Materials and Methods
Animals
Grafting of Mammary Epithelium to Cleared Fat Pads
Antibodies
Immunohistochemistry
Image Acquisition and Processing
Statistics
TGF-β1 Bioassay
Results
TGF-β1 Activation Is Restricted within the Mammary Epithelium of Pubertal Glands
The Pattern of TGF-β1 Activation Is Modulated by the Estrus Cycle
TGF-β1 Activation Is Restricted to a Subpopulation of Epithelial Cells During Early Pregnancy
Tgfβ1+/− Mice Exhibit Accelerated Mammary Ductal Growth and Increased Proliferation During Puberty
% PCNA labeled † Areas for counting were selected at low power using DAPI nuclear counterstain. PCNA staining was scored, without knowledge of genotype, by counting positive cells of three replicate animals in the fluorescein image and total epithelial cells in the DAPI image in 12 fields for each condition (positive/total). | ||||
---|---|---|---|---|
Genotype | % Fatpad filled | Endbuds | Ducts | % Pyknotic endbud nuclei |
Wild type | 27 ± 3.9 | 2.7 | 1.7 | 2.2 |
n = 11 | (34/1265) | (19/1091) | (227/9603) | |
Heterozygote | 61 ± 7.8 | 8.0 | 4.3 | 2.8 |
n = 18 | (117/1460) | (44/1015) | (248/9420) |
The Phenotype in Tgf-β1 Heterozygote Mammary Gland Is Because of Epithelial, Rather than Systemic/Stromal, TGF-β1 Depletion
Tgfβ1+/− Mice Exhibit Increased Cell Turnover in Adult Mammary Epithelium
Genotype | ||
---|---|---|
Estrus cycle status | Wild type | Heterozygote |
% PCNA labeled | ||
Proestrus | 0.14 (3/2211) | 0.05 (1/2091) |
Estrus | 0.57 (18/3142) | 2.3(48/2114) |
Diestrus | 0 (0/1958) | 0.34(7/2085) |
% Pyknotic nuclei | ||
Proestrus | 0.23 (14/6177) | 0.93(59/6311) |
Estrus | 0.47 (30/6370) | 0.23(14/6177) |
Diestrus | 0.45 (27/6064) | 0.60 (36/6034) |
Lobular-Alveolar Development Is Accelerated in Tgfβ1 Null Heterozygotes
Ducts | Alveoli | |||
---|---|---|---|---|
Wild type | Heterozygote | Wild type | Heterozygote | |
% PCNA | ||||
Day 6 | 3.7 (51/1363) | 6.7(75/1113) | 4.8 (53/1101) | 13.8(131/952) |
Day 10 | 3.2 (36/1966) | 5.3(100/1950) | 1.3 (17/1877) | 4.6(79/1830) |
Day 14 | 2.0 (36/1903) | 6.3(177/2507) | 2.8 (36/1835) | 5.1(110/2517) |
% TUNEL | ||||
Day 6 | 0.6 (18/3178) | 1.9(53/2750) | 0.7 (19/2899) | 2.5(50/1979) |
Day 10 | 1.2 (23/1842) | 1.3 (25/1926) | 1.1 (11/1033) | 0.9 (9/976) |
Day 14 | 0.4 (8/1802) | 0.4 (16/2223) | 1.0 (10/997) | 1.7 (35/2026) |
Alveolar area | Wild type | Heterozygote | ||
Day 6 | 5 | 11 | ||
Day 14 | 11 | 42 |
Ovarian Hormones Elicit the Tgfβ1+/− Phenotype in the Mammary Epithelium
Discussion
- Wakefield l
- Colletta AA
- McCune BK
- Sporn MB
Acknowledgements
References
- Transforming growth factor β regulation of cell proliferation.J Cell Physiol. 1987; 5: S1-S7
- Molecular and cell biology of TGF-beta.Miner Electrolyte Metab. 1998; 24: 111-119
- TGFβ inhibition of Cdk4 synthesis is linked to cell cycle arrest.Cell. 1993; 74: 1009-1020
- TGF-beta stabilizes p15INK4B protein, increases p15INK4B/cdk4 complexes and inhibits cyclin D1/cdk4 association in human mammary epithelial cells.Mol Cell Biol. 1997; 17: 2458-2467
- Resistance to inhibition of cell growth by transforming growth factor-β and its role in oncogenesis.Crit Rev Oncog. 1993; 4: 493-540
- TGF-β induction of extracellular matrix associated proteins in normal and transformed human mammary epithelial cells in culture is independent of growth effects.J Cell Physiol. 1993; 155: 210-221
- Mammary tumor suppression by transforming growth factor β1 transgene expression.Proc Natl Acad Sci USA. 1995; 92: 4254-4258
- TGFβ1 inhibits the formation of benign skin tumors, but enhances progression to invasive spindle carcinomas in transgenic mice.Cell. 1996; 86: 531-542
- TGFβ signaling is necessary for carcinoma cell invasiveness and metastasis.Curr Biol. 1998; 8: 1243-1252
- TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells.Genes Dev. 1996; 10: 2462-2477
