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Laboratory of Genetics and Physiology (R.C.H., B.B., L.Z., L.H.), National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; New York University Medical Center (R.R., D.L.), Department of Pathology, New York, New York 10091
Address all correspondence and requests for reprints to: Lothar Hennighausen, Ph.D., Laboratory of Genetics and Physiology National Institutes of Diabetes, Digestive and Kidney Diseases Building 8, Room 101 Bethesda, Maryland 20892. E-mail: hennighausen{at}nih.gov.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inactivation of Stat5 and Stat3 has demonstrated unique roles for these transcription factors in mammary physiology. Inactivation of the genes encoding Stat5a and 5b has revealed their essential role in the proliferation and differentiation of mammary alveolar epithelium during pregnancy (9, 10). In addition, Stat5a is a survival factor for mammary epithelium in the presence of a proliferative growth stimulus (11). Because inactivation of the Stat3 gene results in embryonic lethality, the Cre-loxP recombination system had been used to inactivate this gene in mammary epithelium. The deletion of exon 22, which encodes a critical tyrosine residue, demonstrated a role for Stat3 in the regulation of the first phase of involution (12). Apoptosis of mammary epithelial cells was reduced during the first phase, but involution was able to proceed after a delay of 3 d. This result implicates that Stat3 may not play the dominant role in the regulation of all stages of involution and that additional factors control cell death and tissue remodeling. Alternatively, it is possible that this mutant Stat3, which lacked only 33 amino acids, retained some activity. In T cells, a mutant Stat3 with a loss of the region encoding the tyrosine residue at 705 possessed a dominant negative function (13).
In an attempt to further clarify and define the molecular roles of Stat3 in the process of mammary tissue remodeling, we completely inactivated the Stat3 gene in mammary epithelium using a whey acidic protein (WAP)-Cre transgene (WC) to excise exons 15–21 that had been bracketed by loxP sites (14). This region encodes 245 amino acids including the DNA binding and SH2 domains. These experiments permitted us to address whether functional competence of mammary tissue upon weaning can be maintained in the absence of Stat3, and whether the protease-mediated second stage of involution depends on the presence of Stat3.
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Materials and Methods |
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Generation of STAT 3 fl/fl;WC
Creation of the mice that carry two Stat3 floxed alleles (Stat3 fl/fl) mice has been described previously (14). Briefly, a conditional allele of Stat3 was generated by homologous recombination with loxP sites inserted adjacent to exons 15 and 21 of the mouse Stat3 gene. A single male Stat3 fl/fl (SV129/C57BL6) founder was interbred with WAP-Cre (FvB/C57BL6) transgenic female mice to generate F1 founders heterozygous for the Stat3 floxed allele (Stat3 fl/+) and transgenic for WAP-Cre (Stat3 fl/+;WC). The resulting offspring were backcrossed five times to generate mice homozygous for Stat3 flox and transgenic for WAP-Cre. The same generation nontransgenic Stat3 fl/fl and nontransgenic Stat3 fl/+ littermates (Stat3) were used as controls for all molecular analyses. PCR confirmation of the presence of the Cre transgene and the Stat3 flox and null alleles has been described (14, 15). Genotype of the mice was confirmed after tissue collection by Western analysis for Stat3 expression. 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 if no further time points were needed) mice at d 10 of lactation and d 2, 6, and 9 of involution. Biopsies were performed on anesthetized (isoflurane gas) dams at the desired time point in a survival surgery conducted according to the approved protocol established by the Institutional Animal Care and Use Committee and NIH guidelines. The biopsies have not been found to alter the progression of involution in this or any prior studies using the surgical excision. Day 1 of involution was designated as 24 h after the morning that the pups were removed from the dam. Dams were allowed to lactate for 10 d, and then the pups were removed to induce involution. The mammary lymph node was removed before homogenization of all glands. Tissues were prepared immediately for RNA and protein extraction as described. Inguinal (number 4) mammary glands used for histological analysis were surgically excised, spread on Omniset tissue cages (Fisher Scientific, Pittsburgh, PA), fixed for 5 h in Tellyzinsky’s fixative or overnight at 4 C in 10% neutral buffer formalin. Glands were stored in 70% ethanol until processed by standard embedding and sectioning techniques onto Probe-On Plus slides (Fisher Scientific). Sections were stained with standard hemotoxylin and eosin.
