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Endocrinology Vol. 144, No. 6 2669-2675
Copyright © 2003 by The Endocrine Society

Targeted Expression of the Dominant-Negative Prolactin Receptor in the Mammary Gland of Transgenic Mice Results in Impaired Lactation

Elise Saunier, Fariel Dif, Paul A. Kelly and Marc Edery

Institut National de la Santé et de la Recherche Médicale, Unité 344, Endocrinologie Moléculaire, Faculté de Médecine Necker, 75730 Paris, France

Address all correspondence and requests for reprints to: Dr. Marc Edery, Institut National de la Santé et de la Recherche Médicale, Unité 344, Endocrinologie Moléculaire, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France. E-mail: medery{at}mnhn.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The F3-short form of the rat PRL receptor (F3-SPRLR) form acts as a dominant negative inhibitor in vitro. We have developed a transgenic mouse model in which the rat F3-SPRLR was expressed in mammary epithelium under the control of the mouse mammary tumor virus promoter. Two lines of mice were characterized and shown to express the transgene in the mammary gland. No developmental abnormalities or differences from wild-type littermates were observed on the basis of size, activity, or fertility. Mice with a low level of transgene expression had a mammary phenotype similar to the wild type. However, mice overexpressing the transgene (levels much higher than those of the endogenous long PRLR transcript) had impaired mammary gland differentiation and lactation. In these mice, whole-mount and histological analyses demonstrated normal ductal development, but severely reduced lobuloalveolar outgrowth. signal transducer and activator of transcription-5 phosphorylation and expression of ß-casein and whey acidic protein gene were decreased. In vivo bromodeoxyuridine incorporation at midpregnancy showed that the reduction in mammary development was not due to an inhibition of ductal growth and side-branching. This model demonstrates for the first time in vivo a function of the SPRLR and a local and targeted effect of PRL on the mammary gland that are essential for its function, but not for its development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DEVELOPMENT and functional differentiation of the mammary gland are governed by the coordinated action of steroid and peptide hormones. Estrogen and progesterone are required for ductal outgrowth and alveolar proliferation, respectively (1, 2). The pituitary hormone PRL is known to play a central role in the development, differentiation, and function of the mammary gland. During pregnancy, PRL acts in synergy with insulin and glucocorticoids to induce terminal differentiation and milk production. PRL exerts its actions through the PRL receptor (PRLR), a transmembrane protein of the class 1 cytokine receptor superfamily (3).

Several forms of the PRLR, differing in the length and sequence of their cytoplasmic domains and resulting from alternative splicing of a single PRLR gene (4), have been described in many species, including rats, mice, and humans (5, 6, 7). In the rat these forms consist of a long form (LPRLR) of 591 amino acids (5) and an Nb2 form (an isoform lacking 198 amino acids) (8), both of which are able to transmit a lactogenic signal. In addition, an F3-short form of 291 amino acids (F3-SPRLR) exists (4).

In mice, these receptors have been classified as one long (LPRLR) (9) and three short forms (S1PRLR, S2PRLR, and S3PRLR) (6). The S1PRLR and S2PRLR forms are mouse specific, but a long and a short form receptor have been cloned in other species that are homologous to the mouse LPRLR and S3PRLR. The different cytoplasmic domains of these receptor forms may connect to distinct signaling pathways. Only the long form has been shown to induce transcription of milk protein genes, whereas LPRLR and S1PRLR are capable of mediating PRL-dependent cell proliferation (10). Although a specific biological activity mediated only by an SPRLR has yet to be identified, it is clear that S3PRLR, which is present in mouse, rat, and other species, cannot mediate cell proliferation, but behaves as a dominant negative form by inhibiting the function of the LPRLR (11).

PRLR proteins are expressed ubiquitously, with varying proportions of long and short forms in different tissues. Thus, in rat liver the SPRLR is predominant, whereas the LPRLR is more abundant than the short form in mammary gland. PRLR levels in mammary gland vary at different stages of development depending on the hormonal environment (12).

During hormonal stimulation, the long, but not the rat F3-short, form of the receptor is able to activate signal transducer and activator of transcription-5 (Stat5) to induce the transcription of milk protein genes (13). Interestingly, rat F3-SPRLR acts as a dominant negative inhibitor through the formation of inactive heterodimers, resulting in the absence of Janus kinase/Stat pathway activation in vitro (11, 14). The coexpression in vitro of both forms of PRLR (long and rat F3-short forms), when more short than long form is expressed, results in a block of PRL signal to the milk protein gene promoter as a function of the concentration of the SPRLR (15).

