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Molecular Endocrinology, doi:10.1210/me.2005-0392
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Molecular Endocrinology 20 (3): 675-685
Copyright © 2006 by The Endocrine Society

The Genes Induced by Signal Transducer and Activators of Transcription (STAT)3 and STAT5 in Mammary Epithelial Cells Define the Roles of these STATs in Mammary Development

Richard W. E. Clarkson1, Marion P. Boland1, Ekaterini A. Kritikou1, Jennifer M. Lee, Tom C. Freeman, Paul G. Tiffen and Christine J. Watson

Department of Pathology (R.W.E.C., M.P.B., E.A.K., P.G.T., C.J.W.), University of Cambridge, Cambridge CB2 1QP, United Kingdom; and Medical Research Council-Rosalind Franklin Centre for Genome Research (J.M.L., T.C.F.) Hinxton, Cambridge CB10 1SB, United Kingdom

Address all correspondence and requests for reprints to: Dr. C. J. Watson, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom. E-mail: cjw53{at}mole.bio.cam.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Prolactin and leukemia inhibitory factor (LIF) have different roles in the adult mammary gland, which are mediated in part by the signal transducers and activators of transcription (STAT)5 and STAT3. In vivo studies have shown that STAT5 contributes to prolactin-dependent lobuloalveolar development and lactation whereas STAT3 mediates LIF-dependent epithelial apoptosis during postlactational involution. To understand the molecular basis of these STAT-dependent pathways, we demonstrate the ligand-independent effects of STAT5 and STAT3 in mammary epithelial cells in vitro and also identify the genes regulated by these related transcription factors. Thus, using conditionally active STAT3- or STAT5a-GyraseB fusion proteins, we observed that enforced and specific dimerization of STAT3 induced apoptosis whereas STAT5 induced differentiation of mammary epithelial cells. Furthermore, STAT5 attenuated apoptosis mediated by LIF, the physiological inducer of STAT3. Microarray analysis of STAT3- and STAT5-induced genes using this system demonstrated a marked specificity, which reflected their different physiological effects in vitro and in vivo. STAT5-specific gene targets included the milk protein genes {alpha}-casein and kallikrein-8 and the survival factors prosaposin and Grb10. STAT3-specific genes included the apoptosis regulators CCAAT enhancer binding protein-{delta}, phosphatidylinositol 3-kinase-regulatory subunits, purine nucleoside phosphorylase, and c-fos. These data illustrate that specific activation of STAT3 and STAT5 alone is sufficient to induce and suppress apoptosis, respectively, and that these transcription factors elicit their actions by inducing distinct subsets of target genes in mammary epithelial cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SIGNAL TRANSDUCERS and activators of transcription (STATs) are latent transcription factors that mediate a wide range of cellular responses elicited by specific cytokines, growth factors, or hormones (1). This is exemplified in epithelial cells of the mammary gland in which STAT3 and STAT5 are activated in a reciprocal pattern during the course of a pregnancy cycle (2) in response to leukemia inhibitory factor (LIF) and prolactin, respectively (3, 4). At the onset of postlactational involution, STAT5 activity declines as a direct consequence of a fall in systemic prolactin levels and STAT3 increases in response to LIF. Targeted deletion of prolactin and, more recently, STAT5a in mammary gland demonstrated the importance of this signaling pathway in lobuloalveolar development during pregnancy and the differentiation of mammary epithelial cells before lactation (5, 6). Similarly, using gene knockouts, we have shown that LIF-dependent activation of STAT3 is essential for mammary epithelial cell death after weaning (3), and that this appears to involve the direct suppression of intrinsic cell survival signals mediated by ERK1/2 (3) and Akt (7). Subsequent studies of postlactational involution in STAT5-deficient mice inferred an additional role for STAT5 in cell survival (8, 9); however, these conclusions were confounded by the primary effects on lobuloalveolar development.

Identification of the precise cellular responses to these STAT-dependent pathways and definition of the downstream mechanisms involved has been further hampered by the pleiotropic nature of ligand-mediated signaling and the apparent overlap of residual STAT5 activity with STAT3 activation at the onset of involution (2, 10). Furthermore, there have been no reports distinguishing STAT3- and STAT5-specific pathways in any other cell type.

