Mammary Gland Involution Is Delayed by Activated Akt in Transgenic Mice

Kathryn L. Schwertfeger, Monica M. Richert and Steven M. Anderson

Program in Molecular Biology (K.L.S., S.M.A.) and Department of Pathology (M.M.R., S.M.A.) University of Colorado Health Sciences Center Denver, Colorado 80262


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of the antiapoptotic protein kinase Akt is induced by a number of growth factors that regulate mammary gland development. Akt is expressed during mammary gland development, and expression decreases at the onset of involution. To address Akt actions in mammary gland development, transgenic mice were generated expressing constitutively active Akt in the mammary gland under the control of the mouse mammary tumor virus (MMTV) promoter. Analysis of mammary glands from these mice reveals a delay in both involution and the onset of apoptosis. Expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of matrix metalloproteinases (MMPs), is prolonged and increased in the transgenic mice, suggesting that disruption of the MMP:TIMP ratio may contribute to the delayed mammary gland involution observed in the transgenic mice.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mammary gland involution is characterized by a decrease in milk protein synthesis, extensive apoptosis of the secretory alveolar cells, and structural remodeling of the gland, which requires the activation of matrix metalloproteinases (MMPs) and the inactivation of their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs) (1, 2, 3, 4, 5, 6). It has been reported that 50–80% of the mammary epithelial cells in mice that are present during lactation undergo apoptosis during involution (2). The number of apoptotic cells during involution peaks at 3–4 days post weaning, and remodeling is complete by 6 to 8 days post weaning (1, 7, 8). Mammary gland involution provides an excellent in vivo model for examining mechanisms regulating apoptosis due to the extensive apoptosis of the epithelial cells and the accessibility of the mouse mammary gland.

The use of mouse genetic models has been critical for gaining insight into the molecular processes of apoptosis during mammary gland involution. A conditional knockout of signal transducer and activator of transcription-3 (STAT3) in the mammary gland results in delayed involution and decreased epithelial cell apoptosis, suggesting that STAT3 is critical for the normal process of apoptosis during involution (9). Transgenic mice overexpressing insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein 3 (IGFBP-3) in the mammary gland also exhibit delayed involution and decreased apoptosis (10). IGF-I has been shown to be involved in both proliferation and suppression of apoptosis, and IGFBP-3 may be involved in facilitating the actions of IGF-I in the mammary gland. In addition, mice overexpressing the antiapoptotic protein Bcl-2 in the mammary gland show an inhibition of alveolar cell apoptosis, although the collapse of alveolar structures is not delayed (11). These observations suggest a role for these proteins in regulating mammary gland involution. Finally, the expression patterns of a number of apoptosis-related proteins, including Bcl-x, Bax, p53, transforming growth factor ß1 (TGF-ß1), and sulfated glycoprotein 2 (SGP-2), are regulated during involution (1, 12, 13). The further identification of proteins that regulate the apoptotic process during involution is important for understanding both the regulation of normal mammary gland development and the process of apoptosis in physiological systems.

Akt, also referred to as PKB (protein kinase B) and RAC (related to A and C kinases), is a serine/threonine protein kinase that has been shown to suppress apoptosis in a number of systems. c-Akt (PKB{alpha}, RAC{alpha}) was identified as the cellular homolog of the retroviral oncogene, v-akt (14, 15). In addition to c-Akt (now known as Akt1), two other members of the Akt family have been identified, Akt2 (PKBß, RACß) and Akt3 (PKB{gamma}, RAC{gamma}) (15, 16). The three members of this family are all serine/threonine protein kinases that share similar structural features, including a pleckstrin homology (PH) domain in the amino terminus and two regulatory phosphorylation sites, Thr308 and Ser473 in Akt1, which are critical for activation (17). Numerous studies have shown that Akt is activated in a phosphatidylinositol 3'-kinase (PI3K)-dependent manner (18, 19). The activation of PI3K by growth factor receptors results in the generation of 3'-phosphoinositides, which recruit Akt to the plasma membrane by means of its PH domain binding to these membrane-associated phospholipids (20). Akt is then phosphorylated by phosphatidylinositol-dependent kinase 1 (PDK1), which has been shown to phosphorylate Thr308 (21, 22). Phosphorylation of Ser473 is also required for catalytic activation of Akt, and it has been suggested that this represents an autophosphorylation event (23), although other kinases may also phosphorylate this site. Localization to the membrane appears to be critical for Akt activation because modifications that target Akt to the membrane, such as the gag sequence found in v-Akt (24) or fusion of a myristoylation sequence from src-like kinases to the amino terminus of Akt (25), result in the constitutive activation of the kinase. Activation of Akt results in the suppression of apoptosis induced by a number of stimuli including growth factor withdrawal, detachment from extracellular matrix, UV irradiation, cell cycle discordance, and activation of Fas (25, 26, 27, 28, 29, 30, 31). In addition, expression of gag-Akt, a constitutively active form of Akt, in thymocytes of transgenic mice prevents apoptosis in response to various stimuli (32).

Akt activation can be induced by a number of growth factors, including epidermal growth factor (EGF) (30), IGF-I (17, 33), estrogen (34), and hepatocyte growth factor/scatter factor (35), which all regulate mammary gland development (36, 37, 38, 39, 40). The purpose of this study was to examine the role of Akt as a regulator of apoptosis during mammary gland involution. A constitutively active mutant of Akt, which contains a myristoylation sequence fused to the amino terminus of Akt (myr-Akt), was expressed in mammary glands of transgenic mice using the mouse mammary tumor virus (MMTV) promoter present in the long terminal repeat of MMTV. We demonstrate that the expression of constitutively active Akt in the mammary gland results in delayed involution and a delay in the onset of apoptosis, suggesting that Akt may be involved in regulating normal mammary gland involution.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Akt Is Decreased during Early Involution
Apoptosis of the secretory epithelial cells during mammary gland involution peaks by day 4 of involution and then decreases (7). Therefore, we hypothesized that Akt levels would decrease early during involution to allow apoptosis to occur. To test this hypothesis, we examined the pattern of Akt expression and activation during mammary gland involution. Pregnant FVB female mice gave birth, and the litters were normalized to eight pups each. The pups were allowed to suckle for 9 days to ensure the full establishment of lactation after which time the pups were removed. Forced weaning at day 9 was used in these studies because the onset of involution can be more closely controlled than in natural weaning. The fourth inguinal mammary glands were removed on day 9 of lactation, and days 2, 4, 6, 8, 10, and 14 of involution, and total Akt protein levels were determined by immunoblotting. As shown in Fig. 1AGo, Akt expression levels were high during lactation, but they decreased dramatically by day 2 of involution. The antibody used in this study does not distinguish between Akt1 and Akt2. The immunoblot was reprobed with an antibody to actin to determine whether the observed decrease in protein expression was a result of milk accumulation that normally occurs early in involution. Although actin levels also decrease at involution day 2, normalization of Akt levels to actin levels indicates that Akt expression is decreased after lactation and remains low throughout involution (Fig. 1BGo). These data are consistent with results published by Chodosh et al. (41), in which Northern blot analysis shows highest levels of expression of Akt during lactation, which then decrease by day 2 and remain low through day 7 of involution.