The suppression of PRL synthesis by hypophysectomy or PRL-lowering drugs fails to completely abolish PRL because of extrapituitary PRL production. Moreover, PRL knockout (KO) mice and PRLR KO homozygous mice are sterile (16, 17), with a lack of implantation and pseudopregnancy. Progesterone replacement in PRLR KO null mice allows normal egg development, implantation, and survival of young up to about midgestation, although a few late gestation embryos can be found (18).

In this study to clarify the putative dominant negative effects of the SPRLR in vivo and determine how PRL participates in normal mammary gland development, we developed a transgenic mouse model in which the rat F3-SPRLR (4) was expressed in the mouse mammary epithelium under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR).

Several lines of mice were developed. Three of them were shown to express the transgene in the mammary gland at moderate to high levels. Overexpression of the transgene was associated with null lactational performance. In this mouse model we observed an inhibition of PRL signaling resulting in impaired mammary gland differentiation and lactation. Thus, the SPRLR acts as a dominant negative in vivo. Other lines expressing the transgene at lower levels resemble the wild-type mouse in every respect. In the present study we analyzed in detail two lines of transgenic mice (lines 3 and 8) representative of each phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of transgenic mice
The 1.4-kb Flag-tagged short form of the PRLR cDNA (19) was inserted into the EcoRI site of ß-globin exon 3 of the MMTV-LTR transgene cassette, provided by Dr. H. L. Moses and previously described in detail (20). The 4.1-kb XhoI fragment was purified with S&S Elutip minicolumns (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH) and microinjected into B6/D2 fertilized eggs at a concentration of 2 ng/µl. All mice were handled in an accredited facility in accordance with institutional and European animal care policies. A total of approximately 400 mice were analyzed during this study.

Identification of transgenic mice
Total chromosomal DNA was isolated from mouse tail, digested with proteinase K, and subjected to PCR to identify transgene-positive mice. One oligonucleotide was designed to anneal to the SPRLR cDNA, 5'-CAGATGGGTATCAAATCC-3' (corresponding to nucleotides 817 to 834), and the other oligonucleotide was designed to anneal to the rabbit ß-globin intron between exons 2 and 3 in the transgene cassette, 5'-CACTGTTTGAGATGAGG-3' (corresponding to -70 to -86). The PCR was performed using Ready-To-Go PCR Beads (Pharmacia Biotech, Piscataway, NJ) on a DNA thermal cycler using 35 cycles of the following program: 94 C for 1 min, 52 C for 1 min, and 72 C for 1 min. PCR products were analyzed by electrophoresis on a 1% agarose gel.

PCR identification of transgenic animals was confirmed by Southern blot analysis using standard procedures. The probes used were a 1.4-kb SPRLR cDNA or the 526-bp EcoRI-XhoI fragment of rabbit ß-globin exon 3.

Mammary gland whole-mount and histological analysis
Number four mammary glands were dissected away from sc tissues and fixed in freshly prepared Methacorn solution (60% methanol, 30% chloroform, and 10% acetic acid) for 48 h, dehydrated, skimmed with acetone for 48 h, rehydrated, and stained with iron hematoxylin for 2 h before washing and Histo-Clear conservation (National Diagnostics, Somerville, NJ). Parts of the tissues were fixed and embedded in paraffin, sectioned, and stained with hematoxylin and eosin for regular histological examination.

Northern blot analysis
Tissue samples were collected, frozen immediately in liquid nitrogen, and stored at -80 C until used for isolation of RNA or proteins. Total RNA was prepared from the tissues using TRIzol reagent procedure extraction (Life Technologies, Inc., Grand Island, NY), then mRNA was isolated using Oligotex mRNA Spin-Column from QIAGEN (Chatsworth, CA). Five micrograms of mRNA were separated on a 1% agarose gel and blotted to a Hybond-N+ nylon membrane (Pharmacia Biotech). To detect specific transcripts, [32P]cDNA-labeled probes (NEBlot kit, BioLabs, Northbrook, IL) were used for hybridization of the membranes. The probes used for PRLR detection were the same as those described for the Southern blot analysis. The 530-bp ß-casein probe and the 650-bp whey acidic protein (WAP) probe were obtained from Dr. Lothar Hennighausen (NIH, Bethesda, MD). After initial exposure to film, blots were stripped and rehybridized with a murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe to normalize for loading.

