Figure 7.

Sp1 DNA Binding Is Required for Progestin-Stimulated PR Tethering to Sp1, EGFR Up-Regulation, and S-Phase Entry

A, PR was immunoprecipitated from whole-cell lysates of T47D-YB cells pretreated with MeOH vehicle control or mithramycin A (MyA, 0.4 μm) followed by EtOH or R5020 for 60 min. Immunoprecipitates were subjected to Western blotting with Sp1 or PR Ab. Lysate represents 10% IP input shown for Sp1 and PR. B, T47D-YB cells were treated without or with R5020 in the presence of vehicle control or R5020 for 15 h. EGFR was detected in whole-cell lysates using specific Ab. Total Erk1/2 was included as a loading control. C, Cell cycle analysis of T47D-YB cells treated for 18 h with R5020 in the presence of Sp1 inhibitor mithramycin A (MyA) or MeOH vehicle control. Bars indicate percentage of cells in G₁ (dark gray), S (black), or or G₂/M (light gray) phase of the cell cycle; error bars represent sd (n = 3). D, T47D-YB cells were treated with vehicle or mythramycin A (0.4 μm) for 1 or 15 h. Whole-cell lysates were subjected to Western blotting (upper panel) using total ERα Ab and β-actin as a loading control. Cell cycle analysis (lower panel) was conducted as in C except T47D-YB cells were pretreated for 45 min without (EtOH) or with ICI 182,780 (0.10 μm), followed by 18 h EtOH vehicle (dark gray bars), R5020 (black bars), or E2 (light gray bars). Bars indicate combined percentage of S+G₂/M population of proliferating cells; error bars represent sd (n = 3). Results shown in A–D were repeated in at least three independent experiments. E, Integration of rapid PR signaling and nuclear transcription activity. Classically, ligand-activated PR dimers contact PRE in the promoter regions of target genes such as c-myc and SGK to initiate transcription. Progestin binding to PR-B also mediates nonclassical gene transcription through extranuclear rapid activation of the EGFR, c-Src, and Erk1/2 MAPK cascade to stimulate feed-forward phosphorylation of PR-B Ser345 phosphorylation. Ser345-phosphorylated PR tether to Sp1 to regulate EGFR and p21 transcription. Additionally, rapid Erk1/2 MAPK activation may regulate cyclin D1 transcription independently of PR transcriptional activity (25).