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Figure 1. Akt Expression Decreases during Lactation and Involution

A, Protein extracts were prepared from frozen mammary glands of normal (FVB) mice at day 9 of lactation (L9), and days 2, 4, 6, 8, 10, and 14 of involution (Inv d2, Inv d4, Inv d6, Inv d8, Inv d10, and Inv d14, respectively). The protein extracts were analyzed on an 8% SDS-polyacrylamide gel and immunoblotted with an antibody that recognizes total Akt. B, The Akt and actin immunoblots were scanned on a densitometer and Akt expression was normalized to actin expression. C, Protein extracts were prepared from mammary glands at the indicated times, and the ability of Akt to phosphorylate a GST-GSK3 substrate was examined using a kinase assay. The reactions were analyzed on a 12% polyacrylamide gel and immunoblotted with an antibody that recognizes both phosphorylated isoforms of GSK-3, GSK-3{alpha} and -ß. D, Sections from a lactating day 9 mammary gland were stained using an anti-Akt1-specific antibody (red). Green represents the luminal spaces stained with WGA. Blue represents the DAPI stained nuclei. E, Negative control of panel D; immunostaining was performed using secondary antibody alone. Panels D and E were taken at a magnification of 200x and exposed for the same length of time.

 
Akt activation during involution was determined by examining the ability of immunoprecipitated Akt to phosphorylate a known substrate, glycogen synthase kinase-3 (GSK-3), in vitro (42, 43). The pattern of Akt activity is similar to the pattern of expression; Akt activity is high during lactation followed by a decrease in activity by day 2 of involution and remains low throughout involution (Fig. 1CGo). These data suggest that Akt may be involved in suppression of apoptosis during lactation and that a decrease of Akt expression may be critical for apoptosis to occur during involution.

To determine localization of Akt in the mammary gland, immunofluorescence was performed on paraffin-embedded sections from lactating glands using an anti-Akt1 specific antibody. As shown in Fig. 1DGo, Akt1 is expressed in epithelial cells surrounding the luminal spaces as visualized by the red staining. The luminal spaces are visualized by Oregon-Green 488-conjugated wheat germ agglutinin (WGA), which binds to mucins on the apical surfaces of epithelial cells (44). Blue staining represents the 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei. No staining is observed in the negative control slides that received only the secondary antibody (Fig. 1EGo). These results indicate that Akt is expressed in epithelial cells during lactation and is catalytically activated during lactation, suggesting that the decrease of Akt in these cells at the onset of involution may be necessary for apoptosis to proceed.

Myr-Akt Is Expressed in the Mammary Glands of Transgenic Mice
Because endogenous Akt expression decreases during early involution (Fig. 1Go, A and C), we hypothesized that the inappropriate presence of activated Akt at this stage would result in a change in the involution process. Therefore, we expressed a constitutively active mutant of Akt in the mammary glands of mice under the control of the MMTV promoter, which allows expression of the transgene in the epithelial cells of the mammary gland. Although the MMTV promoter allows expression during all stages of development, the promoter is strongly expressed during lactation. Therefore, we can examine the effect of Akt on the transition between lactation and involution in these transgenic mice. The constitutively active mutant of Akt (myr-Akt) used in these studies contains an amino-terminal myristoylation sequence from c-src fused to the coding sequence of Akt1. This targets the fusion protein to the plasma membrane where it can be constitutively activated after phosphorylation of Thr308 and Ser473 (25). The construct also contains a hemagglutinin (HA) epitope tag at the carboxyl-terminal end of the protein to allow for detection of transgene expression by immunoblotting.

Transgenic mice were produced that express the myr-Akt construct in the mammary gland. Transgene-positive founder mice were identified by PCR of tail DNA (Fig. 2AGo). Immunoblot analysis of mammary gland protein for the HA epitope tag indicates that two lines of mice (designated 1173 and 1176) express detectable levels of the myr-Akt protein (Fig. 2BGo). Expression of the transgene is consistently higher in mice derived from the 1173 founder than expression in mice from the 1176 founder (Fig. 2BGo and data not shown). These data show that two independently generated lines of MMTV-myr-Akt transgenic mice, lines 1173 and 1176, have been produced that express detectable levels of myr-Akt protein in the mammary gland.



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Figure 2. Generation of MMTV-myr-Akt Mice and Expression of the myr-Akt Transgene

A, Tail DNA was extracted from each founder and PCR analysis was performed using primers specific for Akt and the HA epitope tag. Of 39 founders, 9 contained transgene DNA. B, Protein was extracted from mammary glands at day 18 of pregnancy of normal (FVB) and transgenic (1173, 1175, 1176, 1177, 1178, and 1699) mice and analyzed by immunoblot analysis using an anti-HA antibody. Two lines, 1173 and 1176, show expression of the myr-Akt protein in their mammary glands. C, Protein was extracted from mammary glands at L9, Inv d2, Inv d4, Inv d6, Inv d8, Inv d10, and Inv d14 from both normal (FVB) and transgenic (line 1173) mice. The extracted protein was analyzed by immunoblot analysis using an anti-HA antibody. D, Extracts were immunoblotted with an antiphospho-Akt antibody. The lower mol wt protein corresponds to endogenous Akt and the higher mol wt protein corresponds to the myr-Akt transgene. E, Protein extracted from mammary glands at day 18 of pregnancy (P18), day 9 of lactation (L9), and day 6 of involution (Inv d6) were analyzed by immunoblot analysis using the antiphospho-GSK-3{alpha}/ß antibody. F, The immunoblot was reprobed with an antibody to GSK-3{alpha}. G, A section from a normal gland at day 2 of involution immunostained with an anti-Akt1 specific antibody (red). The DAPI-stained nuclei are blue. H, A section from a transgenic gland at day 2 of involution immunostained identically to panel G. Panels G and H were taken at a magnification of 200x and exposed for the same length of time.

 
The pattern of expression of the myr-Akt transgene was examined in the transgenic mice during involution. The fourth inguinal mammary glands were collected during lactation and involution from both normal and transgenic mice. Protein extracts were prepared, and immunoblot analysis was performed to detect the HA epitope tag. As shown in Fig. 2CGo, transgene expression is high during lactation, and expression of myr-Akt persists through day 14 of involution, although transgene levels appear to decrease as involution proceeds. Two bands are apparent on the immunoblot, and this has been consistently observed in all experiments using the anti-HA antibody. Although both bands cross-react with anti-Akt antibodies (data not shown), only the lower mol wt band cross-reacts with the anti-phospho-Akt antibody (Fig. 2DGo). Phosphorylation of Akt is required for its activation; therefore, levels of phosphorylated Akt in the glands were determined by immunoblotting with an antibody specific to the phosphorylated Ser473 residue of Akt. Two bands were observed in mammary glands from transgene-positive mice (Fig. 2DGo). The lower mol wt protein corresponds to endogenous Akt and the higher mol wt protein corresponds to the myr-Akt transgene. Phosphorylation of the myr-Akt transgene is high during lactation and early involution and persists through day 14 of involution, although there is a decrease in the amount of phosphorylated transgene throughout involution corresponding to the decrease in total transgene expression. It appears that phosphorylation of endogenous Akt is suppressed by the presence of the myr-Akt transgene from day 9 of lactation (L9) to day 4 of involution (Inv d4) (Fig. 2DGo, lanes 2–4). To further determine whether the myr-Akt transgene was catalytically active in the mammary gland, the phosphorylation of a known target of Akt, glycogen synthase kinase-3 (GSK-3) (42, 43), was examined. As shown in Fig. 2EGo, phosphorylation of endogenous GSK-3 is higher in mammary glands from transgenic mice compared with those from normal mice during late pregnancy, lactation, and early in involution when transgene expression is high (Fig. 2CGo and data not shown). Reprobing the immunoblot with an anti-GSK-3{alpha} antibody, which also recognizes GSK-3ß, indicates that protein levels of GSK-3{alpha}/ß are not significantly altered in mammary glands from transgenic mice (Fig. 2FGo). The detection of myr-Akt, its phosphorylation, and the increased phosphorylation of GSK-3 in the mammary gland indicate that these transgenic mice can be used to examine the effect of activated Akt upon involution.