Western blot analysis
Four milliliters of lysis buffer (21) were added to each gram of tissue and then homogenized using a Polytron (Brinkmann Instruments, Inc., Westbury, NY). The extracts were cleared by centrifugation at 15,000 rpm at 4 C for 15 min. One microgram of protein was immunoprecipitated with either 25 µl anti-Flag M1 affinity gel (Kodak, Rochester, NY) or 4 µl Stat5a antibody in presence of protein A-G plus agarose (Sigma-Aldrich Corp., St. Louis, MO) for 2 h at room temperature. Proteins were separated on either a 7.5% or 10% acrylamide gel and transferred on a polyvinylidene difluoride transfer membrane (Polyscreen, NEN Life Science Products, Boston, MA). Blots were blocked overnight and incubated with an anti-Flag M2 monoclonal antibody (0.5 µg/ml; Sigma-Aldrich Corp.), an anti-PRLR polyclonal antibody [provided by Prof. F. Talamantes and previously described in detail (22)], an anti-Stat5a polyclonal antibody (L-20, 0.2 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or an antiphosphotyrosine monoclonal antibody (4G10, 2.5 µg/ml; Upstate Biotechnology, Inc., Lake Placid, NY) for 2 h at room temperature. After washing, horseradish peroxidase-conjugated antirabbit (or mouse, depending on the primary antibody used) immunoglobulin G (Sigma-Aldrich Corp.) diluted to 1:4000 (or 1:5000 for mouse antibody) was added and then incubated for another 2 h at room temperature. Proteins were then visualized by enhanced chemiluminescence detection (Pharmacia Biotech).

Bromodeoxyuridine (BrdU) labeling
In vivo BrdU labeling was performed by ip injection of 160 µg BrdU/g body weight 2 h before the mice were killed. Paraffin sections of the fourth inguinal mammary gland were deparaffinized, hydrated, pretreated with 2 N HCl for 1 h at 40 C, and examined by immunostaining with an anti-BrdU rat monoclonal antibody (MAS 250p, diluted 1:50; Accurate Chemical & Scientific Corp., Westbury, NY). The BrdU antibody was detected using the peroxidase system Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA). 3,3'-Diaminobenzidine tetra-hydrochloride was used as chromogen, and sections were counterstained with hematoxylin. A thousand cells were counted for each slide, and ANOVA was performed to check statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of MMTV-SPRLR transgenic mice
Transgenic mice expressing Flag-tagged rat F3-SPRLR (19) under control of the MMTV-LTR promoter were generated by injecting one-cell B6/D2 embryos with the construct shown in Fig. 1AGo. Eight founders were generated, and two lines of mice (lines 3 and 8) were characterized in detail. Female mice in these two lines were studied extensively and have varying mammary phenotypes depending on the level of transgene expression. However, these mice did not show any overt developmental abnormalities and were indistinguishable from their wild-type littermates on the basis of size, activity, and fertility.



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  		Figure 1. A, Structure of the MMTV-SPRLR construct. The hatched arrow represents the 1.5-kb MMTV-LTR. The hatched rectangles correspond to exons 2 and 3 of the rabbit ß-globin gene. The line represents the second intron of the rabbit ß-globin gene. The rat SPRLR cDNA (gray rectangle) was inserted into the third exon of the ß-globin gene. Sense (S) and antisense (AS) arrows represent the positions of primers for PCR. F, N-Terminal flag epitope; X, XhoI; K, KpnI; B, BamHI; EI, EcoRI; EV, EcoRV. B, Southern blot of genomic DNA of transgenic and wild-type mice digested with EcoRV. Genomic DNA was probed for the presence of the transgene.

 
Screening of transgenic mice was performed by Southern blot analysis of genomic DNA isolated from the tail. EcoRV or KpnI digestions of 8 µg DNA from the transgenic mice released the predicted 4.1-kb transgene, resulting from head to tail integration (Fig. 1BGo). PCR of tail DNA using specific primers of the transgene construct was performed to identify the transgenic mice.

