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Endocrinology, doi:10.1210/en.2005-1082
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Endocrinology Vol. 147, No. 4 1819-1829
Copyright © 2006 by The Endocrine Society

Autocrine Growth Hormone Prevents Lactogenic Differentiation of Mouse Mammary Epithelial Cells

Svetlana Mukhina, DongXu Liu, Ke Guo, Mireille Raccurt, Sahra Borges-Bendris, Hichem C. Mertani and Peter E. Lobie

Institute of Molecular and Cell Biology (S.M., K.G.), 117609 Singapore; Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5123, Physiologie Moléculaire (M.R., S.B.-B., H.C.M.), 696222 Villeurbanne Cedex, France; and Liggins Institute (D.X.L., P.E.L.), University of Auckland, Private Bag 92019, Auckland, New Zealand

Address all correspondence and requests for reprints to: Peter E. Lobie, M.D. Ph.D., Liggins Institute, University of Auckland, 2–6 Park Avenue, Private Bag 92019, Auckland, New Zealand. E-mail: p.lobie{at}auckland.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have examined the expression, postnatal ontogeny, and localization of mouse GH (mGH) and its relative expression during pregnancy, lactation, and weaning in the mouse. mGH mRNA and protein was expressed predominantly in the epithelial component of the mammary gland, and maximal expression was observed during the pubertal period. Autocrine mGH expression dramatically decreased during late pregnancy and lactation. Concordantly, autocrine mGH expression is repressed during forced differentiation of mouse HC11 mammary epithelial cells in culture. Forced expression of mGH in HC11 cells abrogated lactogenic differentiation as indicated by reduced expression of ß-casein and reduced expression and loss of lateral epithelial localization of E-cadherin. Forced expression of mGH in mouse mammary epithelial cells increased cell survival and proliferation and consequently increased the size of mammary acinar-like structures formed in three-dimensional Matrigel. Thus, autocrine mGH expression in the mouse mammary epithelial cell is maximal at puberty and prevents mammary epithelial cell differentiation. Autocrine GH will therefore participate in mammary morphogenic processes at puberty.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DEVELOPMENT OF the mammary gland proceeds in distinct stages and is regulated by various factors at each stage. In newborn murines, the mammary rudiment is represented by a small tree of primary ducts ending in small terminal end buds (TEBs). During ductal morphogenesis, which occurs after the onset of puberty at about 4 wk of age, increased mitotic activity in the TEBs causes the ducts to penetrate the stroma (1). Together with an increased rate of branching, it results in filling of the fat pad with the developed epithelial system. When ducts have reached the edge of the fat pads or become surrounded by other ducts, proliferation in TEBs is inhibited (2). Pregnancy hormones induce intensive alveolar proliferation and additional ductal branching, which leads to the filling of the interductal spaces with epithelial acinar structures called lobuloalveoli, the milk-producing elements of the mammary gland. Functional differentiation is accomplished with parturition and lactation (1, 3). After weaning, secretory epithelial cells are lost by apoptosis and the mammary gland undergoes involution (4).

Mammary gland development and lactogenesis are controlled by the functional interaction of systemic and local steroid and polypeptide hormones and growth factors (1, 2, 3). Increasing evidence suggests an important role of GH in the development of the mammary gland. GH is essential for ductal morphogenesis in rodents, acting via stimulation of IGF-I expression (5, 6, 7, 8, 9, 10). Indeed, animals with targeted deletion of the GH receptor display impaired ductal development (11). Recent data also propose a role for GH in the regulation of alveolar proliferation and lactogenesis during pregnancy and after parturition, although the data are controversial. In heterozygous prolactin (PRL) receptor PRLR+/– mice, GH treatment during late pregnancy restored mammary alveolar development (12). GH treatment concomitantly led to a further decrease in acetyl-CoA carboxylase-{alpha} and milk proteins content ({alpha}-casein, ß-casein, and whey acidic protein) when expressed per cell resulting in a relative paucity of milk in the mammary glands of GH-treated animals and the inability of their pups to gain weight (12). These results suggest that GH maintains a proliferative rather than a differentiated mammary gland phenotype. In contrast, other studies have demonstrated that GH synergizes with PRL to maintain lactation and to regulate milk yield and composition in rats (13, 14, 15). Furthermore, mouse GH (mGH) stimulated casein synthesis and {alpha}-lactalbumin secretion in mouse mammary explants and primary cultures of mouse mammary epithelial cells (11, 16, 17).

One potential confounding variable of understanding the function of GH in mammary biology is the potential autocrine/paracrine actions of GH in the mammary gland in contrast to endocrine GH. GH expression has been observed in the mammary epithelial cells of dogs and cats (18, 19, 20) and both stromal and epithelial compartments of the human mammary gland (20, 21). We have recently observed by genome-wide analysis that exogenous and autocrine produced human GH (hGH) differentially regulated gene expression in human mammary carcinoma cells (22). Interestingly, exogenous hGH, but not autocrine hGH, exclusively regulated {alpha}-casein gene expression (22). We have further demonstrated that autocrine production of hGH in immortalized human mammary epithelial cells concomitantly enhances proliferation and offers protection from apoptosis, forming the basis for abnormal mammary acinar morphogenesis, oncogenic transformation, and tumor formation in vivo (23). Furthermore, autocrine production of hGH, in mammary carcinoma cells with epithelial morphology, promotes mesenchymal cellular morphology, increased cell migration, and increased metalloprotease activity with subsequent acquisition of invasive behavior both in vitro and in vivo (24). In stark contrast to the oncogenic and metastatic potential of autocrine hGH, exogenous hGH supports neither tumor formation nor invasion by human mammary epithelial cells.

