Nuclear Factor 1-C2 Is Regulated by Prolactin and Shows a Distinct Expression Pattern in the Mouse Mammary Epithelial Cells during Development

Eva M. Johansson, Marie Kannius-Janson, Amel Gritli-Linde, Gunnar Bjursell and Jeanette Nilsson

Department of Cell and Molecular Biology/Molecular Biology (E.M.J., M.K.-J., G.B., J.N.) and Department of Oral Biochemistry (A.G.-L.), Göteborg University, S-405 30 Göteborg, Sweden

Address all correspondence and requests for reprints to: Jeanette Nilsson, Department of Cell and Molecular Biology/Molecular Biology, Götoborg University, Box 462, S-405 30 Göteborg, Sweden. E-mail: Jeanette.Nilsson{at}molbio.gu.se.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have previously demonstrated that the transcription factor nuclear factor (NF)1-C2 plays an important role in the mammary gland for the activation of the tumor suppressor gene p53. It also activates the milk genes carboxyl ester lipase and whey acidic protein, implying that NF1-C2 participates both in the establishment of a functional gland and in protection of the gland against tumorigenesis during proliferation. In this study, we have developed a new sensitive NF1-C2-specific antiserum for immunohistochemical analyses of the NF1-C2 distribution during mammary gland development. We show that the NF1-C2 protein is present in the epithelial compartment at the virgin stage and throughout mammary gland development. However, in the lactation stage the NF1-C2 protein levels strongly decreased, and many epithelial nuclei stained negative. In situ hybridization shows that NF1-C2 transcripts are expressed in the whole epithelium at pregnancy as well as the lactation stage, indicating that the reduction in protein levels is posttranscriptionally regulated. At involution, the NF1-C2 proteins are back to high levels. Based on studies using NMuMG cells and mammary tissue from heterozygous prolactin receptor knockout mice, we also demonstrate that prolactin has a direct effect in the maintenance of the NF1-C2 protein levels in the mammary epithelial nuclei at the virgin stage and during pregnancy. Hence, we have identified another transcription factor in the mammary gland, besides signal transducer and activator of transcription 5, through which prolactin may control mammary gland development. Furthermore, our data suggest a link between prolactin and p53 in the mammary gland, through NF1-C2.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MAMMARY GLAND MORPHOGENESIS is a complex process that occurs at specific stages during postnatal development. With the onset of puberty, mammary ducts grow and branch into the mammary fat pad to establish a ductal tree that eventually gives rise to lobuloalveolar structures during pregnancy (1). After weaning the gland is remodeled back to a virgin-like stage and is ready for a new round of development. Mammogenesis is governed to a great extent by hormonal stimuli, such as estrogen, progesterone, and prolactin (2). Recent research has to a large extent elucidated the mechanisms behind hormonal regulation of the gland and the signaling pathways involved. Much less is known about the specific transcription factors that are downstream targets of the hormones and that affect development by regulating gene transcription.

The nuclear factor 1 (NF1) family of transcription factors functions both in viral DNA replication and in the regulation of gene expression. Ubiquitously expressed genes, as well as hormonally and developmentally regulated genes, have been demonstrated to be regulated by NF1 proteins. The NF1 family is composed of four members in vertebrates (NF1-A, NF1-B, NF1-C and NF1-X), and the four NFI genes are expressed in unique, but overlapping, patterns during mouse embryogenesis and in the adult. Transcripts of each NFI gene are differentially spliced, yielding as many as 12 distinct proteins from a single gene. Products of the four NFI genes differ in their abilities to either activate or repress transcription, likely through fundamentally different mechanisms (3) (for review, see Ref.4).

We have previously identified the specific NF1 isoform NF1-C2 as an important transcriptional regulator of the tumor suppressor gene p53 in the mammary gland (5). Furthermore, we have shown that NF1-C2 regulates the milk genes carboxyl ester lipase (CEL) and whey acidic protein (WAP) (6). This indicates that NF1-C2 might participate both in the establishment of a functional gland and in protection of the gland against tumorigenesis during proliferation.

Our previous studies regarding NF1-C2 in the mammary gland relied on whole gland homogenates that limited the obtained information, not only because of the multiple cell types in the gland, but also because of the small epithelial compartment relative to stroma. We now examined the cellular localization of NF1-C2 in vivo using immunohistochemistry with a new highly sensitive antiserum specific for NF1-C2. We further investigated possible hormones important for the regulation of NF1-C2 in the mammary gland.

Our analyses demonstrate that the NF1-C2 proteins are present in all the nuclei of the epithelial compartment, both at the virgin stage and during the course of pregnancy. The expression pattern of NF1-C2 thus resembles that of tyrosine phosphorylated pY694/699 signal transducer and activator of transcription 5 (Stat5)a/b, commonly regarded as activated Stat5, in the mouse mammary gland during pregnancy (7). We further show that prolactin plays a direct role in maintaining these nuclear levels of NF1-C2. Hence, we have identified another transcription factor, besides Stat5, through which prolactin could regulate mammary gland development. At lactation, the NF1-C2 protein levels were strongly decreased as a result of posttranscriptional regulation, and several epithelial nuclei stained negative at this stage. Hence, the regulation of NF1-C2 diverges from that of Stat5 after pregnancy, suggesting specific roles for these two transcription factors over the gestation cycle.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Production and Evaluation of an NF1-C2- Specific Antiserum
We have previously shown that the NF1-C2 protein is present in the mouse mammary gland (6). These earlier studies were made on total gland nuclear extract preparations. Because the protein was shown also to be present in the mouse mammary epithelial cell line HC11 and was shown to have an important role in milk gene regulation (6), we suggested that the NF1-C2 in the total gland originated from the mammary epithelium. The precise localization of the NF1-C2 protein would demand immunohistochemistry with a specific antibody. However, the NF1-C-specific antibody (8199) (6) proved not to be useful for analysis by immunohistochemistry, and we hence set out to make a new, NF1-C2-specific antiserum. The specificity is made possible because of the unique characteristics of the NF1-C2 transcript in comparison with other NF1-C isoforms. NF1-C2 lacks exon 9, which causes a frame shift that terminates translation in exon 10 (8). The changed reading frame in the first part of exon 10 encodes a unique 16-amino acid peptide (Fig. 1AGo), which was used for immunization of rabbit. As can be seen in Fig. 1AGo, this peptide is identical in human and mouse, apart from one single amino acid, and hence the produced antiserum should be useful for detection of both human and mouse NF1-C2. The antiserum is referred to as the pNF1-C2 antiserum.



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Fig. 1. Validation of the Specificity of the pNF1-C2 Antiserum

A, Comparison of the amino acid sequences of the C-terminal ends of human and mouse NF1-C splice variants. Note that only a small part of exon 8 is shown. Bars represent missing amino acids. Star represents C-terminal end of amino acid sequence. B, Testing of the specificity of the pNF1-C2 antiserum for NF1-C proteins. MDA-MB 436 cells were transiently transfected with either of the pCHNF1A1.1, pCHNF1B2, pCHNF1C2 or pCHNF1X2 expression plasmids expressing HA-tagged mouse NF1-A1, NF1-B2, NF1-C2, and NF1-X2. Nuclear proteins were prepared and subjected to Western blot analysis. The filter was first incubated with a HA-antibody to show that the NF1 proteins had been expressed at equal levels. It was then stripped and reincubated with the pNF1-C2 antiserum to show that it is specific for NF1-C proteins and does not cross-react with other NF1 family members. C, Testing of specificity of the pNF1-C2 antiserum for NF1-C2 proteins. Human NF1-C2 and NF1-C5 were overexpressed in MDA-MB 436 cells. Nuclear proteins were prepared and subjected to Western blot analysis. The filter was first incubated with a NF1-C-specific antibody (8199) to show that both NF1-C proteins had been expressed at equal levels. It was then stripped and reincubated with the pNF1-C2 antiserum to show that it is specific for the NF1-C2 isoform.

 
To test for the specificity of the antiserum, we overexpressed hemagglutinin (HA)-tagged murine NF1-A1, NF1-B2, NF1-C2, and NF1-X2 in MDA-MB 436 breast epithelial cells. These cells serve as a good system for this purpose because we detected very low levels of endogenous NF1-C2 protein in these cells. A Western blot on nuclear extract preparations with a HA-antibody verified that the four NF1 family members had been expressed at similar levels (Fig. 1BGo, left panel). The filter was stripped and reincubated with the pNF1-C2-specific antiserum. This analysis revealed that the antiserum is specific to NF1-C proteins (Fig. 1BGo, right panel).

To investigate whether the antiserum is specific to the isoform NF1-C2, we overexpressed human NF1-C2 and NF1-C5 in MDA-MB 436 cells. Western blot on nuclear extract preparations was first made with the previously described NF1-C-specific antibody (8199) (6) to verify that both NF1-C isoforms were expressed at equal levels (Fig. 1CGo, left panel). The filter was stripped and then reincubated with the pNF1-C2 antiserum, which demonstrated that the antiserum is NF1-C2 specific as expected (Fig. 1CGo, right panel). Our data also show that the antiserum recognizes both human (Fig. 1CGo) and mouse (Fig. 1BGo) NF1-C2 proteins. We also tested the specificity of the antiserum on native proteins by immunostaining of MDA-MB 436 cells overexpressing NF1-C2 and NF1-C5. Immunostaining with the NF1-C-specific antibody (8199) detected proteins in cells overexpressing both NF1-C2 and NF1-C5, whereas the new pNF1-C2 antiserum only detected proteins in cells overexpressing NF1-C2 (data not shown).

NF1-C2 Proteins Are Present in the Mammary Epithelium at the Virgin Stage and All Gestation Stages, but Are Reduced at Lactation
For localization of NF1-C2 proteins in the mouse mammary gland, we performed immunohistochemistry with the pNF1-C2 antiserum at different stages of development: mature virgin mice (V), pregnant mice at different days (P5, P10, P13, P16), lactating mice at d 1 and 6 (L1, L6), and mice 2 d after weaning (W2). NF1-C2 proteins were detected in the mammary epithelial nuclei at all stages of pregnancy (Fig. 2Go). Both small alveoli and larger ducts showed marked nuclear immunostaining. The nuclear localization of NF1-C2 proteins was expected because earlier studies have shown that NF1 proteins are strongly localized to the nucleus (9). The negative controls represent similar staining on successive sections in which the primary antiserum was omitted (–ab in Fig. 2Go). The positive staining with the new pNF1-C2-specific antiserum also confirms the conclusions drawn earlier by us that the specific NF1-C isoform expressed in the mouse mammary gland is the isoform NF1-C2 (6). Earlier, this conclusion relied on RT-PCR that was set up to distinguish between the different NF1-C isoforms.



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Fig. 2. Localization of NF1-C2 Proteins in the Mouse Mammary Gland during the Gestation Cycle

Immunohistochemistry of NF1-C2 in mouse mammary gland during pregnancy cycle. Mammary glands from mature virgin mice (V); pregnant mice at d 5, 10, 13, and 16 (P5–16); mice at d 1 and 6 of lactation (L1, L6); and weaned mice on d 2 of involution (W2) were formalin-fixed, embedded in paraffin, and subjected to immunohistochemistry with the pNF1-C2 antiserum. The primary antiserum was omitted in the negative controls. The sections were counterstained with methyl blue. Marked positive nuclear staining is observed in the mammary epithelium at all stages of mouse mammary development including the virgin stage. Note the marked decrease in staining at the lactation stages and the scattered positive nuclei at this stage. Note also the intense staining of the involuting gland, indicating that the NF1-C2 proteins are back to high levels. Scale bar, 20 µm.

 
Strikingly, the NF1-C2 protein was also detected at the virgin stage, although possibly at lower levels. We have earlier failed to detect NF1-C2 expression at the virgin stage and early pregnancy stages by EMSA and Western blot using the 8199 NF1-C antibody (6). We presume that the reason for this is the minimal epithelial compartment in nonpregnant mice relative the stromal tissue that make the NF1-C2 protein too diluted for detection with these methods. With the new sensitive antiserum and the immunohistochemical technique, we can now conclude that there is at least basal expression of NF1-C2 at all stages during the establishment of the mammary epithelium. Whether there is a further increased expression of NF1-C2 at midpregnancy needs to be explored by other methods.

At lactation we observed a marked reduction in immunostaining and, in contrast to the pregnancy stages, several scattered epithelial nuclei stained negative at this stage (Fig. 2Go). The background staining with diaminobenzidine at this stage was more pronounced, which to an extent masked the blue counterstaining, and to clearly visualize negatively stained nuclei we performed 4',6-diamidino-2-phenylindole (DAPI) nuclear staining together with diaminobenzidine staining on sections at lactation. This analysis clearly demonstrates that not all epithelial nuclei stained positive for NF1-C2 in secreting alveoli at lactation (Fig. 3AGo). Immunohistochemical staining of mammary glands at d 18 of pregnancy indicates that the reduction in NF1-C2 proteins begins already at midpregnancy as the epithelial cells mature. In these glands, where one can observe both nonsecretory and secretory alveoli, the more mature secretory alveoli stained weaker (Fig. 3BGo).



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Fig. 3. Immunohistochemical Investigation of the Presence of NF1-C2 Proteins at Lactation and Late Pregnancy

A, Mammary glands from d 1 of lactation (L1) were formalin-fixed, embedded in paraffin, and subjected to immunohistochemistry with the pNF1-C2 antiserum. The sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI) to clearly visualize both stained and unstained nuclei. Note the scattered pattern of positive NF1-C2 staining in only a subset of mammary epithelial nuclei. Scale bar, 20 µm. B, Mammary glands from d 18 of pregnancy (P18) were formalin-fixed, embedded in paraffin, and subjected to immunohistochemistry with the pNF1-C2 antiserum. The sections were counterstained with methyl green. Note the marked staining of the epithelial cells of ducts and immature alveoli (black arrows) and the weaker staining of the more mature lipid-filled alveolar epithelial cells (white arrow).

 
Although our immunohistochemical data indicate a decrease at lactation, one should be careful drawing quantitative conclusions from immunohistochemistry. Our previous Western blot analysis with whole gland nuclear extracts indicates only a slight reduction in NF1-C2 levels at lactation, which is probably because of a compensatory effect due to the increased amount of epithelial cells at lactation (5). To get a true understanding of how the NF1-C2 protein levels change in the epithelial compartment at lactation, we investigated, in the present study, the relative levels of NF1-C2 proteins after compensating for the increase of epithelial cells relative stroma during mammogenesis (Fig. 4Go). Nuclear and cytoplasmic proteins were prepared from mouse mammary glands at different stages of development. The relative amounts of epithelial material were estimated by investigating the relative amount of E-cadherin in the cytoplasmic fraction from each preparation by Western blot (data not shown). The loading of nuclear proteins on another Western blot was then adjusted with the attempt to keep the epithelial material constant, and the levels of NF1-C2 proteins were investigated with the pNF1-C2 antiserum. As can be seen in Fig. 4Go, the amount of NF1-C2 proteins is decreased between d 16 of pregnancy and d 1 of lactation, and further decreased at d 6 of lactation. Taking into consideration that the results on Western blot with the E-cadherin antibody show that the extract from d 1 of lactation contains more epithelial material than the other two stages, the reduction between pregnancy and lactation d 1 is even more pronounced. The result clearly demonstrates that there is a true reduction in the amount of NF1-C2 proteins in the epithelial cells at lactation in comparison with pregnancy (Fig. 4Go).



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Fig. 4. Investigation of NF1-C2 Protein Levels during Different Stages of Mammary Gland Development

Nuclear (left) or cytoplasmic (right) extracts from mammary glands at d 16 of pregnancy (P16) and d 1 and 6 of lactation (L1, L6) were subjected to Western blot analysis. The amount of extract loaded in each well was adjusted to the estimated amount of epithelial material of each extract. The filters were incubated with either the pNF1-C2 antiserum (left) or an antibody recognizing E-cadherin (right).

 
As can be seen in Fig. 2Go, 2Go d after weaning, at the involution stage, the NF1-C2 protein is again present at high levels in all epithelial cells. This NF1-C2 protein could be the 74-kDa glycosylated NF1-C2 protein reported earlier to appear at involution (10).

The Reduction of NF1-C2 Proteins at Lactation Is Regulated Posttranscriptionally
In earlier studies with whole gland preparations, the NF1-C2 transcript levels were increased from midpregnancy to lactation, whereas the protein levels seemed constant or rather decreased (5, 6). Taking into consideration the increase of epithelial cells between these stages, the transcript levels seem constant, whereas the amount of proteins dramatically decreases in agreement with our present immunohistochemical data (Fig. 2Go). This suggests that the reduction of NF1-C2 proteins at lactation is regulated at a posttranscriptional level. To determine more carefully the epithelial expression of NF1-C2 transcripts in the mammary gland in the pregnant and lactating gland, in situ hybridization was performed on mouse mammary glands at d 16 of pregnancy (P16), d 6 of lactation (L6), and 2 d after weaning (W2). We used a probe specific for NF1-C transcripts, the same as the probe previously used for Northern blot (6). This revealed expression of the NF1-C gene in a continuous pattern in the mammary epithelial cells in glands of pregnant mice (Fig. 5AbGo) as well as in lactating (Fig. 5AcGo) and involuting glands (Fig. 5AdGo). Sense probe was used on P16 glands as a negative control (Fig. 5AaGo). As a positive control for the NF1-C probe, in situ hybridization was also performed on mouse dental tissue because this tissue has previously been shown to express large amounts of NF1-C transcripts (11). The dental tissue was taken from mouse d 1 postpartum. In agreement with the data from Steele-Perkins et al. (11), we detected NF1-C transcripts in the mesenchymal dental papilla (Fig. 5BGo).



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Fig. 5. Expression of NF1-C2 mRNA in the Mouse Mammary Gland and Molar

A, In situ hybridization with a probe specific to NF1-C transcripts. Mammary glands from mice at d 16 of pregnancy (a and b), d 6 of lactation (c), and 2 d after weaning (d) were formalin-fixed, embedded in paraffin, and subjected to in situ hybridization with the NF1-C-specific probe. Dark-field images represent hybridization with sense probe (a) and antisense probe (b–d). Note the marked positive and similar mRNA staining pattern in the whole mammary epithelium at all stages of mouse mammary development. No staining is seen with the control sense probe (a). B, In situ hybridization with the NF1-C-specific probe on formalin-fixed and paraffin-embedded normal tooth tissue taken from mice d 1 postpartum. The dark-field image represents the second molar at the early bell stage showing expression of NF1-C mRNA in the dental papilla mesenchymal cells (arrow).

 
Prolactin Is Involved in Maintaining the Basal Levels of NF1-C2 Proteins in the Mammary Epithelial Nuclei by Posttranscriptional Control
It has been shown recently that basal levels of activated Stat5 are found in the mouse mammary epithelial nuclei at early stages of development, including the virgin stage (7). The principal factor maintaining these levels was identified as prolactin, present in the circulation at low levels. We hence proceeded to investigate whether prolactin also could play a role in maintaining the nuclear NF1-C2 levels in these stages. As a model for mammary epithelial cells in the nonpregnancy stage, we used mouse mammary NMuMG cells. We found low endogenous levels of nuclear NF1-C2 proteins in these cells when cultured with insulin only, suggesting that these cells lack some of the signals provided by the tissue context. However, immunofluorescent staining of the NMuMG cells with the pNF1-C2 antiserum showed that the nuclear NF1-C2 level is dramatically increased upon prolactin stimulation (Fig. 6AGo). This suggests that exogenous prolactin might be responsible for maintaining the nuclear levels of NF1-C2 protein in the virgin gland. The prolactin effect was observed as quickly as after 15 min, which indicates a direct effect of prolactin on NF1-C2. As a positive control for the prolactin effect, we also immunostained the cells for tyrosine phosphorylated pY694/699 Stat5a/b (phospho-Stat5a/b), because it is well established that Stat5 molecules are tyrosine phosphorylated and translocate to the nucleus within 15 min upon prolactin stimulation (Fig. 6AGo). The similar appearance by staining with the pNF1-C2 antiserum and the Stat5 antibody suggested that prolactin might regulate NF1-C2 proteins in a similar way as it regulates Stat5, by causing nuclear translocation of cytoplasmic proteins. To investigate this, we performed a Western blot analysis on nuclear and cytoplasmic extract preparations of the NMuMG cells. In accordance with the data obtained by immunofluorescent staining, treatment with prolactin resulted in a dramatic increase of NF1-C2 proteins in the NMuMG nuclei (Fig. 6BGo). Interestingly, we detected a slightly smaller NF1-C2 protein species in the cytoplasmic fraction, suggesting that the nuclear NF1-C2 protein might be modified. However, the relative amount of this species was unaffected and did not decrease upon prolactin treatment. Hence, prolactin does not appear to cause nuclear translocation of this smaller species, suggesting that prolactin regulates NF1-C2 in a mechanism distinct from its regulation of Stat5.



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Fig. 6. Effect of Prolactin Signaling on NF1-C2 Proteins in Mammary Epithelial Cells

A, NMuMG cells were treated or not treated with 5 µg/ml prolactin for 15 min and then subjected to immunofluorescence assay with either an antibody recognizing activated tyrosine phosphorylated Stat5 (phospho-Stat5a/b) or the polyclonal NF1-C2-specific antiserum. B, NMuMG cells were treated or not treated with 5 µg/ml prolactin for 15 min. Nuclear (n) or cytoplasmic (c) proteins were prepared and subjected to Western blot analysis with the polyclonal NF1-C2-specific antiserum. C, Immunohistochemical investigation of NF1-C2 expression in mammary glands from mice carrying one defective allele of the PrlR. Mammary gland paraffin sections from the PrlR heterozygotes as well as from wild-type mice at midpregnancy were kindly provided by Dr. Christopher J. Ormandy. Note that the different pattern of staining with the wild-type glands exhibited a more concentrated nuclear staining than the heterozygotes.

 
Ormandy et al. (12) showed that a certain threshold level of the prolactin receptor (PrlR) is necessary in the mouse mammary gland for the prolactin signaling pathway to work properly, because the heterozygote mice with only one functioning PrlR gene, PrlR+/–, showed dramatic defects in functional development of the mammary gland. To investigate the role of prolactin on NF1-C2 proteins in vivo, we performed immunohistochemistry on mammary glands from heterozygous PrlR+/– mice at midpregnancy. Our analysis demonstrated a dramatic difference in the staining for NF1-C2 in the heterozygotes in comparison with the wild-type controls (Fig. 6CGo). Although the staining in the wild-type mice was more concentrated into the nuclei as previously observed (Fig. 2Go), the heterozygotes displayed a staining resembling the untreated NMuMG cells. For a better visualization of the staining differences, staining was performed both with (Fig. 6CGo, top) and without (Fig. 6CGo, bottom) nuclear counterstaining. Together, all these results demonstrate that prolactin directly influences the presence of NF1-C2 proteins in mammary epithelial nuclei. The steady-state levels of NF1-C2 mRNA, assayed by RT-PCR, did not change upon either 15 min or 3 h of prolactin stimulation of NMuMG cells (data not shown). Hence, as expected from the quick and direct effect of prolactin, the increase in NF1-C2 protein levels caused by prolactin is regulated at the posttranscriptional level. The detailed mechanism behind this regulation remains to be explored.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have previously identified the transcription factor NF1-C2 as an important regulator of milk genes as well as the p53 tumor suppressor gene in the mouse mammary gland (5, 6). Our present study demonstrates that NF1-C2 is present continuously in mammary epithelial nuclei of both nonpregnant and pregnant mice. This was somehow surprising because our earlier data have suggested that NF1-C2 proteins are induced at midpregnancy in concordance with the induction of milk genes (6). However, in the previous study we used Western blot and EMSA analysis, and the lack of detection of NF1-C2 in early development stages is probably due to the small epithelial compartment relative to stroma in these stages. In this paper, we used immunohistochemistry with a new and highly sensitive NF1-C2 antiserum for detection of NF1-C2 proteins. It is tempting to draw parallels between our findings concerning NF1-C2 expression and the recent findings by Nevalainen et al. (7) concerning Stat5. They showed that tyrosine phosphorylated pY694/699 Stat5a/b, commonly regarded as activated Stat5, was expressed continuously both at virgin stages and during the gestation cycle, which was unexpected in light of the earlier established view that Stat5 is inducibly activated by lactogenic hormones during pregnancy and lactation (7). The new information was based on sensitive immunohistochemistry for pY694/699 Stat5a/b that allowed detection down to the single cell level.

Nevalainen et al. (7) showed that prolactin, at low circulating levels, is the main hormone responsible for maintaining the basal tyrosine phosphorylation of Stat5 in the virgin mouse mammary gland. Similarly, we demonstrate in this work that prolactin also plays an important role for the basal level of NF1-C2 proteins in mammary epithelial nuclei. When treating cultured mammary epithelial NMuMG cells with prolactin for 15 min, the level of NF1-C2 proteins in the nuclei dramatically increased. The short time of treatment suggests a direct effect of prolactin on NF1-C2. It is well established that Stat5 is activated by prolactin as a result of a cascade of tyrosine phosphorylations via Jak2 (Janus protein tyrosine kinase) upon prolactin binding to its receptor. Phosphorylation of Stat5 by Jak2 causes Stat5 to dimerize and translocate to the nucleus to exert gene regulation (for review, see Ref.13). Interestingly, we detected a slightly smaller NF1-C2 protein species in the cytoplasmic fraction, suggesting that the nuclear NF1-C2 protein might be modified. However, the relative amount of the cytoplasmic species was unaffected and did not decrease upon prolactin treatment, whereas the amount of the nuclear NF1-C2 protein was increased. Hence prolactin does not appear to cause nuclear translocation of this smaller species, suggesting that prolactin regulates NF1-C2 in a mechanism distinct from its regulation of Stat5. PrlR signaling can be mediated through several different pathways besides that of Jak-Stat, such as the MAPK and phosphatidylinositol 3-kinase pathways (14). The mechanism by which prolactin increases the amount of nuclear NF1-C2 remains to be explored. Because there seems to be a net increase in NF1-C2 protein in the whole cell, and not simply a nuclear translocation of already present proteins, prolactin might cause a stabilization of the nuclear NF1-C2 proteins.

In an earlier report, we have shown that NF1-C2 is an important transcriptional regulator of the p53 tumor suppressor gene in the mouse mammary gland (5). The p53 tumor suppressor plays an important role in regulating the balance between growth, differentiation, and cell death in the mammary gland. It is important for the prevention of tumor development as evidenced by the high frequency of mutations and altered expression of the p53 gene in mammary tumors (15). The demonstration in the present study that NF1-C2 proteins are positively regulated by prolactin in mammary epithelial cells implies a link between prolactin and p53, mediated by NF1-C2. This is interesting in the light of prolactin as a proliferative stimulus (16). Apparently, prolactin signals through both proliferative and proliferation-inhibiting pathways, which suggestively provides an appropriate fine balance crucial for the proper regulation of mammary gland development. In the absence of p53, hormonal stimulation is a very potent tumorigenic stimulus (17).

The expression of p53 transcripts follows the expression of NF1-C2 proteins during the whole course of mammary gland development with a peak at midpregnancy, a reduction at lactation, and an increase during involution (5). The tight correlation between levels of NF1-C2 proteins and p53 transcripts during mammary gland development suggests that NF1-C2 might have a direct and independent role in the p53 gene activation. NF1-C2 is also a regulator of the expression of the WAP and CEL milk genes (6). Because expression of these milk genes is dramatically induced at midpregnancy (18, 19, 20), it is obvious that the mere presence of NF1-C2 in the epithelial nuclei is not enough to induce high levels of milk gene expression. Similarly, the presence of pY694/699 Stat5a/b, which is also crucial for WAP gene expression, is not enough to induce the WAP gene on its own (7, 21). These findings suggest a different mechanism for NF1-C2 in activation of the p53 gene from that in activation of milk genes. The idea of different activation mechanisms is further supported by the fact that we observed an effect upon mutation of the NF1 binding site in the CEL promoter only after stable integration of the construct into the genome of mammary epithelial cells (6), whereas an effect upon mutating the site in the p53 promoter construct was observed in transient transfections (5). Milk gene induction needs an additional signal at midpregnancy, presumably induced by higher levels of pregnancy hormones. This signal could potentially be provided by another unidentified factor or by a yet unknown posttranslational modification of the transcription factors. It is also possible that there is a further induction of both pY694/699 Stat5a/b and NF1-C2 levels during pregnancy, which at least in part could explain the induction of milk genes at these stages.

An interesting finding in this work was the markedly decreased levels of NF1-C2 proteins in terminally differentiated epithelial cells at lactation. Hence, the expression pattern no longer followed the one for activated Stat5 that displays sustained and perhaps higher levels at lactation (7). The reason for the reduced NF1-C2 protein levels is presently unknown. One can speculate that the factor has an important role in the establishment of the functional mammary gland and therefore is no longer needed in the terminally differentiated secretory cells. It is clear from earlier studies that NF1-C2 has an important role in milk gene activation at pregnancy (6, 21). However, milk genes are most highly expressed at lactation when the NF1-C2 levels are reduced. We hence propose that NF1-C2 is important for the initiation and not the maintenance of milk gene expression. Unless it can bind during pregnancy, WAP and CEL gene expression is never initiated. Note that the WAP promoter activity is totally abrogated in transgenic mice when the NF1 binding site is mutated (21). However, after the initiation of these genes, the factor might not be needed. How this is accomplished is not known, but one mechanism could be to bring together other transcriptional regulators into a complex important for gene regulation. It is well established that milk gene regulation is conducted through clusters of response elements (CoREs) in milk gene promoters binding several transcription factors. Exogenous NF1 proteins have been shown to be able to synergize the cooperative effect between Stat5 and glucocorticoid receptor in WAP gene regulation (22). NF1 proteins have also been shown to interact with proteins involved in chromatin remodeling such as histones H1 and H3 and the cofactor cAMP response element binding protein-binding protein (CBP)/p300 (23, 24, 25). Hence, another mechanism by which NF1-C2 could initiate milk gene expression could be to open up the chromatin to increase the accessibility for other factors. Furthermore, we cannot exclude the possibility that other NF1 family members are involved in the milk gene regulation at lactation. We have previously demonstrated that NF1-A1 proteins are expressed in the mammary gland at this stage (5). Whether NF1-B or NF1-X proteins are also there is not known today.

Obviously, the increased levels of prolactin at lactation cannot keep the high levels of NF1-C2 in the epithelial nuclei at this stage. It is clear that prolactin has different roles at different stages of mammary development. Under pregnancy, it provides a mitogenic stimulus, whereas at lactation it has more survival properties (16). Prolactin signaling crosstalks with, and is modulated by, different pathways at different stages (26). Furthermore, there exist truncated isoforms of the PrlR that are differentially expressed during mammogenesis, suggesting that they may initiate distinct signaling pathways (26). Prolactin has been shown to increase the expression of cyclin D1 and receptor activator of NF-{kappa}B ligand, which in turn activates NF-{kappa}B (27). All three factors are down-regulated at lactation, and hence it is obvious that there are signals at lactation that counteract the responses mediated by prolactin during pregnancy. Currently, the signals counteracting the prolactin-mediated maintenance of nuclear NF1-C2 protein levels at lactation are unknown.

Although NF1-C2 protein levels decreased at lactation, the mRNA production seemed unaffected. Hence, this reduction in protein levels but not in mRNA levels suggests that NF1-C2 is posttranscriptionally regulated, which also is in accordance with what we showed previously on whole gland preparations (5, 6). Also, the cell culture experiments with prolactin demonstrate that the NF1-C2 protein is regulated at the posttranscriptional level because prolactin treatment resulted in increased nuclear NF1-C2 protein levels but left the NF1-C2 mRNA levels unaffected. These findings suggest that NF1-C2 is a tightly regulated protein.

The regulation of NF1-C2 and Stat5 diverge not only in the lactation stage, but also at involution of the mammary gland. Although the levels of pY694/699 Stat5a/b dramatically decrease at involution (7), we here show that the levels of nuclear NF1-C2 proteins increase. This involution-specific NF1-C2 protein is suggestively the 74-kDa glycosylated NF1-C2 protein described earlier to appear at involution of the gland (10). It is striking that two transcription factors, NF1-C2 and Stat5, that seem to be regulated in a similar way during the virgin and pregnancy stages, diverge in their regulation at lactation and involution. This suggests specific roles for each of them for mammary gland function and remodeling. Both factors might play important roles for the establishment of the gland, but whereas Stat5 acts as a survival factor preventing apoptosis of terminally differentiated epithelial cells (13), NF1-C2 might have opposing effects at these mature stages. Hence, its expression is decreased during lactation. However, after weaning Stat5 is deactivated to allow apoptosis and remodeling of the gland, and NF1-C2 is induced, perhaps to induce expression of apoptosis-related genes. Interestingly, the striking pattern of nuclear NF1-C2 is shared by another transcription factor, NF-{kappa}B, in the mouse mammary gland. NF-{kappa}B is activated during pregnancy, almost completely suppressed during lactation, and reactivated early in involution (28, 29). However, the two factors have fundamentally distinct roles because NF1-C2 activates milk gene expression and NF-{kappa}B has been shown to be an inhibitor of Stat5 activated milk gene induction (30).

It has earlier been shown that disruption of the NF1-C gene in mice causes severe tooth root defects (11). Immunohistochemical staining of tooth tissue with our pNF1-C2 antiserum suggests that NF1-C2 could be the specific isoform important for tooth development (data not shown). An interesting observation is that we found that the expression of NF1-C2 proteins is lower in terminally differentiated odontoblast (data not shown), similar to what we observed in terminally differentiated mammary epithelial cells. It is striking that both teeth and mammary glands are derived from the ectoderm. All ectodermal organs originate from two adjacent tissue layers, the epithelium and the mesenchyme, and their development depends on interactions between these layers (31). Although the organogenesis of these organs is initiated during the embryonic period, morphogenesis does continue postnatally. Perhaps NF1-C2 could play an important role for postnatal organogenesis. It has indeed been shown that the NF1-C gene is up-regulated by thyroid hormones during metamorphic transition in X. laevis development, suggesting that this factor is important for adult organ development (32).

In this study, we have identified NF1-C2 as a prolactin-responsive factor during the development of the mouse mammary gland. Prolactin plays a profound role in establishment of the functional mammary gland (14). To fully understand the mechanism of prolactin action, it is important to elucidate the participation of the multiple signaling pathways and targets activated by prolactin in the mammary gland. The activation of NF1-C2 by prolactin provides new insight into the mechanism of prolactin activation in the mouse mammary gland. Analysis of the mammary gland development in NF1-C knockout mice would be of prime interest to elucidate the function of NF1-C2 during mammary gland development.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Cultures
The human mammary epithelial cell line MDA-MB 436 [American Tissue Culture Collection (ATCC), Mannasas, VA] was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 5 µg/ml insulin. The mouse mammary epithelial cell line NMuMG (ATCC) was grown in DMEM supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 5 µg/ml insulin. All cells were grown at 37 C in a 5% CO2/95% air atmosphere.

Cell Transfections and Plasmids
The MDA-MB 436 cells were transiently transfected using lipofectin in Optimem (Invitrogen, Carlsbad, CA) with 5 µg of either of the pCHNF1A1.1, pCHNF1B2, pCHNF1C2, or pCHNF1X2 expression plasmids (expressing HA-tagged mouse NF1-A1, NF1-B2, NF1-C2, and NF1-X2) [kindly provided by Dr. R. M. Gronostajski, Lerner Research Institute, Cleveland, OH (4)] per 6-cm culture dish. After 24 h, the medium was switched to the RPMI 1640 medium supplemented as described above. After about 40 h, the cells were harvested, and nuclear proteins were prepared.

An expression plasmid for human NF1-C5 was made by cutting out the approximately 1.5-kb hNF1-C5 coding sequence from the expressed sequence tag (EST) clone with GenBank accession no. BM546396 subcloned into pBluescript (Stratagene, La Jolla, CA), with NotI/blunt with Klenow/EcoRI and subcloning it into a pcDNA3.1(–) expression vector (Invitrogen) opened with BamHI/blunt with Klenow/EcoRI. An expression plasmid for human NF1-C2 was made in two steps. First, the BglII/NotI fragment of the previous EST clone in pBluescript was swapped with the BglII/NotI fragment of the EST clone with GenBank accession no. AI760652 to create a clone containing the full hNF1-C2 coding sequence. This sequence was then cut out from pBluescript with NotI/blunt with Klenow/EcoRI and cloned into the pcDNA3.1(–) expression vector opened with BamHI/blunt with Klenow/EcoRI. The MDA-MB 436 cells were stably transfected using lipofectin in Optimem (Invitrogen) with 5 µg of either of the pcDNA3.1(–)/hNF1-C2 or pcDNA3.1(–)hNF1-C5 expression plasmids. Stable transfectants were selected by adding 350 µg/ml Geneticin (G418) (Invitrogen) and harvested for nuclear protein preparations.

Nuclear Protein Preparation
Preparations of nuclear extracts from cells or from the fourth inguinal mammary glands from different stages of development dissected from F1: C57BL6 x CBA mice were carried out as described previously (33). Protein concentrations of the extracts were determined by the method of Bradford (34), and the extracts were stored in aliquots at –70 C before use.

Anti-NF1-C2 Antiserum
An antiserum against NF1-C2 (referred to as pNF1-C2 antiserum) was made by immunization of rabbit with the synthetic peptide (NH2-)CPALRPTRPLQTVPLWD(-COOH). The polyclonal immune serum was affinity purified through a peptide affinity column.

Western Blot Analysis
Nuclear extracts (30 µg) or cytoplasmic extracts (50 µg) from mammary glands, transfected MDA-MB 436 cells, or NMuMG cells (untreated or prolactin stimulated) were electrophoresed through a NuPAGE 4–12% Bis-Tris sodium dodecyl sulfate-polyacrylamide gel (Invitrogen), followed by electroblotting onto Hybond-P filter (Amersham Biosciences, Piscataway, NJ). In Fig. 4Go, the amounts of loaded extracts were adjusted to load equal amounts of epithelial material. To detect overexpressed HA-tagged NF1 proteins in the pCH plasmids, the filter was incubated with mouse anti-HA antibody (Roche, Basel, Switzerland). NF1-C proteins were detected with a 1:1000 dilution of a rabbit polyclonal NF1-C- specific antibody (8199) (6), which was a kind gift from Dr. N. Tanese (NYU Medical Center, New York, NY). NF1-C2 proteins were specifically detected with a 1:5000 dilution of the newly made rabbit pNF1-C2 antiserum. Tyrosine phosphorylated Stat5 proteins were detected with the AX1 antiphospho-Stat 5a/b mouse monoclonal antibody (1:1000 dilution) (Advantex BioReagents LLP, Conroe, TX). The primary antibodies were detected with peroxidase-conjugated antimouse or antirabbit IgG using the ECL Western blotting detection reagents and ECL-films (Amersham Biosciences).

Mice and Tissues
The fourth inguinal mammary glands from different stages of development were dissected from F1: C57BL6 x CBA mice. The tooth tissue was dissected from mice 1 or 3 d postpartum. The tissues were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, processed for paraffin embedding, and used for both immunohistochemistry and in situ hybridization. All animal experiments were conducted with accepted standards of animal care and were approved by the author’s institutional committee on animal care.

Immunohistochemical Analysis of Tissue Sections
Paraffin-embedded sections were cleared in xylene and rehydrated through an alcohol series. Unmasking of epitopes was performed by 10 min pressure-boiling of the sections in Tris-EDTA buffer. Before addition of primary antibodies, the sections were treated with normal goat serum (Vector Laboratories, Burlingame, CA). All sections were blocked with the Avidin/Biotin Blocking kit (Vector Laboratories). The primary antiserum used was the affinity purified rabbit polyclonal pNF1-C2 antiserum (1:10000 dilution). In the negative controls, the primary antiserum was omitted. After incubation with primary antiserum, the sections were incubated with species-appropriate biotinylated secondary antibody (Vector Laboratories). For immunohistochemistry with peroxidase detection, the endogenous peroxidase was inactivated in 3% H2O2 in methanol. Immunoreactive sites were detected using the Vectastain ABC kit (Vector Laboratories) and the DAB-substrate kit (Vector Laboratories) according to the manufacturer’s instructions. Sections were either counterstained with methyl blue or green and mounted with Eukitt, or mounted with 3:2 VectaShield/VectaShield-Dapi (Vector Laboratories). The sections were viewed under a fluorescence-equipped Zeiss Axioplan2 Imaging microscope (Carl Zeiss, Oberkochen, Germany).

In Situ Hybridization
In situ hybridization on sections from paraffin-embedded specimens was performed essentially as described previously (35). A 550-bp fragment from the mouse NF1-C cDNA (the NaeI/BglII fragment from pCHNF1-C2 was kindly provided by Dr. R. M. Gronostajski) was cloned into the pBluescript vector (Stratagene). The vector construct was then linearized at either side of the insert and transcribed with T3 and T7 RNA polymerases to generate [{alpha}-35S]UTP-labeled antisense and sense riboprobes, respectively. Photographs were taken with a Nikon FX-350 DX camera (Nikon, Tokyo, Japan) mounted on Optiphot-2 microscope.

Immunofluorescent Analysis of Cultured Cells
NMuMG cells grown on coverslips were treated with 5 µg/ml ovine prolactin (Sigma, St. Louis, MO) for 15 min. To detect the intracellular localization of NF1-C2 and Stat5 proteins, the cells were fixed in acetone/methanol 50:50 for 10 min. Before addition of primary antibodies, the cells were treated with normal goat serum (for rabbit primary antibody) or MOM immunodetection kit according to instructions from the supplier (for mouse primary antibody) (Vector Laboratories). The primary antibodies used were the rabbit polyclonal NF1-C2 antiserum (1:100 dilution) and the AX1 antiphospho-Stat5a/b mouse monoclonal antibody (1:100 dilution) (Advantex BioReagents LLP). After incubation with primary antibodies, the cells were incubated with species-appropriate biotinylated secondary antibodies followed by fluorescein isothiocyanate conjugated streptavidin (Vector Laboratories). VectaShield/VectaShield-Dapi (3:2) (Vector Laboratories) was used for mounting, and the cells were viewed under a fluorescence equipped Zeiss Axioplan2 Imaging microscope.


    ACKNOWLEDGMENTS
 
We are grateful to Kerstin Dahlenborg and Birgitta Liljenberg for technical assistance. We also thank Dr. C. J. Ormandy (Garvan Institute of Medical Research, Sydney, Australia) for providing us with sections from the PrlR+/– mice, Dr. N. Tanese (NYU Medical Center, New York, NY) for the NF1-C-specific antibody (8199), and Dr. R. M. Gronostajski (Lerner Research Institute, Cleveland, OH) for the pCHNF1 expression plasmids.


    FOOTNOTES
 
This work was supported by grants from the Swedish Cancer Society, the Assar Gabrielsson Foundation, the Lars Hierta Foundation, and the Magnus Bergvall Foundation.

First Published Online January 6, 2005

Abbreviations: CEL, Carboxyl ester lipase; HA, hemagglutinin; L, lactation; NF, nuclear factor; P, pregnancy; PrlR, prolactin receptor; Stat5, signal transducer and activator of transcription 5; V, virgin mice; W, weaning; WAP, whey acidic protein.

Received for publication September 10, 2004. Accepted for publication December 23, 2004.


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