- Transforming growth factor-β and breast cancer: tumor promoting effects of transforming growth factor-β.Breast Cancer Res. 2000; 2: 125-132
- Transforming growth factor-beta: vasculogenesis, angiogenesis, and vessel wall integrity.Cytokine Growth Factor Rev. 1997; 8: 21-43
- Autocrine induction of tumor protease production and invasion by a metallothionein-regulated TGF-β1 (Ser223, 225).EMBO J. 1992; 11: 1599-1605
- Transforming growth factor β1 stimulates contrasting responses in metastatic versus primary mouse prostate cancer-derived cell lines in vitro.Cancer Res. 1996; 56: 3359-3365
- Regulation of immune responses by TGF-β.Annu Rev Immunol. 1998; 16: 137-161
- Gradual phenotypic conversion associated with immortalization of cultured mammary epithelial cells.Mol Biol Cell. 1997; 8: 2391-2405
- Transforming growth factor beta is essential for spindle cell conversion of mouse skin carcinoma in vivo: implications for tumor invasion.Cell Growth Differ. 1998; 9: 393-404
- TGF-b signaling in tumor suppression and cancer progression.Nat Genet. 2001; 29: 117-129
- Altered expression of small proteoglycans, collagen, and transforming growth factor-β1 in developing bleomycin-induced pulmonary fibrosis in rats.J Clin Invest. 1993; 92: 632-637
- Induction of extracellular matrix gene expression in normal human keratinocytes by transforming growth factor β is altered by cellular differentiation.Exp Cell Res. 1991; 193: 93-100
- Transforming growth factor-β in breast cancer: a working hypothesis.Br Cancer Res Treat. 1997; 45: 81-95
- Regulation of mammary growth and function by TGF-β.Mol Reprod Dev. 1992; 32: 145-151
- The role of TGF-β in patterning and growth of the mammary ductal tree.J Mammary Gland Biol Neoplasia. 1996; 1: 331-341
- Regulated expression and growth inhibitory effects of transforming growth factor-β isoforms in mouse mammary gland development.Development. 1991; 113: 867-878
- Inhibition of mammary duct development but not alveolar outgrowth during pregnancy in transgenic mice expressing active TGF-β 1.Genes Dev. 1993; 7: 2308-2317
- Reversible inhibition of mammary gland growth by transforming growth factor-β.Science. 1987; 237: 291-293
- The pro domain of pre-pro-transforming growth factor β1 when independently expressed is a functional binding protein for the mature growth factor.Biochemistry. 1990; 29: 6851-6857
- Human transforming growth factor-β complementary DNA sequence and expression in normal and transformed cells.Nature. 1985; 316: 701-705
- The extracellular regulation of growth factor action.Mol Biol Cell. 1992; 3: 1057-1065
- Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung.J Clin Invest. 1997; 100: 768-776
- Immunohistochemical detection of active TGF-β in situ using engineered tissue.Am J Pathol. 1995; 147: 1228-1237
- Latency and activation in the regulation of TGF-β.J Mammary Gland Biol Neoplasia. 1996; 3: 353-363
- Transforming growth factor-β activation in irradiated murine mammary gland.J Clin Invest. 1994; 93: 892-899
- Latent transforming growth factor-β activation in situ: quantitative and functional evidence following low dose irradiation.FASEB J. 1997; 11: 991-1002
- Immunocytochemical detection of latent transforming growth factor-β activation in cultured macrophages.J Cell Physiol. 1999; 178: 275-283
- Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death.Proc Natl Acad Sci USA. 1993; 90: 770-774
- Transforming growth factor-beta1 is a new form of tumor suppressor with true haploid insufficiency.Nat Med. 1998; 4: 802-807
- Ectopic TGF beta 1 expression in the secretory mammary epithelium induces early senescence of the epithelial stem cell population.Dev Biol. 1995; 168: 47-61
- Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice.Cancer Res. 1959; 19: 515-520