The oncogenic capacity of autocrine hGH presumably represents a recapitulation of a normal developmental function of autocrine GH in the mammary gland. In an effort to determine the physiological role of autocrine GH in the mammary gland, we herein examined the postnatal ontogeny and localization of mGH expression and its relative expression during pregnancy, lactation, and weaning. We also examined the expression of mGH during forced differentiation of mouse mammary epithelial cells in culture and used a three-dimensional in vitro model to delineate the functional effects of autocrine mGH on mouse mammary epithelial cell differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Timed pregnant (day plug found = d 0 of pregnancy) Swiss Albino mice were obtained from the Singapore Animal Center. Animals were housed in a temperature-controlled vivarium (22 C), on 12:12-h light/dark cycle with food and water available ad libitum. Pregnant mice were killed by cervical dislocation at 8, 12, and 18 d of gestation (four animals per group), and the fourth and fifth mammary glands were collected. Other pregnant females were kept until after parturition. Mammary glands of nursing mice were collected at 2, 6, and 12 d of lactation or at d 6 and 14 after weaning. Young female and male mice were separated, and mammary glands of virgin female animals were collected at 2, 3, 6, 8, and 10 wk of age. Mammary glands were either frozen for the later use to assess mGH and mGH receptor (mGHR) mRNA content by RT-PCR and for immunohistochemical analysis or embedded in paraffin for in situ hybridization analysis. All experiments with the animals were conducted with accepted standards of animal care.

Total RNA extraction and RT-PCR analyses
For 2-wk-old virgin mice, mammary glands were pooled (four pools of five to seven animals per pool). The remaining tissue samples were processed independently (four animals for each group). Total RNA was extracted from mouse mammary gland tissue samples using the RNeasy kit (QIAGEN GmbH, Hilden, Germany) and treated with DNase I using the RNase-free DNase kit (QIAGEN) to avoid DNA contamination. To control for extraneous contamination, we substituted water for the test sample. RT-PCR of total RNA (1.28 µg) was performed using OneStep RT-PCR kit (QIAGEN). RNA template was reverse-transcribed into cDNA for 30 min at 50 C and HotStarTaq DNA polymerase was activated by heating the samples for 15 min at 95 C. cDNA templates were amplified by the following cycles: 94 C for 30 sec, 55 C for 60 sec, and 72 C for 60 sec. A final extension was performed for 10 min at 72 C. To compare the RT-PCR products semiquantitatively, 18–40 cycles of PCR were performed to determine the linearity of the PCR amplification. Amplifications occurring in the linear range were used for quantification. Sequences of the oligonucleotide primers were as follows: 5'-ATG GCT ACA GAC TCT CGG ACC-3' and 5'-AGA AGG CAC AGC TGC TTT CC-3' for mGH (649 bp); 5'-AGT TGG AGG AGG TGA ACA CCA T-3' and 5'-GGC ACA AGA GAT CAG CTT CCA T–3' for mGHR (330 bp) (25); 5'-ACA GCT GCA GGC AGA GGA T-3' and 5'-GAA TGT TGT GGA GTG GCA GG-3' for mouse ß-casein (534 bp); and 5'-GTC ACC CAC ACT GTG CCC ATC T-3' and 5'-ACA GAG TAC TTG CGC TCA GGA G-3' for mouse ß-actin (542 bp) (26). The same primers and conditions were used for RT-PCR analyses of cultured mouse mammary epithelial cells. Amplified PCR products were visualized under UV light on a 1.5% agarose gel stained with ethidium bromide. PCR product bands of the expected sizes were then analyzed using National Institutes of Health Image software. Expression level of the mRNA of interest was normalized to ß-actin mRNA content and shown as mean ± SE.

In situ hybridization
In situ hybridization was performed using 30-mer-oligodeoxyribonucleotide probe complementary to nucleotides 341–370 of exons 3–4 of the mGH cDNA sequence (27). This probe showed no homology with the cDNA sequence of the related mouse PRL. The probe has been synthesized at Eurobio (Les Ulis, France) and 3'-end-labeled with digoxigenin (DIG) 11-2'-deoxyuridine-5'-triphosphate and terminal transferase using the DIG oligonucleotide tailing kit, 2nd Generation (Roche Diagnostics, Meylan, France). The probe labeling efficiency was controlled by spotting of serial probe dilutions onto a HyBond nylon membrane followed by immunodetection with anti-DIG-alkaline phosphatase (AP) antibody conjugate and NBT/BCIP substrates according to the manufacturer’s instructions (Roche).

Hybridization with paraffin-embedded tissue sections was carried out as previously described (28) with slight modifications. Briefly, dewaxed 10-µm tissues sections were digested with 1 µg/ml proteinase K (Roche) in Tris-EDTA buffer for 5 min at 37 C, dehydrated, and air dried. Sections were hybridized overnight at 37 C with the hybridization mixture containing 50% deionized formamide, 10% dextran sulfate, 4x standard saline citrate, 1x Denhardt’s solution, 100 µg/ml yeast tRNA, 10 mM dithiothreitol, and 1 µg/ml of labeled probe per ml of hybridization buffer. Sections were sequentially washed in 2x, 1x, and 0.5x standard saline citrate for 1 h at room temperature. Colorimetric detection of hybridized probes was performed in two steps, using a mouse monoclonal antibody to DIG (1/100, 2 h) (Roche) followed by the incubation with goat antimouse-AP conjugate (1/20, 2 h) (Roche) and then revealed using NBT/BCIP substrates (Roche). Controls for the specificity of the in situ hybridization included 1) hybridization with a sense oligonucleotide probe identical to nucleotides 494–523 of mGH cDNA, 2) hybridization with a heterologous labeled proopiomelanocortin oligonucleotide probe, 3) omission of the probe, and 4) hybridization with an excess of unlabeled probe. Mouse pituitary gland sections processed in the same condition as the mammary glands were used as positive control for the mGH hybridization signal.

