PR-B mediates induction of E2F1 expression by R5020. (A and B) The indicated T47D:C42 cells were synchronized and treated with vehicle (veh) or 10 nM R5020 for 18 (A) or 16 (B) h. wt, wild type. (C) Human mammary epithelial cells (HMECs) were infected with a β-gal (negative control)- or PR-B-expressing adenovirus and subsequently treated with vehicle or 10 nM R5020 for 16 h. (A to C) After treatment, cells were lysed and RNA was isolated and reverse transcribed. S100P or E2F1 mRNA levels were quantified using qPCR and normalized to the housekeeping gene 36B4. Results are expressed as mean fold induction over vehicle-treated cells ± SEM (n = 3).

R5020 further amplifies E2F1 transcription by activating a positive feedback loop. (A) Schematic depicting hyperphosphorylation of Rb and subsequent release of E2F, which allows E2F to bind its own promoter and increase transcription in a positive feedback loop. (B and C) Synchronized T47D:A18 cells were treated with vehicle (veh) or 100 pM R5020 (R) for the indicated times (B) or pretreated with vehicle or 10 μM U0126 (U) for 30 min prior to addition of vehicle or 100 pM R5020 for 18 h (C). After treatment, cells were harvested and 20 μg whole-cell extract was resolved by SDS-PAGE, transferred to PVDF, and subjected to immunoblotting for total Rb, Rb phosphorylated on Ser780 (p-Rb Ser 780), Rb phosphorylated on Ser807/811 (p-Rb Ser 807/811), E2F1, or GAPDH or ERK 1/2 as a loading control. ns, nonspecific band. A representative blot is shown (n = 3). (D) Synchronized T47D:A18 cells were treated with vehicle or 100 pM R5020 for the indicated times. Cells were harvested after cross-linking and subjected to immunoprecipitation with either mouse IgG control (mIgG) or E2F1 antibody. Following reversal of cross-linking, DNA was isolated and subjected to qPCR analysis using primers spanning a region in the E2F1 promoter containing E2F binding sites (depicted in Fig. 3B). The results are presented as percent input ± SEM for triplicate amplification reactions from one representative experiment (n = 3).

KLF15 is necessary for maximal PR-mediated regulation of E2F1. (A) T47D:A18 cells were transiently cotransfected with various hE2F1-luc promoter fragment reporters along with increasing amounts of a vector expressing wild-type KLF15 or the KLF15 NΔ291 deletion mutant for 48 h and then were harvested and assayed for luciferase activity. Luciferase values were normalized to a β-galactosidase control. Data are the mean relative light units (RLU) ± SEM for one representative experiment performed in triplicate. Inset, Western blot control confirming equal expression of His-tagged KLF15 variants using GAPDH as a loading control. (B) T47D:A18 cells were transiently transfected with Stealth siRNAs targeting KLF15 (siKLF15 2 and 3) or a negative-control luciferase sequence (siLuc) at a final concentration of 100 nM for 48 h. Cells were synchronized by serum starvation for 24 h and then treated with vehicle (veh) or 100 pM R5020 (R) for 18 h. KLF15, E2F1, and FKBP51 mRNA levels were quantified using qPCR and normalized to the housekeeping gene 36B4. Results are expressed as mean fold induction over vehicle-treated cells ± SEM (n = 3).

Induction of endogenous E2F1 RNA/protein by R5020. (A) Flow chart schematic depicting breakdown of genes analyzed in T47D microarray. (B and C) Synchronized T47D:A18 cells were pretreated with vehicle (veh) or 10 μM U0126 (U) for 30 min prior to addition of vehicle or 100 pM R5020 (R) for 18 h. (C) After treatment, cells were harvested and 20 μg whole-cell extract was resolved by SDS-PAGE; transferred to PVDF; and subjected to immunoblotting for PR, E2F1, or ERK 1/2 as a loading control. A representative blot is shown. (D) Synchronized BT483 cells were treated with vehicle or 10 nM R5020 for 18 h. (B and D) After treatment, cells were lysed and RNA was isolated and reverse transcribed. S100P or E2F1 mRNA levels were quantified using qPCR and normalized to the housekeeping gene 36B4. Results are expressed as mean fold induction over vehicle-treated cells ± SEM (n = 3).

Evidence for direct regulation of E2F1 by PR. (A) The indicated T47D:C42 cells were synchronized and treated with vehicle (veh) or 10 nM R5020 for 24 h. Cells were lysed, and RNA was isolated and reverse transcribed. FKBP51 or E2F1 mRNA levels were quantified using qPCR and normalized to the housekeeping gene 36B4. Results are expressed as mean fold induction over vehicle-treated cells ± SEM (n = 3). (B) Schematic depicting the locations of two proximal enhancer sites located around E2F1 that were identified in ChIP-chip experiments as potential PR-binding sites. (C) Synchronized T47D:A18 cells were treated with vehicle or 10 nM R5020 for the indicated times. Cells were harvested after cross-linking and subjected to immunoprecipitation with either a mouse IgG control (mIgG) or PR antibody. Following reversal of cross-linking, DNA was isolated and subjected to qPCR analysis using primers spanning a region in FKBP51 (positive control), stromelysin (negative control), or the potential PR-binding region proximal to E2F1 (proximal site 1). The results are presented as mean percent input ± SEM for triplicate amplification reactions from one representative experiment (n = 3).

A member of the Sp/KLF family is involved in regulation of E2F1 transcription. (A) Schematic depicting regulatory elements within the E2F1 promoter (13). (B and C) Synchronized T47D:A18 cells were pretreated with vehicle (veh) or 200 nM mithramycin A (MitA) for 30 min and then treated with vehicle or 100 pM R5020 (R) for 18 h (B) or treated with vehicle or 100 pM R5020 for 18 h (C). Cells were lysed, and RNA was isolated and reverse transcribed. SGK, E2F1, SP1, KLF4, KLF9, and KLF15 mRNA levels were quantified using qPCR and normalized to the housekeeping gene 36B4. Results are expressed as mean fold induction over vehicle-treated cells ± SEM (n = 3).

Model of multimodal regulation of E2F1 by progestins. Ligand-bound PR can bind to proximal and distal enhancer sites located near E2F1 and directly regulate E2F1 transcription. PR can also act indirectly through hyperphosphorylation of Rb and induction of KLF15 expression to achieve further progestin-mediated regulation of E2F1 expression in T47D cells.