Targeted Expression of a Dominant Negative Epidermal Growth Factor Receptor in the Mammary Gland of Transgenic Mice Inhibits Pubertal Mammary Duct Development

Wen Xie, Andrew J. Paterson, Edward Chin, Lisle M. Nabell and Jeffrey E. Kudlow

Departments of Medicine and Cell Biology Division of Endocrinology and Metabolism University of Alabama at Birmingham Birmingham, Alabama 35294


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The epidermal growth factor (EGF) system has been thought to play an important role in normal mammary development and carcinogenesis. To study the role of the EGF receptor (EGFR) in mammary development, we developed a transgenic mouse model in which a C-terminal truncated mouse EGFR (EGFR-TR) was expressed in the mouse mammary epithelium under the control of the mouse mammary tumor virus long terminal repeat. The EGFR-TR lacks most of the cytoplasmic domain of the receptor, including the entire protein tyrosine kinase domain. In cultured cells, we show that it acts in a dominant negative manner in EGF-signaled EGFR autophosphorylation. Several lines of mice were characterized and shown to express the transgene at the mRNA and protein levels not only in the mammary gland but also in the salivary glands, epididymis, and prostate. In postpubertal virgin female mice, the expression of the EGFR-TR in the mammary glands was greater than the expression of the endogenous wild type EGFR. In these virgin mice, inhibition in mammary ductal development and a decrease of mammary epithelial DNA synthesis were observed beginning at 5–6 weeks. The inhibition of duct development was most apparent by 15–16 weeks, resulting in a significant defect in ductal branching and outgrowth and an apparent overall decrease in the size of the mammary glands. However, during pregnancy, expression of the endogenous wild type EGFR was markedly increased relative to the EGFR-TR and, at this stage, normal presecretory alveoli developed from the hypoplastic duct tree. Postpartum, normal lactation occurred. Despite EGFR-TR expression in other tissues, no morphological abnormalities were observed. This model demonstrates that the EGFR-TR behaves as a dominant negative regulator of the EGFR system in vivo and that the EGFR system plays an important role in mammary ductal development.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The epidermal growth factor receptor (EGFR) and its ligands are believed to play a vital role in mammary gland development and carcinogenesis (for review, see Ref.1). However, the role of EGFR signaling in mammary development has not been fully explored, partly because EGFR-devoid animals are not viable postnatally (2, 3, 4) when mammary development occurs. Furthermore, there appears to be considerable redundancy in the ligands for the EGFR. These ligands include EGF itself (5), transforming growth factor-{alpha} (TGF{alpha}) (6), amphiregulin (AR) (7), heparin-binding EGF-like growth factor (8), and betacellulin (9). Thus, strategies to eliminate any of these ligands singly could be overcome by this redundancy, a problem suggested by the TGF{alpha} "knockout" studies (10, 11).

We approached blocking EGFR signaling in the mammary gland using a dominant negative strategy. The wild type receptor consists of a ligand-binding extracellular domain, a transmembrane domain, and an intracellular protein tyrosine kinase domain (12). Upon ligand binding, EGFRs oligomerize, the cytoplasmic tyrosine kinase is activated, and the receptor undergoes autophosphorylation. While an intramolecular mechanism of EGFR autophosphorylation has not been ruled out (13), a few lines of evidence suggest that EGFR autophosphorylation occurs in trans as a result of the oligomerization process (for review, see Ref.12). This autophosphorylation of the receptor is a critical component of the signaling process in that the phosphotyrosines in the carboxyl-terminal tail are recognized by a variety of proteins that contain Src homology 2 (SH2) domains, such as phospholipase C-{gamma} (5), SH2-containing sequence (14), or growth factor receptor-bound protein 2 (15). The binding of these SH2-containing proteins to the phosphorylated receptor creates a submembranous signaling complex whose activation leads to the pleiotropic response to the growth factors. In addition, the dimeric EGFR exhibits increased binding affinity for EGF even in the absence of a protein kinase domain (16, 17). We made use of this mechanism of EGFR signaling by overexpressing, in the mammary glands of transgenic mice, a truncated form of the EGFR that lacks the protein kinase and carboxyl-terminal domains. We hypothesized that this mutant receptor would either form nonsignaling dimers with the endogenous wild type receptor or sequester ligands, thereby impairing signaling by all ligands utilizing this receptor. Studies in cultured cells using similarly mutated EGFRs (18, 19) had indicated that this strategy might have an effect in vivo (20).

There is considerable indirect evidence for the role of the EGFR system in mammary ductal development. Most of this development occurs postnatally with a rudimentary ductal pattern having been patterned by the age of 6–7 weeks. At this stage, these minimal ducts terminate in an end bud, which shows evidence of dichotomous branching, while by age 13 weeks the ductal pattern has become quite extensive, forming a tree-like pattern. It is upon these ducts that the secretory alveolar lobules develop during pregnancy. This ductal expansion occurs as a result of cell division of the ductal epithelium in the end buds. EGFR is present in the normal ductal system. The observation that the EGFR concentration in the mammary gland varies through stages of development suggests a role for this receptor in ductal development. Receptor levels are high in the immature pubertal mammary gland and then decrease with age. Receptor levels increase again with the onset of pregnancy, reaching a peak level at 10 days followed by a rapid decline to very low levels during lactation (21). EGF/TGF{alpha} stimulate ductal cell proliferation, but inhibit functional differentiation of mouse mammary epithelial cells in culture (22). Indeed, overexpression of TGF{alpha} in transgenic mice results in mammary carcinoma (23, 24, 25) and impaired milk secretion, partly as a result of the inhibition of the expression of the milk-specific whey acidic protein gene (26). The role of this receptor system in mammary carcinoma is also suggested by the finding of increased TGF{alpha} expression in human breast cancers (27) and amplification of the EGFR gene in some breast cancer cell lines (28) and in some primary breast cancers (29).

