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Development of Mammary Gland Requires Normal Integrin Function |
1. INTRODUCTION
The mammary gland consists of secretory alveoli interconnected by a system of branching ducts. Its development is tightly regulated and needs the concerted action of soluble and extracellular matrix bound factors as well as the correct cell-cell and cell-matrix interactions.
A number of studies in different cell systems, including the mammary gland, have shown that cell-matrix interactions are important regulators of cell growth and programmed cell death. In addition, extracellular matrix (ECM) can regulate the phenotype of mammary epithelium. Particularly, adhesion of mammary epithelial cells to laminin is believed to mediate β-casein gene expression, whereas activity of STAT5 transcription factor, an essential regulator of milk gene transcription, is also controlled by cell-ECM interactions.
Integrins are the major cellular ECM receptors. They are transmembrane heterodimers constituted by non-covalently associated α and β subunits. The large extracellular domain of integrins binds to various ligands, i.e. to the ECM proteins or to other cell surface receptors. The cytoplasmic domain of the receptor interacts with the cytoskeletal complexes triggering a signal transduction cascade in response to ligand binding.
In the mammary gland bilayer, luminal epithelial cells express a2(31, a3(31, a6(31 and a6(34, integrin dimers and basal myoepithelial cells present, in addition, a1(31 (Fig.1). To study the involvement of (31-integrins in the cellular functions of mammary epithelium in vivo, we have targeted the expression of a transgene coding for a chimeric molecule containing the cytoplasmic and the transmembrane domains of the (31-integrin subunit and the extracellular domain of the T-cell differentiation antigen, CD4, to the luminal mammary epithelium using MMTV promoter. Such chimera neither binds to a-integrin subunits, nor interacts with the ECM integrin ligands. However, in vitro, it was shown to be delivered to focal contacts of the adherent cells and, if expressed at high level, to interfere with integnn functions such as adhesion to ECM proteins and FAK phosphorylation following integrin clustering. Thus the chimeric molecule can uncouple adhesion from the intracellular integrin-associated events and acts as a dominant inhibitor of integrin function (Fig. 2A).
2. GENERATION OF TRANSGENIC ANIMALS
Four founders (F0) expressing the βl-chimera under the control of MMTV promoter were generated. The females from 3 transgenic lines were able to feed their litters of normal size, whereas 25% of the females of line 17 lost some or all the pups during the 1st and 2nd days of lactation.Northern blot analysis of glands from different developmental stages has shown that the transgene was already expressed in 8-week-old virgin mice, although its expression was significantly up-regulated in pregnancy, and reach the maximal levels in lactation. The expression of the transgene, was high in line 17, moderate, in line 42, and weak, in lines 44 and 46. Consistent with the transgene expression levels, the morphological differences described below were more drastic in females from line 17, much less pronounced in line 42, and hardly detectable in lines 44 and 46.
3. EFFECTS OF TRANSGENE EXPRESSION ON MAMMARY EPITHELIUM
In the transgenic animals, we have analysed the mammary gland morphology, proliferation and apoptosis rates and differentiation of luminal epithelial cells at different stages of the mammary gland development.
3.1 Delay in the development of mammary glands expressing l-chimera
Morphological analysis of the wild-type and transgenic glands at different developmental stages has shown the first alterations at mid-pregnancy stage. By day 10- 12 of pregnancy the transgenic glands appeared smaller with a branching pattern less complex than the one observed in normal glands (Fig. 2B). Histological analysis of 2-day-lactating wild-type glands has shown a fat pad completely occupied by the secretory epithelium, with small fat stroma islets between the lobules. On the contrary, in the transgenic glands, the alveoli appeared sparse and were surrounded by vast areas of fat stroma.
Later in lactation no significant morphological differences between wild-type and transgenic glands were detected. 
Figure 2. A) Perturbation of β1 integrin functions in the presence of transgene. B) Whole mount staining of mammary glands from wild-type (WT) and βl-transgenic (Tg) animals at12 days of pregnancy.The delayed development of the mammary glands of the transgenic mice might be due to decreased proliferation and/or increased apoptosis rates. We have found that proliferation was significantly diminished in transgenic glands at mid-pregnancy as well as at the second day of lactation (Table 1).
On the other hand, TUNNEL analysis has revealed an increase in the apoptosis rates in 12 and 18-day pregnant and in 2-day lactating transgenic glands whereas later in lactation, similar to wild-type glands, apoptosis rates were low and did not exceed 0.1% (Table 2).Table 1. BrdU incorporation in normal and transgenic mammary glands
Stages Wild-type Transgenic12.10± 2.35 3.21 ± 2.0819.91 ± 1.47 7.96 ± 0.6412-day-pregnant 2-day-lactatingValues presented as mean ± SD.Table 2. Apoptosis in normal and transgenic mammary glands
Stages Wild-type Transgenic | 12-day-pregnant | 1.11 ± 0.11 | 2.46 ± 1.19 | | 18-day-pregnant | 0.42 ± 0.10 | 0.90 ± 0.18 | | 2-day-lactating | 0.28 ± 0.10 | 1.15 ± 0.41 | | 4-day-lactating | < 0.1 | < 0.1 | | 10-day-lactating | < 0.1 | < 0.1 |
Values presented as mean ± SD.
3.2 l-chimera expression affects differentiation of secretory epithelium during lactation
To determine whether the transgene expression affected the differentiation of mammary secretory epithelium, we analysed by Northern blot the expression of WAP and β-casein genes in 2-day-lactating normal and transgenic mammary glands. The amount of mRNA for both proteins are reduced in transgenic glands, however the expression levels varied in different animals. We have observed 40-80% and 20-60% reduction in the amount of mRNA for WAP and β-casein, respectively. These data are in keeping with the results of the in vitro studies suggesting that expression of β-casein gene in mammary epithelial cells requires interaction with laminin mediated by 1-integrin2 and that DNA-binding activity of a transcription factor STAT5, regulator of milk protein gene expression, depends on adhesion to basement membrane.
3.3 l-chimera expression in luminal epithelial cells affects polarity
Polarity is an important property of all organized epithelia and its establishment is mediated by the interaction of the cells with their basement membrane. We have detected evidences of altered cell polarity in the transgenic mammary epithelium as well as in cultured mammary epithelial cells expressing the transgene.
Overall, the mammary epithelium bilayer organization in the transgenic animals appeared normal, and the basement membrane around alveoli appeared continuous as revealed by staining with an anti-laminin antibody. However, surprisingly, in addition to basal localization, at the sites of transgene expression, laminin was accumulated at the lateral surface of luminal epithelial cells. Similar to laminin, distribution of the 4-integrin chain was altered and it was colocalized with laminin at lateral cell surfaces. These observations reveal defects in cell polarization and suggest malformation of cell-cell junctions.
We have isolated epithelial cells from mammary glands of wild-type and transgenic mice and cultured them in Matrigel, in order to compare their ability to establish polarity when interacting with the basement membrane components in vitro. The cells isolated from wild-type animals formed cysts if cultured in Matrigel, while the cells obtained from the transgenic glands remained in aggregates without lumen, failing to establish polarity and to get organized into cysts. In agreement with our results, polarization and organization of normal mammary epithelial cells into cysts in collagen gels as well as in Matrigel were reported to require function of β1-integrins10. In conclusion, our data prove that β1-integrins are involved in growth control and differentiation of mammary epithelium and are essential for normal mammary gland development and function. We are currently searching for the intracellular signalling pathways responsible for the impaired growth and differentiation control in the transgenic glands during pregnancy and lactation.
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Mammary G. Requires