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Molecular Endocrinology, doi:10.1210/me.2003-0345
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Molecular Endocrinology 18 (5): 1171-1184
Copyright © 2004 by The Endocrine Society

Prolactin and Transforming Growth Factor-ß Signaling Exert Opposing Effects on Mammary Gland Morphogenesis, Involution, and the Akt-Forkhead Pathway

Jason P. Bailey, Kathryn M. Nieport, Matthew P. Herbst, Sunil Srivastava, Rosa A. Serra and Nelson D. Horseman

Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: Nelson D. Horseman, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0576. E-mail: nelson.horseman{at}uc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Both prolactin (PRL) and TGF-ß regulate cell survival in mammary epithelial cells, but their mechanisms of interactions are not known. In primary mammary epithelial cells and the HC11 mouse mammary epithelial cell line, PRL prevented TGF-ß-induced apoptosis, as measured by terminal deoxynucleotidyltransferase dUTP nick-end labeling staining and caspase-3 activation. This effect depended on phosphatidyl inositol triphosphate kinase (PI3K). PI3K activates a downstream serine/threonine kinase, Akt; therefore, we investigated the role of Akt in the interaction between PRL and TGF-ß signaling. Akt activity was inhibited by TGF-ß over a 20- to 60-min time course. In TGF-ß-treated cells, PRL disinhibited Akt in a PI3K-dependent manner. Expression of dominant negative Akt blocked the protective effect of PRL in TGF-ß-induced apoptosis. Transgenic mice overexpressing a dominant-negative TGF-ß type II receptor (DNIIR) in the mammary epithelium undergo hyperplastic alveolar development, and this effect was PRL dependent. Involution in response to teat sealing was slowed by overexpression of DNIIR; furthermore, Akt and forkhead phosphorylation increased in the sealed mammary glands of DNIIR mice. Thus, Akt appears to be an essential component of the interaction between PRL and TGF-ß signaling in mammary epithelial cells both in vitro and in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
INVOLUTION IN THE mammary gland consists of two phases: the first phase, which can be reversed by reinitiating suckling, is characterized by an engorgement of the gland with milk followed by widespread epithelial apoptosis (1, 2, 3). The second phase is irreversible and is characterized by an up-regulation of remodeling genes, disruption of the alveolar structure, and remodeling of the basement membrane (3, 4). Several growth factors and proapoptotic genes, such as TGF-ß, proapoptotic Bcl-2 family members, and matrix metalloproteinases are up-regulated during involution (5, 6, 7). Meanwhile, other growth factors and several lactation genes such as prolactin (PRL), ß-casein, and antiapoptotic Bcl-2 family members are down-regulated during involution (1, 6, 8, 9). Recent studies show that PRL plays a critical role in cellular survival (10, 11, 12); however, understanding how these survival factors interact with proapoptotic signals is essential for a more complete understanding of mammary gland development, as well as apoptosis per se.

One known proapoptotic factor is TGF-ß. TGF-ß isoforms are locally produced growth factors that can induce proliferation, growth arrest, and/or apoptosis in a cell type- and dose-dependent manner (13, 14, 15). Typically, TGF-ß can induce apoptosis in epithelial cells and induce proliferation in mesenchymal cells (15). TGF-ß induces apoptosis in a variety of tissues including hepatomas, gastric carcinomas, normal prostate epithelial cells, and uterine epithelial cells (16, 17, 18, 19). Transgenic techniques have allowed a better understanding of how TGF-ß, and which of its isoforms, may affect apoptosis in mammary glands. Whey acidic protein-TGF-ß1 mice have increased levels of apoptosis in pregnant and lactating mammary glands as compared with wild-type mice (20). Furthermore, these mice have a reduced volume of milk and decreased synthesis of milk proteins, both of which are believed to be a result of decreased secretory epithelial cell survival (20). During involution, all three isoforms of TGF-ß (ß1, ß2, and ß3) are up-regulated and act through a common receptor (5, 21); however, TGF-ß3 appears to have the highest increase and may mediate, in part, the first phase of involution (5, 22). Furthermore, mice overexpressing TGF-ß3 in the mammary gland have increased levels of apoptosis during milk stasis, suggesting that TGF-ß3 is a local mediator of cell death during mammary gland involution (22). Mammary explants from TGF-ß3 null mice have lower levels of apoptosis induced by milk stasis, adding more evidence to the critical role of TGF-ß3 in the induction of apoptosis during mammary gland involution (22).

Generation of transgenic mice expressing a dominant-negative form of the TGF-ß type II receptor (DNIIR) has also provided insight into the function of TGF-ßs in mammary gland morphogenesis. The mouse mammary tumor virus (MMTV)-DNIIR mice, expressing the DNIIR transgene in the mammary epithelium, have a phenotype of hyperplasia in the alveolar epithelium and express milk proteins as virgins (23). These data suggest that TGF-ß isoforms may participate in the regulation of mammary epithelial cell death; however, the signaling pathways by which TGF-ß induces apoptosis are unclear. In contrast, the role of PRL as a survival factor is well documented.

PRL activates several signaling pathways that may mediate cellular survival. The Jak/Stat pathway regulates the transcription of milk protein genes but also activates transcription of antiapoptotic genes, such as Bcl-2 and Bcl-XL (24, 25). Another pathway that has been implicated in cell survival is the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway. The hormones and growth factors that can activate this pathway include PRL, insulin, epidermal growth factor (EGF), and IL-6 (10, 26, 27, 28). Recent studies have helped provide a better understanding of the mechanism of PRL-mediated activation of PI3K. Upon tyrosine phosphorylation of the PRL receptor by Janus kinase 2, the p85 subunit of PI3K interacts with the PRL receptor (29). This interaction occurs most likely through Fyn, although insulin receptor substrate-1 and Cbl may also be involved in this interaction (30, 31). Phosphoinositol triphosphates that bind to the pleckstrin homology domains of various kinases, including Akt, are synthesized in response to PI3K activation (32). Akt is a serine/threonine kinase that promotes cell survival in various cell types. Akt requires phosphoinositol triphosphate binding on the pleckstrin homology domain and phosphorylation of both Ser-473 and Thr-308 for full activity (33, 34, 35). Akt phosphorylates several downstream targets, including glycogen synthase kinase 3 (GSK-3), Bad, and the forkhead family of transcription factors (e.g. FKHR) (36, 37, 38, 39). All of these substrates are inhibited by Akt phosphorylation, and this inhibition causes an increase in cell survival. Furthermore, overexpression of Akt in the mammary gland leads to delayed involution (40). All of these data represent an understanding of how Akt can inhibit apoptosis, but little is known about the cross-talk between pro- and antiapoptotic stimuli on Akt activity itself.