To localize expression of the transgenic myr-Akt in the mammary gland, immunofluorescence was performed on mammary tissue at day 2 of involution from normal (Fig. 2GGo) and transgenic (Fig. 2HGo) mice using an anti-Akt1 antibody. Data presented in Fig. 1BGo indicate that the level of endogenous Akt is low at this time point. Red staining represents Akt1 protein and blue staining represents the DAPI-stained nuclei. Some staining is observed in the epithelium of the normal mammary gland (Fig. 2GGo), which is to be expected because a small amount of Akt protein is observed in normal mammary glands at this stage (Fig. 1BGo). However, expression of Akt1 in the mammary gland from the transgenic mouse is much greater and is observed in cells surrounding the luminal spaces (Fig. 2HGo). Identical exposure times were used for photographs in panels G and H of Fig. 2Go. Sections stained with secondary antibody alone show no staining (data not shown). Therefore, it appears that the myr-Akt transgene is expressed in mammary epithelial cells surrounding the alveolar lumina.

Involution Is Delayed in MMTV-myr-Akt Transgenic Mice
Mammary involution can be divided into an early phase (days 1–4) and a late phase (days 5–8) (8). The early phase of involution is characterized by apoptosis of the alveolar epithelial cells (2). Because Akt can suppress apoptosis, we determined the effect of the myr-Akt transgene upon involution. Involution was induced in both FVB and MMTV-myr-Akt female mice using forced involution as described above. The fourth inguinal mammary glands were removed at days 2, 4, 6, 8, 10, and 14 of involution, fixed, and embedded in paraffin.

Histological features of control and myr-Akt-expressing mammary glands were examined in hematoxylin and eosin-stained sections (Fig. 3Go). At day 2 of involution, alveolar structures, comprised of a single layer of epithelial cells surrounding a lumen, are observed in mammary glands from both normal and transgenic mice (Fig. 3Go, A and B). No distinct morphological differences are apparent between the two glands at this stage. At day 4 of involution, the alveolar structures in mammary glands from normal mice have started to collapse, and numerous apoptotic bodies are apparent in the ductal lumens (Fig. 3CGo). In addition, adipocytes have begun to reappear or, alternatively, to reaccumulate lipid. The predominance of adipocytes is typical of mammary glands in virgin mice and postinvolution mice. In the mammary glands from transgenic mice, however, the alveoli have not yet begun to collapse, and they appear distended (Fig. 3DGo). In addition, there is an accumulation of protein in the alveolar lumina, which may represent inspissated milk typically observed during milk stasis. At day 6 of involution, the alveoli in mammary glands from normal mice have collapsed (Fig. 3EGo) and the process of remodeling has continued (8). In mammary glands from transgenic mice, the alveoli have still not collapsed, although some apoptotic bodies are observed in the luminal spaces and some adipocytes have begun to reappear (Fig. 3FGo), suggesting that the process of involution has begun. By day 8 of involution, the normal mammary glands have been completely remodeled and resemble that of a prepregnant gland (Fig. 3GGo). In the mammary glands from transgenic mice, the alveoli are beginning to collapse and numerous apoptotic bodies are visible in the luminal spaces (Fig. 3HGo), similar to the morphology observed at day 4 of involution in the normal gland (Fig. 3CGo). Normal mammary glands at days 10 and 14 of involution are similar to that seen at day 8, as the mammary gland has been completely remodeled (Fig. 3Go, I and K). In contrast, mammary glands from transgenic mice are still undergoing the process of involution through day 10 as indicated by the collapsing alveolar structures and the disorganization of the gland (Fig. 3JGo). By day 14, the mammary glands from transgenic mice appear to have been remodeled, although some alveolar structures still persist (Fig. 3LGo).



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Figure 3. Involution Is Delayed in Mammary Glands of MMTV-myr-Akt Mice

A, C, E, G, I, and K, Mammary glands from normal (FVB) mice. B, D, F, H, J, and L, Mammary glands from MMTV-myr-Akt mice, line 1173. M and N, Mammary glands from MMTV-myr-Akt mice, line 1176. A and B, Day 2 of involution. C and D, Day 4 of involution. E and F, Day 6 of involution. G and H, Day 8 of involution. I and J, Day 10 of involution. K and L, Day 14 of involution. M, Day 4 of involution. N, Day 6 of involution. Magnification bars represent 100 µM.

 
The data shown in Fig. 3Go (panels B, D, F, H, J, and L) were from mice derived from the 1173 founder. To determine that the observed phenotype is not due to an insertional artifact of the transgene, these experiments were confirmed in mice derived from a second founder line, 1176. The mammary glands exhibit a similar phenotype as that seen in line 1173, with alveolar structures persisting through day 4 of involution (Fig. 3MGo). However, alveolar collapse in mammary glands from this line begins by day 6 (Fig. 3NGo) rather than day 8 as seen in the 1173 line (Fig. 3HGo). This difference in the time course of involution is likely due to the lower expression consistently seen in the mammary glands from the 1176 line compared with those from the 1173 line (Fig. 2BGo). These data demonstrate that the presence of a constitutively active Akt in the mammary gland delays involution after forced weaning.

Myr-Akt Delays the Onset of Apoptosis during Involution
To determine whether the delayed involution observed in the MMTV-myr-Akt mice was accompanied by a decrease in apoptosis, the numbers of apoptotic cells were quantified. Figure 4AGo shows the results of quantifying apoptotic bodies in hematoxylin and eosin-stained sections of mammary glands from normal and myr-Akt transgenic mice. Consistent with observations published previously (7), apoptosis peaked at day 4 of involution in the mammary glands from normal mice. However, the peak of apoptosis in the mammary glands from transgenic mice did not occur until day 8 of involution, indicating a delay in the onset of apoptosis in mammary glands from transgenic mice.



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Figure 4. Delayed Onset of Epithelial Cell Apoptosis during Involution in Mammary Glands of MMTV-myr-Akt Mice

A, Percentage of apoptotic cells in normal mammary glands (gray bars) and transgenic mammary glands (black bars) at days 2, 4, 6, 8, 10, and 14 of involution assessed in hematoxylin and eosin-stained sections. B, Percentage of apoptotic cells in normal mammary glands (gray bars) and transgenic mammary glands (black bars) at days 2, 4, 6, 8, and 10 of involution assessed using TUNEL-stained sections. Error bars represent SEM. C, Quantification of the epithelial content of mammary glands from 10 normal mice (gray bar) and 10 transgenic mice (black bar) at day 21 of involution. Error bars represent SEM. D, Hematoxylin and eosin-stained sections of mammary glands at day 21 of involution. The upper panel is a normal mammary gland from an FVB mouse and the lower panel is a section from a mammary gland from line 1173. Magnification bars represent 100 µM.

 
To confirm these results, TUNEL analysis was performed on sections of mammary glands from both normal and transgenic mice. As shown in Fig. 4BGo, apoptosis in mammary glands from normal mice peaked at days 2–4 of involution. Consistent with Fig. 4AGo, the peak of apoptosis in mammary glands from transgenic mice did not occur until day 8 of involution. These data indicate that the presence of the myr-Akt transgene results in a delay in the onset of apoptosis during involution.

To compare the epithelial content of the regressed mammary glands from normal and transgenic mice, the amount of epithelium present in mammary glands at day 21 of involution was quantified. As shown in Fig. 4CGo, epithelial content in mammary glands from transgenic mice was nearly 2-fold higher than that in mammary glands from normal mice. Figure 4DGo shows representative pictures of hematoxylin and eosin-stained sections from normal (upper panel) and transgenic (lower panel) mammary glands at day 21 of involution. These results suggest that less epithelial cells undergo apoptosis in the mammary glands from transgenic mice and that these cells remain until the next pregnancy.