- Immunodetection and quantitation of the two forms of transforming growth factor-beta (TGF-β1 and TGF-β2).J Cell Physiol. 1989; 138: 79-86
- An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.Analyt Biochem. 1994; 216: 276-284
- Roles of estrogen and progesterone in normal mammary gland development: insights from progesterone null receptor mutant mice and in situ localization of receptor.Trends Endocrinol Metab. 1997; 8: 34-39
- Postnatal development of the rodent mammary gland.in: Neville M Daniel CC The Mammary Gland: Development, Regulation and Function. Plenum Press Publishing Corp., New York1987: 3-36
- TGFβ suppresses casein synthesis in mouse mammary explants and may play a role in controlling milk levels.J Cell Biol. 1993; 120: 245-251
- Apoptosis in the terminal endbud of the murine mammary gland: a mechanism of ductal morphogenesis.Development. 1996; 122: 4013-4022
- Laminin and β1 integrins are crucial for normal mammary gland development in the mouse.Dev Biol. 1999; 215: 13-32
- Dominant-negative interference of the transforming growth factor-beta type II receptor in mammary gland epithelium results in alveolar hyperplasia and differentiation in virgin mice.Cell Growth Differ. 1998; 9: 229-238
- Overexpression of a kinase-deficient transforming growth factor-β type II receptor in mouse mammary stroma results in increased epithelial branching.Mol Biol Cell. 1999; 10: 1221-1234
- Role of the stroma in mammary development.Breast Cancer Res. 2001; 3: 218-223
- Murine progesterone receptor expression in proliferating mammary epithelial cells during normal pubertal development and adult estrous cycle. Association with ERα and ERβ status.J Histochem Cytochem. 1999; 47: 1323-1330
- Targeting expression of a transforming growth factor β1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.EMBO J. 1993; 12: 1835-1845
- Topography of DNA synthesis in the mammary gland of the C3H mouse and its control by ovarian hormones: an autoradiographic study.Cell Tissue Kinet. 1968; 1: 51-63
- Progesterone signaling and mammary gland morphogenesis.J Mammary Gland Biol Neoplasia. 1999; 4: 89-104
- Dissociation between steroid receptor expression and cell proliferation in the human breast.Cancer Res. 1997; 57: 4987-4991
- Estrogen receptor-positive proliferating cells in the normal and precancerous breast.Am J Pathol. 1999; 155: 1811-1815
- Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor β 1 precursor.J Biol Chem. 1989; 264: 13660-13664
- Effect of transforming growth factor-β1 on proliferation and death of rat prostatic cells.Endocrinology. 1990; 127: 2963-2968
- Coordinated regulation of apoptosis and cell proliferation by transforming growth factor β 1 in cultured uterine epithelial cells.Proc Natl Acad Sci USA. 1991; 88: 3412-3415
- Transforming growth factor b3 induces cell death during the first stage of mammary gland involution.Development. 2000; 127: 3107-3118
- Apoptotic cell death and tissue remodeling during mouse mammary gland involution.Development. 1992; 115: 49-58
- Roles for transforming growth factors-β in the genesis, prevention and treatment of breast cancer.in: Dickson RB Lippman ME Genes, Oncogens, and Hormones: Advances in Cellular and Molecular Biology of Breast Cancer. Kluwer Academic Publishers, Boston1991: 97-136
- TGF-β and breast cancer: lessons learned from genetically altered mouse models.Breast Cancer Res. 2000; 2: 100-106
- TGF-β signaling in growth control, cancer, and heritable disorders.Cell. 2000; 103: 295-309
- Association between the T29–>C polymorphism in the transforming growth factor beta1 gene and breast cancer among elderly white women: the study of osteoporotic fractures.JAMA. 2001; 285: 2859-2863
- Regulation of transforming growth factor-β subtypes by members of the steroid hormone superfamily.J Cell Sci. 1990; 13: S139-S148