Immunohistochemical detection of mGH
Tissue sections of mouse mammary glands were immunohistochemically stained using Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Frozen sections (10 µm) were fixed in 4% paraformaldehyde/PBS solution for 30 min, washed with 0.1% Triton X-100 in PBS, and incubated with 0.3% H2O2 for 20 min. After blocking of nonspecific binding sites by incubation in 2% BSA and 5% goat serum for 30 min, rabbit antiserum to rat GH (1:200) (kindly provided by Dr. A. F. Parlow, Director of the National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA) was incubated with tissue sections for overnight at 4 C. Then tissue sections were incubated with biotinylated secondary antirabbit antibody for 1 h followed by incubation with conjugated avidin-biotinylated peroxidase complex for another 30 min. Immunoreactivity was then detected with a substrate solution (0.05% diaminobenzidine, 0.02% H2O2). Between each step, the sections were washed with Tris-buffered saline containing 0.05% Tween 20. After immunostaining, tissue sections were counterstained with hematoxylin. In each group, a control section was incubated with anti-rat-GH primary antiserum preadsorbed for 2 h at 4 C with 5 µg mGH protein (Dr. A. F. Parlow).

Cell culture and cell transfection
Mouse mammary epithelial cell line HC11 had been stably transfected with the pUSEamp expression vector (Upstate Biotechnology, Lake Placid, NY) encoding mGH gene (designed HC-mGH cells) or with the control vector (HC-vector cells). A 1.6-kb of genomic fragment containing the open reading frame of mGH was amplified by PCR using the following primers: 5'-AGC ATC CTA GAG TCC AGA TTC C-3' (sense) and 5'-AGG ATT CTC GCA GGC TTC CAG-3' (antisense) and cloned into pUSEamp vector. A sequence coding for two copies of c-Myc tag EQKLISEEDL was inserted in frame with the last amino acid of mGH. For detection of the expression of Myc-tagged mGH mRNA, the sense primer above and an antisense primer specific to the c-Myc tag sequence 5'-AGG ATT CTC GCA GGC TTC CAG-3' was used to amplify the mGH transcript of 785 bp by RT-PCR. HC11 cells were transfected using Effectene transfection reagent (QIAGEN) and selected in G418-containing media for 21 d. Parental HC11 cells, HC-mGH cells, and HC-vector cells were routinely maintained in growth medium: RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml bovine insulin (Sigma-Aldrich Inc., St. Louis, MO), and 10 ng/ml human recombinant epidermal growth factor (EGF) (Upstate Biotechnology). To induce cell differentiation, 2-d confluent cell cultures were treated with RPMI 1640 medium supplemented with 10% FBS and lactogenic hormone mix, 1 µM dexamethasone (Sigma-Aldrich), 5 µg/ml insulin, and 5 µg/ml human recombinant PRL (Sigma-Aldrich) for 4 d as described before (29). Cell cultures treated with medium containing dexamethasone and insulin without PRL were used as controls.

Three-dimensional (3D) acinar formation
The 3D cultures were prepared by growing parental HC11, HC-vector, and HC-mGH cells to confluence as monolayers, followed by trypsinization and embedding into reconstituted basement membrane (Matrigel; Becton Dickinson Labware, Franklin Lakes, NJ) as single cells. Round glass coverslips were placed into 24-well plates (Nunc, Naperville, IL), and 300 µl of cell suspension in Matrigel (2.5 x 105 cells/ml) was added into each well. Gels were allowed to solidify at room temperature for 30 min, and then RPMI 1640 medium supplemented with 2% FBS was added (1 ml per well). The medium was changed every third day, and the cultures were routinely grown in a humidified 5% CO2 incubator at 37 C for 13–16 d.

Immunocytochemistry and confocal laser scanning microscopy
For detection of mGH, parental HC11, HC-vector and HC-mGH cells were cultured on glass coverslips, fixed with 4% paraformaldehyde for 10 min, and incubated with 0.1% Triton X-100 for 10 min. Rabbit antiserum to rat GH (a kind gift of Dr. A. F. Parlow) was added and incubated overnight at 4 C, followed by antirabbit antibody conjugated with Alexa-488 (Molecular Probes, Eugene, OR). mGHR was detected with monoclonal anti-GHR antibody (30), followed by antimouse Alexa-488-conjugated secondary antibody. Control cell cultures were stained with second antibodies only.

The 3D Matrigel cultures on glass coverslips were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 in PBS for 15 min. Cultures were incubated with FITZ-conjugated antibody against E-cadherin (Transduction Laboratories, Lexington, KY) or primary goat antibody against ß-casein (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h followed by the secondary antigoat Alexa-488-conjugated antibody (Molecular Probes) for 60 min. Alternatively, 3D cultures were stained for F-actin with TRITZ-conjugated phalloidin (Sigma-Aldrich) or rhodamine-phalloidin (Molecular Probes). Cell nuclei were stained with Hoechst 33258 (10 µg/ml) for 10 min. The 2D and 3D cell cultures were examined under a Carl Zeiss Axioplan microscope equipped with epifluorescence optics and a Bio-Rad MRC1040 confocal laser system (Bio-Rad, Hercules, CA) or Carl Zeiss LSM 510 Meta microscope equipped with confocal laser system.

Total cell number assay
Equal amounts of parental HC-11, HC-vector, and HC-mGH cells were plated in 24-well plates in complete growth medium (10,000 cells per well), and 24 h later, growth medium was replaced by experimental medium (RPMI supplemented with 2% FBS and 5 µg/ml insulin or 1% FBS with no insulin) or fresh complete medium. After 24 or 48 h incubation, cells were trypsinized and counted in a hemacytometer chamber. The amount of HC-vector cells after 24 h of growth in 1% FBS was arbitrarily set to 1, and the results represent means ± SE of three independent experiments, each performed in quadruplicate.