ß-Casein and Whey Acidic Protein (WAP) Expression Are Sustained in MMTV-myr-Akt Mice throughout Involution
One of the initial events after weaning is the rapid decrease in the expression of milk proteins such as ß-casein and WAP (1). These proteins are highly expressed during lactation, but diminish during the first 5 days of involution (4). We hypothesized that the observed delay in involution would be accompanied by sustained production of ß-casein and WAP RNA. RNA was extracted from the fourth inguinal mammary glands during lactation and involution, and the levels of ß-casein, WAP, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA were examined by Northern blot analysis. Expression of ß-casein and WAP are sustained in the mammary glands from transgenic mice compared with mammary glands from normal mice (Fig. 5Go, A and B). ß-Casein expression decreases dramatically in the normal gland after weaning, and levels are undetectable by day 6 of involution (Fig. 5AGo). WAP expression similarly decreases after weaning, and levels are also undetectable by day 6 (Fig. 5BGo). In contrast, ß-casein expression in the mammary glands from transgenic mice remains high and levels are detectable until day 10 of involution, although they are beginning to decrease by day 6 of involution (Fig. 5AGo). WAP expression is also sustained in the mammary glands from transgenic mice, but decreases by day 8 of involution (Fig. 5BGo). It appears that there is more WAP mRNA production at day 2 of involution than during lactation (Fig. 5BGo); however, there is a corresponding increase in GAPDH (Fig. 5CGo), indicating that this is due to a loading artifact. Similar data were obtained in three different studies using RNA isolated from mammary glands of different mice. These data demonstrate that milk protein gene expression is sustained in the mammary glands of transgenic mice compared with normal mice.



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Figure 5. Milk Protein Expression in MMTV-myr-Akt Mice

Northern blot analysis of total RNA from mammary glands of normal (FVB) and transgenic (line 1173) mice at day 9 of lactation (>NOREF>L9) and days 2, 4, 6, 8, 10, and 14 of involution (Inv). The blots were probed with ß-casein (A) and WAP (B) specific probes. C, Blots were reprobed with a GAPDH-specific probe as a loading control.

 
STAT (Signal Transducer and Activator of Transcription) Expression and Phosphorylation Occur Normally in MMTV-myr-Akt Glands
STATs are a family of transcription factors that are activated by cytokine receptors including the PRL receptor (45, 46). STAT activation requires phosphorylation of a single tyrosine residue, which leads to the formation of homo- and heterodimers that translocate to the nucleus where they regulate the transcription of a number of genes (47). Members of the STAT family have been shown to be involved in normal mammary gland development and function. Specifically, STAT5a is involved in milk production during lactation, and its phosphorylation decreases at the beginning of involution (48, 49). STAT3 is required for normal involution, and its phosphorylation increases at the beginning of involution (9, 49). It has been suggested that this reciprocal regulation may be important for involution to occur (48, 49). The delayed involution in myr-Akt mice suggests a possible disruption in the normal regulation of these two STAT molecules.

To examine STAT phosphorylation, protein was extracted from mammary glands of both normal and MMTV-myr-Akt mice during lactation and involution. The phosphorylated forms of STAT3 and STAT5a were detected by immunoblot analysis using phospho-specific antibodies. STAT3 phosphorylation is low during lactation, but increases by day 2 of involution in mammary glands from both normal and transgenic mice (Fig. 6AGo). This increase is transient and decreases by day 8 of involution in mammary glands from normal mice. Phosphorylated STAT3 persists to day 8 in mammary glands from transgenic mice, possibly reflecting the increased numbers of epithelial cells remaining in the mammary glands from transgenic mice. Overall levels of STAT3 are equivalent in mammary glands from both normal and transgenic mice (Fig. 6BGo).



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Figure 6. STAT Activation in MMTV-myr-Akt Mice

Analysis of STAT3 and STAT5a activation in normal (FVB) and transgenic (line 1173) mice at day 9 of lactation (L9) and days 2, 4, 6, 8, 10, and 14 of involution (Inv). A, Protein extracts were analyzed on an 8% SDS-polyacrylamide gel and immunoblotted with a phospho-specific STAT3 antibody. B, The blot was reprobed with an antibody that recognizes total STAT3. C, Protein extracts were analyzed on an 8% SDS-polyacrylamide gel and immunoblotted with a phospho-specific STAT5 antibody. The arrow indicates phospho-STAT5a. D, The blot was reprobed with an antibody that recognizes total STAT5. The arrow indicates STAT5a.

 
STAT5a phosphorylation is high during lactation, but decreases by day 2 of involution in mammary glands from both normal and transgenic mice (Fig. 6CGo). Although there appears to be less phosphorylated STAT5a in the mammary glands from transgenic mice at day 9 of lactation compared with normal glands, detection of total STAT5a antibody reveals less STAT5a protein in the mammary glands from transgenic mice at lactation day 2, possibly due to a loading artifact (Fig. 6DGo). In addition, there appears to be less STAT5a protein in mammary glands from transgenic mice at days 4 and 6 of involution. However, reprobing this blot with an antiactin antibody reveals that less actin protein is also present (data not shown). This artifact is likely due to the accumulation of milk proteins due to milk stasis that occurs in the transgenic mice through day 6 of involution (Fig. 3Go, D and F) rather than a specific effect of Akt overexpression on STAT5a protein expression. These data demonstrate that although involution is delayed in the mammary glands of the MMTV-myr-Akt mice, the patterns of STAT3 activation and STAT5a inactivation do not change, although STAT3 phosphorylation does persist longer in the transgenic mice. Similar data were obtained in three different studies using extracts prepared from different mice.

Increased and Prolonged TIMP-1 Expression in MMTV-myr-Akt Glands
After apoptosis of the secretory epithelial cells during involution, the mammary gland is remodeled back to a prepregnant state. This process involves activation of MMPs, a family of enzymes that degrades extracellular matrix, and the inactivation or decrease in the concentration of their inhibitors, the TIMPs (4, 6). Expression patterns of the MMP stromelysin-1 (MMP-3) and its inhibitor TIMP-1 during mammary gland involution have been well characterized (4, 50). TIMP-1 is expressed at moderate levels during lactation, but increases in early involution with a peak of expression at 4 days of involution (50). MMP-3 is undetectable during lactation, but increases during involution with a peak of expression at 4–6 days of involution (50). The ratio of MMP to TIMP expression is important for the remodeling process to occur, and disruption of this ratio has been shown to alter involution. Implantation of TIMP-1 pellets in the mammary gland results in delayed involution (4), and overexpression of MMP-3 in transgenic mice results in precocious involution (51, 52).

