NGF-Induced Cell Differentiation and Gene Activation Is Mediated by Integrative Nuclear FGFR1 Signaling (INFS)
Figure 3
Nuclear FGFR1 mediates NGF induced neurite outgrowth and activation of the th gene promoter.
(A) PC12 cells were transfected with two plasmids, one expressing recombinant FGFR1 or control pcDNA3.1 and the second expressing EGFP. EGFP diffuses throughout the cell permitting visualization of the entire neuritic network. More than 90% of cells co-expressed transfected plasmids as reported in previous studies [29], [73]. Twenty-four hours after transfection cultures were switched to 1% horse serum medium with or without (control) 50 ng/ml NGF for an additional 10 days, after which the cells were imaged using fluorescent microscopy. Bar length - 100 µm. (B) The longest process in an individual transfected cell was measured using ImageJ [24].* mark comparison to pCDNA3.1 (-NGF) and # to pCNA3.1 (+NGF). Transfection of constitutive nuclear FGFR1(SP−/NLS) increased neurite outgrowth approximately 3-fold (*p<0.001, One-Way ANOVA, LSD). A similar increase was induced by NGF treatment of pcDNA3.1 transfected cells (*p<0.001). Cells transfected with dominant negative FGFR1(SP−/NLS)(TK-) or FGFR1(TK-) display no significant changes in average neurite length in the absence or presence of NGF. (C) PC12 cells were transfected with a th- Luciferase reported plasmid [37] and dominant negative FGFR1(TK-), FGFR1(SP−/NLS)(TK-) or control pcDNA3.1(-). 24 hours after transfection cells were treated for an additional 24 hours with indicated concentrations of NGF. The th-Luc expression in the presence of NGF was significantly reduced by both FGFR1(TK-) and FGFR1(SP−/NLS)(TK-). Dominant negative FGFR1 constructs had no significant effect on basal promoter activity. One-Way ANOVA, LSD: * (p<0.01) - mark comparison to (-NGF) within individual plasmid transfection groups; #(p<0.05) - comparison to pCDNA3.1+3 ng/ml NGF; •(p<0.01) - comparison to pcDNA3.1+10 ng/ml NGF; and x (p<0.001) - comparison to pCNA3.1+30 ng/ml NGF. Two-Way ANOVA shows interactions (p<0.001) between NGF and plasmid constructs (control, FGFR1(TK-) or FGFR1(SP−/NLS)(TK-)). (D) PC12 cells were transfected with th-Luc and control pcDNA3.1(-) or pcDNA3.1 expressing an active constitutive nuclear FGFR1(SP−/NLS). 24 hours after transfection cells were treated for an additional 6 or 8 hours with NGF. FGFR1(SP−/NLS) increases th promoter activity 2-fold in the absence of NGF but shows no additive stimulation in the presence of NGF. One-Way ANOVA, LSD: * (p<0.001) - comparison to (-NGF) within individual plasmid transfection groups; #(p<0.05) - comparison to pCDNA3.1 (- NGF).