To further elucidate the in vivo role of the EGFR system in mammary development, we created a transgenic mouse model in which a C-terminal truncated EGFR was expressed in the mammary ductal system under the influence of the mouse mammary tumor virus (MMTV) promoter. We found that these mice exhibited considerable suppression of mammary ductal development that was very similar to the phenotype exhibited in mice expressing an activated transforming growth factor-ß1 (TGFß1) under the MMTV promoter (30). These results strongly suggest that the EGFR system is involved in the proliferation and branching that occurs in the ductal system during pubertal development.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation and in Vitro Functional Characterization of a C-Terminal Truncated EGFR
A C-terminal truncated EGFR (EGFR-TR) cDNA was generated by inserting a double-stranded oligonucleotide into the SacI site of the mouse EGFR cDNA sequence (31) and, by doing so, introducing a stop codon (TGA) after the L690 codon. To test the function of this mutant in vitro, the EGFR-TR and the wild type EGFR (EGFR-WT) (32) cDNAs were placed downstream of the cytomegalovirus promoter. These expression vectors, encoding the wild type or truncated EGF receptors, were transiently transfected at various ratios into Chinese hamster ovary (CHO) cells, a cell line that does not express detectable levels of the endogenous EGFR (33). In cells expressing both the EGFR-TR and EGFR-WT, we could detect heterodimer formation between the EGFR-WT and EGFR-TR as revealed by bis(sulfosuccinimidyl)suberate (BS3)-mediated covalent cross-linking experiments with [125I]EGF (data not shown).

EGF-stimulated EGFR autophosphorylation is an essential component of EGFR signaling (for review, see Ref.12). To examine the effects of the EGFR-TR on EGFR autophosphorylation in response to EGF, CHO cells were transiently transfected with EGFR-WT alone or together with increasing amounts of EGFR-TR. After EGF treatment or mock treatment, the EGFR-WT was immunoprecipitated from the transfected cells using an anti-EGFR monoclonal antibody H9B4 (34) that is specific for EGFR intracellular domain. The immunoprecipitates were subjected to immunoblotting to determine the recovery of the wild type EGFR and its degree of tyrosine phosphorylation. The recovery of the receptor was determined by probing the blot with H9B4. Regardless of the ratio of EGFR-WT to EGFR-TR, the recovery of the wild type receptor was uniform (Fig. 1Go). However, when the blot was stripped and reprobed with an anti-phosphotyrosine antibody, 4G10, the level of autophosphorylation of the EGFR-WT was down-regulated, even in the presence of a 1:1 ratio of EGFR-TR to EGFR-WT (Fig. 1Go). At a ratio of 2:1 of EGFR-TR to EGFR-WT, the EGFR-WT autophosphorylation was largely abolished. This down-regulation of EGF-stimulated EGFR autophosphorylation by the EGFR-TR indicated that this C-terminus-truncated EGFR mutant could exert a dominant negative effect on the EGFR-WT auto-phosphorylation.



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Figure 1. The Truncated EGF Receptor (EGFR-TR) Inhibits EGF-Stimulated Autophosphorylation of the Wild-Type EGFR

CHO cells were transfected with various ratios of the expression plasmids encoding the EGFR-WT and the EGFR-TR. The transfected cells were subsequently treated with 10-8 M EGF or mock treated as described in Materials and Methods. Cell lysates were prepared, and the EGFR-WT was immunoprecipitated with a monoclonal antibody, H9B4, directed to the intracellular domain of the EGFR. The precipitated proteins were separated by SDS-PAGE and analyzed by immunoblotting. The filter was first probed with H9B4 and subsequently stripped and reblotted with the monoclonal anti-phosphotyrosine antibody, 4G10. The immunoblotted signals were observed using the ECL detection system. In this assay, untransfected MDA468 human breast cancer cells (28) and empty vector-transfected CHO cells were used as EGFR-positive and -negative control, respectively.

 
Generation of MMTV-EGFR-TR Transgenic Mice
Transgenic mice expressing EGFR-TR under the control of the MMTV long terminal repeat (MMTV-LTR) were generated by injecting one-cell B6xSJL mouse zygotes with the transgene construct shown in Fig. 2Go. Before these injections, we tested the transgene construct in cultured cells. Transfection of the plasmid containing the MMTV-EGFR-TR construct into CHO cells resulted in the expression of the expected 115-kDa EGFR-TR protein as revealed by BS3-mediated chemical cross-linking experiments with [125I]EGF (data not shown). This in vitro study indicated that the ß-globin intron splicing and MMTV promoter functioned normally in conjunction with the EGFR-TR cDNA.



