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Laboratory of Genetics and Physiology, National Institute of Digestive, Diabetes and Kidney Disease, NIH, Bethesda, Maryland 20892
Abstract
The mammary gland undergoes extensive tissue remodeling and cell death at the end of lactation in a process known as involution. We present evidence that the prolactin-activated transcription factor signal transducer and activator of transcription 5a (Stat5a) has a crucial role in the regulation of cell death during mammary gland involution. In a transforming growth factor-
transgenic mouse model that exhibited delayed mammary gland involution, the absence of Stat5a facilitated involution-associated changes in morphology of the gland and the extent and timing of programmed cell death. These Stat5a-dependent changes also affected epidermal growth factor receptor-initiated mammary gland tumorigenesis. Overexpression of the transforming growth factor transgene in the mammary epithelium reproducibly generated mammary hyperplasia and tumors. In the presence of the activated epidermal growth factor receptor, deletion of Stat5a delayed initial hyperplasia and mammary tumor development by 6 weeks. These observations demonstrate that Stat5a is a survival factor, and its presence is required for the epithelium of the mammary gland to resist regression and involution-mediated apoptosis. We also suggest that Stat5a is one of the antecedent, locally acting molecules that initiate the process of epithelial regression and reorganization during involution.
Introduction
Involution is the phase of mammary gland development that remodels the molecular and morphological characteristics of the gland after lactation. The systemic levels of prolactin, one of the dominant hormones in the stimulation and maintenance of lactational competence of the gland, decreases at the onset of involution (1) . The characteristics of the biological activity and regulation of prolactin argue for prominent involvement of this systemic hormone in the induction of mammary gland involution. Prolactin secretion is tightly regulated and is stimulated by suckling. The half-life of prolactin is very short in the blood, and there is evidence that exogenous treatment with prolactin can delay involution at certain stages of development (2 , 3) . Despite the clear role of this systemic hormone in the initiation of involution, the role of its subservient signal transduction pathways in the regulation of the morphological and transcriptional changes of involution is unknown. The prolactin-activated transcription factor Stat5a3 is essential for the development and differentiation of the mammary gland (4) . Stat5a phosphorylation sharply declines within 12 h after weaning, suggesting a role in the onset of involution. In fact, Stat5a has been implicated in the regulation of apoptotic processes in other tissues (5 , 6) .
Mammary gland involution can be separated into two molecularly and morphologically distinct phases. The first phase is defined by changes in the expression patterns of genes involved in milk synthesis (WAP and ornithine decarbox-ylase), cell survival and death (Bax, Bcl-2, Bcl-xs, SGP-2, interleukin-converting enzyme 1, and p53), and proteins that act to regulate potential survival factors (insulin-like growth factor-1, insulin-like growth factor-binding protein 5, and TGF-ß) and modulate interactions with the extracellular matrix (tissue inhibitor of metalloproteinase 1; Refs. 3 and 7, 8, 9, 10, 11) . Importantly, dephosphorylation of the prolactin-dependent transcription factors Stat5a and Stat5b can be detected within 12 h of the initiation of involution (12) . Despite the high levels of apoptosis early in involution, the integrity of the alveolar basement membrane is not disrupted. The second phase of involution is characterized by increases in the expression of proteins and genes involved in reorganizing the extracellular matrix (gelatinase 1, stromelysin 1, and urokinase plasminogen activator) and abrogation of milk gene expression. These molecular changes precede the dramatic degradation of the lobuloalveolar acini and the absorption of apoptotic cells. It is presumed that changes in the concentrations of the systemic hormones prolactin, oxytocin, and progesterone, in concert with changes in the activity of locally acting factors, initiate and stimulate the process of involution.
The importance of the Stat proteins in the growth and differentiation of many cell types is exemplified by their ubiquitous expression and widespread utilization in the signal transduction pathways of multiple cytokines (13) . Stats are capable of transmitting both differentiative and proliferative signals, depending on the cellular and receptor context (14 , 15) . In the mammary gland, Stat5a activation by prolactin is required for functional differentiation of the epithelium during pregnancy and lactation (4 , 16) . Singular tyrosine phosphorylation by the prolactin receptor-associated intracellular kinase Jak-2 is obligatory to maintain Stat5a transcriptional activity (17) . However, novel alternative mechanisms of regulating Stat5a activity have been identified. Prolactin-activated signal transduction can tyrosine- and serine-phosphorylate multiple isoforms of the Stat5a molecules (18) . Furthermore, truncated forms of Stat5a have transcriptional dominant negative activity (19 , 20) and have been linked to the regulation of apoptosis (5) . In the liver, EGF can stimulate phosphorylation and nuclear translocation of the Stat5a isoform, Stat5b (21) . Importantly, serine phosphorylation of Stat5a by the prolactin-independent, MAPK pathway has been identified, (22) including a direct association between Stat5a and the serine kinase ERK-1/2(MAPK) (23) . Serine phosphorylation by the MAPK pathway is required for transcriptional activity of Stat1 and Stat3, but serine phosphorylation is not obligatory for prolactin-dependent transcriptional activity of Stat5 proteins (24) . Taken together, these data imply that Stat5a activity can be regulated by the prolactin and EGF signaling pathways and possesses diverse functional roles within a single cell that are dependent on its phosphorylation state and expressed isoforms.