Expression of the transgene
Northern blots of 5 µg mRNA from mammary glands of 2 d postpartum mice were carried out using a rat PRLR cDNA probe. Our analysis revealed that line 3 had high expression of the transgene visible after 2 h of film exposure (Fig. 2AGo). Line 8 was also characterized and expressed a lower level of the transgene in the mammary gland (Fig. 2EGo). The 1.9-kb transcript was of the predicted size for the transgene and was never detected in any tissues of wild-type littermates (Fig. 2AGo). In line 3, transgene expression was also detected at low levels in epididymis, salivary glands, and lungs after 48 h of film exposure (Fig. 2BGo), which is consistent with previous MMTV promoter-based transgenic studies. Very low transgene expression was observed in liver, seminal vesicle, ovary (Fig. 2BGo), and uterus (data not shown). The SPRLR transgene transcript was detected in the mammary gland of virgin (Fig. 2EGo), pregnant (Fig. 2DGo), and postpartum (Fig. 2AGo) females. As shown in Fig. 2DGo, a similar level of the endogenous LPRLR transcript (6 kb) was also detected with the PRLR probe in mammary gland from wild-type and transgenic mice. The level of transgene expression was much higher than that of the endogenous LPRLR transcript in transgenic mice. As shown in Fig. 1BGo and by others (23), there is no clear correlation between the number of transgene copies determined by Southern blot analysis and the level of transgene expression, determined by Northern blot analysis.



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  		Figure 2. Northern and Western blot analyses of transgene expression from tissues of wild-type (WT) and transgenic mice. A, Mice were biopsied within 36 h after parturition. mRNA was probed for the presence of SPRLR and GAPDH transcripts. The film was developed after a 2-h exposure. 8, Transgenic mouse line 8; 3, transgenic mouse line 3; L, liver; MG, mammary gland. B, mRNA from tissues of virgin transgenic mice of line 3 was probed for the presence of SPRLR and GAPDH transcripts. The film was developed after a 48-h exposure indicative of a much lower level of expression than in A. Ep, Epididymis; SGl, salivary glands; L, liver; SV, seminal vesicle; Lu, lungs; Ov, ovary. C, Flag-tagged SPRLR was immunoprecipitated and blotted with a Flag antibody. The labeling of each lane is identical with that in A. D, Mice were biopsied at midgestation, and mammary gland mRNA was probed for the presence of PRLR and GAPDH transcripts. The film was developed after a 24-h exposure, so that the 6-kb band representing LPRLR could be seen. E, Mammary gland mRNA of wild-type and transgenic virgin mice was probed for the presence of PRLR transcripts.

 
To test for SPRLR transgene protein expression, we performed Western blot analysis. The expressed transgene protein was immunoprecipitated from solubilized tissues of mice 2 d postpartum and analyzed by immunoblot. The Flag antibody used recognizes an epitope located on the N-terminal part of the Flag-tagged SPRLR. Western blot data indicate that the 42-kDa transgene protein is strongly expressed in the mammary gland of transgenic mice of line 3 but is not detected in line 8 (below detection level) or in wild-type mice (Fig. 2CGo). Little or no transgene protein was detected in mouse liver (Fig. 2CGo) or uterus. Thus, the transgene is preferentially expressed in the mammary gland of virgin, pregnant, and postpartum females of line 3 (data not shown).

Impaired mammary gland development and lactation in MMTV-SPRLR transgenic females
Mice with a low level of transgene expression had a phenotype similar to that of wild-type animals (data not shown). However, mice expressing a high level of the transgene had impaired mammary gland development and lactation.

Fertility, length of gestation, and litter size of the transgenic mice were comparable to those in wild-type littermates. However, no signs of successful lactation were obtained in mice highly overexpressing the transgene. After parturition, milk could not be detected in the stomachs of the pups despite vigorous suckling. Upon fostering with wild-type females, these pups thrived, implying that mammary glands of the transgenic mice were unable to produce and deliver milk.

Whole-mount analysis of mammary tissue from postpartum transgenic mice demonstrated incomplete mammopoiesis in mice overexpressing the transgene. Ductal development appeared normal, but lobuloalveolar outgrowth was severely reduced (Fig. 3Go). The first signs of inhibition of mammary gland development were seen at midgestation. Histological examination of the mice showed that lactational performance was correlated with the degree of mammary gland development (Fig. 3Go).



