Article |
Address correspondence to Keiko Miyoshi, Laboratory of Genetics and Physiology NIDDK, National Institutes of Health Bldg. 8, Rm. 101, Bethesda, MD 20892-0822. Tel.: (301) 496-2716. Fax: (301) 480 7312. E-mail: mammary{at}nih.gov
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Abstract |
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Key Words: prolactin receptor; Stat5; mammary gland; cell specification; epithelia
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Introduction |
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Development of the mammary gland occurs predominantly in the postnatal animal and is controlled by steroid and peptide hormones (Hennighausen and Robinson, 1998, 2001). Proliferation and differentiation of mammary alveolar epithelia occurs during pregnancy through prolactin and its receptor (PrlR) (Ormandy et al., 1997b). Stat5a-null mice fail to develop functional mammary tissue during pregnancy, mainly as a result of impaired functional differentiation and not due to the lack of lobulo-alveolar units (Liu et al., 1997). After multiple pregnancies, functional mammary development was attained in Stat5a-null mice (Liu et al., 1998). This phenotype was accompanied by increased levels of active Stat5b, suggesting that Stat5b can partially compensate for the absence of Stat5a. However, Stat5b itself is not required for lactation (Teglund et al., 1998). Because mice carrying inactivated Stat5a and 5b (referred to as Stat5 throughout the text) genes are infertile, the combined function of both Stat5a and 5b during pregnancy had not been investigated.
Components of regulatory pathways are in many cases not exclusive, and may participate in several signaling cascades. In mammary epithelia, Stat5 is activated through the PrlR, but also by GH and the epidermal growth factor receptors (Gallego et al., 2001), and possibly the Src pathway (Kazansky et al., 1999). In addition, the PrlR not only activates Stat5 via Jak2, but also stimulates the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways (Bole-Feysot et al., 1998; Kim and Cochran, 2001). If mammary alveolar epithelial development depends on a pathway that includes the PrlR and Stat5, a similar phenotype should be expected in the respective gene deletion mice. However, if Prl-independent Stat5 activation controls some steps of development, a different phenotype would be expected in the absence of the two components (i.e., PrlR and Stat5).
We have now investigated the relative contributions of Stat5 and the PrlR in pregnancy-induced mammary epithelial development. Although gross morphological analyses have linked the PrlR (Ormandy et al., 1997a) and Stat5a (Liu et al., 1997) to cell proliferation and differentiation, the molecular and cellular mechanisms of prolactin and Stat5 signaling are not understood. Specifically, the combined roles of Stat5a and 5b during pregnancy-induced mammopoiesis are not known. It remains unclear whether the Prl pathway is actually required for the acquisition of a particular cell fate, or whether it controls the differentiation of an already specified cell type. We have used molecular markers that can distinguish between different mammary epithelial cell types in the developing mammary gland to investigate the role of PrlR and Stat5 in the development of alveolar epithelium.
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Results |
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Epidermal growth factor and GH can activate Stat5 in PrlR-null epithelium
Based on histological and electron microscopy studies, Stat5- and PrlR-null epithelia exhibited differences. Whereas Stat5-null epithelium was highly disorganized and did not form open lumina, PrlR-null epithelium formed small open lumina. The observation that Stat5-null epithelium exhibited a more severe phenotype than PrlR-null epithelium, suggested that Stat5 might be activated to some extent by other cytokines in the absence of the PrlR. We have recently demonstrated by Western blot analysis that EGF and GH can activate Stat5 in mammary tissue (Gallego et al., 2001). However, their respective receptors within the epithelial compartment are not required for functional development (Gallego et al., 2001). We now investigated whether Stat5 could be activated in PrlR-null epithelium using immunohistochemistry (Fig. 8). Because Stat5a is more abundant than Stat5b, and Stat5b is not critical for alveolar development, we examined Stat5a activation. Stat5-null epithelia served as a negative control. At parturition, Stat5a was localized within nuclei of wild-type alveolar and ductal cells, which is indicative of its active state (Fig. 8 A). In contrast, only a few cells in PrlR-null epithelia exhibited nuclear, and thus activated Stat5a. However, upon injection with EGF or GH, extensive nuclear translocation of Stat5 was observed in PrlR-null epithelium (Fig. 8 B) indicative of Prl-independent activation of Stat5a.
