Table 1.
PCR primer sequences and conditions.
Figure 1.
Progesterone Receptor stably transfected into mammary epithelial cells upregulates expression of canonical BmpR 1A (Alk3) and non-canonical BmpR Activin-Like Kinase Receptor ActR1A (Alk2).
EpH4 cells stably transfected with empty vector (EV) or the progesterone receptor (PR) were treated+/−10−8 M P for 24 hours. Total RNA was isolated and transcript abundance was evaluated via PCR for either Alk3 (A) or Alk 2 (B). Actin was used as housekeeping gene. C) EpH4-EV and EpH4-PR cells were plated at 2.4×105 cells/well in 6 well dishes and treated the next day+/−10−8 M P for 24 hours. Total cell lysates were analyzed via western blotting with the indicated antibodies then stripped and re-probed for tubulin as a loading control. Representative blots of at least three experiments are shown. Graphs represent mean+/−SD of at least three independent experiments, * P<0.05.
Figure 2.
Phospho-Smads are upregulated in progesterone receptor expressing mammary epithelial cells.
A) EpH4 cells stably transfected with empty vector (EV) or the progesterone receptor (PR) were treated+/−10−8 M P for 6 hours. Total cell lysates were analyzed via western blotting with phospho-Smad 1,5 and then stripped and re-probed for Smad 5 or tubulin. Graphs represent mean+/−SD of at least three independent experiments, * P<0.05. B) EpH4 parental cells were treated+/−300 ng/ml Bmp2 or Bmp4 for 6 hours. EpH4-EV and -PR cells were treated+/−10−8 M P in the presence or absence of 10 nM Noggin for 6 hours. Total cell lysates were analyzed via western blotting with phospho-Smad 1,5, 8, Smad 8 or tubulin antibodies. The blot was stripped between assays.
Figure 3.
Smad 1, 5, 8 translocate preferentially to the nucleus in mammary epithelial cells expressing the progesterone receptor.
EpH4 cells stably transfected with empty vector (EV) or the progesterone receptor (PR) were quiesced by overnight incubation in media containing charcoal stripped serum. A) Nuclear extracts were isolated and immunoprecipitated with Smad 4 antibody that had been cross-linked to Sepharose A beads, separated on a 10% Tris-Glycine gel, and probed with either phospho (P) -Smad 1,5, 8 or Smad 5 antibodies. B) Non-immunoprecipitated samples of cytosolic and nuclear fractions from EpH4-EV and -PR cells were separated and probed with Lamin A/C or tubulin to assure purity of nuclear preparations and equal loading, respectively. cyto = cytosolic, nucl = nuclear. C) EpH4 cells stably transfected with empty vector (EV) or the progesterone receptor (PR) were treated with 10−8 M P for 24 hours, fixed and then subjected to fluorescent immunocytochemistry. Images are representative immunofluorescent staining of phospho-Smad 1, 5, 8 (red) and nuclei were stained with DAPI (blue).
Figure 4.
TNAP/Akp2 is downregulated in progesterone receptor overexpressing mammary epithelial cells.
A) RNA was isolated from EpH4 cells stably transfected with empty vector (EV) or the progesterone receptor (PR) and transcript abundance was analyzed via semi-quantitative PCR. B) Quantitation of transcript abundance. Data represent the mean+/−SD, * P<0.05. C) Luciferase Assay for Akp2 promoter. EpH4-EV and EpH4-PR cells were transfected with an Akp2 promoter-luciferase construct and with the internal control vector, pRL-TK and 24 hours after transfection, media were removed and treated with or without 10−8 M P. Twenty-four hours later, triplicate samples were collected and analyzed. Relative luciferase units are reported as means+/−SE. * P<0.05 between EpH4-EV and EpH4-PR cells.
Figure 5.
Depletion of endogenous TNAP/Akp2 results in upregulation of Alk2 and Msx2 expression.
EpH4 cells were transfected with non-targeting or AKP2 siRNA. Twenty-four to 48 hours post-transfection, total RNA was isolated and transcript abundance was analyzed via semi-quantitative PCR. A) Data represent the mean+/−SD of four independent experiments. B) Representative images from PCR products. NTC = non-targeting control siRNA. *P<0.05.
Figure 6.
Proposed regulation of Msx2 via BMP receptor-mediated PR signaling.
Progesterone receptor signaling upregulates Msx2 transcription via inhibition of the phosphatase Akp2 and upregulation of Alk2 activity, leading to phosphorylation and nuclear translocation of the Smad transcription factor proteins.