In the present study, we have investigated the potential interaction between PRL and TGF-ß signaling in apoptosis. Our results show that PRL can block TGF-ß-induced apoptosis in mammary epithelial cells. Furthermore, this cell survival effect is mediated by disinhibition of Akt because Akt is inhibited by TGF-ß, and this effect can be prevented by PRL in a PI3K-dependent manner. In vivo, TGF-ß and PRL signaling interact during normal morphogenesis, and TGF-ß signaling inhibits Akt and Forkhead phosphorylation during early involution. These data demonstrate a novel homeostatic interaction between PRL and TGF-ß that is important for the regulation of Akt and apoptosis in mammary epithelial cells and, consequently, for normal mammary gland physiology, development, and breast cancer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRL Suppresses TGF-ß-Induced Apoptosis in HC11 Cells in a Dose-Dependent Manner
TGF-ß has been shown to induce apoptosis in a number of tissues, but previous data on mammary epithelial cells have been indirect. HC11 cells were treated with increasing doses of TGF-ß, and apoptosis was measured using the TUNEL (terminal deoxynucleotidyltransferase dUTP nick-end labeling) assay. The lowest dose of porcine (p) TGF-ß (1 ng/ml) did not increase apoptosis compared with the control (9.2 ± 1.8% vs.12.4 ± 2.3%), but 10 ng/ml and 25 ng/ml TGF-ß significantly increased TUNEL-positive cells (36.4 ± 6.8% and 49.1 ± 3.1%, respectively; Fig. 1AGo, panels i–iv). These data demonstrated that TGF-ß acts directly as an inducer of apoptosis in HC11 mammary epithelial cells. Increasing doses of PRL were added to HC11 cells treated with 10 ng/ml TGF-ß, and PRL attenuated the proapoptotic effect of TGF-ß in a dose-dependent manner (Fig. 1CGo, panels v–ix). Therefore, PRL can inhibit TGF-ß-induced apoptosis in mammary cells, suggesting that TGF-ß and PRL interact through at least one common pathway, which controls mammary epithelial cell apoptosis. We next addressed whether the PI3K-Akt pathway might be involved.



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  		Fig. 1. TGF-ß Induces and PRL Inhibits Apoptosis in a Dose-Dependent Manner

HC11 cells, cultured in 1% SCM, were treated with increasing doses of TGF-ß1 to induce apoptosis or 10 ng/ml pTGF-ß1 plus varying concentrations of PRL for 24 h. TGF-ß increased the number of TUNEL-positive cells, and increasing concentrations of PRL reduced the number of TUNEL-positive cells. Confocal microscopy was used to capture images that were used for cell counts in three replicate experiments of TUNEL staining. A, TUNEL staining of HC11 cells treated with the following: i, 1% SCM; ii, 1 ng/ml pTGF-ß1; iii, 10 ng/ml pTGF-ß1; iv, 25 ng/ml pTGF-ß1. B, Quantification of TUNEL-positive cells from panel A. C, TUNEL staining of HC11 cells treated with the following: v, 10 ng/ml pTGF-ß1; vi, 10 ng/ml pTGF-ß1 plus 1 ng/ml oPRL; vii, 10 ng/ml pTGF-ß1 plus 10 ng/ml oPRL; viii, 10 ng/ml pTGF-ß1 plus 100 ng/ml oPRL; ix, 10 ng/ml pTGF-ß1 plus 1 µg/ml oPRL. D, Quantification of TUNEL-positive cells from panel C. Cells were counterstained with propidium iodide (PI) and counted; the percentages reflect the number of TUNEL-positive cells divided by total PI-stained nuclei. Bars with the same letters are not statistically significant; bars with different letters are statistically different, P < 0.05 with n = 3. Error bars represent SEM.

 
PRL Interacts with TGF-ß through PI3K
HC11 cells exposed to 1% serum for 24 h had a low level (8.3 ± 1.2%) of apoptotic cells as defined by TUNEL staining (Fig. 2AGo, panel i). The addition of 1.0 µg/ml PRL maintained the small number (5.0 ± 1.7%) of TUNEL-positive cells (Fig. 2AGo, panel ii); however, the addition of 10 ng/ml TGF-ß alone elicited a strong apoptotic response, inducing 35.9 ± 6.2% of the cells to undergo apoptosis [compared with 1% serum-containing media (SCM), P < 0.001; Fig. 2AGo, panel iii]. Furthermore, 1 µg/ml ovine (o)PRL completely attenuated TGF-ß-induced apoptosis (4.8 ± 0.4% for TGF-ß and PRL vs. 35.9 ± 6.2% for TGF-ß alone, P < 0.001; Fig. 2AGo, panel iv). To determine whether the effect of PRL was mediated through the PI3K pathway, cells were treated with 200 nM wortmannin, a selective PI3K inhibitor. HC11 cells treated with wortmannin alone had 35.9 ± 5% TUNEL-positive cells (Fig. 2AGo, panel vi), and wortmannin induced apoptosis in PRL-treated cells, although at a lower percent as compared with wortmannin alone (25.1 ± 2.5% PRL and wortmannin vs. 35.9 ± 5% for wortmannin alone, P < 0.05; Fig. 2AGo, panel vii). This difference suggested that the antiapoptotic effect of PRL has both PI3K-dependent and PI3K-independent components. Wortmannin completely blocked the protective effect of PRL during TGF-ß-induced apoptosis (34.2 ± 1.3% for PRL, TGF-ß, and wortmannin vs. 4.8 ± 0.4% for PRL and TGF-ß, P < 0.001; Fig. 2AGo, panel viii), suggesting that the primary pathway for TGF-ß/PRL interaction is PI3K dependent. LY294002, another specific PI3K inhibitor, yielded similar TUNEL results as wortmannin (Fig. 2AGo, panels ix–xii).