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Discussion |
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In addition to the mammary gland phenotype observed in the transplant studies described herein, the Stat5-null mice exhibit phenotypes in other tissues and cell types consistent with the inactivation of several cytokine signaling pathways (Teglund et al., 1998). Loss of Stat5 disrupts IL-2 signaling and results in impaired T cell proliferation and a failure to express genes controlling cell cycle progression (Moriggl et al., 1999). In addition, Stat5 has also been linked to cell survival (Humphreys and Hennighausen, 1999; Socolovsky et al., 1999; Schwaller et al., 2000; Ihle, 2001). Lastly, Stat5 has been linked to B cell differentiation induced by IL-4 and IL-7 (Sexl et al., 2000) and Stat5a is required for functional differentiation, but not proliferation, of mammary epithelial cells (Liu et al., 1997).
Cellcell adhesion defects in PrlR- and Stat5-null mammary epithelia
The absence of Stat5 resulted in defective cellcell adhesion as assessed by electron microscopy. Thus, in control samples the basolateral membranes of neighboring cells were in close contact with each other. Conversely, there was evidence of gaps between adjacent cells in the Stat5-null samples. This suggests that Stat5 mediates signals that promote cellcell adhesion. The expression of E-cadherin and ZO-1, proteins known to be involved in cellcell adhesion and associated with tight junctions, respectively (Gumbiner, 1996), revealed normal staining patterns in both the PrlR- and Stat5-null samples. However, it is possible that whereas adjacent cells express detectable E-cadherin, they may not physically contact each other. In addition to E-cadherin, the process of cell adhesion involves additional proteins and it cannot be ruled out that any one of these are responsible for the cell adhesion defects that we observed.
The establishment of cellcell adhesion is not only important for epithelial organization, but also for effective communication between individual cells. Such intercellular communication allows neighboring cells within a defined structural unit to respond in unison to a given signal. For example, the gap junction subunit Cx 43 is expressed in the myoepithelial cell compartment, and it has been suggested that this may help coordinate myoepithelial contraction and milk ejection upon oxytocin stimulation (Plum et al., 2000). The lack of appropriate gap junction protein expression, and by extension intercellular communication, could disrupt the cellular unit (i.e., alveoli) whose formation may be required for their full functional development. It has previously been shown that the mouse mammary gland expresses three connexin isoforms, Cx 26, Cx 32, and Cx 43 (Pozzi et al., 1995). Whereas Cx 43 is expressed in myoepithelial cells, both Cx 26 and Cx 32 are expressed in the epithelial compartment. Interestingly, Cx 32 expression is induced at lactation and cannot readily be detected at other time points, suggesting that it may contribute to the attainment of a secretory phenotype (Locke et al., 2000). Furthermore, Cx 32 has been shown to interact with proteins in the tight junction complex and determine cell polarization in hepatocytes (Kojima et al., 2001). Although we were able to detect Cx 32 expression in control samples at parturition, neither PrlR- nor Stat5-null epithelia expressed detectable levels of Cx 32. It is possible that the lack of Cx 32 expression in PrlR- and Stat5-null epithelia is the result of a lack of secretory differentiation. Alternatively, it is possible that Cx 32 is a Stat5 target gene. In fact, the mouse Cx 32 gene promoter contains an interferon activated sequence site at position 800, suggesting that this promoter may be under direct prolactin control.
We have provided experimental evidence on the level of histology and electron microscopy that cell-cell adhesion and organization is impaired in the absence of Stat5, and to a lesser extent in the absence of the PrlR. Although we have shown apparent normal localization of E-cadherin and ZO-1, this cannot be a true measure for their functional integrity, which needs to be addressed in future studies. There is now widespread interest in the regulation of mammary epithelial cells by cellcell adhesion molecules. It is likely that many proteins, including the transcription factor Stat5, control these processes.
