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Precocious Differentiation of the Virgin Wistar-Kyoto Rat Mammary Gland¹

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53792

Address all correspondence and requests for reprints to: Dr. Michael N. Gould, Department of Oncology, K4/334 Clinical Science Center, 600 Highland Avenue, Madison, Wisconsin 53792. E-mail: gould{at}humonc.wisc.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Wistar-Kyoto (WKY) rat strain expresses high levels of ß-casein in its virgin mammary glands. We found that the onset of ß-casein overexpression (BCO) occurs at 6 weeks of age, with morphological differentiation of the mammary gland detectable at 7 weeks of age. BCO was previously shown to be cell autonomous; however, we found that adrenal and ovarian hormones were permissive and necessary for the expression of the BCO phenotype, indicating that the genetic variation that initiates BCO from within the mammary epithelium can only manifest BCO in the presence of virgin hormone levels. Sequencing of the WKY and Wistar-Furth (WF) rat ß-casein promoters showed them to be identical. Culture of primary rat mammary epithelial cells (RMEC) under lactogenic conditions revealed that expression of ß-casein was independent of epidermal growth factor (EGF) in RMEC from virgin WKY_v, but was dependent in WF_v, RMEC. RMEC from a pregnant WF_p responded similarly to WKY_v RMEC, suggesting that EGF-independent ß-casein expression occurs naturally in differentiated rat mammary epithelium. However, induction of ß-casein expression in RMEC from immature WKY rats was also independent of EGF, indicating that the induction as well as maintenance of BCO do not require EGF. We suggest that an EGF-independent signaling pathway, arising from a trans-acting inherited effector(s), underlies BCO.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
EXPRESSION of the milk protein ß-casein is associated with differentiation of the mammary gland in response to pregnancy and lactation (1). The Wistar-Kyoto (WKY) rat strain was found to be unique in that it substantially expresses ß-casein in the virgin state. ß-Casein is the most abundant of all milk proteins (2) and serves as a marker for differentiation of the mammary gland. This premature differentiation has been shown to be a dominant trait, and cell transplantation assays strongly suggest that it is cell autonomous (3). In the latter assays, Wistar-Furth (WF) rat mammary epithelial cells (RMEC) were transplanted into the interscapular white fat pads of a (WKY x WF)F₁ rat and grown into ductal structures. Despite the presence of WKY hormone levels, the WF mammary glands at the graft sites did not accumulate ß-casein messenger RNA (mRNA). In contrast, similar transplantation of WKY RMEC resulted in expression of ß-casein mRNA at the graft sites.

The rat mammary gland consists of branching ducts that end in terminal end buds, lateral buds, and alveolar buds. At the onset of sexual maturity, rapid proliferation of the gland occurs under hormonal direction, resulting in elongation of the ducts into the fat pad (1). Full development of the gland occurs during pregnancy and lactation, when the terminal end buds and alveolar buds differentiate into extensive lobuloalveolar structures (4). Secretory epithelial cells line the lumina of these alveoli as well as the ducts (1), and surrounding myoepithelial cells perform a contractile function (5, 6). Initial expression of caseins occurs in the mid- to late pregnant gland and peaks during lactation (7); however, trace expression of ß-casein has been reported in the mammary glands of virgin mice cycled in the estrus stage of the estrous cycle (8), and we have occasionally seen similar expression in cycled virgin WF rats (Benton, M. E., and M. N. Gould, unpublished). Based on these data it is clear that abundant expression of ß-casein in the virgin WKY is an anomaly, and an understanding of its mechanism would contribute to a better understanding of the differentiation process in the rat mammary gland.

Alterations in cellular response mechanisms to lactogenic hormones and/or growth factors are probably responsible for ß-casein overexpression (BCO). Expression of ß-casein is regulated by glucocorticoids, insulin, and PRL, which influence the binding of nuclear factors to both positive and negative regulatory elements in the ß-casein gene promoter (9). In addition, this gene is regulated by laminin and tenascin (10, 11), which are components of the extracellular matrix, as well as by certain growth factors (12), including epidermal growth factor (EGF) (12, 13) and transforming growth factor-ß (TGFß) (14, 15). Stat5 (inclusive of homologs 5a and 5b) (16, 17), also known as mammary gland factor (MGF) and a member of the gene family of signal transducers and activators of transcription (Stat), is a nuclear transcription factor that binds to the ß-casein promoter and up-regulates expression by displacing Yy1, a transcriptional repressor (18). The PRL signal transduction pathway stimulates ß-casein expression in the mammary gland by inducing Stat5 activity (19). In addition to Yyl- and Stat5-binding sites, the proximal promoter also contains half-palindromic glucocorticoid receptor (GR)-binding sites and a binding site for CCAAT enhancer-binding proteins (C/EBP) (20). GR has been hypothesized to modulate C/EBP isoforms (20), and GR bound to its half-site has been shown to synergize with Stat5 (21), resulting in enhanced ß-casein expression.