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  		Fig. 2. PRL Blocks TGF-ß-Induced Apoptosis in HC11 Cells in a PI3K-Dependent Manner

HC11 cells were cultured in 1% SCM for 24 h. The cells were then exposed to the respective treatments for an additional 24 h, and the images shown are representative of three replicate experiments. Staining of apoptotic nuclei was greatly increased in TGF-ß-treated cells, and this effect was blocked by PRL in a PI3K-dependent manner. A, Confocal images of TUNEL staining in HC11 cells treated with the following: i, 1% SCM; ii, 1.0 µg/ml oPRL; iii, 10 ng/ml pTGF-ß1; iv, 1.0 µg/ml oPRL plus 10 ng/ml pTGF-ß1; v, 1% SCM plus 200 nM wortmannin (Wort.); vi, 1.0 µg/ml oPRL plus 200 nM wortmannin; vii, 10 ng/ml pTGF-ß1 plus 200 nM wortmannin; viii, 1.0 µg/ml oPRL plus 10 ng/ml pTGF-ß1 plus 200 nM wortmannin; ix, 1% SCM plus 50 µM LY294002 (LY); x, 1.0 µg/ml oPRL plus 50 µM wortmannin; xi, 10 ng/ml pTGF-ß1 plus 50 µM LY294002; xii, 1.0 µg/ml oPRL plus 10 ng/ml pTGF-ß1 plus 50 µM LY294002. B, Quantification of TUNEL-positive cells from panel A. C, Caspase-3 activity assay. Bars with the same letters are not statistically significant; bars with different letters are statistically different; P < 0.05 with n = 3. Error bars represent SEM.

 
A caspase-3 activity assay, another indicator of apoptotic cells, was performed to complement the TUNEL results (Fig. 2CGo). TGF-ß (10 ng/ml) increased caspase-3 activity (143.0 ± 3.1% of control), and addition of 1 µg/ml oPRL inhibited caspase-3 activity (93.5 ± 1.5% of control). Wortmannin (200 nM) increased caspase-3 activity above the TGF-ß-induced level (189.5 ± 7.4% of control), which was partially reversed by 1 µg/ml oPRL (129.3 ± 2.7% of control). As in the TUNEL assay, the PRL effect on caspase-3 in TGF-ß-treated cells was blocked by 200 nM wortmannin (201.4 ± 2.4%). These data suggested that activation of PI3K by PRL is one of the requirements for PRL-mediated cell survival. They also imply that even though there may be multiple pathways by which PRL regulates cell survival and caspase-3 activity, the PI3K pathway is the key mediator for the TGF-ß-PRL interaction.

Akt Activity and Ser-473 Phosphorylation Regulation by PRL and TGF-ß
One of the downstream targets of PI3K is the serine/threonine kinase Akt (35). Therefore, we set out to determine the effects of PRL and TGF-ß on Ser-473 phosphorylation, and in vitro kinase assays using GSK-3 as a substrate were used to ascertain Akt activity. PRL alone (1.0 µg/ml) had a minimal effect on Akt activity in HC11 cells. Transient increases of Ser-473-phosphorylated Akt and Akt activity were seen consistently at 2 min (Fig. 3AGo). Pretreatment for 20 min with 200 nM wortmannin reduced Akt activation to almost undetectable levels (Fig. 3BGo). Pretreatment with 50 µM LY294002 also reduced Akt activation to similar levels (data not shown). TGF-ß (10 ng/ml) inhibited Akt phosphorylation and activity through 20–60 min after treatment (Fig. 3CGo). However, the addition of PRL along with TGF-ß resulted in a maintained level Ser-473 phosphorylation and Akt activity at a steady state for 60 min (Fig. 3DGo), and Akt activity remained constant through 24 h (data not shown). These results suggested that PRL can block TGF-ß inhibition of Akt and, conversely, that TGF-ß prevents the transient activation of Akt by PRL. To determine whether these effects require PI3K activity, PRL and TGF-ß were added together with wortmannin. Under these conditions, Akt activity and Ser-473 phosphorylation were strongly inhibited (Fig. 3EGo).



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  		Fig. 3. PRL Prevents TGF-ß-Induced Dephosphorylation of Ser-473 on Akt and Suppression of Akt Activity in a PI3K-Dependent Manner

Western blot analysis of Akt phosphorylation of HC11 cells in response to PRL, TGF-ß, and wortmannin (Wort.). Blots were probed with anti-Ser-473 Akt, and then stripped and reprobed for total Akt. PRL increased Ser-473 phosphorylation at 2 min, whereas wortmannin blocked this activation. TGF-ß decreased phospho-Ser-473 at 20 min, but the addition of PRL prevented the inhibition, and wortmannin blocked this effect. Akt activity was inhibited by TGF-ß; however, addition of PRL prevented this inhibition. Wortmannin attenuated the effect of PRL on the maintenance of Akt activity both with PRL alone or PRL and TGF-ß. Cells were treated as follows for the indicated times. A, Cells treated with 1.0 µg/ml oPRL; B, cells pretreated for 20 min with 200 nM wortmannin, and then treated with 1.0 µg/ml oPRL for the indicated times; C, cells treated with 10 ng/ml pTGF-ß1; D, cells treated with 10 ng/ml pTGF-ß1 and 1.0 µg/ml oPRL; E, cells pretreated for 20 min with 200 nM wortmannin and then treated with 1.0 µg/ml oPRL and 10 ng/ml pTGF-ß1 for the indicated times (all blots were done in triplicate, n = 3). KA, Kinase assay.

 
PRL Suppression of TGF-ß-Induced Apoptosis Is Blocked by DN-Akt
To determine whether the effects of PRL and TGF-ß were indeed directed through Akt, an adenovirus strategy was used (41). HC11 cells were infected 48 and 72 h before harvesting. Expression of these viruses in HC11 cells was apparent at 48 h post infection (Fig. 4AGo). Immunoblotting with Ser-473 Akt antibody showed that DN-Akt viral expression attenuated the steady-state Ser-473 Akt, confirming that the DN-Akt virus was functional. Furthermore, expression of constitutively active myristolated Akt (Myr-Akt) greatly increased the amount of Ser-473 Akt present in the cells (Fig. 4AGo). Akt kinase assays confirmed the DN-Akt-infected cells lacked active Akt at both 48 and 72 h, and the Myr-Akt-infected cells had high levels of active Akt (Fig. 4AGo).