- Anti-oestrogens induce the secretion of active transforming growth factor beta from human fetal fibroblasts.Br J Cancer. 1990; 62: 405-409
Article info
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Footnotes
Supported by the California Breast Cancer Research Program (grant 4BP-0136), NIH CA66541 (to G.S.), and the Office of Health and Environmental Research, Health Effects Research Division, United States Department of Energy (contract no. DE-AC-03-76SF00098).
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- Figure 1Mammary epithelial LAP and TGF-β1 immunostaining are regulated during ductal morphogenesis. A: Nuclear DAPI staining (top) and dual immunolocalization of active TGF-β1 (middle) and LAP (bottom) of endbuds (left) and distal ducts (right). During ductal morphogenesis, most body cells in endbuds showed LAP and strong TGF-β1 immunostaining, but some lacked active TGF-β1 (arrowhead). Cap cells (arrows) at the interface between the endbud and adipose stroma were negative for TGF-β1 even though they stained with antibodies to LAP. Ductal epithelium proximal to the region of endbuds exhibited less TGF-β1 immunostaining. Tissue was from FVB mice. Scale bar, 20 μm. B: The mean (±SD) intensity of TGF-β1 and LAP were quantified in the peri-epithelial stroma of the ducts (Sd) and endbuds (Se) and for epithelial cells from the cap cell layer (C), endbud body cell (B), and distal ducts (D). Endbud body cells had significantly more TGF-β1 immunoreactivity than any other population. C: To evaluate the frequency of TGF-β1-positive cells in different epithelial populations, cells with immunoreactivity greater than the mean +2 SD of the stromal cell TGF-β1 intensity were defined as TGF-β1-positive. The majority of endbud body and duct epithelial cells were TGF-β1-positive, whereas cap cells were rarely positive.
- Figure 2LAP and TGF-β1 immunostaining is highly heterogeneous during the estrus cycle. A–C: Mammary epithelial immunoreactivity of nulliparous FVB animals as a function of the estrus cycle; DAPI-stained nuclei (A), LAP (B) and active TGF-β1 (TGF-β) (C) immunoreactivity. In diestrus, most epithelial cells stained with both antibodies. During proestrus, a transition occurs in which epithelial ducts show heterogeneous TGF-β1 staining. During estrus, the heterogeneity of TGF-β1 immunoreactivity increased, such that the epithelium contains TGF-β1-negative cells adjacent to positive cells. LAP is more homogeneous. Occasional variation was observed in that a few epithelial ducts from estrus exhibited low homogeneous staining similar to that seen in diestrus (not shown). D: Quantitative image analysis of the intensity of LAP and TGF-β1 immunoreactivity per cell. In diestrus, the relative intensity of LAP per cell was linearly correlated with TGF-β1 in most cells. In proestrus, a population of cells with very low TGF-β1 is evident. At estrus, an additional population appears consisting of cells exhibiting high TGF-β1 and low LAP immunoreactivity. These cells correspond to those with intense TGF-β1 shown above and in E. E: False-color digital micrographs of the dual immunolocalization of antigen-purified TGF-β1 antibodies (red) and LAP antibodies (green) visualized simultaneously with DAPI-stained nuclei (blue). Mammary gland tissue was obtained from animals sacrificed at estrus. LAP immunoreactivity (green) was relatively uniform in the peri-epithelial stroma and adipose stroma, whereas immunoreactive TGF-β1 was not evident in the stroma. Concordant TGF-β1 and LAP staining appeared yellow-orange. Note that all cells stain with LAP. The discordance of LAP and TGF-β1 immunoreactivity suggests that this TGF-β1 epitope is masked in the majority of stromal cells and in some epithelial cells. This highly localized TGF-β1 is indicative of restricted activation. Scale bar, 10 μm.