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  		Figure 3. Whole-mount (WM) and histological (Histo) analyses of mammary gland of wild-type (WT) and transgenic (tg) mice of line 3. Mice in different physiological stages were examined: V, virgin; MG, midgestation; LP, late pregnancy; PP, 2 d postpartum. Whole-mount bar, 200 µm; histological bar, 100 µm.

 
These results demonstrate that overexpression of the SPRLR did not permit lactation and that this phenotype could be attributed primarily to a deficit in the degree of lobular-alveolar development of the gland.

Reduction of milk protein gene expression in transgenic mice
To better understand the absence of lactation in transgenic mice overexpressing the transgene, we performed an analysis of milk protein RNA expression in the mammary gland 36 h after parturition. The levels of ß-casein and WAP gene expression decreased in the mammary gland overexpressing the SPRLR compared with the wild-type mammary gland (Fig. 4Go). No difference was observed in mice expressing the transgene in low amounts compared with wild-type mice (Fig. 4Go).



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  		Figure 4. Northern blot analysis of milk protein gene expression from tissues of wild-type (WT) and SPRLR transgenic mice. Mice were biopsied within 36 h after parturition and probed for the presence of mRNA encoding ß-casein, WAP, and GAPDH. 8, Transgenic mouse line 8; 3, transgenic mouse line 3; L, liver; MG, mammary gland.

 
Inhibition of Stat5a activation in transgenic mice
The presence and activation of Stat5a in mammary gland were verified using immunoprecipitation and Western blot analysis (Fig. 5Go). Stat5a was clearly present in mammary glands from all mice at midgestation. However, Stat5a phosphorylation levels were decreased in mice overexpressing the transgene compared with wild-type mice. No difference was observed in mice expressing low levels of the transgene compared with wild-type mice. Thus, the PRL signaling pathway appears to be repressed in mammary glands overexpressing SPRLR, resulting in an incomplete developmental phenotype.



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  		Figure 5. Western blot analysis of Stat5a protein in the mammary gland of 9-d pregnant, wild-type (WT) and transgenic mice. Stat5a protein was immunoprecipitated with a Stat5a antibody, blotted with an antiphosphotyrosine antibody, stripped, and reblotted with a Stat5a antibody. 8, Transgenic mice line 8; 3, transgenic mice line 3; L, liver; MG, mammary gland.

 
Effects of transgene expression on mammary cell proliferation
To analyze whether the inhibition of mammary development in transgenic mice overexpressing the SPRLR was due to the inhibition of mammary epithelial cell proliferation, we performed in vivo a BrdU incorporation assay. Virgin and midpregnant mice were injected ip with BrdU, and paraffin sections of the fourth inguinal mammary glands were prepared for immunostaining with an anti-BrdU antibody. No difference was observed in transgenic mice compared with wild-type mice (Fig. 6Go). The ratio of proliferating cells (BrdU-positive nuclei) to total cell number was 8.50 ± 1.06% for wild-type vs. 8.83 ± 1.22% and 10.10 ± 1.61% for transgenic lines 8 and 3, respectively. There was no statistical difference between wild-type and transgenic mice; thus, the expression of the transgene does not lead to reduced proliferation of mammary gland cells at midgestation.



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  		Figure 6. In vivo BrdU labeling in mammary gland of transgenic and wild-type mice. A, Sections are from the control animal (WT) and from transgenic mice from lines 8 (8 ) and 3 (3 ) at midgestation. Bar, 20 µm. B, Ratio of proliferating cells (BrdU-positive nuclei). Values are the mean ± SEM of three animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because cloning of PRLR cDNA revealed the existence of multiple receptor forms, extensive studies of the function of each form have been reported. In vitro studies have shown that both the long and intermediate forms of rat PRLR were able to activate a milk protein promoter/reporter gene construct (24), whereas the rat F3-short form was inactive (13). On the other hand, both the long and mouse S1 forms of the PRLR have been shown to transmit a mitogenic signal in transfected NIH-3T3 cells (10). Cotransfection experiments in CHO and 293 cells demonstrated one additional function of the SPRLR in PRL signaling of milk protein gene activation; when overexpressed, it could act as a negative regulator, supporting a novel view in the regulation of PRL actions (11). Furthermore, when the SPRLR is coexpressed with the LPRLR in vitro, PRL signaling to milk protein genes is blocked in a dose-dependent manner (15).