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  		Fig. 4. DN-Akt Prevents PRL Inhibition of TGF-ß-Induced Apoptosis

HC11 cells were infected 48 and 72 h before harvest with CM from 293 cells infected with the following: control CM, DN-Akt adenovirus CM, Myr-Akt adenovirus CM and treated with PRL and/or TGF-ß where indicated. A, Immunostaining for Ser473-Akt, total Akt, and in vitro Akt kinase assay (KA), as labeled at right margin. B, Cells were infected 48 h before fixing with CM from DN-Akt-infected cells or control 293 cells. Cells were treated 24 h before the harvest with 1.0 µg/ml oPRL, 10 ng/ml pTGF-ß1, or both. Confocal images of TUNEL-stained HC11 cells treated as follows: i, 1.0 µg/ml PRL plus control 293 CM; ii, 1.0 µg/ml oPRL plus 10 ng/ml TGF-ß1 plus control 293 CM; iii, 10 ng/ml pTGF-ß1 plus control CM; iv, 1% SCM plus DN-Akt CM; v, 1.0 µg/ml oPRL plus DN-Akt CM; vi, 10 ng/ml pTGF-ß1 plus DN-Akt CM; vii, 1.0 µg/ml oPRL and 10 ng/ml pTGF-ß1 plus DN-Akt CM; viii, 20% FBS plus DN-Akt CM. C, Quantification of TUNEL-positive cells from panel B. Bars with the same letters are not statistically significant; bars with different letters are statistically different, P < 0.05 with n = 3. Error bars represent SEM.

 
To determine the possible role of Akt in the antiapoptotic effect of PRL in HC11 cells, conditioned media (CM) from either control 293 cells or 293 cells infected with DN-Akt adenovirus were added to the culture 48 h before harvest. Then the cultures were treated with PRL and/or TGF-ß 24 h before the cells were harvested. Cells exposed to control CM treated with either PRL alone or PRL and TGF-ß resulted in minimal TUNEL-positive cells (4.8 ± 0.3% and 4.4 ± 0.3%, respectively; Fig. 4BGo, panels I and ii). Cells exposed to control CM and TGF-ß showed 33.2 ± 4% apoptotic cells (Fig. 4BGo, panel iii). Expression of DN-Akt significantly increased TUNEL-positive cells as compared with controls (all values compared with either control, P < 0.001; Fig. 4BGo, panel iv). PRL (1.0 µg/ml) failed to inhibit apoptosis in the DN-Akt-infected cells, suggesting that the cell survival actions of PRL require Akt-dependent mechanisms (Fig. 4BGo, panel v). TGF-ß treatment did not increase the number of TUNEL-positive cells in DN-Akt infected cells (compare Fig. 2AGo, panel iii, with Fig. 4BGo, panel vi). Furthermore, PRL was incapable of inhibiting TGF-ß-induced apoptosis in DN-Akt-infected cells (38.3 ± 4.8% vs. 5.3 ± 1.4%; Fig. 4BGo, panel vii). Incubating the DN-Akt cells with 20% fetal bovine serum (FBS) decreased TUNEL-positive cells to 22.8 ± 2%, suggesting that Akt-independent survival mechanisms can be accessed by serum repletion (Fig. 4BGo, panel viii). These data suggest that active Akt is required for inhibition of apoptosis by PRL.

Myr-Akt Prevents TGF-ß-Induced Apoptosis
Cells were infected with a Myr-Akt adenoviral construct to determine the effect of constitutively active Akt on TGF-ß-induced apoptosis. Cells were infected in the same manner as above. Cells exposed to control CM and TGF-ß showed 33.2 ± 4% apoptotic cells (Fig. 5AGo, panel i). Cells treated with Myr-Akt showed very low levels of apoptosis (4.3 ± 1.1%; Fig. 5AGo, panel ii). Myr-Akt was able to significantly reduce the number of TUNEL-positive cells in TGF-ß treated cultures (8.4 ± 2.3%; Fig. 5Go, panel iv). As a positive control, cells infected with Myr-Akt were treated with 1 µM staurosporine for 24 h, which increased TUNEL-positive cells to 15 ± 2.3% (Fig. 5AGo, panel vi). These data suggest that inhibition of Akt is a prerequisite for TGF-ß-induced apoptosis, although a mechanism by which TGF-ß may inhibit Akt is not known.



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  		Fig. 5. Myr-Akt Prevents TGF-ß-Induced Apoptosis

Cells were infected 48 h before fixing with CM from Myr-Akt-infected cells or control 293 cells. Cells were treated 24 h before harvest with 1.0 µg/ml oPRL, 10 ng/ml pTGF-ß1, or both. Myr-Akt infection prevented TGF-ß-induced apoptosis. A, Confocal images of TUNEL-stained HC11 cells treated as follows: i, 10 ng/ml pTGF-ß plus control CM; ii, 1% SCM plus Myr-Akt CM; iii, 1.0 µg/ml oPRL plus Myr-Akt CM; iv, 10 ng/ml pTGF-ß1 plus Myr-Akt CM; v, 1.0 µg/ml oPRL and 10 ng/ml pTGF-ß1 plus Myr-Akt CM; vi, 1 µM staurosporine (Stauro.) plus Myr-Akt CM. B, Quantification of TUNEL-positive cells for panel A. Bars with the same letters are not statistically significant; bars with different letters are statistically different; P < 0.05 with n = 3. Error bars represent SEM.

 
PRL Prevents TGF-ß-Induced Apoptosis in Primary Mammary Epithelial Cells via an Akt-Dependent Mechanism
Primary mammary epithelial cells (PMECs) were used to expand and validate the findings observed in the HC11 cell line. TGF-ß exerted similar effects on PMECs as it did in HC11 cells. PMECs cultured in complete medium for 24 h contained few TUNEL-positive cells (4.8 ± 1.6%, Fig. 6AGo, panel i). Treatment with 5.0 µg/ml PRL maintained a low number of apoptotic nuclei (1.5 ± 0.7%; Fig. 6AGo, panel 2); in contrast, addition of TGF-ß (10 ng/ml) elicited a substantial apoptotic response, inducing 18.7 ± 3.1% of the cells to undergo apoptosis (Fig. 6AGo, panel iii). Furthermore, 5.0 µg/ml PRL completely attenuated TGF-ß-induced apoptosis (4.6 ± 2.0%; Fig. 6AGo, panel iv). These responses were similar to those seen in HC11 cells; however, the magnitude of the response was lower. To determine the effect of Akt activity on PRL inhibition of TGF-ß-induced apoptosis in PMECs, the adenoviral strategy was used.