Here, we more fully characterize the BCO phenotype both in vivo and in vitro. We show that RMEC from virgin WKY_v rats do not require EGF to initiate ß-casein expression, unlike RMEC from virgin WF rats as well as other rodents (12, 13), which require EGF for milk production. We also show that although the phenotype is mammary cell autonomous, it requires the presence of virgin ovarian and adrenal hormone levels to manifest BCO. Finally, we characterized the morphological and functional differentiation of the virgin WKY mammary gland between the ages of 4–24 weeks to understand the chronology of this unique phenomenon.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Phenol red-free (PRF)-RPMI 1640 was purchased from Life Technologies (Grand Island, NY). Aprotinin, benzamidine, phenylmethylsulfonylfluoride, alum potassium sulfate, carmine (catalogue no. C-6152), fraction V and fatty acid-free BSA, 3,3'-diaminobenzidine peroxidase substrate tablet set, 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS), rabbit antiactin antibody (catalogue no. A-2066), goat antirabbit IgG linked to alkaline phosphatase (catalogue no. A-3812), PRF-DMEM/Ham’s nutrient F-12 (DMEM/F12), hydrocortisone, human transferrin, progesterone, ascorbic acid, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO). FBS was purchased from HyClone Laboratories, Inc. (Logan, UT). Leupeptin was purchased from Calbiochem (La Jolla, CA). Nitex membrane was obtained from Tetko (Depaw, NY). MatriSperse, growth factor-reduced Matrigel, mouse EGF, and bovine insulin were purchased from Collaborative Biomedical (Bedford, MA). Gentamicin and Sequenase 2.0 were obtained from U.S. Biochemical Corp. (Cleveland, OH). Ovine PRL was a gift from Dr. A. F. Parlow through the National Hormone and Pituitary Program, NIDDk (Bethesda, MD). The bicinchoninic acid (BCA) assay was obtained from Pierce Chemical Co. (Rockford, IL). Enzyme catalyzed fluorescence substrate was purchased from Amersham (Arlington Heights, IL). Immobilon-P membrane (polyvinylidene difluoride) was obtained from Millipore Corp. (Bedford, MA). The FluorImager, PhosphorImager, and ImageQuant software were purchased from Molecular Dynamics, Inc. (Sunnyvale, CA). OmniFix II was obtained from An-Con Genetics (Melville, NY). The Vectastain Elite ABC reagent and goat antirabbit secondary antibody were purchased from Vector Laboratories, Inc. (Burlingame, CA). Toluidene blue was obtained from Fluka Chemical Co. Corp. (Ronkonkoma, NY). Permount was obtained from Fisher Scientific International, Inc. (Pittsburgh, PA). Restriction endonucleases and the pSP73 plasmid were purchased from Promega Corp. (Madison, WI). ß-Casein and ß-actin primers were synthesized by the University of Wisconsin-Madison Biotechnology Center.

Animals
Virus-free, virgin, female WKY and WF rats were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). For the pregnancy studies rats were bred in our animal facility, and the length of gestation was judged by the appearance of vaginal plugs, designated day 1 of pregnancy. Female CD2F₁ mice were purchased from Charles River Laboratories, Inc. (Wilmington, MA). Rats and mice were given rat or mouse chow (Teklad, Madison, WI) and acidified water ad libitum. Adrenalectomized rats were given half-physiological saline in addition to acidified water. All animals were housed under a 12-h light, 12-h dark cycle and were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All protocols were approved by the University of Wisconsin-Madison institutional animal care and use committee.

Production of rat ß-casein antibody
A peptide incorporating amino acid residues 55–70 (EDVLQNKFHSGIQSEP) of rat ß-casein was synthesized by Research Genetics, Inc. (Huntsville, AL). Two New Zealand White male rabbits (Covance, Denver, CO) were immunized with 1 mg of the peptide and boosted twice, at 4-week intervals, with 0.5 mg antigen. One rabbit developed a strong response and was boosted every 3–4 months to maintain a high titer.

Whole mounting and staining of rat mammary gland
Abdominal mammary glands were removed and spread onto clean glass slides. Slides were immersed in 70% ethanol for up to 1 week for defatting. Tissue was fixed for 1 h in glacial acetic acid and 100% ethanol (1:3) and then rinsed in 70% followed by 50% ethanol. The glands were rinsed in distilled water and then immersed in filtered alum carmine stain (0.5% alum potassium sulfate and 0.2% carmine boiled in water and cooled to room temperature) for 3–4 days. Glands were dehydrated with 70%, 95%, and 100% ethanol for 30 min each, soaked in xylene overnight, and then stored in mineral oil. Whole mounts were viewed with an Olympus Corp. SZH10 dissecting microscope (Leeds Precision Instruments, Minneapolis, MN). Differentiation was determined by the development of lobuloaveolar clusters.

