Figure 2.

NF-κB-mediated GDNF-signaling activates murine Pitx3 transcription. a, The human, mouse, and rat Pitx3 promoter region contains three highly conserved NF-κB BS (consensus sequence: 5′-GGGRNTYYCC-3′, where R = A or G, Y = C or T, and N = any nucleotide). Position of the first nucleotide of each BS is relative to the transcription start site (arrow). b–j, Immunostaining for NF-κB (b, e, h) and nuclear staining (Hoechst; c, f, i) (merged images in d, g, j) of untreated (b–d) E14 rat primary VM cultures or after treatment with GDNF for 3 h (e–g) or 24 h (h–j). k, Quantification of NF-κB⁺ cells relative to the total number of cells in these cell cultures with or without GDNF-treatment (control: 15.3 ± 1.3%; 3 h GDNF: 57.6 ± 2.5%; 24 h GDNF: 46.1 ± 4.8%, mean ± SEM; **p < 0.005 in the independent t test; n = 3). l, Transfection of SH-SY5Y cells with pGL3-Pitx3 promoter vector (light gray bars) and subsequent treatment of these cells with 10 or 20 ng/ml GDNF resulted in a dose-dependent activation of the Pitx3 promoter as compared to untreated controls. Dark gray bars represent values from pRL-SV40 vector (internal control). GDNF-mediated activation of the Pitx3 promoter was blocked by the simultaneous application of SN50, an inhibitor of NF-κB nuclear translocation (control: SV40 promoter, 96954 ± 2863; Pitx3 promoter, 365722 ± 7460; GDNF at 10 ng/ml: SV40 promoter, 123,900 ± 6980; Pitx3 promoter, 663,831 ± 51,615; GDNF at 20 ng/ml: SV40 promoter, 128,212 ± 7174; Pitx3 promoter, 776,831 ± 62,941; SN50 + GDNF at 20 ng/ml: SV40 promoter, 131,242 ± 7724; Pitx3 promoter, 350,674 ± 9528, mean ± SEM; **p < 0.005 in one-way ANOVA for repeated measurements). Data were derived from three independent experiments. RLU, Relative luciferase unit. Scale bar: (in j), 20 μm.