Effects of FGFR Tyrosine Kinase Inhibition in OLN-93 Oligodendrocytes

Abstract

Fibroblast growth factor (FGF) signaling is involved in the pathogenesis of multiple sclerosis (MS). Data from neuropathology studies suggest that FGF signaling contributes to the failure of remyelination in MS. In MOG_35-55-induced EAE, oligodendrocyte-specific deletion of FGFR1 and FGFR2 resulted in a less severe disease course, reduced inflammation, myelin and axon degeneration and changed FGF/FGFR and BDNF/TrkB signaling. Since signaling cascades in oligodendrocytes could not be investigated in the EAE studies, we here aimed to characterize FGFR-dependent oligodendrocyte-specific signaling in vitro. FGFR inhibition was achieved by application of the multi-kinase-inhibitor dovitinib and the FGFR1/2/3-inhibitor AZD4547. Both substances are potent inhibitors of FGF signaling; they are effective in experimental tumor models and patients with malignancies. Effects of FGFR inhibition in oligodendrocytes were studied by immunofluorescence microscopy, protein and gene analyses. Application of the tyrosine kinase inhibitors reduced FGFR1, phosphorylated ERK and Akt expression, and it enhanced BDNF and TrkB expression. Furthermore, the myelin proteins CNPase and PLP were upregulated by FGFR inhibition. In summary, inhibition of FGFR signaling in oligodendrocytes can be achieved by application of tyrosine kinase inhibitors. Decreased phosphorylation of ERK and Akt is associated with an upregulation of BDNF/TrkB signaling, which may be responsible for the increased production of myelin proteins. Furthermore, these data suggest that application of FGFR inhibitors may have the potential to promote remyelination in the CNS.

Keywords: AZD4547; FGFR signaling; dovitinib; multiple sclerosis; myelin; oligodendrocytes.

Figure 1

Cell viability (A,B) and FGFR expression (C–E) of OLN-93 cells incubated with dovitinib, AZD4547 or FGF2 for 24 h. Both FGFR inhibitors induced anti-proliferative responses in OLN-93 cells (A), while only AZD4547 was cytotoxic to these cells (B). Representative sections of immunofluorescence staining (C). Quantitative analysis of immunofluorescence staining revealed reduced numbers of FGFR1⁺ cells after dovitinib and AZD4547 treatment (D). FGF2 did not affect the number FGFR1⁺ cells. qRT-PCR showed downregulation of FGFR (FGFR1) mRNA only in cells treated with AZD4547 (E). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.005, *** p < 0.001 vs. control; ^## p < 0.005, ^### p < 0.001 vs. FGF2.

Figure 2

Cellular distribution (A,B) and protein expression (C,D) of pAkt. Representative sections of immunofluorescence staining (A). Quantification of fluorescence exposed a reduction in pAkt⁺ cell count for both inhibitors to less than 60% (B). Representative Western blot images of pAkt analysis (C). pAkt levels as quantified by Western blot (D). Data are presented as mean ± SEM. *** p < 0.001 vs. control; ^# p < 0.05, ^### p < 0.001 vs. FGF2.

Figure 3

Cellular staining (A,B) for pERK and its protein expression (C,D). Representative sections for distribution and prevalence of pERK (A). The number of pERK⁺ cells was reduced by more than 40% in all groups treated with FGFR inhibitors (B). Representative images of pERK Western blot (C). pERK protein levels were reduced by dovitinib (D). Data are presented as mean ± SEM. *** p < 0.001 vs. control; ^# p < 0.05, ^### p < 0.001 vs. FGF2.

Figure 4

Cellular distribution (A,B), mRNA expression (C) and total amount of BDNF protein (D,E). Representative images are shown for immunofluorescence staining (A). Quantification of fluorescence showed a 40% enhancement of BDNF⁺ cell numbers by dovitinib (B). BDNF (BDNF) mRNA was abundant after inhibition with AZD4547 (C). Representative Western blot bands of BDNF protein expression (D). BDNF protein expression was increased by dovitinib (E). Data are presented as mean ± SEM. * p < 0.05, *** p < 0.001 vs. control; ^# p < 0.05, ^### p < 0.001 vs. FGF2.

Figure 5

Number of TrkB⁺ cells (A,B), TrkB mRNA (C) and protein levels (D,E). Representative sections of immunofluorescence staining (A). Analysis of immunofluorescence staining revealed increased numbers of TrkB⁺ cells by dovitinib and AZD4547 of more than 20% (B). qRT-PCR showed an upregulation of TrkB (NTRK2) mRNA only in cells treated with AZD4547 (C). Representative Western blot images for TrkB (D). TrkB protein levels were increased by dovitinib (E). Data are presented as mean ± SEM. ** p < 0.005, *** p < 0.001 vs. control; ^## p < 0.005, ^### p < 0.001 vs. FGF2.

Figure 6

Distribution of PLP⁺ cells (A,B), PLP mRNA expression (C) and its amount of total protein (D,E). Representative images for immunofluorescence staining (A). Quantification of fluorescence revealed doubled PLP⁺ cell numbers after treatment with the FGFR inhibitors (B). mRNA expression of PLP (PLP1) was increased by treatment (C). Representative Western blot images of PLP analysis (D). PLP protein was increased by dovitinib (E). Data are presented as mean ± SEM. * p < 0.05, *** p < 0.001 vs. control; ^# p < 0.05, ^### p < 0.001 vs. FGF2.

Figure 7

Immunofluorescence staining for CNPase⁺ cells (A,B), CNPase mRNA (C) and protein levels (D). Depicted representative sections showed increase in distribution and prevalence of CNPase (A). The number of CNPase⁺ cells was increased by more than 60% by AZD4547 or dovitinib treatment (B). mRNA analysis revealed decreased expression of CNPase (CNP) mRNA by FGF2; FGFR inhibitors enhanced mRNA levels (C). Representative Western blot bands for CNPase (D). Quantification of CNPase protein revealed increased expression by the inhibitors (E). Data are presented as mean ± SEM. ** p < 0.005, *** p < 0.001 vs. control; ^## p < 0.005, ^### p < 0.001 vs. FGF2.

Figure 8

mRNA levels of TGFβ1 and SEMA3A. Expression of TGFβ (A) and SEMA3A (B) mRNA was reduced by AZD4547. While dovitinib and FGF2 did not regulate TGFβ (TGFB1) mRNA (A), AZD4547 reduced TGFβ mRNA expression (A) as well as mRNA levels of SEMA3A (SEMA3A) (B). Data are presented as mean ± SEM. * p < 0.05, *** p < 0.001 vs. control; ^## p < 0.005 vs. FGF2.