Immunohistochemical analysis for ß-casein in rat mammary gland
Inguinal (cranial) mammary tissue was fixed for 24 h in Omnifix II and then transferred to 70% ethanol. The tissue was embedded in paraffin, and 3-µm sections were mounted on poly-L-lysine-coated glass slides. The sections were deparaffinized with xylene and rehydrated in a graded series of ethanol. Tissue was blocked for 1 h with PBS containing 5% calf serum and 1% BSA and then incubated 1 h with rat ß-casein antiserum diluted 1:1000 in PBS containing 1% BSA. Preimmune serum, diluted 1:1000, was used as a negative control. Samples were rinsed in PBS and then treated with 1% H₂O₂ in PBS (vol/vol) for 15 min. Tissue was incubated with biotinylated goat antirabbit secondary antibody at a 1:200 dilution in 1% BSA/PBS. After rinsing in PBS, tissue was incubated for 30 min with Vectastain Elite ABC Reagent. Color was developed with diaminobenzidine stain for 8 min and then counterstained for 2 min with toluidene blue. Tissue was dehydrated in xylene and mounted with Permount (Fisher Scientific, Fairlawn, NJ). Slides were viewed using an Olympus BX50 microscope (Leeds Precision Instruments, Minneapolis, MN).

Electron microscopy
Inguinal (cranial) mammary glands from 13-week-old WKY and WF rats were removed and placed in 3% glutaraldehyde on dental wax. Approximately 1-mm cubes of tissue were cut and immersed in 3% glutaraldehyde in cacodylate buffer (0.1 M cacodylate, pH 7.4, at room temperature). Tissue was fixed for 2 h and then washed three times, for 20 min each time, with 0.1 M cacodylate buffer containing 7.5% sucrose. Tissue was postfixed with 1% OsO₄ in 0.1 M cacodylate buffer on ice for 1.5 h. Tissue was then dehydrated in a graded series of cold ethanol (35%, 50%, and 75% for 10 min each) followed by room temperature ethanol (95% for 10 min and then 100% 3 times for 30 min). Tissue was then infiltrated in a 1:1 mixture of propylene oxide-Eponate 12 for 1 h. Eponate 12 was added, and infiltration was carried out overnight. Tissue was subsequently embedded in fresh Eponate. Thin sections were cut with a diamond knife on a Reichert Ultracut E ultramicrotome (Leica Microsystems, Inc., Buffalo, NY) and stained with uranyl acetate and lead citrate. Electron micrographs were taken with a Hitachi H-7000 electron microscope (Hitachi Scientific Instruments, Inc., Tokyo, Japan) operated at 75 kV.

Preparation of mammary tissue for immunoblot
Rats were killed, and 1-cm² sections of inguinal mammary gland were removed and frozen in liquid nitrogen. Tissue was thawed in PBSTDS [10 mM Na₂HPO₄, 154 mM NaCl, 12 mM deoxycholic acid, 1 mM NaF, 3.5 mM SDS, 31 mM NaN₃, and 1% (vol/vol) Triton X-100, pH 7.25, adjusted with 1 M NaH₂PO₄] containing 100 µg/ml phenylmethylsulfonylfluoride, 1 mM benzamidine, 100 µg/ml leupeptin, 25 µg/ml aprotinin, 1 mM sodium vanadate, and 10 mM EDTA. Tissue was homogenized with a Polytron (Brinkmann Instruments, Inc., Westbury, NY), and the homogenates were centrifuged for 15 min at 16,000 x g. The clarified supernatant was carefully removed from below the fat layer and then centrifuged again for 10 min to remove any remaining fat. Samples were boiled in SDS-sample buffer and frozen at -80 C before analysis. Protein concentrations were determined using the BCA protein assay.

Gel electrophoresis and immunoblot analysis
Cultured cells were normalized to cell number using MTT assay data, and mammary tissue samples were normalized to protein content using the BCA protein assay. All samples were resolved on 12.5% SDS-PAGE gels (22) and electroblotted to Immobilon-P membrane for 1.5 h at 270 mA in 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) buffer [10 mM CAPS and 10% (vol/vol) methanol, pH 11] (23). Membranes were blocked overnight in Tris-buffered saline plus Tween [TBST; 25 mM Tris (pH 7.5), 0.9% NaCl, and 0.1% (vol/vol) Tween-20] containing 2% BSA. The blots were probed for 1 h with a 1:5,000 dilution of antirat ß-casein polyclonal antiserum along with a 1:500 dilution of an affinity-purified actin polyclonal antibody (a loading control) in TBST containing 1% BSA. After rinsing in TBST, each blot was subsequently incubated for 1 h with a 1:10,000 dilution of antirabbit IgG linked to alkaline phosphatase in 1% BSA/TBST. Rinsed blots were exposed to enzyme catalyzed fluorescence substrate for 20 min. Blots were scanned using a FluorImager and viewed with ImageQuant software.

Preparation of Engelbreth-Holm-Swarm (EHS)-derived extracellular matrix
EHS sarcoma was provided by Dr. Margot Ip and implanted into both hindlimbs of mature, 4- to 6-week-old, female CD2F₁ mice. Tumors were harvested every 3 weeks, frozen in liquid nitrogen, and stored at -80 C; at the same time, fresh tumor was passaged into a fresh group of mice. EHS-derived extracellular matrix was extracted essentially as described by Hahm and Ip (24) and was stored at -40 C.

