Progesterone receptor activates Msx2 expression by downregulating TNAP/Akp2 and activating the Bmp pathway in EpH4 mouse mammary epithelial cells - PubMed (original) (raw)

Progesterone receptor activates Msx2 expression by downregulating TNAP/Akp2 and activating the Bmp pathway in EpH4 mouse mammary epithelial cells

Jodie M Fleming et al. PLoS One. 2012.

Abstract

Previously we demonstrated that EpH4 mouse mammary epithelial cells induced the homeobox transcription factor Msx2 either when transfected with the progesterone receptor (PR) or when treated with Bmp2/4. Msx2 upregulation was unaffected by Wnt inhibitors s-FRP or Dkk1, but was inhibited by the Bmp antagonist Noggin. We therefore hypothesized that PR signaling to Msx2 acts through the Bmp receptor pathway. Herein, we confirm that transcripts for Alk2/ActR1A, a non-canonical BmpR Type I, are upregulated in mammary epithelial cells overexpressing PR (EpH4-PR). Increased phosphorylation of Smads 1,5, 8, known substrates for Alk2 and other BmpR Type I proteins, was observed as was their translocation to the nucleus in EpH4-PR cells. Analysis also showed that Tissue Non-Specific Alkaline Phosphatase (TNAP/Akp2) was also found to be downregulated in EpH4-PR cells. When an Akp2 promoter-reporter construct containing a ½PRE site was transfected into EpH4-PR cells, its expression was downregulated. Moreover, siRNA mediated knockdown of Akp2 increased both Alk2 and Msx2 expression. Collectively these data suggest that PR inhibition of Akp2 results in increased Alk2 activity, increased phosphorylation of Smads 1,5,8, and ultimately upregulation of Msx2. These studies imply that re-activation of the Akp2 gene could be helpful in downregulating aberrant Msx2 expression in PR+ breast cancers.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

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

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

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

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

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

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.

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