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Figure 2. Schematic Representation of the MMTV-EGFR-TR Transgene Construct

The cross-hatched region corresponds to the 1.5 kb of the MMTV LTR. The solid regions correspond to the second and third exons of the rabbit ß-globin gene. The open region corresponds to the second intron and the 3'-flanking region of the ß-globin gene. The stippled region corresponds to the 2.3-kb segment of the mouse EGF receptor cDNA that encodes codons 1–690 of the receptor. A stop codon was inserted after codon 690, resulting in the expression of the C-terminal truncated mouse EGF receptor (EGFR-TR). The EGFR-TR cDNA (stippled region) was inserted into the third exon of the rabbit ß-globin gene.

 
A total of 10 gene-positive founders, five females and five males, were identified by PCR using a pair of transgene-specific oligonucleotides (data not shown). The integration status of the transgene was tested by Southern blot analysis. There is only one XbaI site in the transgene construct. XbaI digestion of the genomic DNA from the transgenic mice released the predicted 4.9-kb transgene, detected by hybridization with the transgene-specific rabbit ß-globin exon 3 probe (Fig. 3Go). This result indicates that the integrated transgene is intact, and that the multiple copies of the transgene are in a head-to-tail array. Of the 10 founders, one male (31-3) was infertile for unknown reasons, and one male (31-5) was unable to transmit the transgene to its offspring, probably as a result of mosaicism (35). The eight other founders (7-6, 7-8, 21-6, 20-1, 21-5, 31-1, 31-4, 31-7) successfully transmitted the gene in a Mendelian manner, thereby allowing the establishment of eight independent transgenic mouse lines.



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Figure 3. Southern Blot Analysis of MMTV-EGFR-TR Transgenic Mice

Eight micrograms of genomic DNA extracted from a tail biopsy from each transgenic (7-6-6, 31-7-4) or nontransgenic (21-7-4) mouse were digested with XbaI and subjected to Southern blot analysis. The transgene fragment (25 pg) derived from the original plasmid in which it was constructed was loaded as a positive control. The filter was probed with a radiolabeled rabbit ß-globin exon 3 probe that is specific for the transgene. The transgene (4.9-kb) bands were detected in transgenic but not in nontransgenic control mice.

 
Expression of the Transgene
Expression of the transgene was investigated by Northern blot analysis of RNA from the mouse mammary glands using the transgene-specific rabbit ß-globin exon 3 as a probe. Initial Northern blotting of RNA from pregnant female mouse mammary glands revealed that lines 7-6, 31-4, 20-1, and 21-6 had relatively high expression of the transgene. The 2.8-kb transcript was of the predicted size for the transgene and was detected with the ß-globin probe in the mammary glands of transgenic but not wild-type mice. When a cDNA probe encoding the extracellular domain of the EGFR was used, both the endogenous and transgene EGFR transcripts were detected (data not shown). Because these four lines of mice had the highest transgene expression, they were characterized further. Northern blot analysis carried out in line 21-6 using a cDNA probe encoding the extracellular domain of the EGFR revealed that the 2.8-kb transgene was expressed in the mammary glands of both virgin and pregnant transgenic females but not in their nontransgenic siblings (Fig. 4AGo). Of note, in the virgin transgenic females, the steady state level of the EGFR-TR transcripts was higher than that of the endogenous EGFR transcripts (10 and 6 kb). However, in late pregnancy, while no significant change in the level of the transgene transcript was detected, the level of the endogenous EGFR transcripts increased significantly, causing the ratio of EGFR-TR transcripts to the endogenous EGFR transcripts to decrease. When using the rabbit ß-globin exon 3 probe, only the transgene transcripts were detected in the mammary glands of transgenic females (Fig. 4BGo). In line 21-6, expression of the transgene was also detected in salivary glands, lungs, and epididymis (Fig. 4BGo), which is consistent with previous MMTV promoter-based transgenic studies (for review, see Ref.36). Relatively low levels of transgene expression were observed in the placenta and prostate (data not shown). No transgene expression was detected in brain, liver, or kidney. There was no clear correlation between the transgene copy number and the level of transgene expression as judged by Northern blot analysis.



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Figure 4. Northern Blot Analysis of the MMTV-EGFR-TR Expression in Tissues from Female and Male Transgenic Mice

Twenty micrograms of total cellular RNA from each of the indicated tissues from transgenic or nontransgenic control mice were subjected to Northern blot analysis. The filters were hybridized with a [32P]-labeled 2.3-kb EGFR-TR cDNA probe or the rabbit ß-globin exon 3 probe. The filters were subsequently stripped and reprobed with the GAPDH cDNA for the purpose of a loading control. A, Northern blot of mammary gland RNA using the EGFR cDNA probe. Both transgene and endogenous EGFR transcripts were detected. Lane 1 and 2 are control and transgenic (line 21-6) virgin females, respectively; lane 3 and 4 are control and transgenic (line 21-6) pregnant females, respectively. EGFR-TR or endogenous wild type EGFR transcripts are indicated. B, Northern blot of mammary gland RNA using ß-globin probe. Only the transgene transcripts were detected. Lanes 1 and 2 contain mammary gland RNA from control and transgenic (line 21-6) virgin females, respectively. Lanes 3–7 show transcripts from an adult male transgenic mouse (line 21-6); lane 3, epididymis; lane 4, seminal vesicle; lane 5, liver; lane 6, lung; lane 7, salivary gland.