Isolation of primary RMEC
Abdominal and inguinal mammary glands were removed from female virgin rats (ages indicated in the text) or from 14-day pregnant rats. RMEC were prepared as described by Hahm and Ip (24) with some modifications. Briefly, mammary gland was removed from three or four rats per strain for each experiment, except where noted. After enzymatic dispersion of the gland (24), clusters of RMEC were obtained. These clusters are referred to by some investigators as organoids or ductal fragments, but throughout this paper they will be referred to as RMEC. RMEC were pelleted by centrifugation, washed twice with PRF-RPMI 1640, and collected on a 53-µm Nitex membrane, thereby removing single cells. The RMEC were washed with PRF-RPMI 1640 and rinsed off the membrane with PRF-DMEM/F12 containing 5% FBS and 50 µg/ml gentamicin. The isolated cells were pelleted at 300 x g for 5 min, resuspended in 10 ml PRF-DMEM/F12 plus 5% FBS, and then incubated for 2 h at 37 C in a 10-cm tissue culture dish to allow for the attachment and subsequent removal of fibroblasts from the unattached RMEC. RMEC number was determined from duplicate counts of cell nuclei (24).

Primary culture conditions
Isolated RMEC were plated in EHS-derived matrix or in commercially available growth factor-reduced PRF-Matrigel (diluted 1:1 with PRF-DMEM/F12) as previously described (24). Briefly, RMEC were pelleted at 300 x g for 5 min, rinsed in serum-free PRF-DMEM/F12, and suspended in chilled matrix at a density of 1.5 x 10⁶ cells per ml matrix. Approximately 200 µl of the mixture, containing 3 x 10⁵ cells, were plated on top of a 200-µl base of presolidified matrix in 24-well tissue culture plates. The matrix was solidified for 2 h at 37 C before the addition of 1 ml culture medium to each well. Cells were cultured for 4 or 6 days, and medium was changed every 2 days. The optimal serum-free medium used in these studies was developed by Hahm and Ip (24) and contained PRF-DMEM/F12 medium with 1 µg/ml ovine PRL, 10 ng/ml mouse EGF, 1 µg/ml hydrocortisone, 10 µg/ml bovine insulin, 5 µg/ml human transferrin, 1 µg/ml progesterone, 5 µM ascorbic acid, 1 mg/ml fatty acid-free BSA, and 50 µg/ml gentamicin.

Cell harvest
Medium was aspirated from each well, and the wells were rinsed with PBS. The cell/matrix plugs were removed with a p1000 pipette tip, cut off at the tip to widen the opening and prevent shearing of the cells, and transferred to 1.7-ml microfuge tubes. One milliliter of MatriSperse was added to each plug, and the samples were rotated on a rotating arm at 4 C for 1 h to facilitate depolymerization of the matrix. RMEC were centrifuged for 3 min at 735 x g, rinsed in PBS, boiled in 100 µl SDS-sample buffer, and frozen at -80 C. Each experiment was repeated using cells isolated from different groups of animals, and the same trends were observed.

MTT assay
Cell number was determined by a modification of an MTT-based cell proliferation assay that was previously described (25). Briefly, RMEC treated comparably to RMEC used for ß-casein expression analysis were quantified by adding 0.5 mg MTT (100 µl of a 5 mg/ml solution in PRF-RPMI 1640)/ml medium, and the cultures were incubated for 14–15 h at 37 C. Each sample condition was analyzed in duplicate. After removal of the medium, each culture well was rinsed with 1 ml PBS, and then 1 ml 1% (wt/vol) grade II dispase in PBS was added. The EHS-derived matrix was disrupted with a pipette tip and digested for 1 h at 37 C. The RMEC were transferred to 2.2-ml microfuge tubes and centrifuged for 6 min at 16,000 x g. The pellets were dissolved in 1 ml isopropanol, and debris was removed via centrifugation at 16,000 x g for 2 min. The color was analyzed on an ELISA reader (SLT-Labinstruments, Groedig, Austria) at 570 nm while subtracting the background absorbance at 690 nm. A standard curve was developed with freshly isolated RMEC, in triplicate, at the onset of each experiment.

Cloning and sequencing of the rat ß-casein promoter
A rat ß-casein genomic DNA fragment (-524 to +191) was PCR amplified from WKY and WF rats. The sequences of ß-casein oligonucleotides (underlined) used for PCR, including BglII or SacI linkers, were as follows: rat casein-A1, 5'-AACCAGATCTCCTTTTCTCACTTGTTCTAA; and rat casein-R1, 5'-TACCGAGCTCCTGTCCTTGAAAATAATCTC. The PCR conditions were 94 C for 1 min, then 30 cycles of 94 C for 1 min, 49 C for 1 min, 72 C for 2 min and 30 sec, followed by 72 C for 4 min. The PCR products were digested with BglII and SacI, then directionally cloned into the plasmid, pSP73. Multiple clones of the recombinant ß-casein-pSP73 plasmids were sequenced by the dideoxy chain termination method with Sequenase 2.0 following the manufacturer’s protocol.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
ß-Casein is overexpressed in virgin and early pregnant WKY mammary gland
Western blot analysis of ß-casein in different developmental stages of the rat mammary gland demonstrated abundant expression of ß-casein in 10- to 11-week-old virgin WKY rats, and these levels increased steadily throughout pregnancy and into lactation (Fig. 1, A and B). In contrast, the WF virgin and early (5-day) pregnant glands were essentially devoid of ß-casein (Fig. 1A). Expression of the milk protein was initially observed in the pregnant WF on day 10 of gestation, and these levels rapidly increased, such that by day 15 of pregnancy (Fig. 1A) as well as through late pregnancy and early lactation (Fig. 1B), both WKY and WF rats expressed equivalent amounts of the milk protein.

