| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Molecular and Cellular Biology (S.L.G., T.N.S., E.B.K., J.P.L., J.M.R.) and Breast Center (A.V.L.), Department of Medicine, Baylor College of Medicine, Houston, Texas 77030; Molecular and Cellular Endocrinology Section (R.C.H., B.K.V.), Center for Cancer Research, National Cancer Institute, National Institutes of Health, and Laboratory of Genetics and Physiology (K.M., L.H.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; Cancer Research Program (C.J.O.), Garvan Institute of Medical Research, Darlinghurst, Australia; and Department of Neuroscience and Anatomy (M.A.S., T.L.W.), Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: Jeffrey M. Rosen, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: jrosen{at}bcm.tmc.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Previous studies have determined that the CCAAT/enhancer binding protein-ß (C/EBPß) transcription factor is required for normal ductal morphogenesis and lobuloalveolar development during pregnancy (2, 3). The mammary glands of C/EBPßnull mice exhibit enlarged, cystic ducts with decreased side-branching and an inhibition of alveologenesis in response to E + P. In addition, increased levels of progesterone receptor (PR) mRNA and protein were detected in the mammary epithelial cells (MECs) of C/EBPßnull mice, with an altered distribution of PR-expressing cells along the ducts. This alteration in PR expression correlated with a 10-fold decrease in proliferation induced by an acute, 2-d treatment with E + P (4). This was unexpected because progesterone acts a mitogen to stimulate proliferation of MECs (5), and PR in MECs has been demonstrated to be essential for lobuloalveolar development (6). Furthermore, transplantation experiments with chimeras containing PRnull MECs tagged with lacZ along with wild-type MECs indicated that alveolar development in PRnull cells occurred when the two cell types were in close proximity, thus demonstrating that PR acts in a paracrine fashion to stimulate proliferation of neighboring cells (7).
The study of gene-targeted mouse models has helped identify other systemic hormone and local growth factors and their cognate receptors required for mammary gland development (reviewed in Ref. 8). Deletion of many of these genes results in mammary gland phenotypes that exhibit defective lobuloalveolar development. For example, the Prl receptor (PrlR) is required for pregnancy-induced lobuloalveolar development. PrlRnull mice exhibit a phenotype similar to that observed in the PRnull mice (9). This is supported by evidence that PR can regulate PrlR expression (10, 11). The side-branching defect observed in PrlRnull mammary glands can be rescued with P treatment, but lobuloalveolar development still did not occur (12).
Signal transducer and activator of transcription 5 (Stat5) is an essential component of the PrlR signal transduction pathway (13). Stat5a-deficient mice exhibit impaired lobuloalveolar development, which could be partially compensated by Stat5b after multiple pregnancies (14, 15). Stat5b, however, was not essential for lactation, and no significant mammary gland phenotype was observed in Stat5b-deficient mice (16). Because of this potential redundancy, a deletion of both Stat5 genes was generated (16). The mammary gland phenotype of these mice was more severe than in Stat5a-deficient mice, with a complete block of lobuloalveolar development and the absence of functional differentiation (17).
Although PR is required for lobuloalveolar development, E receptor 
(ER) is required at an earlier stage to induce ductal elongation (18). Mammary glands from ERnull mice have a rudimentary ductal tree that does not fill the fat pad, although it can undergo lobuloalveolar development in response to a pituitary isograft or E + P treatment (19). In separate studies of the normal mammary gland, it has been shown that PR and ER are coexpressed in MECs approximately 96% of the time (20). These steroid receptor-positive cells are often located adjacent to proliferating cells but rarely colocalize (4, 20, 21). It is thought that proliferating MECs eventually give rise to more differentiated, steroid receptor-expressing MECs (20, 22).
In the current study, additional changes in gene expression were identified in the mammary glands of C/EBPßnull mice that provide further insight into the mechanisms by which systemic hormones and local growth factors regulate lobuloalveolar development. Alterations in the level and pattern of PrlR concomitant with the changes in PR were observed in C/EBPßnull mice. These studies suggest that the correct distribution of both PR and PrlR is required to facilitate hormone-induced proliferation, and that defects in PR expression and proliferation are common features among several mouse models in which impaired lobuloalveolar development is observed. Changes in IGF binding protein (IGFBP)-5, IGF-II, and insulin receptor substrate-1 (IRS-1) expression and alterations in their distribution also were observed in C/EBPßnull mice, suggesting a role of the IGF axis in the paracrine regulation of lobuloalveolar development. Finally, the inappropriate expression of an epidermal differentiation marker, small proline-rich protein (SPRR2A), and K6 suggests that the germline deletion of C/EBPß not only disrupts the necessary hormone receptor patterning but also results in an alteration of MEC cell fate.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Additional evidence that PrlR expression is mediated through activation of PR is shown in Fig. 1A
. Semiquantitative RT-PCR analysis of the long form of PrlR was performed using mammary gland RNA from either wild-type or PRnull mice at 5 or 12 wk of age. In the mammary glands of wild-type mice there was an approximate 3-fold increase in the amount of PrlR mRNA between 5 and 12 wk of age, coinciding with the increase in circulating ovarian hormones that occurs during this time (1). PRnull mice expressed a much lower level of PrlR at 5 and 12 wk of age, suggesting that PrlR expression depends, at least in part, on PR. These data and previous in situ hybridization (ISH) studies suggest that PR and PrlR may be coregulated during early mammary gland development (24).