To determine whether expression of MMP-3 and TIMP-1 are altered in the MMTV-myr-Akt mice, the amount of TIMP-1 and MMP-3 RNAs was examined using Northern blot analysis. TIMP-1 expression in mammary glands from normal mice increases during early involution, peaking at days 2–4 and decreasing by day 8 of involution (Fig. 7AGo). In mammary glands from transgenic mice, TIMP-1 levels also increase during early involution but remain high until day 10 of involution (Fig. 7AGo). GAPDH levels are equivalent in mammary glands from both normal and transgenic mice (Fig. 7BGo). MMP-3 expression in mammary glands from normal mice is low during lactation and increases by day 4 of involution, peaking at days 4–6, followed by a subsequent decrease in expression by day 10 of involution (Fig. 7CGo). In transgenic mice, MMP-3 levels increase earlier than in normal mice and are detectable on day 2 of involution, peaking at day 6, and are followed by a decrease in expression at day 10 of involution. The observed earlier increase of MMP-3 in mammary glands from transgenic mice is not consistent with a delay in involution. However, since the MMP to TIMP ratio is thought to be critical for MMP activity, the MMP-3 activity is likely being suppressed by the high levels of TIMP-1 at this stage (Fig. 7AGo). GAPDH levels are equivalent in mammary glands from both normal and transgenic mice (Fig. 7DGo). Similar results were obtained in three different studies using tissue extracts prepared from different mice. These data suggest that the increased and sustained levels of TIMP-1 in the mammary glands of transgenic mice may be involved in the delayed involution observed in the myr-Akt transgenic mammary glands.



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Figure 7. TIMP-1 and MMP-3 Expression in MMTV-myr-Akt Mice

Northern blot analysis of total RNA from mammary glands of normal (FVB) and transgenic (Line 1173) mice at day 9 of lactation (L9) and days 2, 4, 6, 8, 10, and 14 of involution (Inv). Blots were probed with a TIMP-1 specific probe (A) and reprobed with a GAPDH specific probe as a loading control (B), a MMP-3-specific probe (C), and re-probed with a GAPDH specific probe as a loading control (D).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The data presented here suggest a role for Akt in normal mammary gland involution and demonstrate suppression of apoptosis by Akt in an in vivo system. Expression of endogenous Akt was examined during involution induced by forced weaning and, after normalization to actin, was found to be high during lactation and decreased at all days following pup withdrawal. Akt1 expression was observed in lactating glands to be primarily located in the luminal epithelial cells. It would be of great interest to determine whether other cell types in the mammary gland express Akt, as well as whether different cell types express different isoforms.

We have generated transgenic mice that express a constitutively active Akt in the mammary gland using the MMTV promoter. These mice were shown to express the transgene during lactation as well as during the first 8 days of involution, with detectable levels remaining at day 14 of involution. Morphologically, the mammary glands of the transgenic mice show a delay in involution that corresponds with a delay in the onset of apoptosis. The mammary epithelial cells eventually undergo apoptosis, and the gland is remodeled suggesting that either transgene expression diminishes and can no longer suppress apoptosis, or that another apoptotic mechanism is present that can override the effects of the myr-Akt transgene.

Understanding the suppression of apoptosis is of great relevance to cancer, as overexpression of genes that suppress apoptosis lead to cancers such as leukemia (53). Although the mammary glands of the MMTV-myr-Akt mice eventually undergo involution, it appears that less epithelial cells are lost than in normal regressed mammary glands. This observation raises the possibility that the presence of the myr-Akt transgene in mammary glands may result in the retention of epithelial structures, particularly in uni- and multiparous mice, which could lead to the development of hyperplasia and/or tumors.

One of the initial characteristics of involution is the decrease in the production of milk proteins in early involution (1). ß-Casein and WAP mRNA expression were sustained in the mammary glands of the transgenic mice compared with those of normal mice. In the histological sections (Fig. 3Go), it is apparent that the alveolar structures are retained in the glands from transgenic mice through day 6 of involution whereas the alveolar structures in glands from normal mice regress after day 2 of involution. It has been demonstrated that association of mammary epithelial cells with extracellular matrix is important for maintaining the differentiation status of the epithelial cells and that disruption of this interaction results in the loss of ß-casein expression (4). Therefore, it is possible that the epithelial cells that have not undergone apoptosis in the mammary glands from transgenic mice are still associated with the extracellular matrix, resulting in the maintenance of their differentiation state as suggested by the prolonged expression of milk protein genes.

STATs have been shown to be important regulators of mammary gland development. STAT5a knockout mice have impaired lobuloalveolar development during pregnancy and fail to lactate (54). STAT5a is activated during pregnancy and is involved in milk protein expression (48, 49, 55), but its activity decreases at the beginning of involution and has been suggested to be involved in the suppression of apoptosis of epithelial cells (9). As mentioned in the Introduction, STAT3 is required for normal involution as demonstrated by delayed involution in conditional knockouts (9). STAT3 activity is low during lactation and increases at the beginning of involution (9, 49). It has been suggested that STAT5 and STAT3 are reciprocally regulated and that this regulation may be important for the normal process of involution (9, 49). Because of the observed delay of involution in the myr-Akt mice, we examined the phosphorylation states of both STAT3 and STAT5a. Although phosphorylated STAT3 persists longer in mammary glands from transgenic mice, this may be due to higher epithelial cell content and the observed delay in involution. Both molecules were expressed equally at the protein level in mammary glands from both normal and transgenic mice. These data may suggest that either Akt functions independently of STAT3 to suppress apoptosis, or that Akt functions downstream of STAT3.

Studies have shown that STAT5a can induce the transcription of the milk proteins such as ß-casein (48, 56). Although ß-casein levels remain high after weaning in the myr-Akt mice, phosphorylated STAT5a levels decrease by day 2 of involution. Therefore, the increased level of ß-casein RNA observed in mammary glands from transgenic mice could reflect either continued transcription of the gene in the absence of phosphorylated STAT5a or an increase in the half-life of the mRNA.

MMPs and their inhibitors, the TIMPs, are critical for the process of remodeling of the gland after apoptosis (4, 6). TIMP-1 has been demonstrated to increase at the beginning of involution, presumably to inhibit the remodeling process until after apoptosis has occurred, and then decrease to allow the MMPs to begin the remodeling process (50). TIMP-1 expression increases during involution in the MMTV-myr-Akt mice, and it remains elevated until day 10 of involution. Interestingly, the expression pattern of MMP-3 in the MMTV-myr-Akt mammary glands is similar to that of the normal glands, although its expression is increased earlier in the mammary glands from transgenic mice when compared with control mice. It is possible that in response to the inability of the epithelial cells to undergo apoptosis, the mammary glands from transgenic mice are up-regulating pathways involved in inducing apoptosis and remodeling in an attempt to overcome this phenotype. It would be interesting to determine the expression patterns of other proteins known to be involved in inducing apoptosis in the mammary gland. Although MMP-3 is expressed earlier in the mammary glands of transgenic mice induced to undergo involution, the increased and sustained levels of TIMP-1 may inhibit MMP-3 activation. It would be interesting to determine whether the expression of other MMPs is altered in our transgenic mice and whether they are responsible for the remodeling that eventually occurs in the mammary glands of myr-Akt transgenic mice.

The coordination of apoptosis and tissue remodeling during involution is not completely understood. One explanation for the delayed involution observed in the mammary glands of transgenic mice is that myr-Akt suppresses apoptosis of the secretory epithelial cells; therefore, the signals required to initiate the remodeling process may not be present. An additional factor contributing to this phenotype may be the observed alteration in MMP to TIMP ratios. The ratio of TIMPs to MMPs is critical for normal involution. Talhouk et al. (4) used pellet implants to demonstrate that the presence of high levels of exogenous TIMP protein during involution resulted in delayed regression of alveolar structures and sustained expression of ß-casein, demonstrating that the TIMP to MMP ratio is important for normal involution and regulation of mammary epithelial function. Therefore, the altered ratio of TIMP to MMP might explain not only the delay in tissue remodeling, but also the sustained expression of ß-casein in the transgenic mammary glands. One possible mechanism for the increased levels of TIMP-1 is that Akt may induce TIMP-1 transcription or alter the molecules that regulate TIMP-1 gene expression. Another possibility is that activated Akt may be indirectly regulating TIMP-1 expression by modulating a signal from the apoptotic mammary epithelial cells that is normally required to down-regulate TIMP-1.