Initiation and progression of breast cancer rely on the inappropriate temporal and qualitative activity of normal cellular signaling pathways (25)
. These alterations in signaling activity often disrupt the normal mechanisms of mammary gland development. In one mouse model (WAP-TGF-), overexpression of TGF-, which binds to and activates the EGFR, delays mammary involution and transforms the mammary epithelium (26)
. The prolactin signaling pathway has been implicated in loss of normal epithelial differentiation (4
, 16)
and transformation (27
, 28)
. The role of Stat5a in tumorigenesis is implicated by the appearance of Stat5a overexpression in leukemic (29
, 30)
and oncogenic (31)
transformed cells. Additionally, constitutive activation of the Jak/Stat pathway occurs in multiple leukemia-, v-abl-, and human T-cell lymphotrophic virus-transformed T cells (32
, 33)
, and specific inhibition of constitutive Jak-2 activity in acute lymphoblastic leukemia can block cell growth and induce cell death (34)
. We used the WAP-TGF- transgenic and Stat5a-null mouse models to explore the in vivo role of Stat5a in mammary gland involution and tumorigenesis.
Results
Delayed Mammary Involution in the TGFTG Mice Is Curtailed in the Absence of Stat5a.
The contribution of Stat5a in regulating involution and the establishment of mammary tumors was investigated in mice that carried the TGF- transgene (TGFTG mice) or were null for the Stat5a gene (Stat5aKO mice) and in Stat5a-null mice that expressed the transgene (Stat5aKOTGF mice). H&E-stained mammary glands from TGFTG, Stat5aKO, Stat5aKOTGF, and WT nontransgenic control mice at day 18 of pregnancy and days 1, 3, and 7 of involution were examined by light microscopy (Fig. 1)
. Morphological differences were observed between the TGFTG and Stat5aKOTGF mammary glands within the first pregnancy and involution. TGFTG mammary glands contained hyperproliferative alveoli and ducts. Alveolar lumina contained heterogenous eosin-positive staining material. A significant increase in the number of stromal cells was evident, in addition to increased deposition of stromal collagen (Fig. 1l)
. Involution was delayed in the TGFTG mice, as reported previously (26)
. Some cell loss and condensation of lobuloalveolar structures was observed during involution, but the gland lacked the dramatic epithelial regression of the WT gland (Fig. 1
, n versus p). Mammary tissue from pregnant Stat5aKOTGF mice was histologically similar to pregnant tissue from TGFTG mice but contained more epithelium than observed in the glands of Stat5aKO mice (Fig. 1, a–d)
. Secretory structures appeared to be more uniform and lacked the observed heterogeneous precipitations in the alveolar lumens. During involution, the Stat5aKOTGF gland remodeled more rapidly and extensively than the TGFTG gland (Fig. 1
, o versus p). This effect was enhanced after subsequent rounds of pregnancy and involution (data not shown). The primary and secondary ducts of the Stat5aKOTGF mice lacked concentrations of hyperplastic cells like those found in the TGFTG mice. On rare occasions, hyperplastic regions were evident after three or more rounds of pregnancy in Stat5aKOTGF mice (data not shown).
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and TGFTG Mice. and TGFTG mice, the levels of apoptosis and proliferation were analyzed by TUNEL and BrdUrd immunohistochemistry, respectively (Tables 1
and 2)
. WT glands exhibited increased levels of apoptosis within 1 day of the initiation of weaning and exhibited maximal levels at day 3, as expected (9
, 35)
. Consistent with an inhibition or delay in involution in the TGFTG gland, the number of apoptotic cells was slightly reduced at day 1 of involution compared with WT and Stat5aKOTGF glands (Fig. 2A)
. Apoptosis was reduced in the TGFTG gland at day 7 when compared with WT gland at day 7. Whereas Stat5aKOTGF mammary glands at day 18 of pregnancy (P = 0.02) and at day 1 of involution (P = 0.015) had significantly higher levels of apo-ptosis than TGFTG and WT mammary glands over the same time period, the levels were similar at days 3 and 7. Interestingly, Stat5aKO mammary glands also displayed significantly high levels of apoptosis at day 18 of pregnancy and day 1 of involution (P = 0.03).
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, TGFTG, Stat5aKO, and WT mice (Fig. 2B)
. Generally, 6–10% of the cells in the pregnant gland were labeled with BrdUrd (Fig. 2B)
. Proliferation was low in involuting tissue from Stat5aKO and WT mice (<1%). In contrast, the TGFTG and Stat5aKOTGF glands had detectable, persistent proliferation levels of 4–7% throughout involution. Apoptotic cells were observed in Stat5aKOTGF mammary glands at day 18 of pregnancy and day 1 of involution in animals labeled exclusively with BrdUrd (Fig. 2C
, arrows). The appearance of apoptotic, BrdUrd-labeled cells demonstrated that DNA synthesis in these cells preceded the initiation of PCD.