|
). E, acting through ER, is known to elevate serum Prl levels (25), and P, acting through PR, increases PrlR mRNA expression (10, 11). A marked increase in the amount of the long form of PrlR mRNA was detected in the mammary glands from C/EBPßnull mice relative to wild-type mice. Hybridization to a keratin 18 (K18) probe demonstrated both similar mRNA loading and levels of mammary epithelium, which was expected after only 2 d of E + P treatment. ISH was also performed to determine the cellular distribution of PrlR mRNA in C/EBPßnull mammary glands (Fig. 1, C and D). Signal was detected in a nonuniform, punctate pattern in MECs along the ducts in mammary glands from wild-type mice, similar to the pattern observed previously for PR (4, 24). However, PrlR mRNA levels were not only increased in C/EBPßnull mice, but the pattern of expression along the ducts was also more uniform, similar again to alterations observed previously for PR expression (4). Based on the results of the semiquantitative ISH, there was an increased level of PrlR mRNA per cell in the C/EBPßnull gland, in addition to a higher percentage of cells expressing PrlR.
Increased PR and Decreased Proliferation in the Mammary Glands of PrlRnull Mice
Because the levels and pattern of PrlR expression were altered in C/EBPßnull mice, and because PR and PrlR appear to be coordinately regulated, the levels of PR were examined in the mammary glands from intact, untreated PrlRnull animals. Between 6 and 9 wk of age, PR expression went from a uniform pattern to a heterogenous, punctate pattern in the MECs of wild-type mice (Fig. 2, A and C), consistent with previous observations (4). By 12 wk, the percentage of PR-positive ductal cells had decreased to approximately 25% (Fig. 2, E and G). In contrast, PR expression was not down-regulated between 6 and 12 wk in PrlRnull mice, retaining a level of approximately 50% positive MECs (Fig. 2, B, D, and F). The difference in the percent of PR-positive MECs between the two genotypes was statistically significant at 9 and 12 wk of age (Fig. 2G).
|
) as compared with wild-type animals (Fig. 3, A and C). When the incorporation of BrdU was measured, there was a significant decrease in the number of proliferating MECs in the PrlRnull mice (Fig. 3, E and F). The finding that 13% of MECs were BrdU-positive in wild-type mice (Fig. 3, D and F) was consistent with previous results (4).
|
). When mammary gland tissue from wild-type mice was transplanted into nude mice and analyzed for PR expression, the percent PR-positive MECs closely resembled the endogenous gland shown in Fig. 4A (data not shown), suggesting that the lowered levels of circulating E + P in nude mice (26) accounted for the higher level of PR in the controls. Despite the higher basal level of PR expression in nude mice, both Stat5ab-deficient and PrlRnull epithelium had increased levels of PR-positive MECs relative to the control (Fig. 4, B and C). There were statistically significant differences between the two gene-deleted outgrowths and the control, as well as between Stat5ab-deficient and PrlRnull outgrowths (Fig. 4D). Proliferation in Stat5ab-deficient outgrowths was also lower relative to the control (Fig. 4, F vs. E), but not to the extent in PrlRnull outgrowths (Fig. 4G). This difference is most likely because PrlR acts through multiple signaling pathways, including Janus kinse (Jak) 2, phosphatidylinositol-3 kinase, and MAPK, as discussed previously (17). In all cases, the amount of PR expressed was inversely proportional to the rate of proliferation.
|
One of the genes identified was IGFBP-5. IGFBP-5 was also identified during a screen using the CLONTECH Laboratories, Inc. (Palo Alto, CA) Atlas array as a gene down-regulated in the mammary glands of C/EBPßnull mice (data not shown), and this observation was confirmed by Northern blot analysis of mammary gland mRNA from untreated C/EBPßnull mice (Fig. 5A). ISH was performed on frozen sections of mammary glands from untreated, nulliparous wild-type or C/EBPßnull mice to determine the cellular distribution of the IGFBP-5 mRNA (Fig. 5, B–E). Opposite to the pattern of PR and PrlR expression, IGFBP-5 mRNA changed from a relatively uniform pattern of expression in wild-type mammary glands to a nonuniform, punctate pattern in C/EBPßnull mice. This effect was more pronounced in the C/EBPßnull after treatment for 2 d with E + P (Fig. 5, C and E).
|
, F–K). At 6 wk, expression of IGF-II was uniform in the ductal epithelium from both wild-type and C/EBPßnull mice (Fig. 5, F and I). By 12 wk, the pattern of expression became punctate in the wild-type (Fig. 5G) but remained uniform in the ducts of C/EBPßnull mice (Fig. 5J), reminiscent of the change in patterning of PR and PrlR expression. The difference in IGF-II distribution became more pronounced in wild-type mice after 2 d of E + P treatment (Fig. 5H).
IRS-1 is a cytoplasmic signaling molecule downstream from the insulin and IGF-I receptors. No significant differences in IRS-1 mRNA levels were observed between untreated or 2-d E + P-treated wild-type and C/EBPßnull mice (Fig. 5L) as determined by ribonuclease protection assay (RPA). However, when the expression of IRS-1 protein was analyzed by Western blot, a 2- to 3-fold decrease between wild-type and C/EBPßnull mice was observed in both the untreated and 2-d E + P-treated animals (Fig. 5M). A change in the expression pattern of IRS-1 from uniform in the wild-type (Fig. 5N) to nonuniform in the ducts of C/EBPßnull mice (Fig. 5O) was observed by immunostaining and correlated with the results obtained by Western blotting.
Hormonally Regulated Signaling Pathways in the Mammary Glands of C/EBPßnull Mice Are Functional
Whereas the levels of PR and PrlR increased in the mammary glands of C/EBPßnull mice, it is not clear if the signaling pathways in these cells are functional. Turnover and down-regulation of PR protein results from prolonged exposure to P (28, 29). Therefore, wild-type and C/EBPßnull mice were implanted with E + P pellets for 21 d to determine if PR levels could be down-regulated after chronic exposure to steroid hormones. Comparing the percentage of PR-positive MECs from untreated animals with those treated for 21 d with E + P showed a 2-fold decrease in the glands from wild-type mice (Fig. 6, A vs. B). Although the level of PR was almost three times greater in C/EBPßnull mice before treatment, the fold down-regulation after hormone treatment was identical (Fig. 6, C vs. D).
|
). The antibody used recognizes both isoforms, but Stat5a is the predominant form in the mammary gland (15). Levels of Stat5 tyrosine phosphorylation were similar in extracts from glands from either untreated or hormone-treated wild-type mice. Interestingly, no Stat5 tyrosine phosphorylation was detected in mammary gland extracts from untreated C/EBPßnull mice, but phosphorylation was observed after acute hormone treatment, which may be due to estrogen-induced Prl expression (25).
SPRR2A, a Marker of Epidermal Differentiation, and K6 Are Expressed in the Mammary Glands of C/EBPßnull Mice
Surprisingly, another gene identified by the SSH-PCR screen was SPRR2A, a marker of epidermal differentiation that is normally expressed in the cornified layer of the skin (30). Northern blot analysis confirmed the differential expression of SPRR2A and demonstrated that the level of SPRR2A mRNA was markedly increased in the mammary glands from nulliparous C/EBPßnull mice (Fig. 7A). Immunohistochemistry was performed to determine the levels and pattern of SPRR2 protein expression in the mammary gland (Fig. 7B). There was no staining in wild-type glands but a punctate, nonuniform pattern of expression was observed in the MECs from C/EBPßnull glands, similar to the pattern seen for PR and PrlR. Although this staining was performed on tissue treated for 2 d with E + P, similar results were seen in tissues from untreated animals (data not shown).
|
). However, K6-positive MECs were readily apparent in ducts from mature C/EBPßnull mice. Whereas the immunoreactivity was more uniform than that observed for SPRR2A, not all MECs were K6-positive. In contrast, no K6-positive cells were identified in ducts from transplanted PrlRnull or Stat5ab-deficient tissue (data not shown). K14 expression is normally detected in the myoepithelial cells surrounding the ducts, and no differences were detected in the staining patterns in the myoepithelium between wild-type and null mice (Fig. 7D). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
More recently, it has been possible to detect the pattern of PR expression in mice using ß-galactosidase staining wherein lacZ has been inserted in place of the PR gene (34). The pattern of lacZ staining observed in mature nulliparous mice was also heterogeneous and was regulated by ovarian steroids during normal development. Furthermore, PR expression has even been observed in the mammary anlage as early as embryonic d 14 (34).
When proliferation rates were quantitated after acute E + P treatment, the Stat5ab-deficient and PrlRnull outgrowths contained fewer BrdU-positive MECs than wild-type mice. However, even with fewer BrdU-positive MECs, the dissociation between PR expression and proliferation was maintained in these gene-targeted mouse models. It has been proposed that p27Kip1 expression in steroid-receptor positive cells may block cell division (35). Our preliminary results show an increase in the number of p27 positive cells along the ducts of the C/EBPßnull glands (Grimm, S. L., and J. M. Rosen, unpublished observation), which correlates both with the increased PR expression and decreased proliferation.
Although the MECs from C/EBPßnull, PrlRnull and Stat5ab-deficient mouse models all display a decreased proliferative response to steroid hormones, the inability of these cells to proliferate is probably not due to defective signaling pathways. The down-regulation of PR protein after prolonged exposure to P is mediated through serine phosphorylation by MAPK, resulting in the ubiquitin-mediated degradation of PR (29). Even though C/EBPßnull mice contain approximately three times the number of PR-positive MECs, chronic exposure to E + P resulted in a 2-fold down-regulation of PR in both wild-type and C/EBPßnull mice. These data rule out the possibility that defects in this pathway account for the increased level of PR in the C/EBPßnull mammary gland. The PrlR/Stat5 pathway also appears to be functional in C/EBPßnull mice. There was no detectable tyrosine phosphorylation of Stat5 observed in the mammary glands of untreated C/EBPßnull mice, most likely due to defective ovarian function in the C/EBPßnull mice (36). However, Stat5 protein was tyrosine phosphorylated after acute hormone treatment, most likely as a consequence of E-induced Prl production (25). However, chronic hormone treatment, which can activate Stat5, was not sufficient to rescue lobuloalveolar development, suggesting that other downstream responses to the Jak/Stat pathway might be altered in C/EBPßnull mice.
These observations have led to the development of a testable autoregulatory model depicted in Fig. 8. During embryonic development, epithelial/mesenchymal interactions required for the development of the mammary anlage may lead to the expression of PR. PR expression then results in the induction of PrlR; whether this occurs via a direct transcriptional mechanism or via an indirect mechanism remains to be determined. PrlR may then act through the Jak/Stat pathway to help regulate the level of ER expression. This is consistent with the observation that Prl can activate ER transcription in corpus luteum via Stat5a or 5b (37). Whether Prl directly activates ER transcription in MECs is not known. Additionally, PrlR may feed back to control PR expression. Finally, ER may then regulate the level of PR gene transcription both via indirect interactions with other transcription factors, such as SP1, as well as binding to ER response element half-sites (38, 39). Although this model helps explain the coregulation of ER, PR, and PrlR observed in MECs in mature, nulliparous mice, the mechanism by which the nonuniform pattern of receptor expression is established in response to E and P remains to be determined. C/EBPß does not appear to play a direct role in this autoregulatory loop; the level of C/EBPß mRNA does not change in the PRnull mammary gland (4), nor does it differ in transplanted PrlRnull or Stat5ab-deficient tissue at parturition (17). Instead, C/EBPß appears to act at a much earlier stage of development and may be required for the specification of mammary epithelial progenitors.
|
However, IGFBP-5 has been shown previously to augment the effects of IGF-I on the migration and proliferation of smooth muscle cells (43). Recent studies have also demonstrated a positive role for IGFBP-5 in Xenopus neural tube induction (44). In this system, it has been suggested that IGFBP-5 potentiates the activity of endogenous IGFs to facilitate signaling through the IGF receptor.
During lactation, Prl is thought to suppress IGFBP-5 expression possibly through the activation of Stat5 (42). The increase in PrlR levels observed in C/EBPßnull mice might, therefore, result in the decreased levels of IGFBP-5 mRNA expression. Changes in the level of IGFBP-5 expression may also reflect the deletion of C/EBPß, which may act as a direct regulator of IGFBP-5 transcription in the mammary gland, as it does in osteoblasts (45). Again, the mechanisms responsible for the observed changes in the spatial pattern of IGFBP-5 expression remain to be established.
Along with changes in IGFBP-5, the expression patterns of other molecules in the IGF axis were altered in C/EBPßnull mice. IGF-II patterning in wild-type mice was reminiscent of PR and PrlR, with a uniform pattern of expression detected initially at 6 wk that became punctate by 12 wk. However, the pattern of IGF-II mRNA expression remained uniform in C/EBPßnull mice throughout this period of development. IGF-II is known to be a mitogen in the mammary gland (reviewed in Ref. 40) and may be a direct target of the Prl-induced Jak/Stat pathway (Ormandy, C., unpublished). Whereas the level of IGF-II mRNA was increased at 12 wk post partum in C/EBPßnull mice as compared with wild-type, the decreased expression of IGFBP-5 and IRS-1 may inhibit the paracrine effects of IGF-II in MECs, as well as IGF-I in the mammary stroma.
IRS-1 expression was also decreased in the mammary glands of C/EBPßnull mice. Again, the change in the pattern of IRS-1 expression resembled that observed for IGFBP-5 expression. The decrease in IRS-1 protein expression appears to be regulated at the post-transcriptional level, as reported previously during normal mammary gland development (46). However, this is probably not the result of ligand-mediated activation of IRS-1 (47), based on the marked inhibition of E + P-induced proliferation in the C/EBPßnull mice. With the availability of epitope-specific antibodies directed against the myriad of phosphotyrosines and phosphoserines in IRS-1, it may be possible to determine directly the mechanisms responsible for the different patterns of IRS-1 expression observed in the mammary gland.
Additional evidence for involvement of the IGF axis with PrlR signaling comes from comparing RNA isolated from transplanted mammary tissue from wild-type and PrlRnull mice using Affymetrix microarrays. IGF-II expression was decreased at d 6 of pregnancy in the PrlRnull transplants, with no change observed at d 2 or 4 of pregnancy (Ormandy, C., unpublished). Thus, it is conceivable that IGF-II may act as one of several local growth factors, including Wnt-4 (48, 49), receptor activator of nuclear factor 
B ligand (50), TGF, and/or amphiregulin (51), all of which may be important mediators of the paracrine/juxtracrine action of steroid hormones and Prl on proliferation during lobuloalveolar development.
Altered Cell Fate in the Mammary Glands of C/EBPßnull Mice
These results suggest that the germline deletion of C/EBPß may result in a change in cell fate preventing ductal epithelial progenitors from responding appropriately to hormone-regulated signal transduction pathways. In this regard, one of the genes identified in the SSH screen that is up-regulated in the mammary gland of C/EBPßnull mice is the sodium potassium chloride (NKCC1) cotransporter, which has been demonstrated previously to represent a marker of the ductal epithelium (17, 52, 53). Ductal morphogenesis is delayed in the mammary glands of NKCClnull mice, and this effect is MEC autonomous (53). Both PrlRnull and Stat5ab-deficient mice fail to undergo lobuloalveolar development, perhaps because of the absence of lobuloalveolar progenitors (17, 54). The expression of NKCC1 is also retained in the ductal epithelium of these mice during pregnancy (53). Thus, it is conceivable that despite the presence of PR and PrlR, their ability to activate the appropriate cellular response is due to a deficiency in the lobuloalveolar progenitors in C/EBPßnull mice.
Additional evidence for a change in cell fate in the mammary glands of C/EBPßnull mice is the misexpression of SPRR2A, a marker of epidermal differentiation. SPRR2A is a protein normally expressed in the cornified layer of the epidermis and is involved in skin barrier function (30). No SPRR2A expression was detected by Northern blot analysis of mRNA from wild-type mice, or by immunohistochemical staining in the ductal epithelium, but high levels of expression were observed in the MECs of C/EBPßnull mice in a nonuniform pattern. K6 expression was also observed in the MECs of C/EBPßnull glands but not in the wild-type MECs or in transplanted tissue from PrlRnull or Stat5ab-deficient mice. The expression of K6 was more uniform than the expression of SPRR2A but was not detected in every cell. It has been proposed that K6/K14-positive MECs exist in mature mammary glands and may represent stem cells (32). Because K6 expression is normally observed in the body cells of terminal end buds (32, 33), this result suggests that deletion of C/EBPß may prevent further differentiation of these ductal progenitors. It appears likely that the expression of these markers may represent a block in the normal cell fate determination and development pathway as a consequence of the germline deletion of C/EBPß, which was not observed in the PrlRnull or Stat5ab-deficient mice.
Overall, these studies have illustrated the importance of appropriate receptor patterning in normal mammary gland development and have helped provide support for the model by which steroid hormones and Prl regulate lobuloalveolar development via a paracrine/juxtracrine mechanism. Disruptions in the pattern of these receptors and diminished proliferative responses were observed after the targeted deletion of several receptors, downstream signaling molecules and transcription factors, suggesting that this is a required mechanism for alveolar development. Although there are common defects between these knockout models, in particular increased PR and decreased proliferation, there are additional alterations in C/EBPßnull mice that suggest an earlier role for this transcription factor in controlling MEC cell fate. The study of C/EBPßnull mice continues to provide useful insights into the steps governing lobuloalveolar development. However, additional experiments are required to understand the precise molecular mechanisms mediating this complex developmental process.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The C/EBPß mice were genotyped by PCR, rather than by Southern blot analysis as described previously (55). The primers for genotyping span the insertion site of the Neo cassette in the 3' end of the C/EBPß gene (55). This results in two different size products, either 367 bp for the wild-type allele or 1.3 kb for the mutant allele. Heterozygous animals will have both size bands. The forward primer was 5'-AGAAGACGGTGGACAAGCTG-3' and the reverse primer was 5'-CTCGGTGCAGGTGCAGGT-3'. The 30-µl PCR contained 1.25 µM MgCl2, 0.2 µM of each primer, and 1/20th vol of dimethylsulfoxide. Thirty cycles were performed using a denaturing step at 94 C for 30 sec, an annealing step at 55 C for 45 sec, and an extension time of 90 sec at 72 C.
Ovary-intact, nulliparous mice were treated for 48 h with a single interscapular sc injection of 17ß-estradiol benzoate (1 µg) and P (1 mg) in 100 µl of sesame oil (all from Sigma, St. Louis, MO). For chronic E + P treatment, mice between 18 and 22 wk of age were treated for 21 d with E + P pellets as previously described (2). The transplantation of Stat5ab-deficient and PrlRnull mammary tissue has been described elsewhere (17). Nine to 10 wk after transplantation into athymic NCr-nu/nu mice, an acute treatment of E + P was given. Two days later, both of the transplanted no. 4 inguinal mammary glands (Stat5ab-deficient and PrlRnull) and an endogenous no. 3 thoracic gland (control) were removed. Two hours before they were euthanized, all E + P-treated animals were injected ip with 0.3 mg BrdU per 10 g body weight (Amersham Pharmacia Biotech, Arlington Heights, IL). Tissues were fixed in 4% paraformaldehyde for 2 h at 4 C. Paraffin-embedded tissues were sectioned (5–7 µm) onto Probe-On Plus charged slides (Fisher Scientific, Pittsburgh, PA). Alternatively, mammary gland tissues were flash frozen in liquid nitrogen and stored at -80 C before cryostat sectioning. Ten-micron frozen sections were collected from all glands, mounted onto Superfrost Plus microscope slides (Fisher Scientific) and stored at -80 C.
RNA Analyses
Details for PrlR semiquantitative RT-PCR and ISH, including RNA preparation, primers and probes, have been described previously (24).
For Northern blot analyses, total RNA was first isolated from frozen mammary gland tissue using RNAzolB reagent (Tel-Test, Inc., Friendswood, TX). Poly(A) RNA isolation from total RNA was performed using the PolyATract I kit (Promega Corp., Madison, WI) according to manufacturer’s instructions. Probes for PrlR-L, K18, SPRR2A, and IGFBP-5 were prepared by digesting the appropriate expression vectors, and the cyclophilin probe was purchased from Ambion, Inc. (Austin, TX). Inserts were labeled with 32P-deoxy-ATP using the Prime-A-Gene kit (Promega Corp.). The blotting protocol was performed as previously described (4).
Radioactive ISH for IGF-II was performed on frozen sections as described previously (56). Nonradioactive ISHs were performed as for the radioactive ISH with the following modifications: the cRNA probe to IGFBP-5 was transcribed according to standard protocols (Roche Molecular Biochemicals, Indianapolis, IN) using a linearized mouse IGFBP-5 cDNA (57). Hybridizations were done overnight at 60 C with 400 ng/ml of digoxigenin-labeled IGFBP-5 cRNA probe. Sections were rinsed in Tris-buffered saline (TBS), blocked in 1x blocking reagent (Roche) in TBS and incubated in anti-DIG AP (1:500; Roche) in blocking reagent for 1 h at 37 C. Sections were then rinsed in TBS and incubated in detection buffer (100 mM Tris; 100 mM NaCl; 50 mM MgCl2, pH 9.5) with levamisole (1.2 mg/ml) for 10 min. Sections were then incubated in nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt substrate system (Sigma) in the dark for 30 min. Slides were rinsed with water for 10 min, dehydrated through ethanols and xylenes, and coverslipped in Permount mounting media (Fisher Scientific).
RPAs were performed as described in the manufacturer’s protocol (Ambion, Inc.). Twenty micrograms of total RNA were incubated with 3 fmol of 32P-uridine triphosphate-labeled antisense IRS-1 (46) or ß-Actin (Ambion, Inc.) riboprobes overnight at 55 C. Samples were run in triplicate and signal intensity was quantitated by PhosphorImager analysis using ImageQuant software (both from Amersham Biosciences, San Diego, CA).
Immunostaining
Sections were deparaffinized in xylenes, then rehydrated through a graded ethanol series. Indirect immunofluorescence for PR and BrdU and IRS-1 immunohistochemistry were performed as previously described (4, 46). Immunohistochemical staining was performed without antigen retrieval and used 5% goat serum (Sigma) in PBS as blocking buffer. Sections were incubated with the following primary antibodies overnight at room temperature: -SPRR2 rabbit polyclonal antibody at 1:3000 (30), -K6 (no. 66) rabbit polyclonal antibody at 1:5000 (kindly provided by Dennis Roop, Baylor College of Medicine, Houston, TX), and -K14 rabbit polyclonal antibody at 1:10,000 (Covance, Richmond, CA). Immunoperoxidase staining was detected using the Vectastain Elite ABC kit and the diaminobenzidine substrate kit according to manufacturer’s instructions (Vector Laboratories, Inc., Burlingame, CA).
Cell Counting and Statistical Analysis
Fluorescent images were digitally captured using an Olympus Corp. BX50 microscope connected to a Hamamatsu C5810 charge-coupled device. At least 6–16 individual 60x microscopic fields per sample were captured using the appropriate fluorescein isothiocyanate, Texas Red, and 4',6 diamidino-2-phenylindole (DAPI) filters. The number of PR- and BrdU-positive MECs in a given field was expressed as a percentage of total number of DAPI-stained MECs. Statistical significance was determined by the Student’s paired t test with all P values below 0.0001.
Immunoprecipitation and Western Blot Analyses
Frozen mammary gland tissues from three to four animals (ages 21–25 wk) for each genotype and treatment were pooled to prepare whole cell extracts as previously described (58). Immunoprecipitation assays were performed as previously described (58) using 1.5 mg of whole cell extract incubated with 800 ng of -Stat5 rabbit polyclonal antibody (N-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antiphospho-tyrosine antibody PY20 (BD Biosciences, San Diego, CA) was used to evaluate the phosphorylation status of Stat5. The blot was then stripped and reprobed with the -Stat5 rabbit polyclonal antibody used for the immunoprecipitation. IRS-1 protein was detected by Western blot analysis using 80 µg of whole cell extract per lane and the same antibody as used for immunohistochemistry, diluted 1:1000. MAPK p42/44 protein was used a loading control (1:1000; Cell Signaling Technology, Beverly, MA). Chemilumiescence was performed using SuperSignal reagents from Pierce Chemical Co. (Rockford, IL).
SSH PCR
The PCR-select cDNA Subtraction kit (CLONTECH Laboratories, Inc.) was used according to the manufacturer’s instructions to screen for genes differentially regulated in the mammary glands of mature, untreated C/EBPßnull mice. Briefly, 2 µg of poly(A) RNA from either wild-type or C/EBPßnull mammary glands (pooled tissue; 3–6 months old) were used to synthesize double-stranded cDNA. After subtraction and PCR amplification using nested primers, the cDNA inserts were cloned into pGEM-T Easy vectors (Promega Corp.), transformed into XL2-Blue ultracompetent cells (Stratagene, La Jolla, CA), and plated for blue/white color selection. Individual colonies (n = 672) were picked randomly and the inserts were PCR-amplified and screened by reverse Southern blot analysis using probes made from either enriched wild-type or C/EBPßnull total cDNA. Approximately 60 clones showing detectable differences in expression levels by reverse Southern were then sequenced (59).
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Present address: Department of Biology, Molecular Biology Section, University of California San Diego, La Jolla, California 92093. 
2 Present address: Department of Animal Science, University of Vermont, Burlington, Vermont 05405.
3 Present address: Department of Biochemistry, School of Dentistry, the University of Tokushima, Tokushima, Tokushima 770-8504, Japan.
Abbreviations: BrdU, Bromo-deoxyuridine; C/EBPß, CCAAT/enhancer binding protein-ß; DAPI, 4',6 diamidino-2-phenylindole; E, estrogen; ER, E receptor; IGFBP, IGF binding protein; IRS, insulin receptor substrate-1; ISH, in situ hybridization; Jak, Janus kinase; K6, K10, K14, or K18, keratin 6, 10, 14, or 18; MECs, mammary epithelial cells; NKCC1, sodium potassium chloride; P, progesterone; PR, progesterone receptor; Prl, prolactin; PrlR, Prl receptor; RPA, ribonuclease protection assay; SSH, suppression subtractive hybridization; SPRR2A, small proline-rich protein; Stat5, signal transducer and activator of transcription 5; TBS, Tris-buffered saline.
Received for publication July 9, 2002. Accepted for publication September 16, 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
, is essential for ductal morphogenesis, lobuloalveolar proliferation, and functional differentiation in the mouse mammary gland. Genes Dev 12:1917–1928 knockout mice. Endocrinology 141:2982–2994 and progesterone receptor in relation to proliferating cells in the mammary gland. Breast Cancer Res Treat 53:217–227[CrossRef][Medline]
gene expression is mediated by Janus kinase 2 (Jak2) while signal transducer and activator of transcription 5b (Stat5b) phosphorylation involves Jak2 and a second tyrosine kinase. Mol Endocrinol 15:1941–1952NURSA Molecule Pages Link:
This article has been cited by other articles:
 |
|
 |
|
  L. M Arendt, L. C Evans, D. E Rugowski, M. J. Garcia-Barchino, H. Rui, and L. A Schuler Ovarian hormones are not required for PRL-induced mammary tumorigenesis, but estrogen enhances neoplastic processes J. Endocrinol., October 1, 2009; 203(1): 99 - 110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Fernandez-Valdivia, A. Mukherjee, C. J. Creighton, A. C. Buser, F. J. DeMayo, D. P. Edwards, and J. P. Lydon Transcriptional Response of the Murine Mammary Gland to Acute Progesterone Exposure Endocrinology, December 1, 2008; 149(12): 6236 - 6250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. P. Pai and N. D. Horseman Biphasic Regulation of Mammary Epithelial Resistance by Serotonin through Activation of Multiple Pathways J. Biol. Chem., November 7, 2008; 283(45): 30901 - 30910. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Muraoka-Cook, M. Sandahl, D. Hunter, L. Miraglia, and H. S. Earp III Prolactin and ErbB4/HER4 Signaling Interact via Janus Kinase 2 to Induce Mammary Epithelial Cell Gene Expression Differentiation Mol. Endocrinol., October 1, 2008; 22(10): 2307 - 2321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. L. LaMarca and J. M. Rosen Hormones and Mammary Cell Fate--What Will I Become When I Grow Up? Endocrinology, September 1, 2008; 149(9): 4317 - 4321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nagy, H. Wei, T.-L. Shen, X. Peng, C.-C. Liang, B. Gan, and J.-L. Guan Mammary Epithelial-specific Deletion of the Focal Adhesion Kinase Gene Leads to Severe Lobulo-Alveolar Hypoplasia and Secretory Immaturity of the Murine Mammary Gland J. Biol. Chem., October 26, 2007; 282(43): 31766 - 31776. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hiremath, J. P. Lydon, and P. Cowin The pattern of {beta}-catenin responsiveness within the mammary gland is regulated by progesterone receptor Development, October 15, 2007; 134(20): 3703 - 3712. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Taneja, S. Ha, N. K. Swenson, H. Y. Huang, P. Lee, J. Melamed, E. Shapiro, M. J. Garabedian, and S. K. Logan Cell-specific Regulation of Androgen Receptor Phosphorylation in Vivo J. Biol. Chem., December 9, 2005; 280(49): 40916 - 40924. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Grimm, A. Contreras, M.-H. Barcellos-Hoff, and J. M. Rosen Cell Cycle Defects Contribute to a Block in Hormone-induced Mammary Gland Proliferation in CCAAT/Enhancer-binding Protein (C/EBP{beta})-null Mice J. Biol. Chem., October 28, 2005; 280(43): 36301 - 36309. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Yang, B. Zhao, V. E. Baracos, and J. J. Kennelly Effects of Bovine Somatotropin on {beta}-Casein mRNA Levels in Mammary Tissue of Lactating Cows J Dairy Sci, August 1, 2005; 88(8): 2806 - 2812. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. B.R. Ewan, H. A. Oketch-Rabah, S. A. Ravani, G. Shyamala, H. L. Moses, and M. H. Barcellos-Hoff Proliferation of Estrogen Receptor-{alpha}-Positive Mammary Epithelial Cells Is Restrained by Transforming Growth Factor-{beta}1 in Adult Mice Am. J. Pathol., August 1, 2005; 167(2): 409 - 417. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. H. Gutzman, S. E. Nikolai, D. E. Rugowski, J. J. Watters, and L. A. Schuler Prolactin and Estrogen Enhance the Activity of Activating Protein 1 in Breast Cancer Cells: Role of Extracellularly Regulated Kinase 1/2-Mediated Signals to c-fos Mol. Endocrinol., July 1, 2005; 19(7): 1765 - 1778. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Behbod and J. M. Rosen Will cancer stem cells provide new therapeutic targets? Carcinogenesis, April 1, 2005; 26(4): 703 - 711. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. Tilli, R. Reiter, A. S. Oh, R. T. Henke, K. McDonnell, G. I. Gallicano, P. A. Furth, and A. T. Riegel Overexpression of an N-Terminally Truncated Isoform of the Nuclear Receptor Coactivator Amplified in Breast Cancer 1 Leads to Altered Proliferation of Mammary Epithelial Cells in Transgenic Mice Mol. Endocrinol., March 1, 2005; 19(3): 644 - 656. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Jackson-Fisher, G. Bellinger, R. Ramabhadran, J. K. Morris, K.-F. Lee, and D. F. Stern ErbB2 is required for ductal morphogenesis of the mammary gland PNAS, December 7, 2004; 101(49): 17138 - 17143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Y. Shi, J. P. Lydon, and M. Zhang Hormonal Defect in Maspin Heterozygous Mice Reveals a Role of Progesterone in Pubertal Ductal Development Mol. Endocrinol., September 1, 2004; 18(9): 2196 - 2207. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Mulac-Jericevic and O. M Conneely Reproductive tissue selective actions of progesterone receptors Reproduction, August 1, 2004; 128(2): 139 - 146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Allar and T. L. Wood Expression of the Insulin-Like Growth Factor Binding Proteins during Postnatal Development of the Murine Mammary Gland Endocrinology, May 1, 2004; 145(5): 2467 - 2477. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Gourdou, J. Paly, C. Hue-Beauvais, L. Pessemesse, J. Clark, and J. Djiane Expression by Transgenesis of a Constitutively Active Mutant Form of the Prolactin Receptor Induces Premature Abnormal Development of the Mouse Mammary Gland and Lactation Failure Biol Reprod, March 1, 2004; 70(3): 718 - 728. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Functional Characterization of Mammary Stem Cells: JEFFREY M. ROSEN,1 BRYAN E. WELM,3 STACEY B. TEPERA,1 SANDRA L. GRIMM,1 TERESA VENEZIA,2 MARGARET A. GOODELL,2 TIMOTHY A. GRAUBERT,4 ZENA WERB,3 YI LI,5, 6 AND HAROLD E. VARMUS,6 1 Department of Molecular and Cellular Biology; 2 Center for Cell and Gene Therapy and Department of Pediatrics; 5 Baylor Breast Center, Baylor College of Medicine, Houston, Texas 77030; 3Department of Anatomy, University of California, San Francisco, California 94143; 4Division of Bone Marrow Transplantation and Stem Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110; and 6 Memorial Sloan Kettering Cancer Center, New York, NY 10021 Toxicol Pathol, January 1, 2004; 32(1): 148 - 149. [PDF]
|
|
|
|
|
|
Y. Li, B. Welm, K. Podsypanina, S. Huang, M. Chamorro, X. Zhang, T. Rowlands, M. Egeblad, P. Cowin, Z. Werb, et al. Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells PNAS, December 23, 2003; 100(26): 15853 - 15858. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Lee, P. Zhang, M. Ivanova, S. Bonnette, S. Oesterreich, J. M. Rosen, S. Grimm, R. C. Hovey, B. K. Vonderhaar, C. R. Kahn, et al. Developmental and Hormonal Signals Dramatically Alter the Localization and Abundance of Insulin Receptor Substrate Proteins in the Mammary Gland Endocrinology, June 1, 2003; 144(6): 2683 - 2694. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