5'-Bromo-2'-deoxyuridine (BrdU) incorporation assay
Progression to the S-phase was determined by measuring the incorporation of BrdU. Cells were plated on glass coverslips in six-well plates. After 24 h incubation in complete growth medium, cells were washed and incubated in RPMI medium supplemented with 2% FBS and 5 µg/ml insulin for an additional 18 h. Cells were pulse labeled with 20 µg/ml BrdU (Sigma) for 30 min, washed twice with PBS, and fixed in 4% paraformaldehyde in PBS (pH 7.4). To denature DNA, cells were treated with 2 N HCl for 1 h at room temperature with gentle shaking. Incorporated BrdU was detected by anti-BrdU antibody conjugated with Alexa Fluor 488 (Molecular Probes). Cell nuclei were counterstained with 10 µg/ml Hoechst 33258 for 10 min at room temperature. Cells were examined under a Carl Zeiss LSM 510 Meta microscope equipped with a confocal laser system. Cells in random microscopic fields were analyzed to determine the percentage of cells with nuclear BrdU incorporation from the total cell number. The results are expressed as means ± SE of three independent experiments, each performed in duplicate (totally, approximately 900 cells for each HC11, HC-vector, and HC-mGH cell line).

Measurement of apoptosis
Apoptotic cell death was measured by fluorescent analysis of condensed and fragmented chromatin stained with Hoechst 33258 (Sigma). Cells were plated on glass coverslips in six-well plates in complete growth medium and cultured for 24 h. Cells were washed and incubated in experimental medium (RPMI serum-free medium or medium supplemented with 1% FBS) for an additional 48 h. After fixation in 4% paraformaldehyde, cells were stained with the karyophilic dye Hoechst 33258 (10 µg/ml) for 10 min at room temperature. Nuclear morphology was examined under a Carl Zeiss LSM 510 Meta microscope equipped with confocal laser system. Apoptotic cells were distinguished from viable cells by their nuclear morphology characterized by nuclear condensation and fragmentation as well as the higher intensity of the blue fluorescence of the nuclei. The results are expressed as means ± SE of triplicate determinations (three independent experiments, one of which was performed in duplicate). Totally, around 500 cells were analyzed for each HC11, HC-vector, and HC-mGH cell line.

Statistics
All experiments were repeated three to five times. All numerical data are expressed as mean ± SE, and P values were determined by unpaired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of mGH and mGHR in the mammary gland at different stages of development
Expression of mGH and mGHR were examined during mouse mammary gland development and differentiation. Thirteen time points within the four stages of mammary gland development were examined: virgin mice (2, 3, 6, 8, and 10 wk old), pregnancy (8, 12, and 18 d), lactation (2, 6, and 21 d), and involution (6 and 14 d). mGH mRNA was detected in the mammary gland samples of 2-wk-old mice and was significantly increased during puberty, reaching the maximal level at 6 wk of age. The expression level of mGH mRNA progressively decreased in the mammary gland of mice to full maturity. The expression level of mGH mRNA further decreased during pregnancy and was subsequently repressed during the lactation period. During these periods, the expression of mGH mRNA was inversely correlated to the expression of ß-casein mRNA. The expression of mGH mRNA remained significantly repressed after involution and did not return to the level observed in the mature virgin female (Fig. 1Go, A and B). In contrast to the dramatic changes observed in mGH mRNA expression, we observed minimal variation in the level of mGHR expression during mammary gland development. mGHR mRNA level slightly increased during puberty, decreased at midpregnancy and lactation, and returned to the prepregnancy level during involution (Fig. 1Go, A and C). Expression of the milk protein ß-casein, used here as a marker of differentiation, followed the pattern described previously (31). Thus, ß-casein mRNA expression appeared in midgestation and persisted through lactation (Fig. 1AGo).


Figure 1
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  		FIG. 1. Expression of mGH and mGHR in mouse mammary gland at different physiological stages. A, RT-PCR analysis for mGH, mGHR, ß-casein, and ß-actin used as a loading control; B and C, densitometric analysis of mGH and mGHR mRNA, respectively. mGH and mGHR mRNA content was normalized to ß-actin mRNA, and the result was expressed as means ± SEM.

 
To determine the cellular localization of mGH gene expression in the mouse mammary gland, we performed nonisotopic in situ hybridization on mammary gland sections of mice starting at age 3 wk. The hybridization signal was evidenced as a brown-purple cellular precipitate, and no counterstain was used on the sections. Figure 2AGo is a representative photomicrograph of the in situ hybridization signal in the 3-wk-old mouse, showing that mGH is specifically detected in the cytoplasm of epithelial cells of terminal ducts and TEBs. A much weaker mGH signal is also associated with some cells of the stromal compartment (Fig. 2AGo). The specificity of the mGH signal was carefully verified on adjacent sections by using several controls, including the use of a sense oligonucleotide probe demonstrating no background staining in either the epithelial or stromal elements (Fig. 2BGo). The site of synthesis of mGH in the mammary gland of 6-wk-old mice was also limited to epithelial cells of the mammary ducts and to scattered cells of the connective stroma (Fig. 2CGo). The overlying mouse skin was not labeled; neither was the adipose tissue (data not shown). Consistent with the results obtained by RT-PCR and the lower sensitivity of in situ hybridization, we were unable to detect the presence of the GH gene in sections of mammary gland from pregnant and lactating animals (data not shown). Additional controls were performed on frozen mouse pituitary gland sections and showed the restricted expression of mGH to the somatotrophs of the anterior lobe and of proopiomelanocortin to cells of the intermediate lobe (Fig. 2DGo).


