stimulus (A, E, I, and M), Shh (B, F, J, and N), bFGF (C, G, K, and O), or bFGF plus Shh (D, H, L, and P). (A–D) Bright-field images of cells cultured for 48 h. (E–H) GFP fluorescence of Math1-GFP⁺ cells cultured for 48 h. (I–L) Ki-67 staining in cells cultured for 48 h. (M–P) MEF2D expression in cells cultured for 72 h. (Magnification: A–D, ×20; E–H, ×20; M–P, ×40.)

Fig. 6.

bFGF accelerates differentiation of GCPs in vivo. Math1-GFP mice were given intracisternal injections of vehicle (Control, A–C) or bFGF (D–F) on P4, P5, and P6, and i.p. injections of BrdU on P6. At P7, cerebella were fixed and stained with anti-GFP (A and D), anti-BrdU (B and E), or anti-NeuN antibodies (C and F) with a DAPI counterstain (blue). Note the reduced number of immature, proliferating (GFP-high, BrdU⁺) cells and the abundance of differentiated (NeuN⁺) cells in the EGL of FGF-treated mice (see arrows in A vs. D, B vs. E, and C vs. F). Data are representative of six mice.

Fig. 7.

bFGF blocks proliferation of medulloblastoma cells from ptc mutant mice. (A and B) FGF inhibits Shh target genes and proliferation. Tumor cells from ptc^+/− mice were pretreated with DMSO or FGFR inhibitor (PD173074, 100 nM), cultured in the absence (Control) or presence of bFGF for 24–48 h, and then harvested for RNA analysis (A) or pulsed and assayed for 3H-Td incorporation (B). Data represent means ± SEM of six samples. ∗, P < 0.01; ∗∗, P < 0.001 (one-way ANOVA). (C–H) FGF promotes differentiation of tumor cells. Tumor cells from Math1-GFP/ptc^+/− mice were cultured with no stimulus (Con) (C, E, and G) or bFGF (D, F, and H) for 24–48 h and then photographed by using bright-field or fluorescent microscopy. (C and D) Bright-field images at 24 h. (E and F) Math1-GFP expression at 24 h. (G and H) Math1-GFP expression at 48 h. (Magnification: ×20).