Figure 6. Interaction of β-catenin/LEF-1 mediates the transcriptional capacity of estradiol.

(A)-β-catenin/TCF estradiol-mediated transcription depends on the phosphorylation of β-catenin. N2a-m cells were transfected with different amounts of the S33Y-βcatenin (S33Y-βcat) expression plasmid as indicated (250 or 500 ng). (B)-β-catenin levels in total extracts from cells transfected with S33Y-βcat or the empty cDNA3.1 plasmid, representative of the analysis of lanes (0.5+); and (0.5−) in A. Under the high levels of S33Y-βcat expression estradiol was virtually unable to further induce reporter expression. No statistical differences are founded between the bars data from different experiments. (C)-Interaction of β-catenin with TCFs is essential for estradiol to induce gene transcription through TCF sites. Endogenous LEF-1 protein levels remain unchanged after estradiol treatment for 60–90 min, as seen with the anti-N terminal LEF-1 antibody. Expression of the Δ56LEF-1 protein was detected in western blots after transfection of increasing amounts of plasmid (400 or 600 ng) using an antibody against the HMG box region of LEF-1. The overexpression of a LEF-1 mutant construct (Δ56LEF-1) prevents estradiol from inducing expression from the TOPFlash reporter plasmid when compared with mock-transfected cells. (D)-Δ56LEF-1 reduces estradiol induced luciferase expression from pENP1-luc. The overexpression of Δ56LEF-1 construct reduced the luciferase activity induced by estradiol from the pENP1-Luc plasmid, when compared with mock-transfected cells (empty-pcDNA3). Estradiol (E) induced luciferase activity to 8–15 fold that of the control levels (C and E), in the pENP1-luc reporter. However, the expression of Δ56LEF-1 diminished this induction (compare pCDNA3+E versus Δ56+E). In both cases (C–D), the graphs show the normalized luciferase activity (RLU) from at least three independent experiments. The P value from the Student's t-test was * (P≤0.05).