Figure 2
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  		FIG. 2. Expression of the mGH gene in mouse mammary gland. The in situ hybridization signal indicating the presence of mGH mRNA appears on photographs as a brown-purple precipitate. A, On a section from a 3-wk-old mouse, mGH gene expression is localized in epithelial cells of the mammary ducts identified by their cuboidal shape and surrounding the luminal space (asterisk) as well as in some cells of the connective tissue stroma (S) mainly composed here of fibroblasts and endothelial cells. The inset represents a high-power photomicrograph of the square area, and shows the discrete cytoplasmic localization of mGH mRNA in the epithelial layer of the duct delineated by the arrows. B, Absence of in situ hybridization signal on a section consecutive to A as identified by the position of luminal spaces (asterisk), and obtained when the experiment was performed using the labeled sense probe. C, On a section from a 6-wk-old mouse, mGH gene expression is similarly restricted to the mammary duct epithelium and to discrete cells of the connective tissue stroma. The apparent multilayering is a result of the sectioning plane through a TEB. The inset represents a high-power photomicrograph of the square area, and arrows point to the cytoplasmic localization of mGH gene expression. D, Frozen mouse pituitary section was used as a positive control to demonstrate that mGH gene expression is restricted to the somatotrophs. The top inset represents a high-power photomicrograph of the anterior lobe showing that only the somatotrophs are labeled, whereas other cells identified by the arrows are clearly unlabeled. The bottom inset is a low-power photomicrograph and represents a control of the in situ hybridization procedure using the proopiomelanocortin antisense probe, which gives a signal specifically restricted to cells of the intermediate lobe (IL) and absent from the anterior (AL) and posterior (PL) lobes. Dots represent capillary spaces. Bar, 50 µm.

 
Immunohistochemical analysis of mGH protein expression and localization was consistent with the pattern of mGH mRNA observed by both RT-PCR and in situ hybridization (Fig. 3Go). The expression of mGH protein was detectable in the mammary glands of 2-wk-old mice (data not shown), was significantly increased during puberty (Fig. 3Go, A, C, and D), and was reduced in adult animals (data not shown). In the mammary glands of 3- and 6-wk-old virgin mice, mGH protein was localized predominantly in the cytoplasm of epithelial cells. mGH protein was also localized to the nucleus of epithelial cells in accord with previously demonstrable nuclear translocation of GH (32). mGH protein was also detected in the stromal compartment but at significantly less intensity compared with the epithelial compartment (Fig. 3Go, A, C, and D). In the mammary gland tissues of pregnant (Fig. 3EGo), lactating (Fig. 3FGo), and weaned females (data not shown), mGH protein was not detectable by immunohistochemistry, concordant with the dramatically repressed level of mGH expression observed by RT-PCR. The specificity of immunohistochemical detection of mGH detection was verified by the lack of signal when primary antiserum was preadsorbed with mGH protein (Fig. 3BGo).


Figure 3
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  		FIG. 3. Immunohistochemical localization of mGH protein in mouse mammary gland. Immunostaining with primary antiserum is shown in 3-wk-old virgin (A), 6-wk-old virgin (C and D), 8-d pregnant (E), and 21-d lactating mammary glands (F). B, Control tissue section of 3-wk-old virgin mouse mammary gland was incubated with primary antiserum preadsorbed with mGH protein. The tissue sections were examined under x630 (D) or under x400 (A–C, E, and F) magnification.

 
Thus, mGH is expressed predominantly in epithelial cells of the mouse mammary gland, and its expression level is regulated during mammary gland development.

Differentiation of mouse mammary epithelial cells HC11 results in decreased mGH expression
The observed decrease in mGH expression in the epithelial component of mouse mammary gland during pregnancy and lactation was suggestive of the possible negative regulation of mGH gene expression during differentiation. To test this hypothesis, we made use of the mouse mammary epithelial cell line HC11, an in vitro cell model suitable to study the regulation of the functional development of the mammary epithelium. This cell line is a clonal derivative of COMMA-1D epithelial cells isolated from mammary glands of midpregnant BALB/c mice (33). HC11 cells display a normal phenotype and retain important characteristics of mammary epithelial cell differentiation, such as induction of the milk protein ß-casein upon treatment with lactogenic hormones in vitro (34, 35). Concordant with the demonstration of mGH expression in epithelial cells of the mouse mammary gland, we observed that HC11 cells also expressed mGH mRNA and protein (Fig. 4Go, A and B). Upon forced differentiation of HC11 cells (monitored by the induction of ß-casein gene expression), the level of mGH mRNA significantly decreased (Fig. 4Go, B and C) similar to that observed in the mammary gland (Fig. 1Go, A and B). We also observed a modest but statistically significant decrease in mGHR expression upon HC11 cell differentiation (Fig. 4Go, A and B), consistent with decreased mGHR expression during lactation (36).


Figure 4
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  		FIG. 4. Forced differentiation of HC11 mouse mammary epithelial cells results in decreased mGH and mGHR expression. A, Immunocytochemical analysis of mGH and mGHR expression in undifferentiated HC11 mouse mammary epithelial cells. Immunostaining with primary antibody followed by the incubation with secondary antibody (antibody) or omission of primary antibody in control slides (control) is demonstrated. B, RT-PCR analysis for mGH, mGHR, ß-casein, and ß-actin mRNA expression in HC11 cell cultures grown under normal (undiff.) and differentiating (diff.) conditions. C, Expression of mGH, mGHR, and ß-casein mRNA was analyzed using densitometry and normalized to ß-actin mRNA content, and the result was expressed as means ± SEM. *, P < 0.05 based on unpaired t test.