WAP-TGF- Transgene Expression Is Present throughout Mammary Gland Development in Stat5aKOTGF and TGFTG Mice.
The WAP gene promoter has been exploited to target high-level expression of transgenes, including TGF-, to the mammary epithelium of pregnant mice (36, 37, 38, 39, 40)
. To assure that the hormonal and morphological changes associated with involution and the absence of Stat5a did not disrupt transgene expression, TGF- expression was evaluated in mammary tissue of pregnant and involuting TGFTG, WT, and Stat5aKOTGF mice. Northern blot analysis of TGF- transgene expression in TGFTG and Stat5aKOTGF mice revealed expression of an appropriately sized transcript at day 18 of pregnancy. Transgene expression persisted through days 1, 3, and 7 of involution in multiple animals (Fig. 3)
. This expression pattern deviates from the expression of the endogenous WAP gene, which declines at the initiation of involution (41)
. Endogenous TGF- expression was only detected in an overloaded lane containing wildtype tissue at day 1 of involution. TGF- transgene expression persisted through multiple rounds of pregnancy (three to nine rounds) in TGFTG and Stat5aKOTGF mice (data not shown).
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to the EGFR initiates receptor dimerization and inherent kinase activation, which stimulates the Ras/Raf/Mek signaling cascade. To establish that the EGFR-dependent pathway was being activated by the expression of the TGF- transgene, Western blot analysis was performed on protein extracts from Stat5aKOTGF, WT, Stat5aKO, and TGFTG animals with an antibody that only recognizes the active (phosphorylated) form of MAPK. Active MAPK was detected in TGFTG and Stat5aKOTGF mammary glands at day 18 of pregnancy and days 1, 3, and 7 of involution (Fig. 4, A and B)
. Increased levels of the active form of this enzyme were detectable after multiple rounds of pregnancy in the TGFTG and Stat5aKOTGF mammary glands. Very low levels of active MAPK were detected in the nontransgenic Stat5aKO gland (Fig. 4C)
. Levels were also very low in pregnant and involuting WT mammary glands (Fig. 4D)
. A significant level of MAPK is present in the lactating, WT mammary gland.
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TG and Stat5aKOTGF Mice.TG mice detected high levels of tyrosine-phosphorylated Stat5a and Stat5b at days 1, 3, and 7 of involution (Fig. 5)
. Conversely, the phosphorylation of Stat5a and Stat5b was decreased at day 1 and remained low to day 7 of involution in WT controls, as reported previously and shown in Fig. 5B
(42)
. Additionally, tyrosine-phosphorylated Stat5a and Stat5b were also detected by immunoprecipitation and Western blot analysis in multiple individual TGFTG tumors (data not shown). Stat5aKOTGF mammary glands, lacking any Stat5a protein, were assayed for the phosphorylation of Stat5b to determine whether there was a compensation for the absence of Stat5a phosphorylation. A slight increase in Stat5b phosphorylation was detected on days 1, 3, and 7 of involution relative to WT controls.
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-initiated Mammary Tumorigenesis Is Delayed in Stat5aKOTGF Mice. transgene under control of the WAP gene promoter induces mammary tumors in mice bred ad lib after three pregnancies (26)
. The contribution of Stat5a to the establishment of mammary hyperplasias and tumors was investigated in TGFTG and Stat5aKOTGF mice. Stat5aKOTGF and TGFTG mice were bred ad lib and examined after each round of pregnancy for palpable hyperplasias and tumors. All 48 TGFTG mice contained palpable hyperplasia in multiple glands by the third pregnancy. TGFTG mammary glands consistently displayed a swollen appearance by the second pregnancy, emblematic of hypertrophic glands and hyperplasias (data not shown). Gross histological examination revealed the presence of multiple hyperplastic alveolar nodules in multiple glands. TGFTG glands also contained fluid-filled cysts and stromal proliferation. Some removed tumors were characterized by abnormal hyperproliferation of glandular structures with relatively little hyperplasia. The latency of mammary hyperplasia and tumor appearance and the histology of the hyperplasias and tumors were consistent with the results described previously (26)
. Hyperplasia and hypertrophy appeared less frequently in the Stat5aKOTGF mammary gland. There was a significant difference in the rate of initial hyperplasia and tumor formation between the TGFTG and Stat5aKOTGF mice, (P = 0.003; Fig. 6
). The initial appearance in the Stat5aKOTGF mice was delayed 1.5 months compared with the TGFTG mice. In Stat5aKOTGF mice that did develop hyperplasias or tumors, usually only a single gland was involved, as compared with multiple gland involvement in the TGFTG mice.
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Loss of Stat5a Activity Is an Early Event in Mouse Mammary Gland Involution.