A recent study by Fata et al. (57) has implicated Akt activity in alveolar development during pregnancy. Impaired alveolar development in osteoprotegerin-ligand (OPGL) knockout mice correlated with loss of phosphorylated Akt. Expression of OPGL by pellet implants in pregnant mammary glands results in an increase in phosphorylated Akt and the restoration of alveolar development. The data presented in this paper focus on the antiapoptotic functions of Akt in the mammary gland and indicate that the loss of Akt at weaning is necessary for normal involution to occur. We have shown that expression of activated Akt in the mammary gland results in suppression of apoptosis and delayed involution. The delayed involution may be a result of increased TIMP-1 expression, and future studies will focus on the regulation of TIMP-1 expression in these mice. In addition, the identification of Akt targets will define other molecules involved in regulating both apoptosis and tissue remodeling. Clearly, the overexpression of signaling molecules in transgenic mice can reveal potential roles in normal developmental processes, but this analysis may be prone to artifacts. Confirmation that Akt is critical in regulating mammary gland involution will require analysis of knock-out mice that eliminate expression of Akt1, Akt2, or both.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of Transgenic Mice
The HA-tagged myr-Akt (Akt1) construct was obtained from Dr. Phil Tsichlis (Kimmel Cancer Center, Temple University, Philadelphia, PA) and the MMTV-SV40-BSSK plasmid was obtained from Dr. William Muller (McMaster University, Ontario, Canada). The HA-tagged myr-Akt construct was isolated from the CMV-6 plasmid using HindIII and EcoRI. The fragment was subcloned into the same sites of the MMTV-SV40-BSSK plasmid. The MMTV-myr-Akt plasmid was digested with SalI/SpeI to remove the plasmid backbone and gel purified using an ElutipD column (Schleicher & Schuell, Inc., Keene, NH) according to the manufacturer’s instructions. Transgenic mice were generated using standard techniques (58). Mice were maintained on an FVB/N background using stock mice from Taconic Farms, Inc. (Germantown, NY). Tail DNA was extracted from the founder mice by Proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation. Transgenic mice were identified using PCR. The primers used were 5'-GCCGCTACTATGCCATGAAGA-3', which is specific to Akt and 5'-GTAATCTGGAACATCGTATGGGTA-3', which is specific to the HA tag.

Tissue Collection
Adult female mice were mated. Litters were normalized to eight pups and were removed after 9 days of lactation. The females were killed by cervical dislocation at day 9 of lactation, or days 2, 4, 6, 8, 10, and 14 of involution. The tissues were either snap frozen in dry ice for RNA and protein extraction or were fixed in 10% neutral buffered formalin and embedded in paraffin. The embedded tissues were sectioned at 5 µM and stained with hematoxylin and eosin. Mammary glands were removed from at least five mice per time point for histological analysis, and all subsequent analyses were performed in duplicate on tissue from at least three mice per time point.

Assessment of Epithelial Cell Apoptosis
Apoptotic cells were quantified using hematoxylin and eosin-stained sections based on morphological criteria (59). The number of apoptotic cells was calculated as a percentage of total cell counts. TUNEL staining was performed on sections using the Apoptag kit (Intergen, Purchase, NY) following the manufacturer’s instructions. The number of apoptotic cells was calculated as a percentage of total cell counts. Hematoxylin and eosin-stained sections were used to calculate epithelial content of glands at day 21 of involution. Epithelial content was quantified by overlaying a grid consisting of 384 squares onto images of mammary glands taken at 100x magnification. The numbers of squares containing epithelium were counted and calculated as a percentage of the total number of squares. The resulting percentage is a representation of the area of the gland containing epithelium rather than a direct count of epithelial cells. A total of 12 mammary glands at day 21 of involution, 6 glands from normal mice and 6 glands from transgenic mice, were quantitated. The study was performed blindly.

Northern Blot Analysis
RNA was extracted from frozen tissue using Trizol (Life Technologies, Gaithersburg, MD). The RNA was then denatured and analyzed on a 1% agarose gel containing 6% formaldehyde and transferred onto GeneScreen (NEN Life Science Products, Boston, MA) according to manufacturer’s protocols. Probes for the Northern blots were radiolabeled using random primers (Prime-It II, Stratagene, San Diego, CA) and [{alpha}-32P]-dCTP (NEN Life Science Products). The ß-casein probe was obtained from Dr. Lothar Hennighausen (NIH, Bethesda, MD). The WAP probe was obtained from Dr. Jeffrey Rosen (Baylor College of Medicine, Houston, TX). The TIMP-1 probe was obtained from Dr. Zena Werb (University of California San Francisco, San Francisco, CA). The stromelysin-1 (MMP-3) probe was obtained from Dr. Lynn Matrisian (Vanderbilt College of Medicine, Nashville, TN). The GAPDH probe was from the collection of this laboratory.

Immunoblot Analysis
Protein was extracted from frozen tissue by homogenizing in RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Triton X-100, 1% DOC, 0.1% SDS, 1 mM dithiothreitol, 5 mM sodium orthovanadate, 100 µg/ml phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (no. P 8340, Sigma, St. Louis, MO). The samples were then boiled for 10 min, chilled on ice, and sonicated until homogeneous. Protein assays were performed using the Pierce Coomassie Plus protein assay reagent (Pierce Chemical Co., Rockford, IL). Total protein (50 µg amounts) was separated on 8% SDS-polyacrylamide gels, transferred to PVDF membrane (Immobilon, Millipore Corp., Bedford, MA), and immunoblotted with various antibodies as described previously (60, 61). The anti-Akt, antiphospho-Akt, antiphospho GSK-3{alpha}/ß, antiphospho-STAT3, and anti-STAT3 rabbit polyclonal antibodies were obtained from New England Biolabs, Inc. (Beverly, MA). The antiactin and anti-GSK-3{alpha} antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antiphospho-STAT5 rabbit polyclonal antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-STAT5 rabbit polyclonal antibody was obtained from Transduction Laboratories, Inc. (Lexington, KY). The horseradish peroxidase-conjugated anti-HA antibody (clone 3F10) was obtained from Roche Molecular Biochemicals (Indianapolis, IN). All antibodies were used at a dilution of 1:1000, except the anti-HA antibody, which was used at a dilution of 1:400. Bound antibodies were detected using horseradish peroxidase-conjugated secondary antibody followed by detection with the ECL system according to manufacturer’s recommendations (Amersham Pharmacia Biotech, Piscataway, NJ). Densitometric analysis was performed on the Computing Densitometer, and images were quantitated using ImageQuant software, both from Molecular Dynamics, Inc. (Sunnyvale, CA).

Akt Kinase Assay
Mammary glands were removed from animals at the indicated times and snap frozen in liquid nitrogen. Lysates were prepared by homogenizing the tissue in lysis buffer [150 mM NaCl, 50 mM HEPES, pH 7.4, 2 mM EGTA, pH 8.0, 1% Triton X-100, 0.25% deoxycholate, and a protease inhibitor cocktail (no. P 8340, Sigma, St. Louis, MO)]. The samples were centrifuged at 13,000 x g for 30 min and the supernatants were used for immunoprecipitation. Protein assays were performed using the Pierce Coomassie Plus protein assay reagent (Pierce Chemical Co.). One milligram amounts of total protein were used for each immunoprecipitation. The immunoprecipitation and kinase assays were performed using the Akt Kinase Assay Kit (no. 9840, New England Biolabs, Inc.) following the manufacturer’s instructions. The reactions were analyzed on a 12% polyacrylamide gel and immunoblotted as described above using the antiphospho-GSK-3{alpha} antibody.

