FIG. 5.

JAK/STAT pathway activation is involved in the hormonal induction of 11β-HSD2 gene expression. (A) T47D cells were cultured in phenol red-free medium under serum-free conditions for 72 h prior to the addition of 10 nM R5020 for 5, 10, or 15 min or 500 ng/ml prolactin (PRL) for 10 min. When indicated, 1 h before hormonal induction, cells were treated with 50 μM AG. Cell lysates were analyzed by Western blotting with antibodies against phosphorylated STAT5A (Tyr694/699) and tubulin. (B) T47D cells, cultured as described for Fig. 1A, were treated with R5020 (10 nM) for 0, 2, or 6 h. When indicated, AG (50 μM) was added 1 h before hormone addition. Cells were harvested, and total RNA was extracted. (Upper panel) 11β-HSD2 and MMTV-Luc mRNA expression levels were analyzed by RT-PCR with specific primers. GAPDH cDNA-specific primers were used as a control. PCR products were run on a 1.2% agarose gel and visualized with ethidium bromide. (Lower panel) 11β-HSD2 mRNA expression was analyzed by RT and real-time PCR with specific primers. GAPDH cDNA-specific primers were used as a control. The values represent the means and ranges of a representative experiment performed in duplicate, with each sample analyzed in duplicate, expressed as numbers of 11β-HSD2/GAPDH relative units. (C) Involvement of MAPK and PI3K pathways in hormone activation of 11β-HSD2. T47D cells, cultured as described for Fig. 1A, were untreated (0) or treated with R5020 (10 nM) for 2, 6, or 12 h. When indicated, PD (50 μM), WM (0.1 μM), or ICI (10 μM) was added 1 h before hormone induction. Cells were harvested, total RNA was prepared, and cDNA was generated by RT. 11β-HSD2 expression was analyzed by real-time PCR with specific primers. To normalize the data, GAPDH expression was used as a control. The values represent the means and standard deviations of two experiments performed in duplicate, expressed as numbers of 11β-HSD2/GAPDH relative units. Asterisks denote significant differences (P < 0.05) between treated (PD or WM) and untreated (only R5020) data sets, as analyzed by Student's t test. (D) T47D-YV cells cotransfected with WT pSG5-PRB and reporter 11β-HSD2-Luc vectors were treated with R5020 (10 nM) for 16 h, and Luc activity was determined. When indicated, cells were treated with AG (50 μM) 1 h before hormone addition. For normalization, equal amounts of cellular extract of each sample were used. The values represent the mean numbers of arbitrary Luc units and ranges of a representative experiment performed in duplicate. EtOH, ethanol. (E) Expression of 11β-HSD2 reporter construct in the presence of a Pro cluster PR mutant. T47D-YV cells cotransfected with WT pSG5-PRB or a Pro cluster mutant and reporter 11β-HSD2-Luc vectors were treated with R5020 (10 nM) for 16 h, and Luc activity was determined. For normalization, equal amounts of cellular extract of each sample were used. The values represent the mean numbers of arbitrary Luc units and ranges of a representative experiment performed in duplicate.