To clearly analyze the role of PRL in normal mammary gland development, we chose strategies aimed at locally blocking the PRL/PRLR pathway at the level of the mammary gland. The dominant negative effect of the rat F3-SPRLR observed in vitro has led to the speculation that its overexpression in the mammary gland could lead to arrest of PRL signaling in vivo. To preferentially express SPRLR in the mammary gland, we used the MMTV-LTR promoter, which allows constitutive expression of the transgene in epithelial cells of the mammary gland (20, 23, 25, 26). Although, as previously described (23), transgene expression can be detected by Northern blot analysis in other tissues, the highest expression level was observed in the mammary gland, because a 2-h film exposure was sufficient to observe a very strong signal of transgene expression in the mammary gland, whereas a 48-h exposure was needed for a weak signal to be detected in other tissues. Indeed, no differences were observed between wild-type and transgenic mice in tissues other than the mammary gland. Particularly, no sign of reduced fertility was noted in female or male transgenic mice.

The mammary gland undergoes development in utero, at puberty (mainly ductal development), and during pregnancy (ductal and alveolar development). The development of the mammary gland during the second half of pregnancy is characterized by the growth of secretory units or alveoli. Lactational problems were expected in females overexpressing the SPRLR if, in fact, it acts as a dominant negative regulator in vivo, due to the essential role of PRL in mammary development. Whole-mount and histological evaluation of the mammary gland of mature virgin SPRLR transgenic mice indicated no difference correlated with the overexpression of SPRLR. Ductal tissues were clearly present, and ductal branching was normal, confirming that PRL signaling was not essential at this stage of development. PRLR KO heterozygous [PRLR KO homozygous females are sterile (17)] and Stat5a-null mice (27, 28) have a mammary phenotype similar to that of SPRLR transgenic mice, with impaired differentiation of lobuloalveolar units and an inability to lactate. Interestingly, female mice carrying only one intact PRLR allele failed to lactate only after their first pregnancy, demonstrating that differentiation was dependent on a threshold level of PRLR (17). More recently, this phenotype was maintained in inbred 129SV mice in the first pregnancy only, but inbred BL6 mice were unable to lactate even after multiple pregnancies (29). Similarly, in Stat5a-null mice after multiple pregnancies, functional mammary development was attained, suggesting that Stat5b can compensate for the absence of Stat5a. The situation in SPRLR transgenic mice resembled the phenotype seen in BL6 PRLR heterozygotes, because no lactation was observed even after several cycles of pregnancy, apparently due to a continued block of PRL signaling. We also demonstrated that alveolar development in postpartum SPRLR transgenic mice is curtailed, and the mammary gland has the appearance of tissue at midpregnancy. Therefore, it appears that the PRL pathway, at least in certain tissues, is dependent on the ratio of expression of the short and long forms of PRLR. Indeed, only the line overexpressing SPRLR exhibits the phenotype, whereas the transgenic line weakly expressing the transgene has a phenotype similar to that of control wild-type mice.

In the mammary gland, the LPRLR is highly expressed both at the end of pregnancy and during lactation. When the biological activity of each form of PRLR was assessed by transient transfection, we found that the long form was able to activate the ß-casein gene promoter, and the short form was inactive (13). Interestingly, the coexpression in vitro of both forms of PRLR resulted in a block of PRL signal to the milk protein gene promoter as a function of the concentration of SPRLR (15).

Milk protein gene expression is regulated by PRL. The level of milk protein mRNA in mammary tissue from immature virgin mice is at the threshold of detection. During pregnancy, expression levels increase several thousand-fold and remained high throughout lactation (30). ß-Casein and WAP genes are expressed in the mammary glands of lactating mice and represent mammary epithelial cell differentiation markers (31). Differential regulation of ß-casein and WAP genes by PRL has been reported. In Stat5a-deficient mammary tissues, the expression of ß-casein is not affected. WAP is the only milk protein with reduced expression in this model (27), which still expresses a reduced level of Stat5b. Both ß-casein and WAP gene and protein expressions were decreased in postpartum mammary tissue from line 3 transgenic mice, which overexpress the transgene, demonstrating a reduction of PRL signaling. In contrast to results obtained with the present model, when the mouse S1PRLR, with an intracellular sequence very different from that of the rat-F3 PRLR used in the present study, is overexpressed in heterozygous PRLR knockout mice, it is able to rescue the mammary phenotype regularly seen in heterozygous (+/-) animals at their first lactation (32). It will be important to understand the mechanisms involved in these two opposing models of mice overexpressing different short forms of PRLR in different backgrounds.