- Figure 3Transition of adult mammary gland from estrus cycle to lobular-alveolar differentiation during pregnancy is accompanied by progressive loss of both active and latent TGF-β. A: During diestrus, chNTGF-β1 immunoreactivity is relatively homogeneous in nulliparous mammary gland. B: In contrast the epithelium was distinctly heterogeneous during estrus. C: Mammary tissue from early (6 day) pregnant mice exhibited less intense LAP immunoreactivity and less chNTGF-β, suggesting that gradual loss of both TGF-β1 production and activation is reduced. D: Epithelium undergoing functional differentiation during late (18 day) pregnancy exhibited barely detectable TGF-β1 and LAP immunostaining. The tissue sections were stained and images acquired together. False-color digital micrographs of the dual immunolocalization of antigen-purified TGF-β1 antibodies (red) and LAP antibodies (green) visualized simultaneously with DAPI-stained nuclei (blue). The images are scaled identically to allow comparison within the figure of the pattern of TGF-β1 and LAP immunoreactivity. Scale bar, 20 μm.
- Figure 4LAP and TGF-β1 immunoreactivity are both decreased in Tgfβ1+/− mice compared to +/+ mice. Comparison of relative LAP (A and C) and TGF-β1 (B and D) immunoreactivity intensity in the epithelium of Tgfβ1+/+ (shaded bars) and +/− (open bars) littermates. A and B: The staining intensity of LAP and TGF-β1 in the distal ducts at puberty was significantly decreased (P < 0.01, Kolmogorov-Smirnov test) in Tgfβ1 heterozygotes. C and D: Similarly, both LAP and TGF-β1 were decreased in Tgfβ1+/− mice in the mammary epithelium of adult mice in estrus.
- Figure 5Accelerated mammary ductal growth in Tgfβ1+/− mice. Mammary gland whole mounts and histology from Tgfβ1+/+ (A, C, and E) and +/− (B, D, and F) littermates. A and B: Low-magnification views of mammary gland whole mounts from 6-week-old mice. In wild-type mice, epithelial outgrowth has reached the lymph node (LN), whereas in the heterozygotes the fat pad is two-thirds full. The nipple is to the left in both cases. C and D: The endbud epithelium of both genotypes is similar and well organized (H&E stain). Asterisks indicate pyknotic nuclei. E and F: High-magnification photomicrographs of mammary gland whole mounts show similar branching patterns in wild-type and heterozygote mice.
- Figure 6Schematic of the pattern of TGF-β1 immunoreactivity and proliferation in mammary epithelium relative to ovarian hormones during the estrus cycle. A: Characteristic patterns of active TGF-β in mammary epithelium are represented as a shaded bar representing the relative intensity of epithelial TGF-β1 as a function of mammary gland development. The pattern is heterogeneous during periods of proliferation and relatively homogeneous during quiescent stages. B: During the estrus cycle, highly heterogeneous TGF-β1 immunoreactivity at estrus correlates with the phenotype of increased proliferation and decreased apoptosis (not shown) in Tfgβ1+/− mice. The relative levels of estrogen (dotted line) and progesterone (solid line) serum concentrations are graphed and the proliferative indices of Tfgβ1+/+ and +/− mammary epithelium are summarized in the bar graph below. The functional link between the role of TGF-β as a growth inhibitor and ovarian hormones regulating proliferation in the mammary gland is supported by the dramatic proliferative response of ovarectomized Tfgβ1+/− mice to estrogen and progesterone.
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