Macrophage migration inhibitory factor promotes proliferation and neuronal differentiation of neural stem/precursor cells through Wnt/β-catenin signal pathway

Abstract

Macrophage migration inhibitory factor (MIF) is a highly conserved and evolutionarily ancient mediator with pleiotropic effects. Recent studies demonstrated that the receptors of MIF, including CD44, CXCR2, CXCR4 and CD74, are expressed in the neural stem/progenitor cells (NSPCs). The potential regulatory effect of MIF on NSPCs proliferation and neuronal differentiation, however, is largely unknown. Here, we investigated the effect of MIF on NSPC proliferation and neuronal differentiation, and further examined the signal pathway by which MIF transduced these signal effects in mouse NSPCs in vitro. The results showed that both Ki67-positive cells and neurosphere volumes were increased in a dose-dependent manner following MIF treatment. Furthermore, the expression of nuclear β-catenin was significantly stronger in MIF-stimulated groups than that in control groups. Conversely, administration of IWR-1, the inhibitor of Wnt/β-catenin pathway, significantly inhibited the proliferative effect of MIF on NSPCs. Immunostaining and Western blot further indicated that doublecortin (DCX) and Tuj 1, two neuronal markers, were evidently increased with MIF stimulation during NSPC differentiation, and there were more Tuj1-positive cells migrated out from neurospheres in MIF-stimulated groups than those in control groups. During NSPC differentiation, MIF increased the activity of β-galactosidase that responds to Wnt/β-catenin signaling. Wnt1 and β-catenin proteins were also up-regulated with MIF stimulation. Moreover, the expression of DCX and Tuj 1 was inhibited significantly by IWR-1. Taken together, the present study indicated that MIF enhances NSPC proliferation and promotes the neuronal differentiation, by activating Wnt/β-catenin signal pathway. The interaction between MIF and Wnt/β-catenin signal pathway may play an important role in modulating NSPC renewal and fate during brain development.

Keywords: MIF; NSPC; Wnt/β-catenin; neuronal differentiation; proliferation.

Figure 1

Identification of NSPCs (neurospheres) derived from mouse brain. The left image of the upper panel shows the cultured NSPCs grow in a manner of neurospheres. The middle and the right of the upper panel show that cells of neurospheres are Nestin-positive and Nestin/Hoechst double staining, respectively. The lower panel shows the Tuj 1- (for neuron, 4 days after differentiation), GFAP- (for astrocyte, 4 days after differentiation), and CNPase- (for oligodendrocyte, 10 days after differentiation) positive cells differentiated from neurospheres, respectively.

Figure 2

The promoting effects of MIF on NSPC proliferation. A. Representative images of immunostaining show Ki67-postive cells (upper panel) and Ki67/Hoechst double staining cells (lower panel) in control and MIF-stimulated groups (MIF 16ng/ml); and the comparison of Ki67-positive cell percentages between control and MIF-stimulated groups (^*p<0.05 vs. control group, n=3 animals in each group). B. Representative images show neurospheres incubated with different concentration of MIF (upper panel); and comparisons of the neurosphere volumes and numbers at different concentration of MIF by ANOVA (^**p<0.0001 vs. MIF 0ng/ml group, n=6 wells in each group, repeated 3 times) (lower panel). CON, control group; MIF, MIF-stimulated group.

Figure 3

The involvement of Wnt/β-catenin pathway in NSPC proliferation induced by MIF. A. Representative images of immunostaining show β-catenin-postive cells (upper panel) and β-catenin/Hoechst double staining cells (lower panel) in control and MIF-stimulated groups; and the comparison of nuclear β-catenin fluorescence intensity between control and MIF-stimulated groups (^*p<0.05 vs. control group, n=3 animals in each group). B. Western blot results show the protein bands of β-catenin in NSPCs and the comparison of Western blot result showing the relative β-catenin levels in control and MIF-stimulated groups (^**p<0.0001 vs. control group, n=3 animals in each group). Results were normalized to β-actin. CON, control group; MIF, MIF-stimulated group. C. Representative images show neurospheres incubated with different concentration of MIF, with or without IWR-1 (upper panel); and the comparisons of neurosphere volumes among different groups by ANOVA (^*p<0.05, ^**p<0.0001, n=6 wells in each group, repeated 3 times) (lower panel). D. The volume comparisons of neurospheres incubated with different concentration of IWR-1 by ANOVA (^*p<0.05, n=8 wells in each group, repeated 3 times)