Western analysis
Preparation of protein extracts for Western analysis has been described (3, 9, 11). 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 and the activated phosphotyrosyl-proteinase inhibitor sodium orthovanadate at 4 mM on ice. Protein lysates were rocked for 1 h at 4 C and then cleared by centrifugation at 14,000 x g for 20 min. Protein concentration in the lysates was determined using Bio-Rad Laboratories, Inc. (Hercules, CA) Protein Assay Kit according to the manufacturer’s protocol. Lysates containing 40 µg of protein were mixed with 2x loading buffer and 2-mercaptoethanol then heated to 90 C for 3 min. Samples were briefly spun, electrophoresed, under denaturing conditions, on 8%, 12%, or 14% precast Tris-glycine gels and transferred to polyvinylidendifluoride membranes according to the manufacturer’s protocol (Novex, San Diego, CA). Western analysis was performed essentially as described with the following exceptions; primary antibody (dilution Stat5a 1:5000; Stat3 1:1000; Bax 1:1000; antiphosphotyrosine 1: 5000; Bcl-x 1:1000, PI3K 1:1000, WAP 1:5000, ß-catenin 1:500, actin 1:5000) was incubated overnight at 4 C with gentle rocking and all incubations with antibodies and initial blocking was performed in 3% nonfat dried milk in 1x TBST (Tris-buffered saline with Tween 20). Detection was performed with the Super Signal Western Detection Kit according to manufacturer’s protocol (Pierce Chemical Co.) and exposed to Kodak MR autoradiography film (Rochester, NY). Exposure times were between 1 sec and 2 min. Immunoprecipitations with anti-Stat5a and anti-Stat3 antibodies were carried out essentially as previously described (11). Stripping was performed by incubating blots for 25 min at room temperature in Antibody Stripping Solution from the Western Blot Recycling Kit according to the manufacturer’s protocol (Alpha Diagnostic, San Antonio, TX). Blots were washed in TBST and then blocked with 3% nonfat dried milk in TBST. Membranes were stripped and reprobed up to four successive times. A loading control gel was generated from a parallel SDS-PAGE gel that was fixed for 2 h in 50% methanol, 10% acetic acid, stained overnight in 50% methanol, 0.05% Brilliant Cresyl Blue. Gel was destained in 5% methanol, 7% acetic acid until the background cleared. Gel was dried according to the manufacturer’s protocol (Research Products International, Mount Prospect, IL). The Western data shown are representative of separate analyses that have been performed in triplicate using different sample sets (12 Stat3-null and 6 control mice). Stat3 and Stat5a analyses have been performed on every blot to ensure deletion and compare the relative loading between blots.
Immunohistochemistry of STAT3 and STAT5a
Immunohistochemical detection of Stat3 and Stat5a has been described previously. Briefly, 4% neutral buffered formalin fixed, 5-µm tissue sections were deparaffinized, incubated in tissue unmasking fluid (Vector Laboratories, Inc., Burlingame, CA), and heated in a microwave for 2 min. Sections were allowed to cool for 10 min and then washed in PBS, blocked with 10% normal goat serum for 30 min at room temperature and then incubated overnight at 4 C with primary antibody at a dilution of 1:200 for Stat3 and 1:600 for Stat5a. A standard secondary antibody detection protocol with biotin avidin HRP antibodies identified the presence of antigen. Sections were counterstained with hemotoxylin and dehydrated according to standard protocols.
Zymogram gel analysis
Coomassie stained gelatin zymogram SDS-PAGE was carried out using 40 µg of protein from whole gland lysate essentially as described in the manufacturer’s protocol (Novex/Invitrogen). Gelatin zymography was performed using unstained 10% gelatin embedded Tris-glycine gels from Invitrogen. After electrophoresis, renaturing, and developing the gelatin embedded Tris-glycine SDS-PAGE gels were fixed for 2 h in 50% methanol, 10% acetic acid, stained overnight in 50% methanol, 0.05% Brilliant Cresyl Blue. Gel was destained in 5% methanol, 7% acetic acid until the background cleared. Gel was dried according to the manufacturer’s protocol (Research Products International, Mount Prospect, IL). Preparation and quantitation of protein lysates were described in the previous section describing the Western analysis. The image was captured in grayscale to demonstrate a clear contrast in loading and protease activity.
TUNEL assays
Protocols for TUNEL (terminal deoxyuridine triphosphate nick end labeling) analysis have been previously described (11). Tissue sections from mammary glands analyzed for Stat3 deletion by Western analysis were used for the TUNEL analysis. Each apoptosis sample counted represents a minimum of three random fields (at x200 magnification) and a minimum of 1000 total cells per tissue section for each mouse. A minimum of three mice per time point for each genotype were collected and analyzed. We were able to use a Z-distribution for the statistical analysis of TUNEL positive cells with the data sets that included three mice each contributing 1000 individual units (cells counted), and a total of 3000 cells counted for each time point and genotype.