 
The EGFR-TR protein, encoded by the transgene, was expressed in tissues of the transgenic animals. To detect the EGFR-TR, BS3-mediated chemical cross-linking was performed on microsomes purified from the examined tissues. This approach had the advantage of detecting the functional ligand-binding domain of the receptor and its dimerization partners while adding only 6 kDa to the molecular mass of the receptors. Figure 5Go shows that, in addition to the endogenous EGFR (170 kDa), the 115 kDa EGFR-TR protein was detected in the mammary glands of pregnant transgenic mice. EGFR-TR protein was also detected in the salivary gland (Fig. 5Go), prostate, and epididymis of transgenic animals (data not shown). Cross-link labeling of these EGF receptor proteins by [125I]EGF was completely inhibited in the presence of excess unlabeled EGF. We were not able to detect either EGFR-TR or the endogenous wild type EGFR protein in the mammary glands of virgin animals, probably because of the low mammary epithelial cellularity at this stage.



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Figure 5. Expression of the Truncated EGF Receptor (EGFR-TR) Protein in Transgenic Mice

Microsomes prepared from the indicated mouse tissues were analyzed for EGF binding using a cross-linking assay with [125I]EGF. The positions corresponding to the EGFR-TR and the endogenous wild type EGFR are indicated. Lanes 1 and 2 show the EGF receptors in the mammary glands of control and transgenic (line 20-1) pregnant females, respectively. Lanes 3 and 4 show the receptors in transgenic male mouse tissues (line 7-6); lane 3, testis; lane 4, epididymis.

 
Consequences of MMTV-EGFR-TR Transgene Expression in Female Mice
The mammary glands of the MMTV-EGFR-TR transgenic mice were examined to determine whether the expression of the truncated receptor resulted in developmental changes in this tissue. Comparison of whole-mount preparations of the fourth inguinal mammary glands from virgin transgenic and wild type mice indicated that the expression of EGFR-TR was associated with the inhibition of mammary duct development. The first signs of ductal development inhibition could be seen in the virgin transgenic mice as early as 5–6 weeks of age when puberty begins. As shown in Fig. 6Go, A and B, there were dramatically fewer end buds in a 5-week-old transgenic animal (Fig. 6BGo) as compared with its control littermate (Fig. 6AGo). The end buds show clear signs of dichotomous branching in the control, but not in the transgenic mice. Furthermore, the size of the end buds was considerably smaller in the transgenics. At this early stage, however, no significant differences were observed in the rudimentary duct pattern or density. The ductal inhibition in the transgenics progressed with age. By 10 weeks, the transgenic mammary glands exhibited a much simpler, sparser duct system, with a dramatic reduction in both side branching and terminal branching (Fig. 6Go, C and D). The reduction in side- and terminal branching was even more striking at higher magnification as depicted in Fig. 6Go, E and F. Figure 6Go, G and H, shows the mammary whole-mounts of 17-week-old mice. At this age, while the wild type virgin animals exhibited extensive dichotomous branching, complete ductal arborization and extension of the duct tree to the limit of the mammary fat pad (Fig. 6GGo), the transgenic virgin animals exhibited a profound defect in dichotomous branching (Fig. 6HGo). Beginning at 10 weeks, the size of the fourth inguinal mammary glands was also apparently smaller in the transgenics than in the control animals (Fig. 7Go), probably as a result of lesser fad pad invasion by the ductal tree. This inhibition of ductal development was observed consistently in multiple transgenic mice, indicating that the observed inhibition was not due to sampling at different stages of the estrous cycle. Similar inhibition of mammary duct development was also observed in each of the four transgenic mouse lines (7-6, 31-4, 21-6, and 20-1), in which the expression of the transgene in the mammary glands had been proven at both the mRNA and protein levels. Therefore, the phenotype was not dependent on the site of transgene integration. A correlation between the level of transgene expression and the extent of mammary duct inhibition was sustained. The expression of the EGFR-TR in line 31-7 was one third of that observed in line 31-4 as determined by Northern blot analysis normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Correspondingly, the low-expression line 31-7 exhibited only subtle inhibition of mammary duct development as compared with the high-expression line 31-4 (data not shown).



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Figure 6. Whole Mounts of Mammary Glands from Transgenic Females and Nontransgenic Control Mice

Whole mounts of fourth inguinal mammary glands from control (A, C, E, and G) or transgenic (line 21-6) (B, D, F, and H) virgin females were prepared and stained with hematoxylin. Mice from three age groups were examined. A and B, 5-week-old; notice the difference in number and size of the end buds (larger arrowhead indicates a typical normal end bud in wild type animal) and degree of dichotomous branching (smaller arrowhead); C and D, 10-week-old; notice the difference in side- and terminal branching; E and F, higher magnification of the periphery of the glands shown in C and D; G and H, 17-week-old; notice the difference in mammary duct outgrowth, density, and branching. The dark, elliptical masses in E and F are lymph nodes (arrowheads). Original magnification A to D, 50x; E and F, 200x; G and H, 15x.

 


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Figure 7. Gross Morphology of the Mammary Glands from Transgenic and Nontransgenic Control Mice

The fourth inguinal mammary glands from a 10-week-old transgenic (line 7-6) (bottom) and its nontransgenic litter mate (top). Notice the smaller size of the transgenic mammary gland.

 
Despite the inhibition of mammary duct development in virgin mice, normal alveolar development was observed in the transgenic mice during pregnancy as judged by whole-mount preparations (data not shown). All transgenic females, including those mice that were homozygous for the transgene, were able to suckle their young successfully even with litters of as many as 12 pups. Consistent with this observation, no significant difference in the lobuloalveolar structure was observed on mammary gland whole-mount examination between transgenic mice and wild type animals during lactation (data not shown).