 
Therefore, expression of mGH is decreased in the mouse mammary epithelial cell line HC11 upon differentiation.

mGH overexpression in HC11 cells results in their decreased competency for differentiation, decreased apoptosis, and increased proliferation
We therefore next analyzed whether the forced expression of mGH in mammary epithelial cells would inhibit the differentiation process. For this purpose, we used the HC11 cell line, which expresses mGHR (Fig. 4Go, A and B) and therefore is suitable to study the effects of autocrine mGH on cell behavior. HC11 cells were stably transfected with either the mGH gene (HC-mGH cells) or with control vector (HC-vector cells). The forced expression of mGH in HC-mGH cells was confirmed by immunocytochemical localization (Fig. 5AGo) and by RT-PCR analysis using myc-specific primers against the myc-tagged introduced mGH gene (Fig. 5CGo). Synthesized mGH was secreted into culture medium (Fig. 5BGo) and therefore was able to act in an autocrine or paracrine manner. RT-PCR analysis demonstrated that HC11 cells did not express IGF-I mRNA in the absence or presence of autocrine mGH in either the pre- or postdifferentiated state. IGF-II mRNA was detected in HC11 cells, although its expression was not altered by autocrine mGH or by differentiation status (data not shown).


Figure 5
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  		FIG. 5. Forced expression of mGH in HC11 mammary epithelial cells abrogates differentiation. A, Immunocytochemical analysis of mGH expression in HC-vector and HC-mGH mouse mammary epithelial cells. GH was detected by rabbit antiserum to rat GH followed by antirabbit secondary antibody conjugated with Alexa Fluor 488. B, Western blot analysis of forced expression and secretion of mGH in HC-mGH cells by anti-c-myc antibody against c-Myc tag inserted into the mGH expression vector. ß-Actin was used as loading control for cell lysates. C, RT-PCR analysis of ß-casein expression in HC-vector and HC-mGH cells grown under normal (undiff.) and differentiating (diff.) conditions. The forced expression of mGH in HC-mGH cells was confirmed by using myc-specific primers against c-Myc tag inserted into the mGH expression vector. D, Expression of ß-casein mRNA was analyzed using densitometry and normalized to ß-actin mRNA content, and the result was expressed as means ± SE. *, P < 0.05 based on unpaired t test.

 
To determine differentiation of HC11 cells, we used ß-casein as a marker, because its regulation in cultured HC11 cells has been found to reflect the in vivo regulation of this milk protein expression in the mammary gland during pregnancy (34). We observed that induction of ß-casein in response to the lactogenic hormone mix was significantly reduced in HC-mGH cells in comparison with HC-vector cells (Fig. 5Go, C and D), indicative of an impaired ability to differentiate.

We had previously demonstrated that autocrine production of hGH by immortalized human mammary epithelial cells MCF-10A and human carcinoma cells MCF-7 resulted in increased proliferation and decreased apoptosis (23, 37, 38). Therefore, we compared growth and survival characteristics of the HC-vector and HC-mGH cell lines. We observed that the forced expression of mGH in mouse mammary epithelial cells led to an increase in total cell number under all examined conditions in comparison with HC-vector cells (Fig. 6AGo). The increase in cell number may be because of an accelerated cell cycle, increased cell survival, or both. To determine cell cycle progression, we analyzed nuclear BrdU incorporation. Forced expression of mGH in mammary epithelial cells led to a significant increase in BrdU incorporation in comparison with HC-vector cells (Fig. 6BGo). Moreover, apoptotic cell death was also decreased in HC-mGH cells under both serum-free and serum-reduced conditions (Fig. 6CGo). In all experiments, growth and survival characteristics of HC-vector cells were identical to the parental HC11 cell line (data not shown). Forced expression of mGH in mammary epithelial cells therefore results in increased cell proliferation and survival and a decreased ability to differentiate.


Figure 6
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  		FIG. 6. Forced expression of mGH in HC11 mammary epithelial cells stimulates proliferation and decreases apoptosis. A, Equal amount of HC-vector and HC-mGH cells were plated in 24-well plates, and total cell number was determined after 24 or 48 h incubation under the indicated conditions as described in Materials and Methods. The number of HC-vector cells after 24 h of growth in 1% FBS was arbitrarily set to 1, and the results represent means ± SE. B, BrdU incorporation was determined for HC-vector and HC-mGH cells incubated in RPMI medium supplemented with 2% FBS and 5 µg/ml insulin. The results represent means ± SE. The representative micrographs of HC-vector and HC-mGH cells stained with anti-BrdU antibody conjugated with Alexa-488 and Hoechst 33258 to counterstain the nuclei are demonstrated on the right. Bar, 20 µm. C, Apoptotic cell death was determined for cells incubated in RPMI serum-free media (SFM) and media supplemented with 1% FBS for 48 h by staining with Hoechst 33258. The results represent means ± SE. The representative micrographs of HC-vector and HC-mGH cells in 1% FBS are demonstrated on the right. Bar, 20 µm. *, P < 0.001; **, P < 0.0001 based on unpaired t test.

