Figure 2

4ICD is required for estrogen stimulated expression of PgR. (A) Suppression of endogenous HER4 expression was performed in MCF-7 cells using erbB-4/Her4 siRNA SMARTpool or Nonspecific siRNA Negative Control Pool and siIMPORTER transfection reagent (Upstate Biotechnology, Lake Placid, NY) as described elsewhere [5]. Transfected cells were cultured in growth media supplemented with 5% CS-FBS for 48 hrs and incubated in the presence or absence of 100 pM 17-β-estradiol (Estrogen) for an additional 16 hrs. Total RNA was extracted and PgR expression was analyzed by RT-PCR. β-actin RNA was amplified as a control for RNA quantitation. (B) The MCF-7 variant TamR [23] lacking endogenous HER4 expression [6] was stably transfected with an EGFP vector, HER4-EGFP, or the γ-secretase processing mutant HER4V673I-EGFP which fails to generate 4ICD. Each cell line was incubated in growth media supplemented with 5% CS-FBS for 48 hrs and incubated in the presence or absence of 100 pM 17-β-estradiol (Estrogen) for 2 or 16 hrs. Total RNA was extracted and (B) PgR or (C) SDF-1 expression was analyzed by RT-PCR. β-actin RNA was amplified as a control for RNA quantitation. All RT-PCR reactions and conditions including primer sequences have been described elsewhere [5]. Results indicate that the 4ICD coactivator is essential for estrogen stimulated PgR and SDF-1 expression in the MCF-7 variant.