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  		Fig. 6. PRL Blocks TGF-ß-Induced Apoptosis in PMECs in an Akt-Dependent Manner

PMECs were cultured in growth medium for 24 h. The cells were then exposed to the respective treatments for an additional 24 h, and the images shown are representative of three replicate experiments. Staining of apoptotic nuclei was greatly increased in TGF-ß-treated cells, and this effect was blocked by PRL in an Akt-dependent manner. A, Confocal images of TUNEL staining in PMECs treated with the following: i, 1% SCM; ii, 1.0 µg/ml oPRL; iii, 10 ng/ml pTGF-ß1; iv, 1.0 µg/ml oPRL plus 10 ng/ml pTGF-ß1. B, Quantification of TUNEL-positive cells for panel A. Bars with the same letters are not statistically significant; bars with different letters are statistically different; P < 0.05 with n = 3. Error bars represent SEM. C, Confocal images of TUNEL staining in PMECs infected with DN-Akt adenovirus for 24 h, and then treated with the following for an additional 24 h: v, 1.0 µg/ml PRL plus control 293 CM; vi, 10 ng/ml pTGF-ß1 plus control CM; vii, 1% SCM plus DN-Akt CM; viii, 1.0 µg/ml oPRL plus DN-Akt CM; ix, 10 ng/ml pTGF-ß1 plus DN-Akt CM; x, 1.0 µg/ml oPRL and 10 ng/ml pTGF-ß1 plus DN-Akt CM; xi, 20% FBS plus DN-Akt. D, Quantification of TUNEL-positive cells for panel C. E, Confocal images of TUNEL staining in PMECs infected with DN-Akt adenovirus for 24 h, and then treated with the following for an additional 24 h: xii, 10 ng/ml pTGF-ß plus control CM; xiii, 1% SCM plus Myr-Akt CM; xiv, 1.0 µg/ml oPRL plus Myr-Akt CM; xv, 10 ng/ml pTGF-ß1 plus Myr-Akt CM; xvi, 1.0 µg/ml oPRL and 10 ng/ml pTGF-ß1 plus Myr-Akt CM; xvii, 1 µM staurosporine (Stauro.) plus Myr-Akt CM. F, Quantification of TUNEL-positive cells for panel E.

 
Cells were infected with DN-Akt-CM for 24 h before treatment with PRL and/or TGF-ß. Cells exposed to control CM treated with PRL alone resulted in minimal TUNEL-positive cells (2.6 ± 1.6%; Fig. 6CGo, panel v) whereas, cells exposed to control CM and TGF-ß showed 16.6 ± 2.8% apoptotic cells (Fig. 6CGo, panel vi). Expression of DN-Akt significantly increased TUNEL-positive cells as compared with PRL-treated CM (all DN-Akt treatments vs. PRL-treated control, P < 0.05; Fig. 6CGo, panel vii). PRL (5.0 µg/ml) failed to inhibit apoptosis in the DN-Akt-infected cells, suggesting that the cell survival actions of PRL require Akt-dependent mechanisms (Fig. 6CGo, panel viii). TGF-ß treatment did not increase the number of TUNEL-positive cells in DN-Akt infected cells (compare Fig. 2AGo, panel iii, with Fig. 6CGo, panel ix). Furthermore, PRL was incapable of inhibiting TGF-ß-induced apoptosis in DN-Akt-infected cells (26.2 ± 3.3% vs. 4.6 ± 2.0%; Fig. 6CGo, panel x, and Fig. 6AGo, panel iv). Incubating the DN-Akt cells with 20% FBS decreased TUNEL-positive cells to 14 ± 2.6%, suggesting that Akt-independent survival mechanisms can be accessed by serum repletion (Fig. 6CGo, panel xi). These data suggest that active Akt is required for inhibition of apoptosis by PRL.

Furthermore, Akt inhibition is required for TGF-ß-induced apoptosis in PMECs as seen in Fig. 6EGo. Cells exposed to control CM and TGF-ß showed 16.4 ± 2.9% apoptotic cells (Fig. 6EGo, panel xii). Cells treated with Myr-Akt showed very low levels of apoptosis (1.3 ± 0.4%; Fig. 6EGo, panel xiii). Myr-Akt was able to significantly reduce the number of TUNEL-positive cells in TGF-ß-treated cultures (0.7 ± 0.4%; Fig. 6EGo, panel xv). As a positive control, cells infected with Myr-Akt were treated with 1 µM staurosporine for 24 h, which increased TUNEL-positive cells to 10 ± 1.9% (Fig. 6EGo, panel xvii).

Hyperplastic Development of MMTV-DNIIR Mammary Glands Is PRL Dependent
To determine whether PRL has a role in the hyperplastic phenotype present in mice lacking TGF-ß signaling in mammary epithelium (MMTV-DNIIR), MMTV-DNIIR mice were crossed with PRL knockout mice (23). MMTV-DNIIR mice undergo hyperplastic development, characterized by extensive branching and proliferation of lobuloalveolar structures (Fig. 7AGo, panel ii). PRL –/– mice (Fig. 7AGo, panel iii), as previously reported (1), lacked subordinate branching and alveolar buds, as compared with the wild-type virgin mice. The double transgenic MMTV-DNIIR/PRL–/– (Fig. 7AGo, panel iv) lacked the exaggerated alveolar structures and branching of the MMTV-DNIIR mouse with a functional PRL gene. Furthermore, DNIIR gene expression does not change with the lack of PRL expression, thereby demonstrating this effect is a direct result of PRL insufficiency and not decreased DNIIR expression (data not shown). Western blot analyses (Fig. 7BGo) showed that Akt phosphorylation was reduced in mice lacking PRL. Important downstream effectors of Akt, the forkhead transcription factors FHKR and AFX, were phosphorylated in mice exposed to PRL (wild-type or pituitary-grafted animals). Furthermore, immunohistochemistry using a Ser-473 antibody confirmed the results of the Western blot analysis, that elevated levels of activated Akt are dependent on the presence of the PRL gene, but independent of DNIIR expression (Fig. 7CGo). These data suggest that PRL activates the Akt pathway in the mammary gland in vivo. DNIIR had no perceptible effect on steady-state Akt or forkhead phosphorylation in virgin mice, presumably because TGF-ß exposure is low in virgin mice. To further investigate the role of TGF-ß signaling and Akt activity at a time when TGF-ß signaling is activated, involution studies were performed.