Immunofluorescence
Mammary glands were removed from animals at the indicated times, fixed in 10% neutral buffered formalin, and embedded in paraffin. Sections were cut at 5 µm. After dehydration with graded alcohols, microwave antigen retrieval was performed for 20 min in 10 mM sodium citrate, pH 6.0. The sections were permeabilized with 0.2% glycine in PBS, blocked with 5% normal goat serum, and incubated with the anti-Akt1 antibody kindly provided by Dr. Morrie Birnbaum (University of Pennsylvania, Philadelphia, PA) at a dilution of 1:200. The sections were then incubated with Cy3-conjugated antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), Oregon-Green 488-conjugated WGA (Molecular Probes, Inc., Eugene, OR), and 0.6 µg/ml DAPI (Sigma). Images were collected using SlideBook software (Intelligent Imaging Innovations, Inc., Denver, CO) on a Diaphot TMD microscope (Nikon, Melville, NY) equipped for fluorescence with a Xenon lamp and filter wheels (Sutter Instruments, Novato, CA), fluorescent filters (Chroma, Brattleboro, VT), cooled CCD camera (Cooke, Tonawanda, NY), and stepper motor (Intelligent Imaging Innovations, Inc., Denver, CO).


    ACKNOWLEDGMENTS
 
The authors would like to thank Drs. Robert Strange, Michael Lewis, Janet Stephens, John Ryder, and Margaret Neville for their contributions to this work. We also thank the members of the Mammary Gland Biology Group at the University of Colorado Health Sciences Center for discussions and comments on this research.


    FOOTNOTES
 
Address requests for reprints to: Steven M. Anderson, Department of Pathology, Box B216, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262. E-mail: steve.anderson{at}uchsc.edu

This research was supported by a University of Colorado Cancer Center "Wines for Life" Seed Grant and NIH Grants CA-085736, DK-53858, and DK-48845. K.L.S. and M.M.R. are predoctoral and postdoctoral fellows supported by grants from the US Army Breast Cancer Research Program (BC980097 and BC990978, respectively). The University of Colorado Cancer Center Transgenic Mouse Core Facility is supported by a Cancer Center grant from the National Cancer Institute (CA-46934).