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Results |
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). At d 18 of pregnancy the recombined allele was predominant, suggesting efficient deletion of exons 15–21 of the Stat3 gene. The presence of an unrecombined allele is indicative of the presence of stroma, where the WAP-Cre gene is not active and the presence of the wild-type allele.
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). No Stat3 was detected in mammary tissue from Stat3 fl/fl;WC mice. In contrast Stat3 was detected in mammary tissue from wild-type mice at d 10 of lactation and increased levels at d 2 of involution (Fig. 1B). Stat3 is preferentially expressed in mammary epithelium and the stroma contributes little to the signal seen in normal mammary tissue. We also examined the expression of Stat1 and Stat5 that could potentially compensate for the absence of Stat3 (Fig. 1B). Stat5 levels decreased after weaning and Stat1 levels increased. The expression patterns of these STATs were similar in both Stat3 fl/fl;WC and control mice.
Expression of WAP and sulfated glycoprotein 2 (SGP-2) are maintained in Stat3-null mammary epithelium during involution
We examined the presence of milk proteins and other proteins linked to the maintenance and regression of mammary tissue. The milk protein WAP was detected in Stat3-null mammary tissue at d 10 of lactation followed by an increase at d 2 of involution (Fig. 1C). In contrast, WAP was not detected at d 2 of involution in wild-type controls. SGP-2 was detected during lactation in Stat3 fl/fl tissues, and its level was increased at d 2 of involution followed by a decrease at d 6 of involution (Fig. 1C). In contrast, SGP-2 levels remained high at d 6 of involution in Stat3fl/fl;WC mammary tissue (Fig. 1C). We also examined the expression patterns of proteins linked to cell survival, cell death and cell integrity. The expression patterns of Bcl-x, Bax, and PI3K were comparable between control and Stat3-null mammary tissue (Fig. 1D).
Loss of Stat3 prolongs mammary involution
Stat3 fl/fl;WC and Stat3 fl/fl mice were mated and at d 10 of lactation the pups were removed. The integrity of mammary tissue was analyzed at d 10 of lactation and d 2, 6, and 9 of involution (Fig. 2). There was lack of epithelial remodeling in Stat3-null mammary tissue during these stages and an apparent failure to cease secretion at a time that corresponded to the second, irreversible stage of involution. The presence of lipid droplets and eosinophilic material within the alveoli at d 6 (Fig. 2F) suggested that the majority of alveolar structures had retained a functional lactogenic phenotype. We did not observe any significant epithelial regression until d 14 after the initiation of involution (data not shown). The regression at d 14 of involution in the Stat3 null gland resembled the control samples at d 6 of involution. After 45 d of involution, the mutant glands were completely remodeled. Dams had no apparent difficulty nursing their pups during the second litter, and no significant difference was noted when the pregnancy occurred during the postpartum estrous. However, after their third pregnancy dams often displayed symptoms of mastitis and invasion of the mammary tissues by lymphocytes unless they had been allowed to involute for at least 3 wk before the third pregnancy. The symptoms of apparent infection and mastitis are similar to results observed in the previous Stat3 study (12).
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, panel d). Western blot analysis of these glands revealed that WAP levels had also increased after 5 d of suckling (Fig. 4C). These data demonstrate that the Stat3-null epithelium could reinitiate lactation after 6 d of forced involution, and display appropriate physiological markers.
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, panels b and d). In contrast nuclear Stat3 was observed in Stat3 fl/fl mammary epithelium (Fig. 4B, panels a and c).
Reduced levels of apoptosis in the absence of Stat3
One of the hallmarks of mammary gland involution is the increase in programmed cell death and an increase in the expression of genes involved in the regulation of programmed cell death (2). We examined the level of apoptosis in mammary tissue from Stat3 fl/fl;WC and Stat3 fl/fl control mice by TUNEL analysis (Fig. 5). The percentages of TUNEL positive cells were determined for Stat3 fl/fl;WC and Stat3 fl/fl tissues at 10 d of lactation and d 2 and d 6 of involution. The percentages obtained were 0.14 (± 0.03), 0.86 (± 0.03), and 1.16 (± 0.11) for Stat3 fl/fl;WC at 10 d of lactation, d 2 and d 6 of involution, respectively. The percentages obtained for the Stat3 fl/fl control tissues were 0.20 (± 0.10), 2.73 (± 0.19), and 2.52 (± 0.71) for 10 d of lactation, d 2 and d 6 of involution, respectively. The level of apoptosis was significantly reduced (P < 0.05) at d 2 of involution (significance determined using a Z-distribution, n=3 mice with 1000 cells counted per mouse, and a total of 3000 cells counted at each time point for both Stat3 fl/fl and Stat3 fl/fl;WC using random fields for analysis). At 6 d of involution no significant difference could be determined within an appropriate statistical confidence interval between the Stat3 fl/fl and Stat3 fl/fl;WC tissues. Levels of TUNEL positive cells at d 2 or 6 of involution in the Stat3 null tissues were very low but not entirely absent. We conclude from this data that the program that initiates programmed cell death in the mammary gland is debilitated in the absence of Stat3.
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). MMP9 activity (92k) in Stat3-null samples during involution was delayed (Fig. 6, arrow 2). In contrast, bands corresponding to the 72k proform and 62k active MMP2 did not show significant difference between the Stat3-null and control samples (Fig. 6, arrows 3 and 4, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our study confirms and extends previous research on the role of Stat3 in mammary gland involution (12). Although both studies point to a critical role of Stat3 in the process of involution, there are distinct differences between the observed phenotypes. We demonstrate that mammary tissue retained functional competence, whereas in the previous study the deletion of 33 amino acids resulted in a delay of involution and no functional rescue was reported. The differences could be the result of the targeting strategy and the level of Cre-mediated excision of Stat3. Although the previous study generated a Stat3 that left the DNA binding and SH2 domains intact, no Stat3 protein was detected in our study. The truncated Stat3 protein used by Chapman et al. (12) has been associated with a dominant negative effect in T cells (13). Alternatively, it is possible that the Cre transgenes used in the two studies account for some of the differences.
Programed cell death is induced in the mammary gland within 12 h of the end of suckling (20) and increases sharply during the first phase of involution. Alterations in the levels of programmed cell death have been demonstrated in other transgenic and knockout mouse models that elicit a disruption of involution (12, 21, 22, 23, 24). We were able to report a significant decrease in the level of apoptosis at d 2. However, there was not a complete absence of programmed cell death at either d 2 or 6 of involution, implying that the regulation of apoptosis is not entirely dependent on the presence of Stat3. We were able to show an increase in the level and duration of SGP-2 expression, although it is a protein of unknown function with respect to direct biological significance during mammary development or involution. Many possible mechanisms and roles have been suggested for SGP-2; however, the expression of SGP-2 is not necessary for the apoptotic process to proceed (25). SGP-2 is a secreted protein that has been shown to adhere to cells near the point of secretion and may infer a cytoprotection against certain apoptotic stimuli (26, 27, 28). Cell survival of mammary epithelium at the interface of lactation and involution has been shown to depend on the presence of the Bcl-x protein (16). Because the Bcl-x levels were unaltered in the absence of Stat3, cell survival must depend on other proteins.
The second stage of involution is characterized by the destruction of the lobuloalveolar unit and its surrounding extracellular matrix followed by a remodeling of the epithelial and stromal components (1, 29, 30, 31). MMPs are possible mediators of this process (32). A critical role for the MMPs and their inhibitors, tissue inhibitors of metalloproteinases, has been demonstrated in the development and maintenance of the mammary tissue (33, 34) and in the progression of involution (17, 22, 33). This function agrees with the disrupted protease pattern observed in the absence of Stat3. Therefore, we suggest that the loss of Stat3 causes a delay in the activity of the proteases and thereby alters the initiation and progression of the second irreversible stage during mammary involution. Stat3 and the IL6 family of cytokines that activate Stat3 can regulate the expression of MMP-3 and tissue inhibitor of metalloproteinase-1 (35, 36) in vitro and in vivo. However, the mechanism may be of indirect nature. Based on this and other studies (12), it is clear that functional loss of mammary epithelium after weaning requires the presence of Stat3, and we now show that Stat3 is also required for the activation of proteases in vivo. At this point, the molecular and mechanistic link between Stat3 and protease-mediated remodeling is not known.
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Acknowledgments |
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Footnotes |
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2 Current Address: Regeneron Pharmaceuticals, Tarrytown, New York 10591.
Abbreviations: EGF, Epidermal growth factor; MMP, matrix metalloprotease; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; PRL, prolactin; SG-P-2, sulfated glycoprotein 2; STAT, signal transducer and activators of transcription; TBST, Tris-buffered saline with Tween 20; TUNEL, terminal deoxyuridine triphosphate nick end labeling; WAP, whey acidic protein; WC, WAP-Cre transgene.
Received February 26, 2002.
Accepted for publication May 2, 2002.
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B inhibits apoptosis in murine mammary epithelia. J Biol Chem 275:12737–12742
in LNCaP cells by overexpression of sulfated glycoprotein-2 (clusterin). Cancer Res 55:2431–2437This article has been cited by other articles:
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