Analysis of paraffin sections of fourth inguinal mammary glands confirmed that expression of the truncated receptor impaired ductal development in the postpubertal animals. Compared with the wild type virgin mice (Fig. 8AGo), the duct density in the virgin transgenic animals (line 31–4) (Fig. 8BGo) was significantly reduced at the age of 17 weeks. Under higher magnification, the apparent mammary duct branching observed in wild type animals (Fig. 8CGo) was markedly reduced in the transgenic mice (Fig. 8DGo). Consistent with the whole-mount results, the mammary glands from pregnant transgenic animals (Fig. 8FGo) exhibited only subtle histological differences from pregnant wild type animals (Fig. 8EGo). The alveoli in the transgenic animals appeared somewhat hypertrophied, perhaps in compensation for the hypoplastic ductal tree. Similarly, during lactation, slightly more secretory alveoli in the wild type animal (Fig. 8GGo) and slightly compensatory enlargement of the alveoli in the transgenic female were observed (Fig. 8HGo).



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Figure 8. Histological Sections from the Mammary Glands of Transgenic and Nontransgenic Controls

Mammary glands from control (A, C, E, and G) or transgenic (line 7-6) (B, D, F, and H) female animals were fixed, paraffin-embedded, and sectioned, followed by routine hematoxylin and eosin staining. Mice in different physiological stages were examined. A and B, 17-week-old virgins; C and D, higher magnification views of 17-week-old virgins; E and F, late pregnancy (day 19–20); G and H, lactation day 1. Original magnification, A and B, 100x; C and D, 400x, E to H, 100x.

 
Finally, no consistent structure or function changes were observed in transgenic mice in tissues other than the mammary glands, despite high levels of transgene expression. The examined tissues included salivary glands, lungs, epididymis, and prostates. No tumor has been found in MMTV-EGFR-TR transgenic mice after observation for 1.5 yr.

Effects of Transgene Expression on Mammary Epithelial Cell DNA Synthesis
We performed in vivo bromodeoxyuridine (BrdU) incorporation assay to determine whether the inhibition of mammary duct development in MMTV-EGFR-TR mice resulted from the inhibition of mammary epithelial cell proliferation. Inhibition of the EGF-induced mitogenic response by a similarly truncated EGFR has been reported in cultured cells (19). Ten-week-old wild type and transgenic animals were injected intraperitoneally with BrdU, and paraffin sections of the fourth inguinal mammary glands were prepared for immunostaining with an anti-BrdU antibody. Three transgenic virgin females from line 21-6 and two of their nontransgenic litter mates were examined. Figure 9Go shows representative ducts stained with BrdU. It is significant that consistently more BrdU-positive mammary epithelial cells were observed in control (Fig. 9AGo) than transgenic (Fig. 9BGo) animals. At this age, because end buds were not readily seen in the sections, only the terminal ducts were scored. Five representative mammary terminal ducts from each transgenic animal or control mouse were scored for the BrdU-labeling index. This index is the percentage of BrdU-positive epithelial cells in relation to the total cells scored of individual duct. Table 1Go shows that the BrdU labeling index in the 10-week-old transgenic virgin females of line 21-6 was about one third of that seen in the control wild type mice, indicating that expression of the EGFR-TR does inhibit mammary ductal epithelial cell proliferation in vivo. A similar degree of inhibition of mammary epithelial BrdU labeling was also observed in transgenic line 31-4 (data not shown). The inhibition of BrdU labeling in the transgenic animals was specific for the mammary duct epithelial cells, in that the transgene had no impact on the BrdU staining in lymphocytes in the mammary lymph nodes (data not shown).



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Figure 9. In vivo BrdU Labeling of the Mammary Glands of Transgenic and Nontransgenic Controls

Ten-week-old transgenic mice (line 21–6) or their nontransgenic litter mates were subjected to in vivo BrdU labeling. The animals received an intraperitoneal injection of BrdU 2 h before sacrifice. Paraffin sections of the fourth inguinal mammary gland were examined by immunostaining with an anti-BrdU monoclonal antibody detected using the peroxidase system. The sections were counterstained with hematoxylin. Sections are from the control animal (A) and from the transgenic animal (B). Original magnification 400x.

 

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Table 1. BrdU Labeling Index in Mammary Terminal Duct Epithelial Cells of Wild Type and MMTV-EGFR-TR Transgenic (Line 21-6) Mice at 10 Weeks

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of a C-terminal truncated EGFR in transgenic animals under the control of the MMTV promoter caused moderate inhibition of mammary ductal development. This observation is compatible with the notion that the truncated receptor behaves in a dominant negative manner, thereby blocking signaling through this receptor. Our own data with the EGFR-TR and published data using a similarly truncated receptor (18, 19) indicate that the EGFR-TR, when expressed in cultured cells at sufficiently high levels, result in a blockade of EGF signaling through the wild type receptor. Both EGF-stimulated receptor autophosphorylation and EGF induction of c-fos gene expression are down-regulated by the EGFR-TR. More recently, expression of a truncated EGFR in the skin under the control of a keratin promoter was shown to result in developmental abnormalities in the skin (20) presumed to result from the dominant negative action of the truncated receptor. However, interpretation of this skin model was equivocal, in that areas of hyperplasia were also observed, raising the possibility of a compensatory response from another growth factor system or an action of the truncated receptor other than signal blockade. In the mammary gland system described in this paper, only a hypoproliferative response was observed in the ductal cells, completely compatible with a dominant negative mechanism for the EGFR-TR. This dominant negative strategy using receptor truncation or kinase domain mutation has also been applied to study the role of some other receptor systems including the fibroblast growth factor receptor (37, 38), vascular endothelial growth factor receptor Flk-1 (39), and insulin receptor (40).