 
To further characterize the effect of forced expression of mGH on mammary epithelial cell proliferation, apoptosis and differentiation, we examined the behavior of HC-vector and HC-mGH cells in 3D reconstituted basement membrane Matrigel. Although, to our knowledge, the ability of HC11 cells to form organotypical structures in reconstituted basement membrane has not been addressed, other immortalized human and mouse mammary epithelial cell lines (39, 40, 41) and primary mouse mammary epithelial cells isolated from midpregnant mice (42) have been demonstrated to form polarized acini-like structures and to express milk proteins. We demonstrated that parental HC11 cells (data not shown) and HC-vector cells (Figs. 7Go, A and B, and 8AGo) were able to form acinar structures when embedded in Matrigel as single cells. Using 3D reconstruction, we estimated the number of cells in the individual colonies. Concordant with the increased proliferation of HC-mGH cells in comparison with HC-vector cells cultivated on plastic dishes, we observed an increased number of cells per colony in colonies formed by HC-mGH cells after both 8 and 16 d of cultivation in Matrigel (Fig. 7CGo). After 13–16 d of cultivation, HC-vector cells formed spherical acinar structures, with a single layer of epithelial cells surrounding a hollow lumen (Figs. 7BGo and 8AGo). The acini formed by HC-vector cells possessed a mean diameter of 25.58 ± 0.65 µm (Fig. 7DGo). Forced expression of mGH in HC11 cells led to an increased acinar size up to 46.43 ± 1.45 µm (Fig. 7DGo). Moreover, HC-mGH colonies demonstrated delayed formation of the lumen (data not shown), similar to that observed with another antiapoptotic molecule, Bcl-2, in studies on acinar formation in 3D Matrigel (40). Therefore, the increased number of cells in HC-mGH colonies can be attributed to a mGH-stimulated increase in proliferation and survival. Furthermore, by confocal laser scanning microscopy, we also confirmed that the expression of ß-casein was dramatically abrogated in acini formed by HC-mGH cells in comparison with control HC-vector acinar structures (Fig. 8BGo). Another marker of the differentiated epithelial phenotype is the lateral membranous expression of E-cadherin in the adherens junctions mediating intercellular adhesion (43). We observed a more dispersed localization of adherens junctions component E-cadherin in HC-mGH-formed acini, whereas its localization in colonies formed by HC-vector cells was restricted, as expected, to the lateral cell membrane (Fig. 8CGo). Thus, forced expression of mGH in mouse mammary epithelial cells inhibits lactogenic differentiation and concomitantly promotes cell proliferation and survival.


Figure 7
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  		FIG. 7. Forced expression of mGH in HC11 mammary epithelial cells promotes proliferation and prevents differentiation in 3D cell culture assay. Cell colonies were stained with rhodamine-phalloidin (F-actin) and Hoechst 33258 (nuclei). A and B, The 3D reconstruction of the colonies formed by HC-vector and HC-mGH cells at 8 d (A) and single section through the middle of the colonies at 16 d (B) after seeding in Matrigel as single cells. Bar, 10 µm. C, Number of cells per colony was estimated for 8- and 16-d colonies formed by HC-vector and HC-mGH cells. D, The size of acinar structures formed by HC-vector and HC-mGH cells. *, P < 0.01; **, P < 0.0001 based on unpaired t test.

 

Figure 8
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  		FIG. 8. Confocal laser scanning microscopic analysis of the acinar structures formed by HC-vector and HC-mGH cells with TRITC-conjugated phalloidin (A), anti-ß-casein (B) and anti-E-cadherin (C) antibodies.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated here that in the mouse mammary gland, mGH is expressed predominantly in the epithelial cell, and its expression level is differentially regulated during ontogenic and physiological stages of mammary gland development. Autocrine mGH was demonstrated to be maximally expressed during the pubertal period, concordant with previous observations demonstrating a primary role for GH in the formation of TEBs (5, 6), highly proliferative structures that lead the entire process of ductal morphogenesis during puberty. Although intensive proliferation of epithelial cells occurs during both puberty and pregnancy, the underlying mechanisms are different, and these two processes are regulated by independent factors (44). The dichotomous branching occurring through bifurcation of actively growing TEBs during pubertal development of the mammary gland is different from a process of budding from previously existing ducts, taking place at recurrent estrous cycles and during pregnancy, when a subset of the mitotically quiescent cells lining the ductal lumen initiate proliferation to generate a new duct (44). The distinct temporally specific expression of mGH in the mammary gland is indicative that mGH expressed by mammary epithelial cells is involved in mammary morphogenic processes during puberty but not in the proliferative phase of lobuloalveolar development during pregnancy.

Herein we also observe a modest increase in GHR expression in the mammary gland of pubertal and mature 10-wk-old virgin mice and decrease during late pregnancy and lactation. These observations are concordant with a previous report, suggesting an important role of GHR during mammary development; mammary ductal development was greatly retarded in 11-wk-old GHR knockout mice (11). The observations are also consistent with previous data (36, 45), demonstrating that mGHR expression level was highest in intact mammary glands of virgin mice and decreased during late pregnancy (18 d) with the lowest level in the lactating gland (6 d lactation).