The presence of Stat5a is mandatory for mammopoiesis and lactogenesis. Milk protein genes have a {gamma}-interferon activation site (GAS) in their promoter regions, which is necessary for their expression in mammary cell lines (33) and in transgenic animals (34). Phosphorylated Stat5a is able to bind to this GAS site. Tyrosine phosphorylation of Stat5a is essential for dimerization, translocation to the nucleus, binding to the GAS site, and gene activation (35). The presence of phosphorylated Stat5a provides a biochemical indicator for the activity of this protein. Previous studies have shown that a sharp increase in Stat5a phosphorylation occurs at midpregnancy (36). Suppression of Stat5a phosphorylation in mammary glands of SPRLR transgenic mice (line 3) during gestation is consistent with the failure of mammary development and lactation seen in Stat5a KO mice (27). These observations again confirm that the PRL pathway is blocked in mammary glands of line 3 transgenic mice.

During the first half of pregnancy, ductal expansion occurs in the mammary gland as a result of cell division of the ductal epithelium in the end buds. The epidermal growth factor receptor system plays an important role at this stage of development (23), whereas the PRL system is implicated in the later stages of mammary gland differentiation. Maximal proliferation appears to occur in the alveolar progenitors in the ducts during the first few days of pregnancy (37). Here we found that a BrdU incorporation assay shows no difference in proliferation of mammary epithelial cells in wild-type vs. the two lines of transgenic mice at midgestation, confirming that activation of the PRL pathway is not required for ductal growth and side-branching (38). Furthermore, overexpression of rat F3-SPRLR does not induce proliferation of the mammary epithelial cells at this stage.

PRL plays important roles in normal mammary gland development and may also act during the induction and progression of breast cancer. PRLRs are widely expressed in human breast cancer and are up-regulated in tumor tissues (39). In 1972, a treatment of a small group of breast cancer patients with ergot drugs, which lower circulating levels of PRL, failed to show any beneficial effects (40), leading oncologists to the conclusion that PRL had no functional role in human breast cancer. More recently, it has been shown that PRL can act as an autocrine-paracrine factor within the breast. It is thus necessary to redefine the role of PRL in breast cancer by using new strategies, such as specific receptor antagonists, aimed at locally blocking the PRL/PRLR axis at the level of the mammary gland (41).

Using transgenic mice, the present study provides evidence that the short from of the rat PRLR behaves as a dominant negative regulator of the PRL system in vivo, and that the lack of PRL signaling in the mammary gland of mice results in impaired mammary development and lactation. Thus, this animal model can be viewed as a tool to better understand mammary development and the potential influence of PRLR signaling on the incidence of mammary tumors in specific model systems.


   Acknowledgments
 
We respectively thank Dr. H. L. Moses for kindly providing the MMTV-LTR transgene cassette, Dr. L. Hennighausen, for the ß-casein and WAP cDNA, and Dr. F. Talamantes for the polyclonal antiserum against the mouse PRLR. We also thank Dr. Alain Pezet and René Duchateau for their help in generating transgenic mice.


   Footnotes
 
This work was supported in part by a grant from the Association pour la Recherche sur le Cancer (France), grants from Institut National de la Santé et de la Recherche Médicale, a Yamagiwa-Yoshida Memorial UICC International Cancer Study grant (to M.E.), and a grant from la Ligue Nationale contre le Cancer (France; to E.S.).

Abbreviations: BrdU, Bromodeoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GAS, {gamma}-interferon activation site; KO, knockout; LPRLR, long form of PRL receptor; LTR, long terminal repeat; MMTV, mouse mammary tumor virus; PRLR, PRL receptor; SPRLR, short form of PRL receptor; Stat, signal transducer and activator of transcription; WAP, whey acidic protein.

Received October 8, 2002.

Accepted for publication February 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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