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  		Fig. 7. PRL Is Required For Manifestation of Hyperplastic Mammary Gland Development in MMTV-DNIIR Mice

A, Whole mounts of 20-wk-old mice of the following genotypes: i, PRL+/–, DNIIR–; ii, PRL+/–, DNIIR+; iii, PRL–/–, DNIIR–; iv, PRL–/–, DNIIR+. B, Western blots from virgin 20-wk-old mammary gland protein extracts, using antibodies identified in the right margin. 1, PRL–/–, DNIIR+; 2, PRL+/–, DNIIR+; 3, PRL–/–, DNIIR–; 4, PRL+/–, DNIIR–; 5, PRL–/–, DNIIR+ with pituitary graft from 18 d. C, Ser473-Akt immunohistochemistry of sections of virgin 20-wk-old mammary glands: v, PRL+/–, DNIIR–; vi, PRL–/–, DNIIR; vii, PRL+/–, DNIIR+; viii, PRL–/–, DNIIR+.

 
DNIIR Mice Have a Defect in Mammary Gland Involution
TGF-ß expression is increased significantly at 24–72 h after involution initiated by milk stasis or removal of pups (5, 22). Mammary glands were sealed for 72 h and tissues were harvested. The morphology of wild-type and DNIIR glands were similar in active lactation (Fig. 8AGo, panels i and iii, respectively). However, after teats were sealed for 72 h, the wild-type gland lost the integrity of the alveolar structures, and adipose tissue began to return (Fig. 8AGo, panel ii). The sealed DNIIR glands remained intact, and the alveolar structures had not collapsed (Fig. 8AGo, panel iv). These data suggest that TGF-ß signaling is essential for milk stasis-induced mammary gland involution (the first stage of involution). Immunoblots of protein extracts for these sealed and lactating glands revealed a large increase in Akt and FKHR phosphorylation in MMTV-DNIIR mice compared with wild-type controls (Fig. 8BGo), suggesting one of the actions of TGF-ß during milk stasis may be to restrain Akt and FKHR phosphorylation. Furthermore, caspase-3 activation was prevented in the DNIIR mice during milk stasis-induced involution (Fig. 8CGo).



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  		Fig. 8. Blockade of TGF-ß Signaling Causes a Delay in Involution in Sealed Mammary Glands

A, Hematoxylin and eosin-stained sections of sealed and lactating control mammary glands. i, Wild-type (WT) lactating d 13; ii, WT 3-d sealed; iii, MMTV-DNIIR lactating d 13; iv, MMTV-DNIIR 3-d sealed. B, Western blots of mammary gland protein extracts from indicated treatment groups, as identified above each lane. Antibodies used for each Western blot are identified in the right margin. C, Activated caspase-3 immunohistochemistry of sections of the following: i, WT lactating d 13; ii, WT 3-d sealed; iii, MMTV-DNIIR lactating d 13; iv, MMTV-DNIIR 3-d sealed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
PRL effects on milk protein gene expression have been studied extensively. However, the role of PRL as a cellular survival signal is less well understood. It has been documented in Nb2 cells that PRL prevents dexamethasone-induced apoptosis by activating Akt (10); however, PRL as a survival factor in mammary epithelial cells has yet to be substantially investigated. Here we report that PRL and TGF-ß interact with each other through PI3K regulation of the serine/threonine kinase Akt. Our results also suggest that PI3K is essential for the survival effect of PRL in TGF-ß-treated mammary cells. TUNEL and caspase-3 activity assays demonstrate PRL can inhibit TGF-ß-induced apoptosis in a PI3K- and Akt-dependent manner in both HC11 cells and PMECs. DN-Akt prevented the protective effect of PRL on TGF-ß-induced apoptosis, whereas Myr-Akt suppressed TGF-ß-induced apoptosis altogether in two different mammary epithelial cell culture systems. These data demonstrate a role of Akt in the interaction between PRL and TGF-ß signaling. A similar mechanism was recently reported between IL-6 and TGF-ß in CH3 10T1/2cells (28). It has been reported that low doses of TGF-ß1 (2 ng/ml) can activate Akt and prevent apoptosis in another mouse mammary epithelial cell line, NmuMG (13). However, Yu et al. (14) demonstrated recently that a low dose (5 ng/ml) of TGF-ß1 slightly activated Akt, but apoptosis was induced at time points similar to what was seen in the HC11 cells. These paradoxical results in NmuMG cells are not consistent with the effects of TGF-ß in transgenic mouse models, or the induction of TGF-ß expression during involution (5, 22), and suggest that TGF-ß signaling is subverted in NmuMG cells. In the HC11 cells no apoptosis was seen at low (1 ng/ml) doses and 5 ng/ml TGF-ß increased apoptosis slightly (Fig. 1Go).

PRL appears to inhibit apoptosis through both PI3K-dependent and PI3K-independent mechanisms, although the PI3K-independent mechanisms appear to play a smaller role in our system. Although the mechanism of PRL inhibition of apoptosis has both PI3K-dependent and -independent components, both of these mechanisms required Akt activity. The TGF-ß activation of apoptosis appeared to affect only the PI3K-dependent pathway, because there was no PI3K-independent effect of PRL when the cells are treated with PRL, TGF-ß, and a PI3K inhibitor. These data present a novel and interesting mechanism of interaction between PRL, TGF-ß, Akt, and cell fate in mammary epithelial cells.

PRL activation of Akt has been studied extensively in lymphocytes (10, 11). PRL protects Nb2 lymphocytes, decidual cells, and thymocytes from apoptosis in a PI3K/Akt-dependent manner (10, 11, 12, 42). The mechanisms by which TGF-ß inhibits PI3K/Akt signaling are not known. One possible target would be a phosphatase that is activated by TGF-ß. The phosphatase, Src homology 2 domain-containing 5-inositol phosphatase, is transcriptionally up-regulated and activated by TGF-ß1 (43). Furthermore, TGF-ß1 inhibited Akt phosphorylation in MPC-11 cells at 20 min, which is the same timeframe in which we see maximal inhibition of Akt by TGF-ß in HC11 cells (43). TGF-ß was also shown to activate protein phosphatase 2A, a serine/threonine phosphatase that can dephosphorylate Akt (44, 45). Phosphatase and tensin analog is another phosphatase that can inhibit Akt activity (18, 33, 34). Also, some breast tumors lack phosphatase and tensin analog and have constitutively active Akt (46, 47). The exact mechanism by which TGF-ß inhibits Akt and induces apoptosis in mammary epithelial cells remains to be elucidated; however these phosphatases appear to fit the criteria as possible targets in this pathway. PRL may also inhibit one of the above phosphatases, but there is no evidence that PRL can inhibit these classes of phosphatases. Another possible mechanism for TGF-ß to regulate Akt is through cAMP-dependent protein kinase A (PKA). PKA is up-regulated by d 1 of weaning, a time when TGF-ß has also been up-regulated (48). TGF-ß activates PKA, which negatively regulates Akt in a PI3K-independent manner (28). PKA activation by TGF-ß is maximal at 15 min (49), which is the same relative time course that we see in TGF-ß inhibition of Akt. Our results provide a well-characterized biological context in which to address these possible TGF-ß mechanisms.