View larger version (34K): [in this window] [in a new window]   Figure 1. Western blot showing ß-casein expression in different developmental stages of the WKY and WF rat mammary glands. A, Mammary tissue from 10- to 11-week-old virgin (V) and 5-, 10-, and 15-day-pregnant (P) rats. Five micrograms of total protein were loaded into each lane. B, Mammary tissue from 18-day pregnant and 2-day lactating (2L) rats. One microgram of total protein was loaded into each lane. Rat milk was used as a positive control, and actin was used as a loading control. C, Western blot control of mammary tissue. Antiserum preabsorbed with rat ß-casein peptide immunogen (residues 55–70) and preimmune serum were used to show the specificity of the antirat ß-casein antibody. Lanes 1 and 6, Eighteen-day pregnant WKY; lanes 2 and 7, 2-day lactating WKY; lanes 3 and 8, 18-day pregnant WF; lanes 4 and 9, 2-day lactating WF; lanes 5, 10, and 11, rat milk. Each lane contains 1 µg total protein. Lane 11 was probed with ß-casein antiserum as a positive control.

Virgin WKY mammary gland is morphologically more differentiated than control, virgin WF mammary gland
To define the morphological development of the WKY mammary gland, whole mounts were prepared from the abdominal mammary glands of rats ranging in age from 4–24 weeks. Age-matched WF rats were used as controls. The degree of differentiation of each gland was scored from 0 (no differentiation) to 2++ (extensive differentiation). As shown in Table 1, the glands in both rat strains were undifferentiated at 4 and 6 weeks of age and appeared identical. At 7 weeks of age, a small amount of differentiated epithelium was apparent in a few of the WKY glands (2 of 10). This differentiation was localized to central ductal regions. By 8 weeks of age, similar localized development was seen, except it was more widespread (4 of 6 rats were positive). Figure 2A illustrates the extent of epithelial differentiation in a 12-week-old WKY gland compared with that in an age-matched WF gland (Fig. 2B). The alveolar sacs in the WKY are clearly much larger and more dense than those in the WF. At this age, differentiation was more extensive, encompassing the entire gland, and was observed not only in all 12-week-old WKY glands, but also in all 18- and 24-week-old WKY glands. Occasionally, WF glands exhibited a differentiated morphology; however, this was only seen in 2 of 10 rats each at 7 and 12 weeks of age.

View larger version (85K): [in this window] [in a new window]   Figure 2. Whole mounts of 12-week-old virgin WKY (A) and WF (B) mammary glands. Glands were visualized with a dissecting microscope at x25 magnification. The levels of ß-casein in WKY (C) and WF (D) mammary glands were determined by immunohistochemical analysis. Tissue sections of the mammary glands shown in A and B were analyzed for ß-casein expression with an antirat ß-casein antibody. Tissue sections were visualized with a light microscope at x100 magnification. Rats were in the estrus stage of the estrous cycle.

View larger version (135K): [in this window] [in a new window]   Figure 3. Electron microscopy of 12- to 13-week-old virgin WKY (A) and WF (B) mammary glands. Rats were in diestrus of the estrous cycle. Epithelial cells (E) line the ductal lumens (L), and myoepithelial cells (ME) surround them. Key features supporting a differentiating phenotype include secretory granules (SG) carrying milk proteins, microvilli (MV), and fat droplets (F). Magnification, x2800 (A) and x2500 (B).

View larger version (55K): [in this window] [in a new window]   Figure 4. Western blot showing the effect of ovariectomy (OVX) on ß-casein expression in WKY rat mammary gland. A, Rats were ovariectomized before the onset of BCO, at 4 weeks of age, and killed along with age-matched control rats at 10 weeks of age. B, Rats were ovariectomized at 10 weeks of age, after the onset of BCO, and were killed along with age-matched control rats 3 weeks later. Each lane contains 5 µg total mammary protein. Rat milk was used as a positive control for ß-casein.

View larger version (54K): [in this window] [in a new window]   Figure 5. Western blot showing the effect of adrenalectomy on ß-casein expression in WKY rat mammary gland. Rats were age matched and killed at 12 weeks of age, 10 days after adrenalectomy. All lanes contain 5 µg total mammary protein. Rat milk was used as a positive control, and actin was used as a loading control.