Pathway redundancy
There were notable differences when comparing the individual phenotypes on the histological level. In particular, epithelial development in the absence of the PrlR was more inhibited than in the absence of Stat5. This could in part be explained by a reduction in the proliferative capacity of PrlR-null epithelium relative to Stat5-null epithelium. Such differences may be due to the activation of compensatory signaling pathways. For example, mitogen-activated protein kinase and PI3K can be activated upon Prl stimulation. It is also possible that other Stats (i.e., Stat1 and/or Stat3) may be recruited to Jak2 in the absence of Stat5, thus resulting in additional stimulation of epithelial development.
There was evidence of open lumina in the PrlR-, but not in the Stat5-null, transplants. Furthermore, the ductoli present in the PrlR-null epithelia were more organized compared with Stat5-null epithelia, suggesting that Stat5 is necessary for appropriate organization of individual cells into cohesive cellular structures. Whereas Prl is probably the key cytokine responsible for the activation of Stat5, we demonstrated that Stat5 has some residual activity in the absence of the PrlR. Because both GH and EGF activate Stat5 in the absence of the PrlR, it is likely that these two cytokines contribute to the formation of cellcell contacts and thus a lumen.
Pathways controlling alveolar epithelial development
We have established that the PrlR and Stat5 are each essential for the attainment of functional alveologenesis. Several other genes and signaling pathways that control alveolar development have been identified, including ErbB2 (Jones and Stern, 1999) and ErbB4 (Jones et al., 1999), cyclin D1 (Fantl et al., 1995; Sicinski et al., 1995), C/EBPß (Robinson et al., 1998; Seagroves et al., 1998), the osteoclast differentiation factor RANKL and its receptor RANK (Fata et al., 2000), and the helix-loop-helix protein Id2 (Mori et al., 2000). Data from these mouse models suggests that alveologenesis is a complex process requiring the functional cooperation of numerous molecules. Interestingly, comparable phenotypes were observed in some of these mice, i.e., lack of alveolar development. Developmental roles for ErbB2 and 4 have been suggested based on transgenic mice that express dominant negative forms under control of a mouse mamary tumor virus long terminal repeat. Expression of a dominant negative ErbB2 resulted in condensed alveoli and reduced luminal secretion at parturition (Jones and Stern, 1999). ErbB4-dominant negative epithelium formed condensed alveoli and failed to expand at mid lactation, which correlated with reduce expression of -lactalbumin and WAP and a loss of Stat5 activity (Jones et al., 1999).
Similar to the models described here, C/EBPß-null mice possess undifferentiated alveolar epithelium; in contrast, branching morphogenesis was also impaired. C/EBPß mRNA levels in PrlR- and Stat5-null transplanted epithelia at parturition were similar to those seen in wild-type tissue (unpublished). These results suggest that C/EBPß expression is essentially independent of the PrlRStat5 pathway, although they may converge at the ß-casein promoter (Wyszomierski and Rosen, 2001). In the absence of RANKL, a growth factor produced by mammary epithelia in the second half of pregnancy, mammary epithelia also fail to develop (Fata et al., 2000). Because Prl can activate RANKL expression (Fata et al., 2000), it may be downstream of Stat5. However, both RANKL and RANK are expressed at high levels in PrlR- and Stat5-null transplanted epithelia at parturition (unpublished data), suggesting that these pathways are parallel and not dependent on each other. Id2-deficient mice also show severely impaired mammary gland development (Mori et al., 2000). Furthermore, Id2-deficient mammary epithelia exhibit reduced phosphorylation of Stat5. Normal levels of Id2 mRNA were detected in PrlR- and Stat5-null epithelia at parturition (unpublished data), suggesting that Id-2 is not downstream of Stat5. The presence of several, and apparently parallel, pathways controlling mammary alveolar development further emphasizes that distinct signals contribute to alveologenesis. At this point it is not clear whether these pathways have unique molecular targets leading to the formation of functional alveoli. The understanding of signaling pathways that are required for the formation of mammary epithelia but are dispensable for life of the organism itself provides a unique opportunity to develop molecular interventions and prevention for breast cancer.
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Materials and methods |
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Antibodies
Polyclonal antirabbit Stat5a antibodies have been described previously (Liu et al., 1996). Mouse monoclonal E-cadherin and smooth muscle actin antibodies were obtained from Transduction Laboratories, polyclonal antirabbit ZO-1 antibodies were purchased from Zymed Laboratories, and polyclonal antirabbit NKCC1 antibodies (Moore-Hoon and Turner, 1998) were a gift from Dr. Jim Turner (National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD). The polyclonal antirabbit Npt2b antibodies (Hilfiker et al., 1998) were a gift from Dr. Jurg Biber (Department of Physiology, University of Zurich, Zurich, Switzerland).
Transplantation of adult mammary epithelia into the cleared fat pad of nude mice
The transplantation was performed as previously described (DeOme et al., 1959). In brief, small pieces of mammary tissue were excised from mature virgin female wild-type, Stat5-, or PrlR-null mice. Athymic nude mice (3-wk-old) were anesthetized with an intraperitoneally injection of avertin and the proximal part of the inguinal gland containing the mammary epithelium was excised. Pieces of mammary tissue from a PrlR- and a Stat5-null mouse were grafted into contralateral cleared fat pads of recipients. The other combinations of transplants were Stat5:wild type and PrlR:wild type. To assess the completeness of clearing, the excised endogenous glands were processed for whole mount staining according to standard protocols. 8 wk after transplantation, fat pads containing transplants were harvested from virgin hosts. Alternatively, the hosts were bred and tissue was harvested on the day of parturition. For whole mounts, mammary glands were removed, fixed in Carnoy's fixative overnight and stained in carmine alum.
Immunofluorescence
After fixation in Tellyesniczky's fixative for 4 h at room temperature, tissues were embedded in paraffin and sectioned at 5 µm. Sections were cleared in xylene and rehydrated. Antigen retrieval was performed by heat treatment using an antigen unmasking solution (Vector Laboratories) and tissue sections were blocked for 30 min in PBST containing 10% goat or horse serum. For ZO-1 detection, antigen retrieval was performed by protease treatment (Auto/Zyme Reagent set; Biomeda Corp.) at 37°C for 10 min. Sections were incubated with E-cadherin (1:1,000) and ZO-1 (1:500) antibodies, E-cadherin (1:1,000) and Npt2b (1:100) antibodies, smooth muscle actin (1:1,000) and NKCC1 (1:1,000) antibodies or E-cadherin (1:1,000), and Stat5a (1:250) antibodies. The primary antibodies were allowed to bind for 60 min at 37°C except for Stat5a:E-cadherin and ZO-1:E-cadherin (4°C, overnight). Nonspecifically bound antibody was removed by rinsing in PBST before the addition of both antimouse FITC-conjugated (1:250) and antirabbit Texas redconjugated (1:250) secondary antibodies. Sections were incubated in the dark for 30 min, washed in two changes of PBST, and mounted in Vectashield (Vector Laboratories, Inc.). Fluorescence was visualized with a Zeiss Axioscop microscope equipped with FITC, TRITC, and FITC:TRITC filters. Images were captured using a Sony DKC5000 digital camera.
Analysis of cellular proliferation
After 9 wk, the transplant recipients were treated for 48 h with 1 µg ß-estradiol (E) (Sigma-Aldrich) and 1 mg progesterone (P) (Sigma-Aldrich) in 100 µl sesame oil via a single interscapular subcutaneous injection behind the neck. After acute hormone treatment, both of the transplanted number 4 inguinal mammary glands and an endogenous number 3 gland (control) were removed. 2 h before sacrifice, mice were injected with 0.3 mg BrdU per 10 g body weight (Amersham Pharmacia Biotech). Tissue was fixed in 4% paraformaldehyde in PBS for 2 h at 4°C. Immunofluorescence and BrdU-positive cell counting were performed as previously described (Seagroves et al., 2000). In brief, paraffin sections (57 µm) were dewaxed and subjected to microwave antigen retrieval in 10 mM citrate buffer, pH 6.0. After blocking in 5% BSA/0.5% Tween 20 for 4 h at room temperature, sections were incubated with antiBrdU-FITCconjugated antibody (1:5; Becton Dickinson) in blocking solution overnight at room temperature. After PBS washes, slides were mounted in Vectashield + DAPI medium (Vector Laboratories). At least four glands per genotype were used for each experiment (endogenous control #3, n = 5; PrlR-null epithelia transplanted gland, n = 5; Stat5-null epithelia transplanted gland, n = 4). Cells from 16 fields at 60x magnification were counted from each sample. The number of BrdU-positive cells in a given field was expressed as a percentage of the total number of DAPI-stained cells. Statistical significance was determined by Mann-Whitney paired t test.
Gene expression analysis
Total RNA was isolated from fresh or frozen tissues and Northern blots were performed as described previously (Robinson et al., 1995). In brief, 10 µg of total RNA was loaded in each lane. Membranes were hybridized with random-primed [-32P] dCTP-labeled probes in QuickHyb solution for 3 h at 65°C. Washes were performed in 0.1 x SSC/0.1% SDS at 65°C. At first, the membrane was hybridized with WAP and ß-casein probes together and exposed to x-ray films. After stripping, membranes were rehybridized with WDNM1 and keratin 18 probes together. A 415-bp WAP-specific probe was generated by RT-PCR with primers 5'-GTA-CCA-TGC-GTT-GCC-TCA-TC-3' and 5'-GCT-GCT-CAC-TGA-AGG-GTT-ATC-3'. A 577-bp ß-casein-specific probe was generated by RT-PCR with primers 5'-CTA-AAG-TTC-ACT-CCA-GCA-TCC-3' and 5'-CAT-TTC-CAG-TTT-CAG-TCA-GTT-C-3'. A full-length cDNA for WDNM1 was used (Robinson et al., 1995). The keratin 18specific probe was a 1.1-kb EcoRI cDNA fragment (Singer et al., 1986).
Electron microscopy
Small pieces of mammary tissue were cut, minced into 1-mm cubes, and fixed in a 0.025% solution of glutaraldehyde and 3.8% paraformaldehyde in PBS, pH 7.2, for 2 h. The tissue was rinsed in PBS and postfixed in 2% osmium tetroxide in 0.5 M sodium cacodylate buffer for 2 h, and dehydrated in a graded series of acetone solutions (Blanchette-Mackie and Scow, 1971). Temperature was maintained at 4°C from excision through dehydration and tissues were embedded in epon at room temperature (Luft, 1961). Sections were cut on a Reichert Om U2 ultramicrotome. Thick sections were stained with Toluidine blue in 1% sodium borate (pH 8.3) (Trump et al., 1961). Thin sections were stained with Karnovsky's lead hydroxide (Karnovsky, 1961) and uranyl acetate (Zobel and Beer, 1961) and examined with a JEOL 1010 electron microscope.
RT-PCR assays
Total RNA (1 µg) was transcribed into cDNA using Thermoscript reverse transcriptase (Life Technologies, Inc.) according to the manufacturer's protocol. Total RNA (1 µg) was first incubated with dNTPs and an oligo-dT(1218) primer at 65°C for 5 min. All components were added except the reverse transcriptase, and the reaction was incubated at 42°C for 2 min. Thermoscript RT (50 units) was added to each reaction and incubated for a further 50 min. For controls, the samples without RT reactions were amplified. Single-stranded RNA was degraded by treating the reaction with Escherichia coli RNase H for 20 min at 37°C. PCR assays were performed for Cx 32 and GAPDH cDNA. Cx 32 gene-specific primers were 5'-GTT-GCA-ACC-AGG-TGT-GGC-AGT-G-3' and 5'-CGG-AGG-CTG-CGA-GCA-TAA-AGA-C-3'. GAPDH gene-specific primers were 5'-CAA-CGG-GAA-GGG-CCC-CCA-TAC-CAT-C-3' and 5'-ACG-ACG-GAC-ACA-TTG-GGG-GTA-G-3'. The template was first denatured at 94°C for 2 min followed by 35 cycles (Cx 32) or 25 cycles (GAPDH) of denaturation (94°C, 40 s), annealing (65°C, 40 s), and extention (72°C, 1 min). A final extention at 72°C for 10 min was performed.
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Footnotes |
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Acknowledgments |
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J. Shillingford is funded by Department of Defense fellowship (17-00-1-0246). S.L. Grimm is supported by Department of Defense fellowship 17-00-1-0138.
Submitted: 13 July 2001
Revised: 8 October 2001
Accepted: 8 October 2001
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