The phenotype of the MMTV-EGFR-TR transgenic animals has features in common with animals overexpressing activated TGFß1 in the mammary gland (30) and animals in which the estrogen receptor gene has been disrupted (for review, see Ref.41). In the case of the MMTV-TGFß1 mice, it was demonstrated that the lack of ductal elongation and branching in the postpubertal, nonpregnant animals resulted from the ability of the TGFß1 to block duct epithelial cell proliferation (30). The inhibition of mammary ductal epithelial proliferation by TGFß1 is consistent with the known ability of TGFß1 to block the proliferation of most nontransformed epithelial cells (for review, see Ref.42). This antiproliferative effect of TGFß1 could either be direct (43) or result, in part, from TGFß1 down-regulation of expression of the EGFR and TGF{alpha} (44). That ductal development and BrdU labeling of ductal cells were also partially blocked by the expression of the EGFR-TR in the mammary epithelium suggests that the truncated receptor also inhibited epithelial cell proliferation, thereby resulting in a similar phenotype. Another similarity shared between the MMTV-TGFß1 and MMTV-EGFR-TR models was the developmental stage at which the phenotype was most apparent. In both models, it was the postpubertal elaboration of the ductal system that was most affected (30). The ability of the EGFR-TR to block some degree of postpubertal growth of the ductal system implies that the EGFR and its ligands play an important role in this stage of mammary gland development.

Most mammary gland development occurs postpubertally, implying an important role for gonadal steroids. Classic endocrinological experiments using gonadectomy and hormone replacement had indicated a crucial role for estrogen and progesterone in this process. Estrogen promotes proliferation of normal (45, 46) and cancerous mammary epithelial cells. More recently, the role for estrogen was further supported by the development of an animal model in which the endogenous estrogen receptor gene was disrupted (41). These animals display very limited mammary ductal growth and branching, implying that estrogen plays a crucial role in the control of the proliferation of the mammary epithelium. There is considerable evidence that estrogen and the EGFR signaling interact in the control of this proliferation. TGF{alpha}, a ligand for the EGFR, is expressed in the normal mammary gland (27) and in other estrogen- responsive tissues such as the pituitary lactotrophs (47) and uterus (48). In each of these tissues, TGF{alpha} gene expression has been shown to be stimulated by estrogen. Furthermore, the TGF{alpha} gene promoter contains an atypical estrogen response element that confers an estrogen response to a reporter gene (49, 50). Thus, estrogen might stimulate growth of the ductal cells by increasing the local expression of TGF{alpha} and, consequently, the autocrine stimulation of the EGFR in the ductal epithelial cells. There is also some evidence that estrogen and EGF act synergistically in the activation of estrogen-responsive genes (51, 52, 53). Thus, this transgenic animal model, with impaired EGFR signaling in the mammary gland, may exhibit suboptimal estrogen action, thereby impairing other actions of estrogen that are required for ductal development. However, the inhibition of duct development by the EGFR-TR is unlikely to relate just to the estrogen effects. Mammary duct development also depends on many other factors. Indeed, estrogen replacement in prepubertally ovariectomized mice is not sufficient to allow normal duct development, and other hormones, such as progesterone, GH, PRL, and adrenal steroids, also contribute to this process (54, 55, 56). Whether the EGFR-TR also affects the action of these other hormones or whether the partial development of the mammary duct system, despite the EGFR-TR, results from continued action of these other hormones remains to be seen.

In addition to controlling mammary epithelial proliferation, the EGFR system may also play a role in the control of mammary gland-differentiated function. Transgenic animals overexpressing TGF{alpha} in the mammary gland exhibited a failure to lactate (24). This antilactogenic effect of EGF/TGF{alpha} may relate to the observation that EGFR activation inhibits the expression of milk-specific ß-casein and whey acidic protein both in cultured cells and in transgenic animals (26). In concert with these known effects of EGF/TGF{alpha}, the MMTV-EGFR-TR animals were functionally normal during lactation. These animals exhibited normal alveolar outgrowth during pregnancy and were able to sustain sufficient lactation to support large litters. This result suggests that either the EGFR system is not crucial for alveolar development during pregnancy and lactation or that the level of expression of the EGFR-TR was insufficient to block these stages of mammary development. In midgestation, maternal expression of the endogenous EGFR in the mammary gland has been shown to be at its highest (21). Since the dominant negative effect of the EGFR-TR largely relies on a relatively high ratio of mutant over wild type (Fig. 1Go and Refs. 17 and 36), the mammary gland could escape from this EGFR blockade in midgestation. Indeed, Northern blot analysis and [125I]EGF cross-linking studies performed on the mammary glands of pregnant transgenic mice indicated that the expression of the endogenous receptor had increased greatly while the expression of the truncated receptor did not. Therefore, this observed change in the EGFR to EGFR-TR ratio could explain why mammary alveolar development during pregnancy is practically normal in this transgenic model. The involvement of estrogen, progesterone, and PRL in alveolar development has also been well established (55, 56). Alveolar development is markedly impaired in animals in which the progesterone (54) or PRL (57) receptor gene has been disrupted. The role of EGF signaling in conjunction with these hormones has not been resolved by this model because of the failure of the MMTV promoter to drive expression of the EGFR-TR to levels greater than that of the endogenous receptor. However, the high level of expression of the endogenous EGFR during pregnancy does suggest some role for this receptor at this maternal developmental stage.

In contrast to the situation in pregnancy, during lactation, endogenous EGFR expression is low (21). This low receptor number and the lack of an observed effect of the EGFR-TR during lactation suggests that EGFR signaling at this time plays only a minor role and that other hormones or other growth factors dominate in the establishment of lactation. Interestingly, the waved-2 mouse, a line that expresses a mutant EGFR with partial loss of protein tyrosine kinase activity, does exhibit impaired lactation (58). While this lactation defect might result from a direct effect of the mutant EGFR on mammary development, it also remains possible that the defect in the EGFR gene of waved-2 mice, which occurs in all cell types including the other endocrine glands, exerts its effect on lactation indirectly by altering the complex hormonal milieu that is required to establish and maintain lactation.

In addition to directing expression to the mammary gland, the MMTV-LTR has been reported to direct transgene expression to salivary epithelial cells, lung, kidney, and seminal vesicles (for review, see Ref.36). Expression of oncogenes in transgenic animals under the control of the MMTV-LTR therefore often results in hyperplasia or neoplasia at these nonmammary sites (36). In the present study, we observed high levels of extramammary EGFR-TR expression in the lung (line 21-6), salivary gland, epididymis, and prostate. However, thus far, no significant phenotype has been observed in these tissues. Interestingly, the MMTV-TGF{alpha} mice, with similar extramammary transgene expression, also exhibited no histological abnormalities in these extramammary tissues (24). The lack of effect of EGFR-TR and TGF{alpha} expression in these tissues suggests that the EGFR system is less important for development in these tissues than it is in the mammary gland.

Other growth factor receptor systems, such as other members of the erbB family of receptor tyrosine kinases, may play a role in mammary development. The neu/erbB2 receptor tyrosine kinase, when overexpressed in the mammary gland, results in carcinogenesis (59, 60). Furthermore, abnormalities in erbB2 have been detected in spontaneous human breast carcinomas (61). Recently, there is some evidence that erbB2 interacts with the EGFR through the formation of signaling heterodimers (62, 63). It remains possible that the EGFR-TR could also interact with erbB2, perhaps impairing signaling through this receptor. Thus, the phenotype observed in the MMTV-EGFR-TR transgenic mice may also relate to interactions, not only with the endogenous EGFR, but other growth factor receptor systems. The transgenic model described in this manuscript could be used to explore these interactions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of EGFR-TR Expression Vector
A 2.3-kb C-terminal truncated mouse EGFR (EGFR-TR) cDNA encoding amino acid 1 to 690 (L) was generated by inserting a double-stranded oligonucleotide 5'-GAGCTG-TGAATTCTGATCACAGCTC-3' into the SacI site at nucleotide 2224 of the mouse EGFR cDNA (31) and, by doing so, introduced a stop codon (TGA) after L690 followed by an EcoRI site. EGFR-TR or EGFR-WT cDNA (32) was placed downstream of the cytomegalovirus promoter of plasmid pGFP (Clontech, Palo Alto, CA) after removal of the GFP cDNA using standard cloning strategies.

Transient Transfection and Western Blot Assay
Electroporation-mediated transient transfection was carried out as described (64). CHO cells were transfected with EGFR-WT alone (7.5 µg plasmid) or together with increasing amounts of EGFR-TR (7.5 µg or 15 µg plasmid). Thirty six hours after transfection, the cells were treated with 10-8 M EGF for 5 min at 37 C or mock treated. Cells were subsequently collected and lysed in RIPA buffer [50 mM HEPES, pH 7.2, 150 mM NaCl, 1.5 mM MgCl2, 1.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% deoxycholic acid, 10 µg/ml leupeptin, 1 mM phenylmethylsulfony fluoride (PMSF), 1 mM sodium vanadate, 50 mM NaF]. Equivalent amounts of lysate were immunoprecipitated with an anti-EGFR monoclonal antibody H9B4 (34), which is specific for the EGFR intracellular domain. The immunoprecipitates were resolved in 7.5% SDS-PAGE, followed by Western transfer, and blotted with H9B4 using enhanced chemiluminsescence (ECL) detection system (Amersham, Arlington Heights, IL). The same filter was subsequently stripped and reblotted with an anti-phosphotyrosine antibody, 4G10 (Upstate Biotechnology, Inc, Lake Placid, NY).

Generation of MMTV-EGFR-TR Transgenic Mice
The MMTV-LTR transgene cassette was kindly provided by Dr. H. L. Moses and was described in detail elsewhere (30). The EGFR-TR cDNA encoding amino acids 1 to 690 (L) was inserted into the EcoRI site of ß-globin exon 3. The 4.9-kb XhoI fragment containing the entire transgene cassette was isolated by electroelution from a 1.0% agarose gel followed by purification with GlassMax (GIBCO-BRL, Grand Island, NY). All mice were handled in an accredited university facility in accordance with the institutional animal care policies. Microinjection into one-cell B6xSJL mouse zygotes at a concentration of 2 ng/ml was carried out at the University of Alabama at Birmingham transgenic animal facility under the direction of Dr. Carl Pinkert.