Expression of ß-casein is higher in WKY_v vs. WF_v RMEC cultured in vitro under lactogenic conditions
To study specific hormone and growth factor interactions in the WKY and WF mammary glands, an extracellular matrix-based cell culture system was adapted (24) that promotes the growth and differentiation of primary RMEC in a defined, serum-free medium. The matrix used was a reconstituted basement membrane matrix prepared in our laboratory using tumors derived from the EHS mouse sarcoma. RMEC from virgin rats were embedded in this EHS-derived matrix and cultured in estrogen-free optimal medium that, in addition to progesterone and EGF, contained the lactogenic hormones PRL and insulin as well as the lactation-promoting steroid hormone hydrocortisone. Under these conditions, ß-casein was detectable in WF_v RMEC within 4 days (Fig. 6A). At this time point, WKY_v RMEC were found to produce approximately 20-fold higher levels of ß-casein (normalized to actin) than WF_v RMEC (Fig. 6A, lanes 1 vs. 5). Although ß-casein was seen in virgin WKY mammary gland before tissue dispersion, it was not present upon initial plating of the cells, indicating that most of the intracellular ß-casein was degraded during cell dispersion of the mammary tissue (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 6. A, Effect of EGF on ß-casein expression in WKYv vs. WFv RMEC. The Western blot shows ß-casein levels in RMEC from 8-week-old virgin WKY and WF rats that were cultured for 4 days in EHS-derived matrix with serum-free, lactogenic medium containing the concentrations of EGF shown. Detectable levels of ß-casein expression in the WF RMEC are noted by arrows. B, Effect of EGF on proliferation of RMECv. Cell growth was measured using a MTT assay carried out in wells treated comparably and in parallel to those in A. Each data point represents the mean ± SEM of duplicate wells.

View larger version (53K): [in this window] [in a new window]   Figure 7. Effect of EGF on ß-casein expression when cells are cultured in growth factor-reduced Matrigel. RMEC from 10- to 11-week-old virgin WKY and WF rats were cultured for 6 days in commercial Matrigel with serum-free, lactogenic medium containing less than 0.5 ng/ml EGF. The Western blot shows ß-casein expression in duplicate wells cultured with or without exogenous EGF (10 ng/ml) as shown.

ß-Casein expression and cell growth are dependent on PRL in both WKY_v and WF_v RMEC
PRL is a lactogenic hormone that stimulates ß-casein transcription (19). A possible effect of PRL on BCO was examined by culturing RMEC in EHS-derived matrix in the absence and presence of 0.2–1 µg/ml PRL for 4 days (Fig. 8A). Western blot analysis revealed dose-dependent expression of ß-casein in WF_v RMEC, with detectable accumulation at and above 0.4 µg/ml. Like WF_v RMEC, WKY_v RMEC did not express the milk protein in the absence of PRL; however, abundant accumulation of the milk protein was detected at 0.2 µg/ml PRL, and similar expression levels were seen at all higher concentrations tested. The effect of suboptimal concentrations of PRL on BCO was also tested (Fig. 9). WKY_v RMEC were cultured in growth factor-reduced Matrigel in the presence and absence of EGF with less than 0.1 µg/ml PRL for 6 days. The expression of ß-casein was detectable and dose dependent on PRL at and above 0.05 µg/ml PRL in the presence of EGF and at and above 0.075 µg/ml PRL in the absence of the growth factor. Parallel measurements of cell proliferation revealed that the growth of both WKY_v and WF_v RMEC occurred at the same rate and was dose dependent on PRL (Fig. 8B).

View larger version (25K): [in this window] [in a new window]   Figure 8. A, Effect of PRL on the expression of ß-casein in RMECv. The Western blot shows ß-casein levels in RMEC from 8-week-old virgin WKY and WF rats that were cultured for 4 days in EHS-derived matrix with serum-free, lactogenic medium containing the concentrations of PRL shown. B, Effect of PRL on the proliferation of RMECv. Cell growth was measured using a MTT assay carried out in wells treated comparably and in parallel to those in A. Each data point represents the mean ± SEM of duplicate wells.

View larger version (61K): [in this window] [in a new window]   Figure 9. Effects of suboptimal concentrations of PRL on ß-casein expression in RMEC. RMEC from 10- to 11-week-old virgin rats were cultured for 6 days in growth factor-reduced Matrigel, with or without 10 ng/ml EGF. Serum-free, lactogenic medium was used, containing the concentrations of PRL indicated. The Western blot shows the expression of ß-casein.

View larger version (34K): [in this window] [in a new window]   Figure 10. Effects of EGF (A) and PRL (B) on ß-casein expression in RMECp. RMEC were prepared from 14-day midpregnant WKY and WF rats (one rat of each strain). Cells from 12-week-old, virgin, age-matched rats were used as controls. The Western blot shows ß-casein levels in RMEC that were cultured in EHS-derived matrix with serum-free, lactogenic medium containing the concentrations of EGF or PRL as shown. C and D, Effects of EGF and PRL, respectively, on proliferation of WKYp and WFp RMEC. Cell growth was measured using a MTT assay carried out in wells treated comparably and in parallel to those in A and B, respectively. Each data point represents the mean ± SEM of duplicate wells.