Our in vitro and 3D culture experiments suggest that the ability of epithelial cells to differentiate and the level of mGH expression are inversely correlated. Namely, we have demonstrated that mGH expression is decreased in the mammary epithelial cell line HC11 upon differentiation in vitro, and differentiation of HC11 cells is abrogated by forced expression of mGH. The latter is in apparent contradiction with previous reports on the lactogenic properties of GH (11, 13, 14, 15). It has been observed that incubation of mouse mammary explants from PRL receptor-null mice in media supplemented with rat GH and lactogenic hormone mix resulted in the induction of ß-casein expression, suggesting that GH was able to activate milk protein expression independent of PRL signaling (11). In contrast, we demonstrate here that autocrine mGH significantly decreases ß-casein expression induced by the same lactogenic stimulation. This apparent discrepancy may be similar to the bipolar effect of another mitogenic agent, EGF, on HC11 cell differentiation. HC11 cells were demonstrated to synthesize high levels of ß-casein only in a medium containing EGF before treatment with lactogenic hormone mix (46). However, simultaneous treatment of HC11 cells with EGF and lactogenic hormone mix dramatically decreased ß-casein expression (47). We have previously demonstrated that exogenously added and autocrine GH stimulate the expression of both common and distinct sets of genes (22), presumably by differential temporal activation of the same signaling pathways (48). Such differential gene expression between exogenous and autocrine GH consequently produces distinct functional effects on mammary cell behavior (24, 38, 48, 49). It is interesting to note that the Ras-Mek-MAPK pathway was required for the inhibition of HC11 differentiation by EGF during simultaneous treatment by EGF and lactogenic hormone mix (47). We have also observed that autocrine hGH stimulated the prolonged (48 h) activation of p42/44 MAPK activity in mammary carcinoma cells (48). Exogenous hGH also increased p42/44 MAPK activity but in a temporally limited manner (10 min). Therefore, sustained activation of p42/44 MAPK activity by autocrine GH, compared with transient activation by exogenous GH, may be responsible for the discordant effects of exogenous vs. autocrine GH in mammary epithelial cells. In any case, autocrine mGH (this study) and autocrine hGH (23) both inhibit mammary epithelial cell differentiation, providing a potential platform for tumorigenesis. Although a decrease in autocrine GH in the HC11 cell is required for full differentiation, it is apparent from data generated in our laboratory (Mukhina, S., D. X. Liu, and P. E. Lobie, unpublished observation) that that is not sufficient. Inhibition of mGH functional activity by expression of a mGH antagonist (mGH-G118R), in the absence of the differentiation protocol, is insufficient to result in differentiation of the HC11 cell. Thus, decreased autocrine mGH is necessary but not sufficient for HC11 mammary cell differentiation.

Interestingly, we have observed that mGH expression in the mammary gland decreased during late pregnancy, and its level remained repressed after weaning, being significantly reduced in comparison with virgin 10-wk-old females. A protective effect of early full-term pregnancy against subsequent development of mammary carcinoma has been demonstrated in numerous epidemiological studies on human subjects (3, 50, 51) and in mouse (52) and rat (53, 54) model systems. Data suggest that a decreased level of circulating GH in parous females may contribute to the protective effect of early full-term pregnancy against mammary carcinoma (55), demonstrated in numerous epidemiological studies on human subjects (3, 50, 51) and in mouse (52) and rat (53, 54) model systems. Moreover, the decreased mammary carcinogenesis in parous rats as compared with age-matched virgins was accompanied by significantly decreased GH serum titers (54). Given that GH-deficient (56, 57) or functionally GH-deficient (58) rodents are refractory to the development of mammary adenocarcinoma in response to carcinogen, it is tempting to speculate that, together with lower circulating GH, the reduced local mGH expression in mammary epithelial cells may be an important factor involved in the pregnancy-associated protection against mammary carcinogenesis. Such a hypothesis gains further momentum considering that forced expression of autocrine hGH in human mammary epithelial cells is sufficient to support oncogenic transformation (23). Although species specificity in the response of the mammary epithelial cell to autocrine GH is apparent (this study vs. Ref.23), higher levels of autocrine GH in the rodent, although not stimulating oncogenic transformation by itself, may support carcinogen-induced oncogenic transformation. One potential mechanism for the repression of GH gene transcription after pregnancy may be methylation of the mammary GH promoter. Indeed, the pituitary GH promoter is methylated, and changes in methylation status are associated with pituitary neoplasia (59). Work is currently in progress to test these hypotheses.

In accord with our recent demonstration of the oncogenic potential of hGH in human mammary epithelial cells (23), Isaksson and colleagues (60) had previously demonstrated that mice transgenic for hGH develop mammary adenocarcinoma. However, primate GHs used in nonprimates activate both the nonprimate GH and PRL receptor (61). Later work from the same group using lactogenic- and somatogenic-specific ligands demonstrated that the development of adenocarcinoma in transgenic animals was mediated via the PRL receptor and not via the GHR (62). Our results here, in which forced expression of mGH, although inhibiting lactogenic differentiation of mouse mammary epithelial cells, does not result in their oncogenic transformation, are in agreement with the inability of a somatogenic-specific ligand to promote the development of spontaneous mammary adenocarcinoma in transgenic mice (62). However, these observations must be rationalized with the documented resistance of GH-deficient rodents to carcinogen-induced mammary carcinoma (56, 57, 58). In any case, a direct comparison of the effect of species homologous ligands in mammary epithelial cells (this study and Ref.23) is indicative that mGH, in contrast to hGH, will not result in oncogenic transformation of mammary epithelial cells.

In conclusion, we have demonstrated a distinct ontogenically regulated expression of the mGH gene predominantly in epithelial cells of the mouse mammary gland. Autocrine mGH functions to prevent terminal differentiation of mammary epithelial cells and will therefore be involved in mammary morphogenetic processes.


   Acknowledgments
 
We thank Dr. A. F. Parlow, Director of National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Peptide Program (Torrance, CA) for providing us with rabbit antiserum to rat GH and mGH peptide. S.M. acknowledges Temasek Life Sciences Laboratory for the facilities.


   Footnotes
 
This work was supported by the Marsden Fund, Royal Society of New Zealand, Agency for Science, Technology and Research of Singapore, and The National Research Centre in Growth and Development (New Zealand).

Present address for S.M.: Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, 117604 Singapore.

P.E.L. consults for and has equity interest in Neuren Pharmaceuticals Inc.

First Published Online January 19, 2006

Abbreviations: AP, Alkaline phosphatase; BrdU, 5'-bromo-2'-deoxyuridine; 3D, three-dimensional; DIG, digoxigenin; EGF, epidermal growth factor; FBS, fetal bovine serum; GHR, GH receptor; hGH, human GH; mGH, mouse GH; PRL, prolactin; TEB, terminal end bud.

Received August 24, 2005.

Accepted for publication January 10, 2006.


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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals