Article Figures & Data
Figures
- FIG. 1.
Expression of Akt1 is regulated by Jak2/Stat5 signaling in mammary epithelial cells. (A) Western blot analysis using immortalized mammary epithelial cells (HC11; control) that express Stat5a, a constitutively active prolactin receptor (caPRLR), or both. Cells were cultured in the absence or presence of growth factors (GF), i.e., insulin, EGF, and serum. Alternatively, cells were treated for 20 min with PRL 24 h after the removal of all growth factors (MG, lactating mammary gland control). (B) Immunoprecipitation (IP)/Western blot analysis to compare the expression and tyrosine phosphorylation levels of the prolactin receptor (PRLR) in normal HC11 cells and in the lactating mammary gland (MG). (C) Quantitative real-time RT-PCR analysis to assess the relative expression of Cish mRNA in HC11 cells that express Stat5a, caPRLR, or both. Cish is a known transcriptional target of active Stat5 and negative regulator of Jak2/Stat5 signaling, which explains its higher expression in cells with active PRLR/Stat5 signaling in the absence of additional growth factors (normalized expression of cells with active PRLR/Stat5 that were maintained in the presence of GF was set to 1). (D) Quantitative real-time RT-PCR analysis to determine the relative expression of Akt1 mRNA in HC11 cells expressing exogenous Stat5a, caPRLR, or both (normalized expression of −GF control was set to 1). All expression values shown in panels C and D were normalized to Gapdh. Bars indicate the standard error (SE).
- FIG. 2.
A mammary gland-specific transcript of Akt1 originates from a distinct promoter. (A) Genomic structure of the mouse Akt1 gene; exon 1 represents the commonly known 5′-untranslated region of the mature mRNA. A novel noncoding exon (1m), which originates from downstream regulatory elements, was identified by using 5′RACE in a mammary gland-derived mRNA pool. Arrows indicate the location of RT-PCR primers that were used in panel B. (B) RT-PCR to determine the tissue-specific transcriptional activation of the newly identified exon 1m. PCR primers that amplify the coding region of Akt1 (exons 2 and 3), as well as beta-actin (ActB), were used as controls. (C) The upper panels show the results of quantitative real-time RT-PCR to determine Akt1 and Akt1m mRNA levels in tissues from a lactating female, as well as an epithelium-free mammary fat pad in comparison to cultured primary mammary epithelial cells (MECs, their relative expression level set to 1). The lower panels show Akt1 and Akt1m mRNA expression levels at various stages of mammary gland development (V, virgin; P12, pregnancy day 12; L10, lactation day 10; I2, involution day 2). The relative expression levels of the virgin gland were set to 1. In all four panels, expression was normalized to Gapdh. Bars represent the SE.
- FIG. 3.
Stat5 binds to consensus sites within Akt1 and greatly enhances the transcriptional activation of Akt1m. (A) Quantitative real-time RT-PCR analysis to assess the activation of the Akt1m transcript in HC11 cells expressing Stat5, caPRLR, or both. Cells were maintained in the presence or absence of growth factors (GF); normalized expression of the −GF control was set to 1. (B) qRT-PCR analysis to assess the transcriptional activation of Akt1m in the mammary gland of a Jak2-deficient female (MMTV-Cre Jak2fl/fl) and her wild-type littermate control (Jak2fl/fl) at day 12.5 of gestation. All expression values shown in panels A and B were normalized to Gapdh. Bars represent the SE. (C) Location of putative binding sites for Stat5 homodimers (TTCYNRGAA) within the 5′ region of the Akt1 locus and the Wap gene. (D and E) PCR and quantitative real-time PCR analysis on chromatin that was bound to endogenous Stat5 and coprecipitated with an antibody against Stat5a (ChIP). HC11 cells with or without exogenous caPRLR were maintained in the presence or absence of growth factors (GF). The promoter of Wap, which is a known Stat5 target gene, was used as a positive control for this ChIP assay. (F) Stat5 ChIP combined with qPCR analysis to assess the binding of Stat5 to interferon gamma activation sites (GAS) within the promoters of Akt1 and Wap in mammary tissues of females at day 10 of lactation and day 2 of involution. Quantitative real-time values of amplified Stat5 consensus sites within Akt1 and Wap shown in panels E and F were normalized against precipitated DNA using an IgG control antibody, as well as unspecific amplicons from nonconsensus binding regions. Bars represent the SE.
- FIG. 4.
Expression of myristolated Akt1 increases cyclin D1 levels and accelerates the proliferation of Jak2-deficient MECs. (A) Immunoprecipitation (IP) and Western blot analysis to verify the expression of HA-tagged myr-Akt1 in MECs that lack Jak2. (B) Western blot analysis to assess the expression of Akt1 and cyclin D1 (Ccnd1) in MECs lacking Jak2 with or without exogenous Akt1, as well as isogenic Jak2-expressing control cells. Phosphorylated Foxo3a was used as another positive control to verify that exogenous Akt1 is functional in Jak2-deficient cells. Beta-actin (ActB) was used as a loading control. (C) Viable cell count over a 5-day period to determine growth rates of Jak2-deficient MECs with or without expression of exogenous Akt1. All time points were analyzed in triplicate. Bars represent the SE.
- FIG. 5.
Doxycycline (Dox)-controlled overexpression of hyperactive Stat5a delays mammary gland involution in vivo. (A) Experimental design. Double transgenic Wap-rtTA/TetO-Stat5(S710F) mice were analyzed for ligand-inducible coexpression of luciferase using bioluminescence imaging (IVIS200) prior to treatment with Dox on lactation day 7 (left panel). After confirming the correct temporal and spatial activation of the TetO-Stat5 transgene in Dox-treated females on lactation day 10, we sealed the nipples of the left mammary glands 3, 4, and 5 to induce milk stasis (middle panel). All sealed (closed) mammary glands and their lactating (open) controls were collected between the first and fifth days after closing the nipples (right panel). (B) Comparative histological analysis of H&E-stained sections of mammary glands of Wap-rtTA/TetO-Stat5(S710F) double-transgenic females. These involuting (closed) mammary glands and their lactating (open) intra-individual control tissues were examined 2 and 5 days after sealing the nipples. Bars represent 500 μm (50 μm in the insets).
- FIG. 6.
Doxycycline (Dox)-controlled overexpression of hyperactive Stat5a leads to sustained transcription of Akt1. (A) Immunoprecipitation (IP)/Western blot analysis to assess the expression of active Stat5 on the second day after sealing the nipples (lanes C, closed) of lactating Wap-rtTA/TetO-Stat5(S710F) double-transgenic females that were treated with Dox and their untreated controls. The lactating (lanes O, open) glands of the same animals served as intraindividual controls. The protein levels of Cish, as well as total and phosphorylated Akt1, were determined by conventional immunoblotting. Beta-actin (ActB) was used as a general loading control, and unchanged expression levels of E-cadherin (Cadh1) indicate equal amounts of epithelial cells in all samples prior to programmed cell death and remodeling. (B and C) Quantitative real-time PCR analysis to assess the relative expression levels of Akt1 and Akt1m mRNA in involuting mammary glands between 1 and 3 days after sealing the nipples (I1 to I3) of Dox-treated females and their untreated controls. These relative expression values were compared to lactating control tissues of open glands (L10). The expression values of lactating tissues of untreated mice, which are represented by open bars, were set to 1.0. All expression values were normalized to Gapdh. Bars represent the SE.
- FIG. 7.
Gain of function of Stat5 does not initiate aberrant proliferation of differentiated epithelial cells but promotes their survival during involution. (A) Immunostaining for Ki67 (red) on involuting mammary glands of Dox-treated Wap-rtTA/TetO-Stat5 double-transgenic females and their untreated (−Dox) controls. A mammary gland from a female at day 12.5 of gestation was used as a positive control. Sections were counterstained with DAPI (blue). The bar represents 50 μm. (B) TUNEL analysis of apoptotic nuclei was performed on histological sections from lactating (open) and involuting mammary glands (closed) of doxycycline (Dox)-treated Wap-rtTA/TetO-Stat5(S710F) females and their untreated controls on day 3 and day 5 after sealing the nipples of the left glands. Sections were counterstained with methyl green. The bar represents 50 μm. (C) Relative number of TUNEL-positive cells on days 2, 3, and 5 after sealing the nipples. One-thousand cells from three representative areas of the sections were counted for each time point. Bars represent the SE, and the P value was calculated by using a paired Student t test.
- FIG. 8.
Proapoptotic signaling events are initiated despite Stat5-mediated sustained cell survival. (A) Immunohistochemistry to determine the activation and nuclear localization of Stat3 in Stat5-expressing, involuting tissues (+Dox, closed), and their controls 48 h after sealing the nipples. Slides were counterstained with hematoxylin. The bar represents 50 μm. (B) Western blot analysis to quantitatively assess the expression and activation of Stat3, as well as the Stat3-induced expression of the p50α/p55α PI3K subunits during the first and second days after sealing the nipples (lanes C, closed) of lactating Wap-rtTA/TetO-Stat5(S710F) double-transgenic females that were treated with Dox and their untreated controls. The lactating (lanes O, open) glands of the same animals served as intra-individual controls. (C) Stat3 ChIP assay combined with qPCR analysis to assess the binding of this transcription factor to the promoters of p50α and p55α in lactating and involuting mammary tissues of Wap-rtTA/TetO-Stat5(S710F) double-transgenic females that were treated with Dox (+Dox) and their untreated controls (−Dox). Tissues were collected on the second day after sealing the nipples (columns C, closed), and the lactating (columns O, open) glands of the same animals served as intra-individual controls. qPCR values of amplified Stat3 consensus sites within p55α and p55α were normalized against precipitated DNA using an IgG control antibody, as well as unspecific amplicons from nonconsensus binding regions, bars represent the SE. Note that activation of Stat3 and expression of p50/p55, which herald the onset of apoptosis beginning at 24 h after milk stasis, occur independently of Stat5-mediated suppression of programmed cell death.
- FIG. 9.
Divergent roles of Stat5 and Stat3 in the survival and death of secretory mammary epithelial cells in vivo.