The mechanism for protecting cells from apoptosis driven by TGF-ß may be a common and vital mechanism in epithelial cells. In the mammary gland this mechanism may be relevant at the interface between involution and lactation, where levels of TGF-ß and PRL are in flux. The lactating gland is exposed to high levels of PRL, with low TGF-ß, and does not undergo apoptosis (1, 5, 22). However, after weaning, PRL levels drop, TGF-ß levels increase, and there is widespread apoptosis in the mammary epithelium (3, 5, 7, 22). The levels of these factors may determine whether or when involution takes place. At other stages of mammary gland development, the relative levels of exposure to PRL and TGF-ß may be regulated in specific compartments of the developing gland so that branching, alveolar development, and terminal end bud regression are coordinated. For example, in branching morphogenesis, TGF-ß complexes with the extracellular matrix at ductal branch points and inhibits growth (50). In vivo experiments in MMTV-DNIIR and PRL –/– mice provide insight into the physiological function of Akt regulation and mammary gland morphogenesis by PRL and TGF-ß. Although no effect on Akt phosphorylation was seen in DNIIR-expressing virgin mice, dramatic increases in Akt and FKHR phosphorylation were seen in sealed DNIIR-expressing mammary glands. This suggests a vital function of TGF-ß signaling in maintaining a low level of Akt activity during the initial lactogen-resistant phase of mammary gland involution. Recently, forced weaning experiments were conducted in the MMTV-DNIIR mice and showed a similar delay in involution (51).

Blockade of TGF-ß signaling by a dominant-negative receptor prevented the morphological changes of the mammary gland during milk stasis, once again supporting the role of TGF-ß as a modulator of cell survival, and of the involution process itself. The milk stasis data presented here correlate well with similar results in the BLG-TGF-ß3 mice in terms of the critical role for TGF-ß signaling in general, specifically TGF-ß3, in milk stasis-induced apoptosis during mammary gland involution (22).

Our data are the first to demonstrate phosphorylation of forkhead transcription factors (FHKR and AFX) by PRL in the mammary gland. FHKR and AFX are members of a family of transcription factors that regulate several proapoptotic genes. These data suggest a novel homeostatic interaction between TGF-ß and PRL in cell survival wherein PRL may enhance cell survival by counteracting the actions of TGF-ß on Akt and forkhead.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents and Antibodies
Porcine TGF-ß (pTGF-ß) was obtained from R&D Systems (Minneapolis, MN). oPRL (NIDDK oPRL-20, AFP10677C) was obtained from the National Hormone and Pituitary Program (Bethesda, MD). Wortmannin was purchased from Sigma Chemical Co. (St. Louis, MO). Rabbit polyclonal antibodies for Akt, Ser-473-specific Akt, phospho-GSK-3, and LY294002 were purchased from Cell Signaling Technologies (Beverly, MA). Rabbit monoclonal antikeratin-18 was purchased from BD Biosciences Pharmingen (San Diego, CA).

Animals
PRL–/– and MMTV-DNIIR mice were described previously and maintained in the University of Cincinnati Animal Care Facility (23, 52). The surgeries and other procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee. Donor pituitaries were harvested from littermate PRL+/– females and inserted under the kidney capsule of recipients. PRL –/– males were mated to PRL +/– DNIIR females to generate all four genotypes used in these experiments.

Cell Culture
HC11 cells were maintained in RPMI 1640 supplemented with the following: 10% fetal bovine serum (Harlan Bioproducts for Science, Inc., Indianapolis, IN), 1x L-glutamine (Life Technologies, Gaithersburg, MD), 1x antibiotic/antimycotic (Life Technologies), 5 µg/ml insulin (Sigma), 20 ng/ml EGF (Sigma). During experimental procedures, cells were in the following medium: 1% SCM consisted of RPMI 1640, 1x L-glutamine, 1x antibiotic/antimycotic, 500 µg/ml fetuin (Life Technologies), and 50 µg/ml transferrin (Sigma) and 1% FBS. 293 cells were maintained in DMEM (Life Technologies), supplemented with 10% FBS and 10 µg/ml gentamycin (Sigma). Primary mouse mammary epithelial cells (PMEC) were prepared from virgin mice (Swiss Black) by enzymatic digestion and differential trypsinization, a modification of the method of Imagawa (53). In brief, excised mammary glands were minced with a razor blade, transferred into 50-ml conical tubes, and digested in M199 medium containing BSA (2.5 mg/ml, fraction V; Sigma) and collagenase type III (0.1%; Worthington Biochemical Corp., Freehold, NJ), penicillin-streptomycin (1x pen-strep, 50 U/ml and 50 µg/ml, respectively; Life Technologies), and amphotericin B (2 µg/ml; Sigma) for 3 h at 37 C. Organoids were allowed to settle, washed twice in PBS, and incubated under 5% CO2 in DMEM/F12 (1:1) containing insulin (5 µg/ml), EGF (10 ng/ml), 1% FBS (Harlan Bioproducts for Science, Inc.) and 1x pen/strep (complete medium) for 5–7 d to allow the cells to attach to rat tail collagen-coated dishes (BD Biosciences, Palo Alto, CA). Fibroblasts were removed by four rounds of partial trypsinization. Resulting concentrated epithelial cells were plated on CC2-treated eight-well slides (Fisher Scientific, Pittsburgh, PA). The cells were allowed to sit for 3 d, complete medium without insulin or EGF was added to the cells for 24 h, and then specific treatments were carried out for an additional 24 h.