In this study we use conditionally active forms of STAT3 and STAT5, in a mammary epithelial cell line (KIM-2) that can be induced to undergo differentiation or apoptosis (11), to determine 1) the direct, ligand-independent, effects of these transcription factors on cell viability; and 2) the transcriptional specificity of STAT3 and STAT5 signaling in mammary epithelial cells. We show that forced dimerization of these STAT transcription factors recapitulated the cytokine-mediated effects observed in vivo. Specifically, activation of STAT3 resulted in apoptosis, whereas STAT5 induced differentiation, of KIM-2 cells. We also show, for the first time, that STAT5 directly protected cells against physiological death signals mediated by STAT3. Microarray analysis identified STAT3-specific and STAT5-specific genes that correlated with these respective roles and highlighted an additional immunoregulatory role for STAT3 during involution. The relevance of these transcription targets is discussed in relation to previous reports of gene expression in the mouse mammary gland.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Derivation of Mammary Epithelial Cell Lines Expressing STAT-GyraseB Chimeric Proteins
To investigate the molecular basis of STAT3 and STAT5 action in mammary epithelial cells, we expressed conditionally active forms of these transcription factors (STAT-Gyrase fusion proteins) in KIM-2 cells using a retroviral delivery system as described previously (12). Using this system, the addition of the bifunctional agent coumermycin to transduced cells resulted in the chemical dimerization of two GyrB domains located at the carboxy terminus of the STAT transcription factor in question. This was sufficient to target the transcription factor to the nucleus (Fig. 1Go). The expression of the fusion protein was confirmed in KIM-2 cells by Western blot analysis using either anti-STAT3 or anti-STAT5 antibodies (Fig. 1Go, A and B). Fusion and endogenous STAT proteins were expressed to similar levels in both sets of retrovirally transduced cells (STAT5-Gyrase and STAT3-Gyrase). Nuclear translocation of STAT protein in response to coumermycin occurred in the majority of the cell population (Fig. 1CGo) but the extent of the induction observed was relatively modest (Fig. 1Go, lanes 4 and 6) compared with the exogenous ligands prolactin and LIF (data not shown). The monomeric antibiotic novobiocin (2 µM) did not induce nuclear translocation of STAT5-GyraseB or STAT3-GyraseB (data not shown).


Figure 1
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  		Fig. 1. Expression of STAT-GyraseB Fusion Proteins in KIM-2 Murine Mammary Epithelial Cells %A, Expression of STAT3-GyraseB and (B) STAT5-GyraseB fusion proteins were determined by Western blotting of total cell extracts from KIM-2 cells transduced with retroviral expression constructs (lanes 2) and control (lanes 1). After coumermycin (coum) treatment (1 µM) (lanes 5 and 6) nuclear (N) and cytosolic (C) extracts were analyzed. DMSO was employed as vehicle control (lanes 3 and 4). The positions of the expressed STAT3/5-GyraseB fusion protein (S3f, S5f) and endogenous STAT proteins (S3e, S5e) are indicated. C, STAT5-GyraseB transfected cells were treated with coumermycin (1 µM) or DMSO (vehicle control) for 1 h and probed with an anti-STAT5 antibody and FITC-conjugated secondary antibody. Nuclear localization was visualized by immunofluorescence microscopy. Magnification, x63.

 
Effect of STAT5 and STAT3 Activation on Epithelial Differentiation and Cell Death
STAT5, activated in response to prolactin, is essential for mammary epithelial cell differentiation and milk protein production during lactation (5). Previously we have shown that prolactin induces the differentiation of KIM-2 cells, characterized by modest expression of the milk protein genes ß-casein (Csnb) and whey acidic protein (Wap) (11). We examined whether Csnb and Wap expression was induced by STAT5, in the absence of prolactin, after coumermycin stimulation of STAT5-GyraseB cells (Fig. 2Go, A and B). Both milk protein genes were expressed 4 d after induction of dimerization, which was similar in kinetics to the induction seen with prolactin (11), although the amplitude of the induction reflected the comparatively weak translocation of STAT5 in the presence of coumermycin. Coumermycin treatment of STAT3-GyraseB cells, however, did not induce Wap, nor did it affect its expression mediated by STAT5, activated in response to prolactin (data not shown). Rather, specific activation of STAT3 in complete medium induced a 2.2-fold increase in apoptosis, compared with a 3.3-fold increase observed after serum depletion alone (Fig. 2CGo). These data support our previous study demonstrating a role for STAT3 in mammary epithelial cell death in vivo (13). Furthermore, STAT3 dimerization in combination with serum depletion for 24 h induced a further increase in apoptosis, as measured by annexinV binding and cleaved caspase 3 levels (Fig. 2Go, C and D). This effect was additive, suggesting that apoptosis was induced by distinct (but nonoverlapping) pathways in response to STAT3 activation and serum withdrawal.