Figure 4

MIF promotes NSPC differentiation to neuron lineage. A. Representative images of immunostaining show DCX-positive cells in control and MIF-stimulated groups (MIF 16ng/ml) and the comparison of the areas of DCX-positive cells between control and MIF-stimulated groups (^*p<0.05 vs. control group, 7 days after differentiation, n=3 animals in each group). The inserts at the top right corners are high magnification of the corresponding areas in the boxes in the images, respectively. B. Western blot results show the protein bands of DCX in differentiated NSPCs and the comparison of Western blot result showing the relative DCX levels in control and MIF-stimulated groups (^*p<0.05 vs. control group, 7 days after differentiation, n=3 animals in each group). Results were normalized to β-actin. C. Representative images of immunostaining show Tuj 1-positive cells in control and MIF-stimulated groups (MIF 16ng/ml) and the comparisons of the total Tuj 1-positive cells and the Tuj 1-positive cells migrated out from neurospheres between control and MIF-stimulated groups. (^*p<0.05 vs. control group, 7 days after differentiation, n=3 animals in each group). The inserts at the top right corners are high magnification of the corresponding areas in the boxes in the images, respectively. D. Western blot results show Tuj 1 protein bands in differentiated NSPCs and the comparison of Western blot result showing the relative Tuj 1 levels in control and MIF-stimulated groups (^*p<0.05 vs. control group, 7 days after differentiation, n=3 animals in each group). Results were normalized to β-actin. CON, control group; MIF, MIF-stimulated group.

Figure 5

The activation of Wnt/β-catenin signal pathway by MIF during NSPC differentiation. A. Representative images show β-galactosidase-positive cells (upper panel) and β-galactosidase /Hoechst double staining cells (lower panel) and the comparison of β-galactosidase-positive fluorescence intensities between control and MIF-stimulated groups (^*p<0.05 vs. control group, 2 days after differentiation, n=3 animals in each group). B. Western blot results show Wnt1 and β-catenin protein expressions in differentiated NSPCs in control and MIF-stimulated groups and the comparison of Western blot results showing the relative Wnt1 and β-catenin levels in control and MIF-stimulated groups (^*p<0.05 vs. control group, 7 days after differentiation, n=3 animals in each group). CON, control group; MIF, MIF-stimulated group; β-gal, β-galactosidase

Figure 6

Effects of MIF and IWR-1 on neuronal differentiation of NSPC. A. Representative images of DCX immunostaining show the effect of 1μM of IWR-1 on DCX expression and the comparison of DCX expression among the 4 groups by ANOVA (^**p<0.0001, n=3 animals in each group). The arrows show some of the DCX-positive processes. B. Representative images of DCX immunostaining show the effect of 10μM of IWR-1 on DCX expression and the comparison of DCX expression among the 4 groups by ANOVA (^**p<0.0001, n=3 animals in each group). The arrows show some of the DCX-positive processes. C. Representative images of Tuj 1 immunostaining show the effect of 1μM of IWR-1 on Tuj1 expression and the comparison of Tuj 1-positive cells among the 4 groups by ANOVA (^**p<0.0001, n=3 animals in each group). D. Representative images of Tuj 1 immunostaining show the effect of 10μM of IWR-1 on Tuj1 expression and the comparison of Tuj 1-positive cells among the 4 groups by ANOVA (^**p<0.0001, n=3 animals in each group). CON, control group; MIF, MIF-stimulated group. E. Western blot results show DCX and Tuj 1 protein expressions in differentiated NSPCs in control and MIF-stimulated groups with or without IWR-1 (10μM) and the comparison of Western blot results showing the relative DCX and Tuj 1 levels in four groups (^**p<0.001, 7 days after differentiation, n=3 animals in each group). CON, control group; MIF, MIF-stimulated group.