TUNEL Staining
Cells were maintained in growth medium until they reached confluence. The cells were then maintained for 3 d at confluence with the medium being changed every 24 h. At 3 d post confluence, the cells were incubated in 1% SCM for 24 h; then media were changed to the desired treatment groups for an additional 24 h. After 24 h of treatment, the cells were fixed in 4% paraformaldehyde for 1 h and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate on ice for 2 min. The cells were then stained according to the manufacturer’s protocol of the In Situ Cell Death Detection Kit (Roche Clinical Laboratories, Indianapolis, IN). Slides were counterstained with 1 µg/ml propidium iodide (Sigma) to assess total cell number. Images were produced on a Zeiss LSM510 confocal microscope (Carl Zeiss, Thornwood, NY) at the Microscopy Imaging Core of the Cell Biology, Neurobiology, and Anatomy Department (University of Cincinnati). All of the confocal images were made using the identical parameters (scan mode, scale, average, and pinhole).

Western Blotting
Cells were grown to confluence and then maintained at confluence for 3 d. At 3 d post confluence, the media were replaced with 1% serum containing media for 24 h. After 24 h the cells were treated with experimental media for the indicated times. Cells were washed with ice-cold 1x PBS, and then lysed with 1x lysis buffer (Cell Signaling Technologies). Protein concentrations were determined using the Coomassie Plus Protein Assay Reagent (Pierce Chemical Co., Rockford, IL) 50 µg protein was loaded into a 12% acrylamide gel, transferred onto nitrocellulose membrane, and blocked for 1 h in 5% nonfat milk in 1x Tris-buffered saline with 0.1% Tween 20 (TBST). Membranes were incubated with primary antibodies overnight, incubated with horseradish peroxidase-conjugated secondary antibody, and detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL). Densitometry analysis was performed using Gel-Doc imager and software (Bio-Rad, Hercules, CA). All immunoblots were done in triplicate and one-way ANOVA with Tukey’s post hoc tests were done to determine statistical difference.

Akt Kinase Assays
Protein extracts (200 µg) were used for the Nonradioactive Akt Kinase Assay per manufacturer’s instructions (Cell Signaling Technologies, Beverly, MA). Cell extracts were shaken overnight with Akt-sepharose beads. After washing the beads the kinase reaction was performed using GSK-3 fusion protein (Cell Signaling Technologies, Beverly, MA) for 30 min, and samples were then run on a 12% acrylamide gel. Transfer, incubation with antibodies, and analysis of membranes were the same as described above.

Adenovirus production and infection.
DN-Akt and Myr-Akt were constructed as previously described (41). Virus was introduced to 80–90% confluent 293 cells for 3 d, and then the CM was collected and spun to remove cellular debris. Uninfected 293 cells were maintained under the same conditions to produce control CM. The multiplicity of infection was determined using an OD260 of the CM. HC11 cells were infected at the indicated times by adding 10% of the CM (with a multiplicity of infection of 5.5) to the cells for 24 h.

Caspase-3 Assay
Apoptosis was detected using the fluorimetric immunosorbent enzyme caspase-3 assay (Roche). Cell lysates from HC11 cells were treated with PRL, TGF-ß, and wortmannin. Experiments were carried out using the manufacturer’s instructions. Plates (96-well) were coated with anticaspase-3 antibody for 2 h at 37 C, nonspecific binding was blocked, lysates were incubated on plates for 2 h, and the substrate Ac-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin was added for 2 h. The activity was quantified at an excitation wavelength of 360 nm and an emission wavelength of 530 nm.

Teat Sealing Experiment
The teat of the right fourth (inguinal) gland was sealed using a surgical adhesive agent. The remaining glands were left open. Litter size was standardized to six mice per litter. Mice were observed after sealing to ensure complete teat closure and monitored every 8–12 h to ensure that the teat remained sealed. Paired open and closed glands were harvested 72 h after teat closure. The glands were divided for sectioning, protein extracts, and mRNA isolation. Three to four wild-type and MMTV-DNIIR (DNIIR) mice were used in these experiments. A portion of each mammary gland was used for RNA extraction, using TRI Reagent (Molecular Research Center, Inc. Cincinnati, OH). Another portion was used to make protein extracts using the lysis buffer described above. The rest of the gland was fixed for sectioning.

Histochemical and Whole-Mount Analysis
Mammary glands were fixed in 4% paraformaldehyde overnight and embedded in wax after a graded alcohol dehydration, two xylene-clearing stages, and wax series. Paraffin sections (5–10 µm) were dewaxed and rehydrated in graded ethanol series and stained in hematoxylin and eosin. For whole-mount histology of the mammary glands, mammary glands from each animal was excised and processed as previously described (52).

Immunohistochemistry
Sections of mammary glands were deparaffinized with xylene and rehydrated in a graded series of ethanol. Saponin (0.05%, 30 min) was used to permeabilize the tissue sections. Nonspecific binding was blocked by preincubation with normal goat serum (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA). The anticleaved caspase-3 antibody or Anti-Ser473 Akt (Cell Signaling Technologies) was used at a 1:200 dilution in TBST. The protocol for the Vectastain kit was followed as directed by the manufacturer, and peroxidase substrate was used to visualize the caspase-3 staining. Slides were coverslipped, and digital images were taken with Spot camera and software (Sciscope Instrument Co. of Iowa, Iowa City, IA).

Cell Counting and Statistical Analysis
Each of the TUNEL experiments was done in triplicate. Each image was counted for total cell number (propidium iodide) and for TUNEL-positive cells. The percentage of apoptotic cells was averaged for the three experiments. A one-way ANOVA was performed on these data with Tukey’s post hoc test to determine significant differences between groups.


   ACKNOWLEDGMENTS
 
We thank Dr. Kenneth Walsh for providing the DN-Akt and Myr-Akt adenoviral constructs.


   FOOTNOTES
 
This work was supported by grants from the National Institutes of Health (DK52134) and Shriners Hospital for Children (Award 8520).

Present address for R.S.: Department of Cell Biology, University of Alabama-Birmingham, Birmingham, Alabama 35294.

Abbreviations: CM, conditioned media; DN-Akt, dominant negative Akt; DNIIR, transgenic mice expressing a dominant-negative form of the TGF-ß type II receptor; EGF, epidermal growth factor; FBS, fetal bovine serum; GSK-3, glycogen synthase kinase 3; MMTV, mouse mammary tumor virus; Myr-Akt, myristolated Akt; PI3K, phosphatidyl inositol triphosphate kinase; PKA, protein kinase A; PMEC, primary mouse mammary epithelial cell; PRL, prolactin; SCM, serum-containing media; TUNEL, terminal deoxynucleotidyltransferase dUTP nick-end labeling.

Received for publication September 8, 2003. Accepted for publication January 27, 2004.


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