Dephosphorylation and hence inactivation of the transcription factors Stat5a and Stat5b and de novo phosphorylation of Stat3 occur in the first phase within several hours of pup removal (42)
, implicating the Stats and prolactin as early regulatory molecules in the regression of the gland. Using a combination of transgenic and gene deletion mouse models, we have now demonstrated a role for Stat5a in involution that is dependent on its phosphorylation and can be regulated by constitutive EGFR signal transduction. Involution is abrogated on continued activation of the EGFR (and Stat5a) through the expression of a TGF- transgene. In mammary tissue of TGFTG mice, Stat5a remained phosphorylated, as did Stat5b. However, on deletion of the Stat5a gene, involution proceeded unabated, despite expression of the TGF- transgene. Rapid involution in the absence of Stat5a was accompanied by increased levels of apoptotic cells. Our experiments not only demonstrate that Stat5a is a survival factor in vivo but also show that its presence is mandatory to avoid the initiation of PCD in mammary epithelial cells.
Despite the lack of known genetic targets for Stat5a in the involuting gland, there is evidence to suggest that prolactin and Stat5a can control cell survival. Activation of Stat5a has been implicated in the inhibition of apoptosis in T cells through interleukin 2 (6)
and interleukin 9 (43)
signaling. In addition, selection of apoptosis-resistant immature T cells leads to the activation of a dominant negative form of Stat5a (5)
. Prolactin can also stimulate cell survival in mammary epithelial cells in vitro (44)
, and prolactin and growth hormone can promote cell survival in the involuting rat mammary gland (2
, 3)
. The presence of apoptotic cells in the mammary tissue of pregnant mice that lack the Stat5a gene and express the TGF- transgene suggests that cell dependence on the presence of phosphorylated Stat5a precedes the actual systemic hormonal switch into involution. This observation also implies that the Stat5a-dependent mechanism of apoptosis induction may be hormonally independent. We did not observe this apoptotic phenomena in the WT and TGF- transgenic mice. This increased number of apoptotic cells during pregnancy was also detected in BrdUrd-labeled Stat5aKOTGF mice. Analysis of these glands revealed the presence of BrdUrd-labeled apoptotic cells at day 18 of pregnancy and at days 1, 3, and 7 of involution. These cells replicated their DNA and then proceeded directly into PCD. This association between the cell’s ability to replicate its DNA and the progression into the apoptotic program has been reported previously (45)
. Cells may be programmed to initiate PCD before they enter the cell cycle; consequently, BrdUrd-labeled cells undergoing apoptosis are detected. Interestingly, not all the cells in the Stat5aKOTGF pregnant gland undergo PCD. This suggests that some cells have obviated the requirement for Stat5a, possibly through an unknown compensatory mechanism. Alternatively, these cells may have a unique differentiation state that precludes them from entering the apoptotic pathway.
Despite persistent phosphorylation of Stat5a in involuting mammary tissue, TGFTG mice did undergo involution-dependent epithelial reorganization and activation of PCD. This morphological change was characterized by moderate condensation of alveolar structures and loss of cells through the induction of PCD. We propose that the dephosphorylation of Stat5a is not the only guiding factor for apoptosis progression in the involuting mammary gland and that other parallel signals may be required for the complete induction of both phases of involution (12)
.
Transgenic TGF- Activates the EGFR and Ensures Persistent Phosphorylation of Stat5a.
TGF- stimulates the EGFR and activates MAPK through the Ras/Raf/Mek signaling cascade. This is exemplified by the presence of the activated form of MAPK during involution in the TGFTG and Stat5aKOTGF mice. Persistent activation and phosphorylation of Stat5a in the TGFTG mice could occur through three distinct mechanisms. The EGFR can directly phosphorylate Stat1 and Stat3 in the absence of Jak activation (46, 47, 48)
. Although direct phosphorylation of Stat5a by the EGFR cannot be ruled out, it was not possible to coimmunoprecipitate this receptor with anti-Stat5a antibodies in the TGFTG mammary gland (data not shown). Secondly, stimulation of MAPK by EGF can lead to phosphorylation and nuclear translocation of the Stat5a isoform Stat5b in the liver (21)
. Support for this mechanism comes from results describing that Stat5a and its shorter isoforms can be tyrosine- and serine-phosphorylated by prolactin (18)
, and Stat5a and Stat5b can be tyrosine- and serine-phosphorylated by prolactin-independent, MAPK-dependent mechanisms (22
, 49)
. A direct interaction was recently demonstrated between MAPK(ERK1/2) and Stat5a in growth hormone-dependent signaling (23)
. Growth hormone is known to influence mammary gland involution, although this effect is not as dominant as that observed for prolactin (2)
. Thirdly, EGFR could activate Stat5a through Jak-2. The EGFR is known to use Jak-1 to activate Stat1 and Stat3 (14
, 50)
and can modulate Stat5 expression in mammary epithelial cells (51)
. Theoretically, the constitutively active EGFR in the TGFTG and Stat5aKOTGF mice could stimulate Jak-2, although this was not formally proven. Alternatively, EGFR could stimulate a secondary Jak, which inappropriately uses Stat5a as a substrate that has been demonstrated in IFN-
signaling (52
, 53)
. Presumably, the stimulation of the EGFR kinase in the presence of the TGF- transgene leads to phosphorylation and activation of a downstream kinase, possibly including a Jak, that maintains Stat5a in its phosphorylated state. We suggest that one or more of these mechanisms regulate the phosphorylation of Stat5a and control apoptosis in the mouse mammary gland.
Persistent Stat5a Activity Contributes to Mouse Mammary Tumorigenesis, and Its Absence Acts to Delay Hyperplasitic Epithelial Formation.
One of the dominant pathways commonly altered in breast cancer is the EGF signal transduction pathway (25
, 54)
. Gene deletion studies in mice have demonstrated that the EGFR is not dispensable for normal mammary development (55
, 56)
, and that aberrant signaling through this receptor induces neoplastic growth and frank tumors (26
, 57, 58, 59)
. Using an EGF-dependent transgenic mouse model, we can now show that the persistent activation of Stat5a is linked to the transformation of the mammary gland. We suggest that the transformation of the mammary epithelium occurs at the expense of a loss of cell survival regulation, which is contingent on the persistent phosphorylation of Stat5a and permits the inappropriate survival of epithelial cells. The appearance of tumors and hyperplasias in Stat5aKOTGF mice suggests that the absence of Stat5a will not completely block mammary epithelial transformation, and that the TGF- transgene can activate alternative (non-Stat5a) pathways to bypass PCD and involution and support tumor formation. Such pathways may include Stat5b, which, under specific circumstances, substitutes for Stat5a (60)
. The involvement of a single gland in a majority of the Stat5aKOTGF tumors suggests that significant numbers of potentially transformed cells are destroyed during involution. This loss of cells probably contributes to the observed lower rate of tumor formation and the delay in initial tumor appearance. In spite of single gland involvement, survival of the Stat5aKOTGF mice was no greater than that observed for the TGFTG mice after initial tumor appearance.
Our findings that Stat5a is a survival factor and that its absence reduces mammary tumorigenesis support findings from other settings. Prolactin and other type 1 cytokines do possess mitogenic activity, implicating the associated Stat molecules with contributing to the mitogenesis of these organs (13) . Constitutive Stat activity has been observed in cells transformed by oncogenic viruses (32) and oncogenes (61) and in leukemia cells (29) . Moreover, constitutive activation of Jak expression can lead to transformation (27 , 62) .
Stat5a Is a Survival Factor for the Mammary Epithelium.
The absence of Stat5a disrupts the prolactin-mediated survival of the mammary epithelium. Whereas overexpression of TGF- in the presence of an intact Stat5a results in delayed involution, apoptosis and involution in the absence of Stat5a lead to elimination of the majority of the epithelium. The dephosphorylation or lack of de novo phosphorylation and inactivation of Stat5a that occur during involution in the WT gland are mimicked by the absence of Stat5a in the Stat5aKOTGF mice. Therefore, we propose that the presence of the activated form of this transcription factor prolongs cell survival, and its inactivation or deletion permits PCD to occur. These experiments demonstrate that Stat5a can act as a survival factor for the epithelium of the mammary gland. In addition, they illustrate that the inhibition and/or disruption of the mechanisms that regulate this involution process are critical in mammary gland tumorigenesis. The conclusions from this study lead us to hypothesize that the inhibition or disruption of Stat5a phosphorylation can lead to protection from transformation in the mammary gland. The application of Stat and/or Jak-specific tyrosine or serine kinase inhibitors may be an approach to blunt the development of mammary tumors originating from EGFR deregulation.
Materials and Methods
Materials.
The TGF- cDNA probe was a kind gift from Dr. David Lee (Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC). The generation of the Stat5a antibodies has been described previously (63)
. Phosphorylated MAPK antibody was purchased from Promega (Madison, WI). Anti-phosphotyrosine antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Goat antirabbit and rabbit antimouse secondary antibodies were purchased from Transduction Laboratories (Lexington, KY).
Generation of TGFTG and Stat5aKOTGF Mice.
WAP-TGFTG mice were a gift of Dr. Eric Sandgren (University of Wisconsin, Madison, WI Ref. 26
). The generation of the Stat5a-null mice has been described previously (4)
. TGFTG mice were interbred with Stat5a-null mice (SvEv129/C57B6) to generate F1 founders that were hemizygous for Stat5a deletion (Stat5aHT) and transgenic for TGF- expression. Female Stat5aHTTGF mice were bred with Stat5aHTTGF males to generate offspring that were transgenic for TGF- and homozygous for deletion of the Stat5a gene. These mice were backcrossed for five generations to generate a pure inbred Stat5aKOTGF strain of mice. The same generation TGFTG and control WT littermates was used for molecular analyses. Confirmation of the presence of the TGF- transgene was performed by PCR analysis with TGF- forward primer 5''-TGTCAGGCTCTGGAGAACAGC-3' and reverse primer 5'-CACAGCGAACACCCACGTACC-3'. Stat5a PCR was performed with two sets of primers to knockout (null) and WT alleles: (a) Stat5a forward (5'-CTGGATTGACGTTTCTTACCTG-3') and Stat5a reverse (5'-TGGAGTCAACTAGTCTGTCTCT-3') and (b) Neo forward (5'-AGAGGCTATTCGGCTATGACTG-3') and Neo reverse 5'-TTCGTCCAGATCATCCTGATC-3'. PCR for all primers was performed with a denaturing step of 94°C for 3 min, followed by 30 cycles of 94°C for 40 s, 56°C for 40 s, and 68°C for 40 s, followed by 10 min at 68°C. Genotype of the mice was confirmed after tissue collection by Northern and Western blot analysis for TGF- gene expression and absence of Stat5a protein expression, respectively. All animals were housed and handled according to the approved protocol established by the Institutional Animal Care and Use Committee and NIH guidelines.
Mammary Gland Collection.
Mammary glands were surgically removed from anesthetized and cervically dislocated mice at day 18 of pregnancy and days 1, 3, and 7 of involution. Day 1 of involution was designated as 24 h after the morning that the pups were born. Pups were immediately removed from the dam after birth and fostered onto a WT mother. The mammary lymph node was removed before homogenization of all glands. Tissues were prepared immediately for RNA and protein extraction, as described previously (42)
. Whole (number 4) inguinal or (number 3) thoracic mammary glands were surgically excised from mice, spread on Omniset tissue cages (Fisher Scientific, Pittsburgh, PA), fixed for 5 h in Tellyzinckys fixative, and stored in 70% ethanol until processed by standard embedding and sectioning techniques onto Probe-On Plus slides (Fisher Scientific). Sections were stained with H&E.
Immunoprecipitations and Western Blot Analysis.
Preparation of protein extracts and immunoprecipitations have been described previously (42)
. Briefly, 2 mg of fresh and frozen tissue were homogenized in 2 ml of lysis buffer with protease inhibitors; phenylmethylsulfonyl fluoride, leupeptin, and aprotinin at 50 µg/ml on ice. Protein lysates were rocked for 1 h at 4°C and then cleared by centrifugation at 14,000 x g for 15 min. The supernatants were removed, mixed with 2x loading buffer, and heated to 90°C for 3 min. Samples were spun briefly, electrophoresed under denaturing conditions on 8% precast tris-glycine gels, and transferred to polyvinylidene difluoride membranes according to manufacturer’s protocol (Novex, San Diego, CA). Western blot analysis was performed essentially as described with the following exceptions; primary antibody (Stat5a, 1:20,000 dilution; Stat5b, 1:10,000 dilution; anti-phosphotyrosine, 1:5,000 dilution; anti-MAPK, 1:5,000 dilution) was incubated overnight at 4°C with gentle rocking, and all incubations with antibodies and initial blocking were performed with 3% nonfat dried milk in 1x TBST. Detection was performed with the enhanced chemiluminescence kit according to manufacturer’s protocol (Amersham) and exposed to Kodak (Rochester, NY) MR autoradiography film. Exposure times are between 1 s and 2 min. Immunoprecipitations with anti-Stat5a antibodies were carried out as described previously (4)
. Stripping was performed by incubating blots at 56°C in 6.25 mM Tris-HCl (pH 6.8), 2% SDS, and 1% ß-mercaptoethanol for 30 min. Blots were washed extensively in TBST and then blocked with 3% nonfat dried milk in TBST.
Northern Blot Analysis.
Total RNA was isolated from fresh tissue by homogenization in lysis buffer as described previously (63)
. RNA was quantified by spectrophotometry and prepared for Northern blot analysis by heating in loading buffer at 65°C. Total RNA was separated by 1.5% agarose gel electrophoresis and transferred to Hybond N+ (Amersham) nylon membrane by capillary transfer with 10 x SSC. After overnight transfer, the membrane was UV-irradiated and hybridized to each cDNA probe in Quikhyb (Stratagene, La Jolla, CA). Hybridization for the TGF- cDNA probe was performed in Quikhyb (Stratagene) at 65°C for 16 h, followed by two washes in 1 x SSC and 0.5% SDS for 30 min, followed by one wash in 0.1 x SSC and 0.5% SDS for 30 min at 56°C. Blots were hybridized for 16 h and then washed twice in 1 x SSC and 0.5% SDS for 30 min, followed by one wash in 0.1 x SSC and 0.5% SDS for 30 min. cDNA and oligo probes were random-primed and Klenow-labeled with [-32P]dCTP. After hybridization and washing, blots were exposed from 30 min to 24 h, as specifically described in the figure legends, to Kodak MR autoradiography film at -70°C.
BrdUrd and TUNEL Assays.
Protocols for BrdUrd and TUNEL analysis have been described elsewhere (64)
. Mice were injected 2 h before sacrifice with 20 µg/g body weight BrdUrd labeling reagent, as described by the manufacturer (Amersham). Each proliferation and apoptosis sample counted represents a minimum of three random fields (at x200) and a minimum of 1000 total cells/section for each mouse. A minimum of three mice per timepoint were collected and analyzed. The total number of cells counted for proliferation assay is 6.5 x 105. The total number of cells counted for the apoptosis assay is 7.5 x 105. The number of mice collected and the number of mice analyzed are listed by genotype and by timepoints (pregnancy day 18/involution day 1/involution day 3/involution day 7) for apoptosis and proliferation, respectively: KOTG, 4/4/4/3 and 4/3/3/3; TG, 3/5/3/5 and 3/3/3/5; WT, 3/4/5/3/ and 3/3/3/3; NTGKO, 3/3/2/3 and 3/3/3/3.
Hyperplasia and Tumor Analysis.
Mice were palpated at the 18th day of pregnancy for mammary hyperplasia, hypertrophy, and tumors. Animals were scored for the presence of either hyperplasia or tumor at every pregnancy. Most animals were sacrificed at the third pregnancy by anesthesia followed by cervical dislocation. In studies that required long-term breeding for tumor development, dams were rebred within 2 days after birth and removal of the pups.
Acknowledgments
We thank Dr. Eric Sandgren for the kind gift of TGF- transgenic mice. We thank Drs. J. Shillingford and E. Rucker for critical reading of the manuscript and Drs. P. Furth and B. Gusterson for essential discussions. We thank Ashton Garett, Bill Kemp, and Sandra Price for animal husbandry and technical support and Dr. J. Shillingford for developing the Stat5a PCR assay.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by Department of Defense, Medical Research and Material Command Grant DAMD17-99-1-9328 (to R. C. H.). 
2 To whom requests for reprints should be addressed, at Laboratory of Genetics and Physiology, National Institute of Digestive, Diabetes and Kidney Disease, NIH, Building 8, Room 111, Bethesda MD 20892. Phone: (301) 435-6634; Fax: (301) 480-7312; E-mail: robinh{at}box-r.nih.gov
3 The abbreviations used are: Stat, signal transducer and activator of transcription; TGF, transforming growth factor; Jak, janus kinase; MAPK, mitogen-activated protein kinase; WAP, whey acidic protein; EGFR, epidermal growth factor receptor; BrdUrd, bromodeoxy uridine; WT, wild-type; PCD, programmed cell death; EGF, epidermal growth factor; TBST, Tris buffered saline Tween; TUNEL, terminal deoxynucleotidyl transferase-mediated uridine nick end labeling; ERK, extracellular signal-regulated kinase.
Received for publication 5/11/99. Revision received 8/ 4/99. Accepted for publication 8/10/99.
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 P. Koppikar, V. W. Y. Lui, D. Man, S. Xi, R. L. Chai, E. Nelson, A. B.J. Tobey, and J. R. Grandis Constitutive Activation of Signal Transducer and Activator of Transcription 5 Contributes to Tumor Growth, Epithelial-Mesenchymal Transition, and Resistance to Epidermal Growth Factor Receptor Targeting Clin. Cancer Res., December 1, 2008; 14(23): 7682 - 7690. [Abstract] [Full Text] [PDF]
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 L. Hennighausen and G. W. Robinson Interpretation of cytokine signaling through the transcription factors STAT5A and STAT5B Genes & Dev., March 15, 2008; 22(6): 711 - 721. [Abstract] [Full Text] [PDF]
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 A. J Brennan, J. A Sharp, E. Khalil, M. R Digby, S. L Mailer, C. M Lefevre, and K. R Nicholas A population of mammary epithelial cells do not require hormones or growth factors to survive J. Endocrinol., March 1, 2008; 196(3): 483 - 496. [Abstract] [Full Text] [PDF]
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F. Sotgia, H. Rui, G. Bonuccelli, I. Mercier, R. G. Pestell, and M. P. Lisanti Caveolin-1, Mammary Stem Cells, and Estrogen-Dependent Breast Cancers. Cancer Res., November 15, 2006; 66(22): 10647 - 10651. [Abstract] [Full Text] [PDF]
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 R. W. E. Clarkson, M. P. Boland, E. A. Kritikou, J. M. Lee, T. C. Freeman, P. G. Tiffen, and C. J. Watson 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 Mol. Endocrinol., March 1, 2006; 20(3): 675 - 685. [Abstract] [Full Text] [PDF]
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 C. M. Blakely, L. Sintasath, C. M. D'Cruz, K. T. Hahn, K. D. Dugan, G. K. Belka, and L. A. Chodosh Developmental stage determines the effects of MYC in the mammary epithelium Development, March 1, 2005; 132(5): 1147 - 1160. [Abstract] [Full Text] [PDF]
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 M. T. Nevalainen, J. Xie, J. Torhorst, L. Bubendorf, P. Haas, J. Kononen, G. Sauter, and H. Rui Signal Transducer and Activator of Transcription-5 Activation and Breast Cancer Prognosis J. Clin. Oncol., June 1, 2004; 22(11): 2053 - 2060. [Abstract] [Full Text] [PDF]
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M. H. Faulds, H. Olsen, L. A. Helguero, J.-A. Gustafsson, and L.-A. Haldosen Estrogen Receptor Functional Activity Changes during Differentiation of Mammary Epithelial Cells Mol. Endocrinol., February 1, 2004; 18(2): 412 - 421. [Abstract] [Full Text] [PDF]
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 S. P. Suchyta, S. Sipkovsky, R. G. Halgren, R. Kruska, M. Elftman, M. Weber-Nielsen, M. J. Vandehaar, L. Xiao, R. J. Tempelman, and P. M. Coussens Bovine mammary gene expression profiling using a cDNA microarray enhanced for mammary-specific transcripts Physiol Genomics, December 16, 2003; 16(1): 8 - 18. [Abstract] [Full Text] [PDF]
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 C. V. Clevenger, P. A. Furth, S. E. Hankinson, and L. A. Schuler The Role of Prolactin in Mammary Carcinoma Endocr. Rev., February 1, 2003; 24(1): 1 - 27. [Abstract] [Full Text] [PDF]
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 M. T. Kloth, K. K. Laughlin, J. S. Biscardi, J. L. Boerner, S. J. Parsons, and C. M. Silva STAT5b, a Mediator of Synergism between c-Src and the Epidermal Growth Factor Receptor J. Biol. Chem., January 10, 2003; 278(3): 1671 - 1679. [Abstract] [Full Text] [PDF]
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 E. Iavnilovitch, B. Groner, and I. Barash Overexpression and Forced Activation of Stat5 in Mammary Gland of Transgenic Mice Promotes Cellular Proliferation, Enhances Differentiation, and Delays Postlactational Apoptosis Mol. Cancer Res., November 1, 2002; 1(1): 32 - 47. [Abstract] [Full Text] [PDF]
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 L. Shan, M. He, M. Yu, C. Qiu, N. H. Lee, E. T. Liu, and E. G. Snyderwine cDNA microarray profiling of rat mammary gland carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 7,12-dimethylbenz[a]anthracene Carcinogenesis, October 1, 2002; 23(10): 1561 - 1568. [Abstract] [Full Text] [PDF]
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 R. C. Humphreys, B. Bierie, L. Zhao, R. Raz, D. Levy, and L. Hennighausen Deletion of Stat3 Blocks Mammary Gland Involution and Extends Functional Competence of the Secretory Epithelium in the Absence of Lactogenic Stimuli Endocrinology, September 1, 2002; 143(9): 3641 - 3650. [Abstract] [Full Text] [PDF]
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 B. S. Wiseman and Z. Werb Stromal Effects on Mammary Gland Development and Breast Cancer Science, May 10, 2002; 296(5570): 1046 - 1049. [Abstract] [Full Text] [PDF]
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M. T. Nevalainen, J. Xie, L. Bubendorf, K.-U. Wagner, and H. Rui Basal Activation of Transcription Factor Signal Transducer and Activator of Transcription (Stat5) in Nonpregnant Mouse and Human Breast Epithelium Mol. Endocrinol., May 1, 2002; 16(5): 1108 - 1124. [Abstract] [Full Text] [PDF]
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 A. V. Capuco, M. Li, E. Long, S. Ren, K. S. Hruska, K. Schorr, and P. A. Furth Concurrent Pregnancy Retards Mammary Involution: Effects on Apoptosis and Proliferation of the Mammary Epithelium after Forced Weaning of Mice Biol Reprod, May 1, 2002; 66(5): 1471 - 1476. [Abstract] [Full Text]
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 L. Shan, S. A. Rouhani, H. A. J. Schut, and E. G. Snyderwine 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) Modulates Lactogenic Hormone-mediated Differentiation and Gene Expression in HC11 Mouse Mammary Epithelial Cells Cell Growth Differ., December 1, 2001; 12(12): 649 - 656. [Abstract] [Full Text] [PDF]
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 K. Miyoshi, J. M. Shillingford, G. H. Smith, S. L. Grimm, K.-U. Wagner, T. Oka, J. M. Rosen, G. W. Robinson, and L. Hennighausen Signal transducer and activator of transcription (Stat) 5 controls the proliferation and differentiation of mammary alveolar epithelium J. Cell Biol., November 12, 2001; 155(4): 531 - 542. [Abstract] [Full Text] [PDF]
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M. H. Faulds, K. Pettersson, J.-A. Gustafsson, and L.-A. Haldosen Cross-Talk Between ERs and Signal Transducer and Activator of Transcription 5 Is E2 Dependent and Involves Two Functionally Separate Mechanisms Mol. Endocrinol., November 1, 2001; 15(11): 1929 - 1940. [Abstract] [Full Text] [PDF]
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A. V. Kazansky and J. M. Rosen Signal Transducers and Activators of Transcription 5B Potentiates v-Src-mediated Transformation of NIH-3T3 Cells Cell Growth Differ., January 1, 2001; 12(1): 1 - 7. [Abstract] [Full Text]
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