Identification of Transgenic Mice
Genomic DNA was isolated by phenol/chloroform extractions as described (65). The PCR was used to screen for transgene-positive mice with one oligonucleotide designed to anneal to the EGFR-TR cDNA, 5'-CTGGGCACAGATGATTT-3' (corresponding to nucleotides 795 to 811) (31), and the other oligonucleotide was designed to anneal to the rabbit ß-globin intron between exon 2 and exon 3 in the transgene cassette, 5'-CACTGTTTGAGATGAGG-3' (corresponding to Il -70 to -86) (66) The PCR was carried out on a DNA thermal cycler (Perkin-Elmer/Cetus, Norwalk, CT) using the following program: 94 C for 1 min, 57 C for 2 min, and 72 C for 3 min. PCR products were analyzed by electrophoresis on a 1% agarose gel.

PCR identification of the transgenic animals was confirmed by Southern blot analysis. Eight micrograms of mouse tail genomic DNA was digested with KpnI or XbaI restriction enzymes and subjected to Southern blotting and hybridization using Hybond-N+ nylon membrane (Amersham), following the protocol of the membrane manufacturer. The probes used were a 2.3-kb EGFR-TR cDNA (EcoRI-EcoRI fragment) or the 526 bp EcoRI-XhoI fragment of rabbit ß-globin exon 3.

Northern Blot Analysis
Total RNA was prepared from the tissues using acid guanidinium thiocyanate-phenol-chloroform extraction as described (65). RNA was separated on 1.25% agarose-6% formaldehyde gel and transferred to a Hybond-N+ nylon membrane (Amersham). To detect specific transcripts, [32P]cDNA probes labeled by Deca Prime II kit from Ambion (Austin, TX) were used for hybridization on the membranes. The probes used were the same as described in Southern blot. The filters were cohybridized or subsequently rehybridized with a plasmid containing the murine GAPDH cDNA for normalization of loading.

Tissue Microsome Preparation and [125I]EGF-Mediated EGF Receptor Cross-Linking Assay
The tissues were homogenized in buffer A (5 ml/g tissue) containing 10 mM HEPES, pH 7.5, 1 mM PMSF, 1 mM dithiothreitol, 0.3 M sucrose using a Polytron. After centrifugation at 10,000 x g for 30 min at 4 C, the supernatant was transferred to a new tube and further centrifuged at 95,000 x g for 75 min at 4 C. The crude microsome pellet was resuspend in 5 ml of buffer B (buffer A containing 0.6 M KCl), followed by centrifugation at 95,000 x g for 75 min at 4 C. The microsomes were subjected to the cross-linking assay, or resuspended in 200 µl of buffer A and stored in liquid nitrogen until use. For the cross-linking assay, the microsome pellet was resuspended in 200 µl of binding buffer (PBS with 0.1% BSA and 1 mM PMSF), and incubated with [125I]EGF (final concentration about 1.4 x 10-8 M EGF) at 4 C for 5 h. The microsomes were recovered by centrifugation at 50,000 x g for 60 min at 4 C. After washing with 1 ml of binding buffer twice, the microsomes were resuspended in 250 µl PBS containing 2.0 mM BS3 (Pierce, Rockford, IL). Cross-linking was carried out at room temperature for 30 min. The cross-linking reaction was quenched with 250 mM glycine, and the microsomes were recovered by centrifugation. The 125I-labeled proteins were subjected to 4.5% SDS-PAGE and visualized by autoradiography.

Mammary Gland Whole-Mount and Histological Analysis
Inguinal mammary fat pads were removed from transgene-positive and control animals and fixed in formaldehyde (4% in PBS) at 4 C for at least 24 h, followed by whole-mount preparation and hematoxylin staining as described (30). The whole mounts were analyzed in methyl salicylate under a dissecting microscope. Parts of the fixed tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin for regular histological examination.

BrdU Labeling and Immunohistochemistry
In vivo BrdU labeling was performed by intraperitoneal injection of BrdU as described (30). Paraffin sections were deparaffinized, hydrated, pretreated with 2 N HCl for 20 min at 37 C, and exposed to 0.01% trypsin at 37 C for 3 min, followed by BrdU immunostaining using a rat monoclonal anti-BrdU antibody MSA250p (Accurate, Westbury, NY) at 1:100 dilution and Vectastain Elite ABC Kit (Vector, Burlingame, CA). 3,3'-Diaminobenzidine tetrahydrochloride was used as chromogen, and sections were counterstained with Gill’s Hematoxylin (Vector).


    ACKNOWLEDGMENTS
 
The authors wish to thank Dr. H. L. Moses for the MMTV-LTR transgene cassette, Drs. A. Aviv and D. Givol for the mouse EGF receptor cDNA, and Dr. A. Wells for wild type human EGF receptor cDNA. We also gratefully acknowledge Drs. S. J. Frank and A. Wells for helpful discussions.


    FOOTNOTES
 
Address requests for reprints to: Jeffrey E. Kudlow, Division of Endocrinology and Metabolism, University of Alabama at Birmingham, Birmingham, Alabama 35294.

This work was supported by Public Health Service Grant DK-43652 and NIH Grant DK-48882.

Received for publication June 19, 1997. Revision received August 1, 1997. Accepted for publication August 5, 1997.


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