Figure 2
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  		Fig. 2. Functional Analysis of STAT3/5-GyraseB Expression in KIM-2 Mammary Epithelial Cells Confluent cultures of STAT5-GyraseB cells were induced to differentiate in the presence of coumermycin (1 µM) and dexamethasone. Total cell extracts were prepared from confluent cells (0) and 1, 4, and 6 d treatment and probed for (A) ß-casein (Csnb) by Western blot analysis with ß-actin (Actb) control and (B) Wap expression by RT-PCR using primers directed against Wap (182 bp) ß-actin (Actb, 300 bp) and cyclophilin (Ppia, 410 bp). C, Confluent cultures of STAT3-GyraseB in KIM-2 mammary epithelial cells were treated with MM (–) or for 24 h in the presence of DMSO (D), 1 µM novobiocin (NV), 1 µM coumermycin (CM), or serum-depleted MM (3%) with and without coumermycin (3% + CM). Cells were stained with annexin V (ann. V)and counted by flow cytometry. The graph represents the fold induction of annexin V positive cells compared with unstimulated controls (MM alone) ± SEM from 12 experiments. Student’s t test: *, P < 0.001. D, Confluent cultures of STAT3-GyraseB KIM-2 cells were stimulated with coumermycin for 24 h in MM or serum-depleted medium (3%) with and without coumermycin. Total cell extracts were probed for cleaved caspase 3 by Western blot analysis. Protein (15 µg per lane) was loaded.

 
Taken together, these data illustrate that ligand-independent dimerization of STAT5 or STAT3 is sufficient to induce the differentiation and cell death associated with these signaling molecules.

STAT5 Protects against STAT3-Mediated Apoptosis
A role for STAT5 in preventing apoptosis and/or remodeling in mammary gland has been inferred from its rapid decline at the onset of involution and from elevated levels of cell death observed in STAT5a-deficient mammary glands (8, 9). Overexpression of STAT5 in vivo has been shown to delay involution (14), but there has been no direct evidence that STAT5 prevents cell death in mammary epithelial cells. Here we show that coumermycin-mediated activation of STAT5 in STAT5-gyrase cells protected against hormone withdrawal-induced apoptosis, as shown by a diminished level of caspase 3 cleavage (Fig. 3AGo).


Figure 3
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  		Fig. 3. STAT5 Protects against Apoptosis in Response to Hormone Withdrawal or LIF %A, Fully differentiated cultures of STAT5-GyraseB transfected KIM-2 cells were induced to undergo apoptosis after the withdrawal of lactogenic hormones (AM), in the presence and absence of coumermycin (CM). Cells were harvested after 24 h, and extracts were prepared and probed for cleaved caspase 3 (c-C3) by Western blot analysis (15 µg/lane). Confluent cultures of STAT5-GyraseB cells were induced to undergo apoptosis after treatment with LIF (10 ng/ml), in the presence and absence of coumermycin (dose indicated). B, Cells were harvested after 24 h, extracts were prepared and probed for cleaved caspase 3 (c-C3) by Western blot analysis (15 µg/lane). C, Cells were stained in situ with annexin V and analyzed by immunofluorescence microscopy. The number of cells staining positively for annexin V are expressed as an average per randomly selected field. Data are representative of three separate experiments ± SEM. Student’s t test: *, P < 0.05.

 
Because residual but diminishing STAT5 activity remains in mammary epithelial cells at the onset of involution and coexists for a period with activated STAT3 (2, 10), we addressed the consequences of activating a STAT3-specific proapoptotic stimulus in mammary epithelial cells expressing active STAT5. First we confirmed that the LIF pathway remained intact in STAT5-gyrase cells by demonstrating activation of STAT3 in response to LIF (data not shown) and subsequent induction of apoptosis (Fig. 3BGo). Prior activation of STAT5 with coumermycin resulted in a dose-responsive protection of LIF-STAT3-mediated apoptosis as indicated by the diminished level of cleaved caspase 3 (Fig. 3BGo). These data were supported by a reduction in the number of annexin V-positive cells treated with both LIF and coumermycin compared with LIF stimulation alone (Fig. 3CGo). The protective effect of STAT5 was confirmed in STAT3-gyrase KIM-2 cells. Pretreatment of these cells with prolactin protected against STAT3-mediated induction of apoptosis (Fig. 4Go).


Figure 4
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  		Fig. 4. Prolactin Protects against STAT3-Mediated Apoptosis %Confluent cultures of STAT3-GyraseB cells were treated in MM (–) with coumermycin (CM), with 5 mg/ml prolactin (Prl) or with CM in the presence or absence of prolactin. A, Cells were harvested after 24 h, and extracts were prepared and probed for cleaved caspase 3 (c-C3) by Western blot analysis (15 mg/lane). B, Cells were stained in situ with annexin V and analyzed by immunofluorescence microscopy. The number of cells staining positively for annexin V are expressed as an average per randomly selected field. Data are representative of three separate experiments ± SEM. *, Student’s t test: P < 0.05 for prolactin/CM vs. CM. Actb, ß-Actin.

 
Collectively, these data support the hypothesis that STAT5 activation is a survival signal in mammary epithelial cells, and that it suppresses STAT3-mediated apoptosis.

Microarray Analysis of STAT5 and STAT3 Transcriptional Targets
We have identified opposing apoptosis-related functions of two STAT factors in mammary epithelial cells. In an attempt to understand the mechanism underlying these differences, we have performed microarray analyses to compare the transcriptional targets of STAT5 and STAT3 in KIM-2 cells.

Total RNA was isolated from KIM-2 cells in which either STAT3-GyraseB or STAT5-GyraseB had been specifically activated for 4 h with coumermycin under the same conditions described above. Four hours was chosen to identify early transcriptional changes in response to STAT activation, as previously described (15). Paired negative (vehicle only) controls were also prepared. Northern blot analysis carried out on CCAAT enhancer binding protein-{delta} (C/ebp{delta}), a known STAT3 target (16) and suppressor of cytokine signaling 2 (SOCS2), a known STAT5 target (17), confirmed that transcriptional changes were evident at this time point (Fig. 5AGo).


Figure 5
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  		Fig. 5. Validation of STAT3 and STAT5 Target Genes Identified by Microarray Analysis %A, STAT5-GyraseB or STAT3-GyraseB cells were treated for 4 h with coumermycin (+) or DMSO (–). RNA was prepared and analyzed by Northern blotting for known gene targets, C/ebp{delta} in the case of STAT3 and SOCS2 in the case of STAT5. A DNA probe to 18S rRNA was used as loading control. B, Quantitative RT-PCR was performed on five gene targets selected from the lists presented in Tables 1Go and 2Go. Expression levels were compared between STAT5-GyraseB and STAT3-GyraseB cells treated for 4 h with coumermycin (+) or DMSO (–). Each PCR was normalized against an internal PCR reference (cyclophilin) and the housekeeping gene Ywhaz (see Materials and Methods). Each graph represents the mean (± SEM) relative amount (compared with Ywhaz) in at least three experiments. Student’s t test: *, P < 0.05; **, P < 0.01. Gene definitions can be found in Tables 1Go and 2Go.

 
Duplicate samples were labeled and hybridized to separate Affymetrix U74av2 oligonucleotide arrays representing approximately 8500 mouse genes. Genes induced by STAT3 and STAT5 were ranked according to change confidence P values based on fold-change and expression level (see Materials and Methods). The 25 most significantly up-regulated transcripts for STAT3 and STAT5 are listed in Tables 1Go and 2Go respectively. Complete lists of all 12,400 transcripts, ranked according to change confidence scores following STAT3 induction and STAT5 induction are provided as supplemental data published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org. Of the genes illustrated in Tables 1Go and 2Go, 5 STAT3 targets and 2 STAT5 targets have been previously described as STAT responsive genes (indicated by asterisks) (18, 19). To provide an indication of the physiological relevance of these STAT responsive genes in vivo, the relative expression of each transcript in Table 1Go was plotted on a time course of mammary gland development using data from a parallel microarray study of the complete mammary pregnancy cycle (18). A majority of the STAT targets listed (23 of 25 STAT3 and 18 of 25 STAT5) exhibited elevated gene expression profiles correlating with periods of known STAT5 or STAT3 activity in the mammary gland during a pregnancy cycle (Tables 1Go and 2Go). Five genes from each list were also confirmed as STAT targets by real-time quantitative PCR (Q-PCR) (Fig. 5BGo).


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  		Table 1. Ranked List of the Principal 25 Transcripts Induced by STAT3-GyraseB

 

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  		Table 2. Ranked List of the 25 Principal Transcripts Induced by STAT5-GyraseB

 
A comparison of the change confidence scores for each of the STAT targets from Tables 1Go and 2Go demonstrate that STAT3 and STAT5 exhibit a marked specificity for their respective target genes (also represented as comparative Rank scores in Supplemental Table 1). This was confirmed in Q-PCR analysis of selected STAT targets, all of which demonstrated STAT3 or STAT5 specificity (Fig. 5BGo). Furthermore, only one principal STAT target gene [secretory leukocyte protease inhibitor (Slpi)] was common to both STAT5 and STAT3 lists. This gene is represented by two alternate transcripts on the MGU742a affymetrix array (GenBank accession nos. AV087239 and AF002719). Each transcript was STAT specific, suggesting that these alternate transcripts were under the control of different STATs.

The STAT5-specific gene list contained a higher proportion of mammary epithelial differentiation markers (6/25 vs. 0/25 in the STAT3 list) and cell survival genes (2/25 vs. 0/25) whereas the STAT3-specific list contained a higher proportion of proapoptotic genes (10/25 vs. 0/25 in the STAT5 list) and antiinflammatory mediators (6/25 vs. 1/25) (see Supplemental Table 1).

The functional implications of these gene targets are discussed below


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
STAT proteins regulate growth, differentiation, and cell death in a variety of cell types (1). Like many other latent transcription factors, a variety of cytokines and growth factors can lead to simultaneous activation of two or more STAT factors. This activation is tightly coordinated in the mammary gland, where distinct and largely reciprocal regulation of individual STAT family members suggests specific developmental roles. This is exemplified by the phenotypes of null (STAT5a) and tissue-specific knockouts (STAT3), which show impaired differentiation/lactation and delayed involution, respectively (3, 5, 6).

To further characterize STAT function in mammary epithelium, we describe the derivation of two mammary epithelial cell lines in which specific activation of distinct STAT factors can be induced. This activation is independent of ligand and circumvents the problems associated with collateral activation of other STAT factors and other signaling pathways. This culture system has allowed us to specifically address the effect of STAT3 and STAT5 signaling in mammary epithelial cells, to establish the role of STAT5 as a differentiation/survival signal in mammary epithelium, and to identify gene targets downstream of these STAT factors.

We show for the first time that STAT5a dimerization alone is sufficient to induce a modest increase in the expression of Wap, a marker of differentiation in mammary epithelium. Relatively few mammary cell lines are capable of expressing this late marker of differentiation, and none are capable of expressing Wap at the levels observed in vivo. The production of milk proteins in vivo (WAP, ß-casein, {alpha}-lactalbumin) is regulated by the concerted action of STAT5 with a number of proteins including nuclear factor (NF)1 (20), Nuclear receptor coactivator 1/steroid receptor coactivator 1 (21), and the glucocorticoid receptor (22). We have identified Ets-1 as a primary target of STAT5 in mammary epithelial cells (Table 2Go). This transcription factor has been shown to play a role in Wap expression during pregnancy and forms complexes with STAT5 to potentiate its transcriptional activity (23). This may explain the relatively weak induction of Wap by STAT5 alone. In addition to WAP and caseinß, the milk protein casein{alpha} was induced by STAT5 (Table 2Go). The kinetics of this induction (4 h after coumermycin treatment) compared with Wap and casein-ß (up to 4 d) suggests a difference in their response to STAT5 in vitro, which may reflect differences in their dependency for additional transcription factors such as those described above.

In a recent microarray study of three mouse models of failed lactation, a core set of 35 genes were identified as potential regulators of lactation (24). The fact that none of these genes appear here as principal STAT5 targets in vitro highlights the complexity of prolactin-related gene expression in vivo. This is supported by the relatively weak correlation between the activity of the STAT5 targets and STAT5 activity in the mammary gland (see final column, Table 2Go). Thus although this in vitro system provides a useful insight into the specific role of STAT5 in mammary epithelial cells, it does not accurately predict the key genes involved in lactation. A recent report describes the use of chromatin immunoprecipitation to identify direct STAT5 targets in breast cancers (25). Although specific STAT target genes have yet to be defined, this technique offers a valuable complementary approach to the identification of STAT targets in vivo.

Using knockout mouse lines, we have previously demonstrated that LIF-induced STAT3 was required for appropriate induction of apoptosis in the early involuting mammary gland. Here we have complemented this model by conditionally activating STAT3 in vitro to demonstrate that STAT3 alone was sufficient to induce apoptosis in mammary epithelial cells. Indeed, unlike STAT5, STAT3 targets strongly correlated with STAT3 activity in vivo (see final column, Table 1Go). Furthermore, identification of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K) as one of the principal, specific gene targets of STAT3 confirms our recent observations in vivo that Akt activity is directly suppressed by STAT3 in involution (7). Because Akt is a key survival factor in the mammary gland (26), these results confirm our hypothesis that STAT3 promotes apoptosis by actively suppressing pAkt.

Although this underlines the importance of STAT3 in eliminating key survival signals before mammary involution, the question remains whether STAT3 also provides a proapoptotic stimulus. We have confirmed that C/ebp{delta} is a primary transcriptional target of STAT3. This transcription factor has recently been shown to contribute to apoptosis in mammary epithelia in vivo (27) and is therefore likely to have an important role in mediating the proapoptotic effects of STAT3. A number of other STAT3-specific gene targets identified in this study have also been implicated in apoptosis. Among these were: c-Fos, a component of the transcription factor activator protein 1, which is activated early in involution (10) and is associated with stress-induced apoptosis in other cell types (28); XBP-1, a stress-related transcription factor that responds to accumulation of unfolded proteins in the estrogen receptor (29); the TGFß target, Sma and Mad-related protein 1 (30), which along with other Sma and Mad-related protein family members is differentially regulated during involution (17, 18); and IFI16, a BRCA1-associated protein involved in p53-mediated apoptosis (31). Intriguingly Bcl3, a member of the NF-{kappa}B/I{kappa}B family of proteins (32), was found to be a STAT3 target. We have previously shown that NF-{kappa}B is rapidly activated at the onset of involution and that this activity is protective to mammary epithelial cells in culture (33). This is the first reported link between these two transcription factor families in the mammary gland.

A recent report describes the specific activation of STAT3 in a melanoma cell line using a conditional Stat3-ER fusion protein induced by addition of 4-hydroxytamoxifen (15). Sustained STAT3 activity (24 h) promoted apoptosis in these cells, and three target genes, Fos, Osmr, and C/ebp{delta}, induced within 4 h of conditional STAT3 induction, are common with specific targets described here. Interestingly, a large proportion of STAT3 targets are not common to both cell types, despite the similar cellular outcome, indicating an additional level of specificity in STAT signaling, which is presumably due to cell type specificity. This may reflect, in part, the difference between transformed and nontransformed cell types, which would have implications for the role of STAT3 in those breast cancers known to exhibit elevated levels of STAT3 (34, 35, 36).

We have demonstrated that STAT5 alone partially protects mammary epithelial cells from apoptosis, which is consistent with studies in other cell types (37, 38) and supports previous reports suggesting that prolactin/STAT5 signaling promotes the survival of differentiated epithelial cells during pregnancy (8, 9) and in mammary cancers (8, 39). We have identified specific STAT5 gene targets that might be relevant to its antiapoptotic effects in vivo. For example, prosaposin is a natural component of milk and a key regulatory factor in the ceramide-S-1-P rheostat, which mediates its effects by promoting DNA synthesis and inhibiting apoptosis (40). Interestingly, prosaposin is also the most significantly down-regulated gene after STAT3 activation, as measured by change confidence scores from our microarray data (see Supplemental Table 2). Further work is required to assess the significance of this differential regulation with respect to the differentiation and/or cell survival role of prosaposin in the mammary gland. The STAT5 target, Grb10, is a component of the PI3K/Akt signaling pathway acting downstream of insulin receptor substrate (41). This gene is down-regulated at the onset of involution, thus coinciding with the decline in STAT5 activity (Table 2Go) (17) and induction of the PI3K regulatory subunits by STAT3 (Table 1Go) (7, 17). In the light of the importance of PI3K/pAkt signaling in preventing cell death before the onset of involution, the balance between STAT3 and STAT5 may be important in regulating the net activity of pAkt in mammary epithelial cells during the transition from lactation to involution. The relevance of these in vitro STAT5 targets in the context of the multitude signaling pathways in the lactating mammary gland, remains to be determined. Clearly, enforced dimerization of STATs in the absence of upstream signals could circumvent additional protein modifications that might normally influence STAT target specificity. Consequently this has implications for the identification of STAT targets using this system. However, as dimerization alone leads to apoptosis (STAT3) and survival/differentiation (STAT5) in this cell model, at least a proportion of the relevant STAT targets appear to be activated. What remains clear from this study is that STAT3 and STAT5 alone are sufficient to induce their respective effects on mammary epithelial cell survival.

Interestingly, we show here that transcription of SOCS2 is stimulated by STAT5 whereas SOCS3 is specifically up-regulated by STAT3. Up-regulation of SOCS2 is dependent on active STAT5, whereas SOCS3 expression is STAT3 dependent (42). However, SOCS3, but not SOCS2, is capable of abrogating prolactin-dependent transactivation of STAT5 targets in vivo (43). The specificity of these STAT-dependent SOCS activities therefore suggests specialized SOCS function at different stages of the mammary cycle. This raises the intriguing possibility that STAT5 activity may be suppressed at the onset of involution by the transcriptional effects of STAT3 as part of the apoptotic program.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
All cell culture reagents were obtained from Life Technologies (Paisley, Scotland, UK). Insulin, prolactin, linoleic acid, epidermal growth factor, dexamethasone, and coumermycin A1 were obtained from Sigma-Aldrich Co. Ltd. (Dorset, UK). Fluorescein-conjugated annexin-V and propidium iodide were from R&D Systems (Abingdon, UK). Antibodies to STAT5 and phospho-STAT5 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Upstate Biotechnology, Inc. (Lake Placid, NY), respectively. Antibodies to phosphotyrosine STAT3, STAT3, and cleaved caspase 3 were from Cell Signaling (Hitchin, UK). Antibodies to ß-actin were from Sigma-Aldrich Co. Ltd., and anti-ß casein antibodies were a gift from (Dr. B. Binas, Texas A&M University, College Station, TX). Recombinant LIF was from Peprotech (London, UK). cDNA probes for Northern analysis were generously supplied by Dr. Jim Johnston (Department of Microbiology and Immunobiology, Queens University Belfast, Northern Ireland).

Cell Culture and Transfections
The conditionally immortalized murine mammary epithelial cell line, KIM-2, was maintained as previously described (11). Cells were grown to confluency with maintenance medium (MM) [Ham’s F12-DMEM, 10% fetal calf serum (FCS), 5 µg/ml insulin, 5 µg/ml epidermal growth factor, 5 µg/ml linoleic acid] maintained at confluency for 1 d and transferred to differentiation medium, (Ham’s F12-DMEM, 10% FCS, 1 µg/ml prolactin, 5 µg/ml insulin, 40 ng/ml dexamethasone, 5 µg/ml linoleic acid) for 8 d. For apoptosis studies, undifferentiated KIM-2 cells were maintained at 100% confluency for 1 d and then incubated with either MM or MM with reduced serum (3%) or LIF for 24 h. Fully differentiated cells were incubated with differentiation medium or apoptotic medium (Ham’s F12-DMEM, 10% FCS, 5 µg/ml linoleic acid).

Retroviral Constructs and Transfections
STAT3- or STAT5-GyraseB fusion proteins subcloned into the retroviral vector pMXpuro were generous gifts from Dr. Alice Mui (Department of Surgery, University of British Columbia, Vancouver, British Columbia, Canada). Initially, the packaging cell line, Phoenix, obtained from Dr. Gary Nolan (Department of Microbiology and Immunology, Stanford University, Stanford, CA) was transfected with either the STAT3- or STAT5-GyraseB pMX construct employing Calcium Phosphate precipitation as described (http://www.stanford.edu /group/nolan/ retroviral_systems/phx.html). Supernatants containing active retrovirus were used to infect subconfluent cultures of KIM-2 mammary epithelial cells in the presence of polybrene. Polybrene was removed after 3 h, and cells were selected after a further 24 h in the presence of puromycin (1.5 µg/ml). Positive selection was confirmed by Western blot analysis of the STAT3/STAT5-GyraseB fusion protein compared with endogenous STAT3/STAT5 protein. Dimerization (and subsequent nuclear localization) of the STAT3/STAT5-GyraseB protein was induced after the addition of the antibiotic, coumermycin, in which between 1–2 µM was determined to be optimal and nontoxic to the cells.

Western Blotting
Confluent cell cultures were stimulated with LIF or coumermycin and cell extracts prepared with RIPA buffer containing phenylmethlysulfonyl fluoride, sodium orthovanadate, aprotinin, pepstatin, and leupeptin. Protein concentrations were measured (Pierce Chemical Co., Rockford, IL) and between 5–15 µg of each sample was run on 8% or 12% sodium dodecyl sulfate-polyacrylamide gels. Membranes were blocked with blocking buffer (5% Marvel in Tris-buffered saline with 0.1% Tween 20) for 1 h and then incubated with primary antibodies overnight at 4 C at the following dilutions: antiphosphotyrosine STAT3 antibody, STAT5, and STAT3 all at 1:1000; antiphosphotyrosine STAT5 antibody at 1.5 µg/ml; anticleaved caspase-3 at 1:1000; anti-ß-actin at 1:2000. Specifically bound antibody was detected with a horseradish peroxidase-conjugated secondary antibody (DAKO Corp., Copenhagen, Denmark) and enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL) and recorded after exposure to x-ray film.

Annexin V Apoptosis Assays
For flow cytometry, KIM-2 and STAT3-GyraseB KIM-2 cells were harvested as previously described (11). Cells (1 x 105) were resuspended in 100 µl annexin V binding buffer, 5 µg/ml propidium iodide, and 1 µg/ml fluorescein isothiocyanate (FITC)-annexin V and incubated for 15 min in the dark. Cells were analyzed in a fluorescence-activated cell sorter (Becton Dickinson and Co., San Jose, CA).

Immunofluorescent Microscopy
After treatment with coumermycin or vehicle control [dimethylsulfoxide (DMSO)], STAT5- or STAT3-gyrB KIM-2 cells were fixed in methanol (100%) at –20 C for 10 min followed by washes with 1x Tris-buffered saline (Tris-buffered saline, three times for 5 min). After incubation with block solution containing 5.5% normal calf serum and 0.1% Triton X-100 at room temperature to reduce nonspecific binding, cells were incubated with primary antibody (anti-STAT5, 1:100) overnight at 4 C, followed by rinses and further incubation with FITC-labeled goat antirabbit secondary antibody for 1 h at room temperature. Slides were mounted in Vectashield (Vector Laboratories, Inc., Burlingame, CA). Fluorescence was monitored using a Zeiss Axiovert 200M inverted microscope (Carl Ziess Ltd.; Hertfordshire, UK), with FITC filter sets, and an objective setting of x63.

Microarray Analysis of STAT5 and STAT3 Transcriptional Targets
Two 75-cm2 flasks of confluent STAT3-GyraseB cells and two flasks of STAT5-GyraseB cells were incubated in the presence of coumermycin (2 µM) for 4 h. Duplicate cultures were also treated with DMSO only (vehicle control). Total RNA was isolated from each flask individually as previously described (18). Each RNA sample was labeled and hybridized to separate MGU74aVer2 Affymetrix oligonucleotide arrays (total of eight arrays). After normalization by MAS5 Affymetrix algorithm, the mean intensity data from biological replicates were ranked according to change confidence values (MAS5) based on the comparison between test (coumermycin) and control (vehicle) data sets. Normalized and raw data files, fulfilling Minimum Information about a Microarray Experiment (MIAME) criteria, are available from (http://www.ebi.ac.uk/arrayexpress/) and are also available through GeNet (http://genet.hgmp.mrc.ac.uk:8080/servlet/GeNet) for online analysis and download.

Northern Blot Analysis
Total RNA (10 mg) was resolved on formaldehyde denaturing gels. Following overnight transfer onto nylon membranes, hybridization was performed with probes labeled as previously described. Bands were visualized after autoradiography. 18S RNA was employed as loading control.

Semiquantitative PCR and Q-PCR
Cells were harvested at various time points and RNA prepared as previously described (17). cDNA was generated from RNA samples using Superscript II (Promega UK Ltd., Southampton, UK). Semiquantitative PCR was performed using primers to WAP, cyclophilin B, and ß-actin [synthesized by MWG Biotech Ltd., Milton Keynes, UK]. Quantitative detection of STAT3 and STAT5 target cDNA was performed using iCycler supermix (Bio-Rad Laboratories, Hercules, CA) with the addition of fluorescein and SYBR-green according to supplier’s recommendations. PCRs were run in triplicate. The expression values obtained were normalized against those obtained from control cyclophilin via standard curves generated by serial dilution of pooled mammary gland cDNA. Relative changes in each transcript were expressed as a proportion of the housekeeping gene tyrosine 3-monooxygenase (Ywhaz).


   ACKNOWLEDGMENTS
 
We thank M. Wayland for technical assistance.


   FOOTNOTES
 
First Published Online November 17, 2005

1 R.W.E.C., M.P.B., and E.A.K. contributed equally to this study. Back

Abbreviations: C/ebp, CCAAT enhancer binding protein; DMSO, dimethylsulfoxide; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; LIF, leukemia-inhibitory factor; MM, maintenance medium; NF, nuclear factor; PI3K, phosphatidylinositol 3-kinase; Q-PCR, quantitative PCR; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; WAP, whey acidic protein.

This work was supported by the Biotechnology and Biological Services Research Council, UK.

Present address for R.W.E.C.: School of Biosciences, University of Cardiff, Cardiff CF10 3US, Wales, United Kingdom.

R.C., M.B., E.K., J.L., T.F., P.T., and C.W. have nothing to declare.

Received for publication September 23, 2005. Accepted for publication November 10, 2005.


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