View larger version (52K): [in this window] [in a new window]   Figure 11. Effect of EGF on ß-casein expression in RMEC from immature WKY and WF rats. The Western blot shows ß-casein levels in RMEC from age-matched, 4-week-old WKY and WF rats (12 rats in each pool) that were cultured for 4 days in EHS-derived matrix with serum-free lactogenic medium containing the concentrations of EGF shown.

View larger version (38K): [in this window] [in a new window]   Figure 12. A, Effect of progesterone (Pg) on ß-casein expression in WKYv and WFv RMEC. The Western blot shows ß-casein levels in RMEC from 10-week-old virgin rats that were cultured for 4 days in growth factor-reduced Matrigel. Serum-free, lactogenic medium was used with and without 1 µg/ml progesterone and 10 ng/ml EGF. B, Effect of 1 µg/ml progesterone (P) and 10 ng/ml EGF (E) on the proliferation of RMECv. Cell growth was measured using a MTT assay carried out in wells treated comparably and in parallel to those in A. Each data point represents the mean ± SEM of triplicate wells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Preliminary characterization of this phenotype previously revealed that expression of ß-casein in the WKY is mammary epithelial cell autonomous (3). In that study a transplantation assay was performed in which WF mammary epithelial cells were transplanted into the interscapular white fat pads of both WF and (WKY x WF)F₁ hosts. Transplanted cells grew into mammary ductal structures, which, in both hosts, were not found to contain significant levels of ß-casein mRNA. This indicates that WF RMEC cannot respond to a virgin WKY hormonal environment. In contrast, similar studies with WKY donor cells resulted in substantial ß-casein mRNA accumulation in an (WKY x WF)F₁ rat. We extended this study to determine whether the cell autonomous inherited factor(s) responsible for EGF-independent ß-casein expression in the WKY MEC could also obviate the need for circulating ovarian and adrenal hormones that normally support ß-casein expression in pregnancy and lactation.

To determine the effects of adrenal and ovarian hormones on the BCO phenotype, we performed ablation experiments. We found that ovariectomy of immature WKY rats prevented the onset of BCO, and ovariectomy of sexually mature WKY rats abolished BCO. Thus, functional ovaries are necessary for the WKY to initiate and maintain the BCO phenotype. Like the ovaries, the adrenals have also been shown to be essential for casein gene expression in the rat (1, 29), and a glucocorticoid signaling pathway has been identified that directly stimulates the ß-casein promoter (21). After adrenalectomy of mature, ß-casein-producing WKY rats, the phenotype was not observed, indicating that adrenal hormones must be present to maintain BCO. Furthermore, hematoxylin and eosin staining of paraffin sections of the mammary gland revealed that control WKY rats had multiple alveolar sacs full of milk, whereas the ADX rats displayed diminished alveolar development, and the remaining alveolar sacs were collapsed and devoid of milk (data not shown). These data suggest that the virgin levels of ovarian and adrenal hormones in virgin WKY rats are permissive and required for the BCO phenotype.

To identify inherited variations in the WKY that induce precocious expression of ß-casein, we initially examined the promoter elements of the ß-casein gene in WKY and WF rats. The proximal promoters of both strains were sequenced and found to be identical. Furthermore, genetic mapping of the BCO phenotype did not reveal a quantitative trait locus in the region of the casein gene family; hence, distal cis-elements are not linked to BCO (Benton, M. E., and M. N. Gould, unpublished). Together these data probably indicate that one or more trans-acting factors underlie the phenotype.

To identify signal transduction pathways affecting BCO, a primary cell culture system using EHS-derived matrix was adapted (24) that allowed us to study factors differentially influencing ß-casein expression in RMEC from virgin WKY and WF rats. The cells were cultured in a defined serum- and estrogen-free medium containing lactogenic hormones and the growth factor EGF. The expression of ß-casein was independent of EGF in RMEC from sexually mature virgin WKY rats, in which production of the milk protein in culture is a maintained phenomenon. In contrast, we found that EGF, in addition to lactogenic hormones, is clearly required for the accumulation of ß-casein in WF_v RMEC, where expression of the milk protein is an induced phenomenon. These data identified a clear difference between the two rat strains. Interestingly, RMEC from a midpregnant WF, which had initiated milk production, were found to produce ß-casein independently of EGF. These data indicate that EGF-independent expression of ß-casein is a molecular event normally induced by pregnancy but that exists prematurely in the virgin WKY. At this point it was not clear whether EGF-independent signaling was a primary inherited consequence of the BCO phenotype or merely a secondary response to differentiating mammary gland. To identify an inherited role for EGF-independent signaling in BCO, RMEC from uninduced (i.e. non-ß-casein-producing) immature WKY rats, were cultured under lactogenic conditions both with and without exogenous EGF. Expression of the milk protein was equivalent at all but the highest concentration of EGF tested. These data clearly show that BCO arises from an inherited alteration resulting in EGF-independent signaling. This alteration may lie within the EGF or an EGF-related signal transduction pathway. Alternatively, it may result from altered signaling in another pathway. We will consider the former possibility here.