Received for publication January 8, 2001. Accepted for publication March 22, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Strange R, Li F, Saures S, Burkhardt A, Friis R 1992 Apoptotic cell death and tissue remodelling during mouse mammary gland involution. Development 115:49–58[Abstract]
  2. Walker NI, Bennett RE, Kerr JFR 1989 Cell death by apoptosis during involution of the lactating breast in mice and rats. Am J Anat 185:19–32[Medline]
  3. Talhouk R, Chin J, Unemori E, Werb Z, Bissell M 1991 Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture. Development 112:439–449[Abstract]
  4. Talhouk R, Bissell M, Werb Z 1992 Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution. J Cell Biol 118:1271–1282[Abstract]
  5. Wilde CJ, Knight CH, Flint DJ 1999 Control of milk secretion and apoptosis during mammary involution. J Mammary Gland Biol Neoplasia 4:129–136[CrossRef][Medline]
  6. Rudolph-Owen LA, Matrisian LM 1998 Matrix metalloproteinases in remodeling of the normal and neoplastic mammary gland. J Mammary Gland Biol Neoplasia 3:177–189[CrossRef][Medline]
  7. Quarrie L, Addey C, Wilde CJ 1995 Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labeling. Cell Tissue Res 281:413–419[CrossRef][Medline]
  8. Lund LR, Romer J, Thomasset N, Solberg H, Pyke C, Bissell MJ, Dano K 1996 Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways. Development 122:181–193[Abstract/Free Full Text]
  9. Chapman R, Lourenco P, Tonner E, Flint D, Selbert S, Takeda K, Akira S, Clarke A, Watson C 1999 Suppression of epithelial apoptosis and delayed mammary gland involution in mice with a conditional knockout of Stat3. Genes Dev 13:2604–2616[Abstract/Free Full Text]
  10. Neuenschwander S, Schwartz A, Wood T, Roberts Jr C, Henninghausen L, LeRoith D 1996 Involution of the lactating mammary gland is inhibited by the IGF system in a transgenic mouse model. J Clin Invest 97:2225–2232[Abstract/Free Full Text]
  11. Jager R, Herzer U, Schenkel J, Weiher H 1997 Overexpression of Bcl-2 inhibits alveolar cell apoptosis during involution and accelerates c-myc-induced tumorigenesis of the mammary gland in transenic mice. Oncogene 15:1787–1795[CrossRef][Medline]
  12. Li M, Liu X, Robinson G, Bar-Peled U, Wagner KU, Young WS, Hennighausen L, Furth PA 1997 Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution. Proc Natl Acad Sci USA 94:3425–3430[Abstract/Free Full Text]
  13. Schorr K, Li M, Krajewski S, Reed JC, Furth P 1999 Bcl-2 gene family and related proteins in mammary gland involution and breast cancer. J Mammary Gland Biol Neoplasia 4:153–164[CrossRef][Medline]
  14. Bellacosa A, Testa J, Stall S, Tsichlis PN 1991 A retroviral oncogene. akt, encoding a serine-threonine kinase containing an SH 2-like domain. Science 254:274–277[Medline]
  15. Staal S 1987 Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma. Proc Natl Acad Sci USA 84:5034–5037[Abstract]
  16. Konishi H, Kuroda S, Tanaka M, Matsuzaki H, Ono Y, Kameyama K, Haga T, Kikkawa U 1995 Molecular cloning and characterization of a new member of the RAC protein kinase family: association of the pleckstrin homology domain of three types of RAC protein kinase with protein kinase C subspecies and ß {gamma} subunits of G proteins. Biochem Biophys Res Commun 216:526–534[CrossRef][Medline]
  17. Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA 1996 Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15:6541–6551[Abstract]
  18. Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR, Tsichlis PN 1995 The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81:727–736[Medline]
  19. Burgering B, Coffer P 1995 Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376:599–602[CrossRef][Medline]
  20. Andjelkovic M, Alessi DR, Meier R, Fernandez A, Lamb NJC, Frech M, Cron P, Cohen P, Hemmings BA 1998 Role of translocation in the activation and function of protein kinase B. J Biol Chem 272:31515–31524[Abstract/Free Full Text]
  21. Alessi DR, James S, Downes C, Holmes A, Gaffney P, Reese C, Cohen P 1997 Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B{alpha}. Curr Biol 7:261–269[Medline]
  22. Stokoe D, Stephens L, Copeland T, Gaffney P, Reese C, Painter G, Holmes A, McCormick F, Hawkins P 1997 Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277:567–570[Abstract/Free Full Text]
  23. Toker A, Newton AC 2000 Akt/protein kinase B is regulated by autophosphorylation at the hypothetical PDK-2 site. J Biol Chem 275:8271–8274[Abstract/Free Full Text]
  24. Ahmed NN, Franke TF, Bellacosa A, Datta K, Gonzalez-Portal ME, Taguchi T, Testa J, Tsichlis PN 1993 The proteins encoded by c-akt and v-akt differ in post-translational modification, subcellular localization and oncogenic potential. Oncogene 8:1957–1963[Medline]
  25. Kennedy SG, Wagner A, Conzen S, Jordan J, Bellacosa A, Tsichlis PN, Hay N 1997 The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev 11:701–713[Abstract]
  26. Crowder RJ, Freeman RS 1998 Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons. J Neurosci 18:2933–2943[Abstract/Free Full Text]
  27. Khwaja A, Downward J 1997 Lack of correlation between activation of Jun-NH2-terminal kinase and induction of apoptosis after detachment of epithelial cells. J Cell Biol 139:1017–1023[Abstract/Free Full Text]
  28. Kennedy SG, Kandel ES, Cross TK, Hay N 1999 Akt/Protein kinase B inhibits cell death by preventing the release of cytochrome c from the mitochondria. Mol Cell Biol 19:5800–5810[Abstract/Free Full Text]
  29. Kauffmann-Zeh A, Rodriguez-Viciana P 1997 Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature 385:544–548[CrossRef][Medline]
  30. Gibson S, Tu S, Oyer R, Anderson S, Johnson G 1999 Epidermal growth factor protects cells against Fas-induced apoptosis. Requirement for Akt activation. J Biol Chem 274:17612–17618[Abstract/Free Full Text]
  31. Datta SR, Brunet A, Greenberg ME 1999 Cellular survival: a play in three Akts. Genes Dev 13:2905–2927[Free Full Text]
  32. Jones R, Parsons M, Bonnard M, Chan V, Yeh W-C, Woodgett J, Ohashi P 2000 Protein kinase B regulated T lymphocyte survival, nuclear factor kB activation, and Bcl-XL levels in vivo. J Exp Med 191:1721–1733[Abstract/Free Full Text]
  33. Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, Segal RA, Kaplan DR, Greenberg ME 1997 Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275:661–665[Abstract/Free Full Text]
  34. Simoncini T, Hafezi-Moghadam A, Brazil D, Ley K, Chin W, Liao J 2000 Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 407:538–541[CrossRef][Medline]
  35. Bowers DC, Fan S, Walter KA, Abounader R, Williams JA, Rosen EM, Laterra J 2000 Scatter factor/hepatocyte growth factor protects against cytotoxic death in human glioblastoma via phosphatidylinositol 3-kinase- and Akt-dependent pathways. Cancer Res 60:4277–4283[Abstract/Free Full Text]
  36. Coleman S, Silberstein GB, Daniel CW 1988 Ductal morphogenesis in the mouse mammary gland: evidence supporting a role for epidermal growth factor. Dev Biol 127:304–315[Medline]
  37. Kleinberg D 1998 Role of IGF-I in normal mammary development. Breast Cancer Res Treat 47:201–208[CrossRef][Medline]
  38. Yang Y, Spitzer E, Meyer D, Sachs M, Niemann C, Hartmann G, Weidner M, Birchmeieir C, Birchmeier W 1998 Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland. J Cell Biol 131:215–226[Abstract]
  39. Fendrick J, Raafat A, Haslam S 1998 Mammary gland growth and development from the postnatal period to postmenopause: ovarian steroid receptor ontogeny and regulation in the mouse. J Mammary Gland Biol Neoplasia 3:7–21[CrossRef][Medline]
  40. Richert M, Wood T 1998 Expression and regulation of insulin-like growth factors and their binding proteins in the normal breast. In: Manni A (ed) Contemporary Endocrinology: Endocrinology of Breast Cancer. Humana Press, Totowa, NJ, pp 39–52
  41. Chodosh L, Gardner H, Rajan J, Stairs D, Marquis S, Leder P 2001 Protein kinase expression during murine mammary development. Dev Biol 219:259–276
  42. Cross D, Alessi D, Cohen P, Andjelkovic M, Hemmings B 1995 Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785–789[CrossRef][Medline]
  43. van Weeren P, de Bruyn K, de Vries-Smits A, van Lint J, Burgering B 1998 Essential role for protein kinase B (PKB) in insulin-induced glycogen synthase kinase 3 inactivation. Characterization of dominant-negative mutant of PKB. J Biol Chem 273:13150–13156[Abstract/Free Full Text]
  44. Valdizan M, Julian J, Carson D 1992 WGA-binding, mucin glycoproteins protect the apical cell surface of mouse uterine epithelial cells. J Cell Physiol 151:451–465[Medline]
  45. Gouilleux F, Wakao H, Mundt V, Groner B 1994 Prolactin induces phosphorylation of Tyr694 of STAT5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J 13:4361–4369[Abstract]
  46. Ihle JN, Kerr IM 1995 Jaks and Stats in signaling by the cytokine receptor superfamily. Trends Genet 11:69–74[CrossRef][Medline]
  47. Darnell Jr JE 1997 STATs and gene regulation. Science 277:1630–1635[Abstract/Free Full Text]
  48. Liu X, Robinson G, Hennighausen L 1996 Activation of Stat5a and Stat5b by tyrosine phosphorylation is tightly linked to mammary gland differentiation. Mol Endocrinol 10:1496–1506[Abstract]
  49. Philp J, Burdon T, Watson C 1996 Differential activation of Stats 3 and 5 during mammary gland development. FEBS Lett 396:77–80[CrossRef][Medline]
  50. Li F, Strange R, Friis R, Djonov V, Altermatt HJ, Saurer S, Niemann H, Andres A-C 1994 Expression of stromelysin-1 and TIMP-1 in the involuting mammary gland and in early invasive tumors of the mouse. Int J Cancer 59:560–568[Medline]
  51. Alexander CM, Howard EW, Bissell MJ, Werb Z 1996 Rescue of mammary epithelial cell apoptosis and entactin degradation by a tissue inhibitor of metalloproteinases-1 transgene. J Cell Biol 135:1669–1677[Abstract]
  52. Wiesen JF, Werb Z 1996 The role of stromelysin-1 in stromal-epithelial interactions and cancer. Enzyme Protein 49:174–181[Medline]
  53. Kondo E, Nakamura S, Onoue H, Matsuo Y, Yoshino T 1992 Detection of bcl-2 protein and bcl-2 messenger RNA in normal and neoplastic lymphoid tissues by immunohistochemistry and in situ hybridization. Blood 80:2044–2051[Abstract]
  54. Liu X, Robinson G, Wagner KU, Garrett L, Wynshaw-Boris A, Hennighausen L 1997 STAT5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev 11:179–186[Abstract]
  55. Schmitt-Ney M, Happ B, Ball R, Groner B 1992 Developmental and environmental regulation of a mammary gland-specific nuclear factor essential for the transcription of the gene encoding ß-casein. Proc Natl Acad Sci USA 89:3130–3134[Abstract]
  56. Ball R, Friis R, Schoenenberger C, Doppler W, Groner B 1988 Prolactin regulation of ß-casein gene expression and of a cytosolic 120-kD protein in a cloned mouse mammary epithelial cell line. EMBO J 7:2089–2095[Abstract]
  57. Fata JE, Kong Y-Y, Li J, Sasaki T, Irie-Sasaki J, Moorehead RA, Elliott R, Scully S, Voura EB, Lacey DL, Boyle WJ, Khokha R, Penninger J 2000 The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell 103:41–50[Medline]
  58. Hogan B, Beddington R, Costantini F, Lacy E 1994 Manipulating the Mouse Embryo: A Laboratory Manual, ed. 2. Cold Spring Harbor Press, Cold Spring Harbor, NY
  59. Wyllie AH, Kerr JF, Currie AR 1980 Cell death: the significance of apoptosis. Int Rev Cytol 68:251–306[Medline]
  60. Anderson SM, Burton EA, Koch BL 1997 Phosphorylation of Cbl following stimulation with interleukin-3 and its association with Grb2, Fyn, and phosphatidylinositol 3-kinase. J Biol Chem 272:739–745[Abstract/Free Full Text]
  61. Burton EA, Hunter S, Wu SC, Anderson SM 1997 Binding of src-like kinases to the ß-subunit of the interleukin-3 receptor. J Biol Chem 272:16189–16195[Abstract/Free Full Text]