Whether EGF is a direct acting lactogen or simply primes mammary cells to respond to lactogenic hormones is currently controversial. Several studies have demonstrated a role for EGF in promoting MEC competence for differentiation. Maximal induction of ß-casein gene expression and/or casein synthesis have been observed when HC11 mouse MEC (12) and midpregnant mouse mammary gland explants (30, 31) were cultured in the presence of EGF and then subsequently cultured in medium containing PRL, hydrocortisone, and insulin. EGF-induced competency for differentiation has been linked to modulation of the extracellular matrix glycoproteins laminin and tenascin-C. EGF has been shown to alter the assembly of endogenous laminin, which stimulates ß-casein expression (10, 32), and to down-regulate tenascin-C, an inhibitor of ß-casein expression in HC11 cells, thereby committing the cells to respond to lactogens (11). Interestingly, EGF has been shown to inhibit ß-casein mRNA levels when added simultaneously with lactogenic hormones to primary mouse MEC cultured in EHS-derived matrix (33) and to HC11 cells (34). These mouse MEC studies are clearly different from the rat system used here, where EGF is shown to promote ß-casein accumulation in the presence of lactogenic hormones. Furthermore, RMEC from CD rats cultured in the same system that we have described here have also been shown to accumulate ß-casein in the presence of EGF (13, 24); interestingly, this lactogenic effect could not be reversed by removing EGF from the lactogenic medium (13). Based on the latter finding, EGF probably serves a competency role in our system, such that the EGF pathway simply primes WKY RMEC to respond to lactogens. A role for EGF as a direct acting lactogen cannot be ruled out, however, as EGF transcripts have been observed in the lactating mammary gland (35), and plasma levels of EGF have been shown to be elevated during lactation, at which time the growth factor can be found in milk (36).

EGF is also a potent mitogen. It has been shown to induce extensive alveolar and multilobular branching morphogenesis of RMEC cultured in EHS-derived matrix under lactogenic conditions (13), to promote ductal development in virgin mice (37), and to stimulate lobuloalveolar development and epithelial cell proliferation in whole organ culture of mouse mammary gland (38). In our culture system, the proliferation of both WF and WKY RMEC was found to be dose dependent on EGF. These data indicate that EGF-induced proliferation and differentiation signaling pathways either bifurcate or are independent from one another. This concept is not new. EGF regulation of ß-casein expression and mammary cell growth in the mouse has been reported to occur by independent mechanisms (33).

EGF is one of several growth factors that signal through the same pathway. EGF as well as TGF and amphiregulin are synthesized in virgin, pregnant, and lactating mammary epithelia (35, 37), and signal through the cell surface-bound EGF receptor (37, 38). Upon binding, receptor dimerization, activation of tyrosine kinase activity, and receptor autophosphorylation take place, inducing multiple signaling pathways (38). Very little is known about the signaling mechanisms of EGF in the rodent mammary gland; EGF in combination with insulin was shown to increase the activity of a double stranded DNA-binding multiprotein complex in pregnant mice that is critical for lactogenic hormone-induced ß-casein gene transcription (39), and a study performed in mouse liver found that EGF induces tyrosine phosphorylation and nuclear translocation of Stat5 (40), a transcriptional activator of the ß-casein gene in mammary gland. However, studies carried out with HC11 cells have shown that EGF suppresses Stat5 activity (41).

EGF-like, but EGF receptor-independent, signaling pathways may also account for BCO. These include the EGF-like growth factors Cripto-1 and Neu differentiation factor/heregulin, which have been shown to promote responsiveness of HC11 cells to lactogenic hormones (42, 43). A polymorphism in one or more of these EGF-like pathways could result in EGF-independent signaling in WKY RMEC and promote BCO.

PRL signaling does not appear to influence BCO. The lactogenic hormone was required for ß-casein expression in cultured WKY_p and WKY_v RMEC, although much lower concentrations of the hormone were necessary to induce ß-casein expression in WKY RMEC compared with WF RMEC. PRL signals through Stat5 in the mammary gland; the PRL receptor dimerizes in response to PRL, and the receptor-associated tyrosine kinase, Jak2, subsequently phosphorylates Stat5, thereby activating the transcription factor to translocate to the nucleus, where it stimulates ß-casein gene transcription (44). Based on our culture data, the response of WKY cells to PRL is enhanced relative to that of WF cells; however, this may be a secondary effect resulting from the primary mechanism underlying the phenotype. Furthermore, immunoprecipitation of the tyrosine-phosphorylated proteins from 13-week-old WKY and WF mammary tissue followed by Western blot analysis with a Stat5 antibody revealed equivalent levels of the activated transcription factor in both strains (data not shown). In combination, these data indicate that the PRL pathway is not altered in the WKY. A complete understanding of the mechanism underlying BCO will require further investigation. In addition to biochemical studies, we are currently using genetic linkage analysis to identify primary inherited effectors of the BCO phenotype.

	Acknowledgments

 
We thank Jane Barnes for adrenalectomizing and ovariectomizing the rats.

	Footnotes

 
¹ This work was supported by a grant from the DHHS, NIH Grant CA-77494, as well as Postdoctoral Training Grant CA09471.

Received July 28, 1998.

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