Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when beta-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when beta-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1 beta converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin-1 and uPA were weakly induced, if at all, in hydrocortisone-treated mice. Furthermore, mRNA for membrane-type matrix metalloproteinase decreased after hydrocortisone treatment and paralleled the almost complete inhibition of activation of latent gelatinase A. Concomitantly, the gland filled with an overabundance of milk. Our data support the hypothesis that there are at least two distinct phases of involution: an initial phase, characterized by induction of the apoptosis-associated genes SGP-2 and ICE and apoptosis of fully differentiated mammary epithelial cells without visible degradation of the extracellular matrix, and a second phase, characterized by extracellular matrix remodeling and altered mesenchymal-epithelial interactions, followed by apoptosis of cells that are losing differentiated functions.

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Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments

Journal of Cell Science ◽

10.1242/jcs.108.2.519 ◽

1995 ◽

Vol 108 (2) ◽

pp. 519-527 ◽

Cited By ~ 6

Author(s):

P.L. Jones ◽

N. Boudreau ◽

C.A. Myers ◽

H.P. Erickson ◽

M.J. Bissell

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Protein Synthesis ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Active Regions ◽

Mammary Epithelial ◽

Dependent Manner ◽

Beta Casein ◽

Casein Protein

The physiological role of tenascin in vivo has remained obscure. Although tenascin is regulated in a stage and tissue-dependent manner, knock-out mice appear normal. When tenascin expression was examined in the normal adult mouse mammary gland, little or none was present during lactation, when epithelial cells actively synthesize and secrete milk proteins in an extracellular matrix/lactogenic hormone-dependent manner. In contrast, tenascin was prominently expressed during involution, a stage characterized by the degradation of the extracellular matrix and the subsequent loss of milk production. Studies with mammary cell lines indicated that tenascin expression was high on plastic, but was suppressed in the presence of the laminin-rich, Engelbreth-Holm-Swarm (EHS) tumour biomatrix. When exogenous tenascin was added together with EHS to mammary epithelial cells, beta-casein protein synthesis and steady-state mRNA levels were inhibited in a concentration-dependent manner. Moreover, this inhibition by tenascin could be segregated from its effects on cell morphology. Using two beta-casein promoter constructs attached to the chloramphenicol acetyltransferase reporter gene we showed that tenascin selectively suppressed extracellular matrix/prolactin-dependent transcription of the beta-casein gene in three-dimensional cultures. Finally, we mapped the active regions within the fibronectin type III repeat region of the tenascin molecule that are capable of inhibiting beta-casein protein synthesis. Our data are consistent with a model where both the loss of a laminin-rich basement membrane by extracellular matrix-degrading enzymes and the induction of tenascin contribute to the loss of tissue-specific gene expression and thus the involuting process.

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Progesterone and EGF inhibit mouse mammary gland prolactin receptor and beta-casein gene expression

AJP Cell Physiology ◽

10.1152/ajpcell.1994.267.5.c1467 ◽

1994 ◽

Vol 267 (5) ◽

pp. C1467-C1472 ◽

Cited By ~ 32

Author(s):

S. Nishikawa ◽

R. C. Moore ◽

N. Nonomura ◽

T. Oka

Keyword(s):

Gene Expression ◽

Mammary Gland ◽

Prolactin Receptor ◽

Mammary Epithelium ◽

Mouse Mammary Gland ◽

Mrna Levels ◽

Casein Gene ◽

Beta Casein

Regulation of mouse mammary gland long-form prolactin receptor (PRL-RL) mRNA levels by progesterone and epidermal growth factor (EGF) and the relationship between PRL-RL and beta-casein gene expression were examined in vivo and in vitro. PRL-RL and beta-casein mRNA levels increased approximately 6- and 15-fold from the pregnant to the lactating period, respectively, when normalized to the level of beta-actin mRNA. Ovariectomy of pregnant mice rapidly reduced the serum concentration of progesterone and increased the level of PRL-RL and beta-casein mRNAs approximately three- and fourfold compared with sham-operated animals 24 h after the operation. Injection of progesterone, but not estrogen, inhibited the increase in both mRNA levels. PRL-RL and beta-casein mRNA levels in cultured mammary epithelium increased in response to insulin, hydrocortisone, and prolactin, whereas progesterone or EGF caused inhibition. The combination of EGF and progesterone produced a greater inhibition than either hormone alone. These results indicate that both progesterone and EGF serve as negative regulators of lactogenesis.

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Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.560-565.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 560-565

Author(s):

K F Lee ◽

S H Atiee ◽

J M Rosen

Keyword(s):

Gene Expression ◽

Mammary Gland ◽

Transgenic Mice ◽

Fusion Gene ◽

Chloramphenicol Acetyltransferase ◽

Fusion Genes ◽

Specific Expression ◽

Casein Gene ◽

Noncoding Exon ◽

Beta Casein

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.

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Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.560 ◽

1989 ◽

Vol 9 (2) ◽

pp. 560-565 ◽

Cited By ~ 32

Author(s):

K F Lee ◽

S H Atiee ◽

J M Rosen

Keyword(s):

Gene Expression ◽

Mammary Gland ◽

Transgenic Mice ◽

Fusion Gene ◽

Chloramphenicol Acetyltransferase ◽

Fusion Genes ◽

Specific Expression ◽

Casein Gene ◽

Noncoding Exon ◽

Beta Casein

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Induction of Osteopontin Gene Expression during Mammary Gland Involution and Effects of Glucocorticoid on its Expression in Mammary Epithelial Cells

Bioscience Biotechnology and Biochemistry ◽

10.1271/bbb.64.2225 ◽

2000 ◽

Vol 64 (10) ◽

pp. 2225-2228 ◽

Cited By ~ 5

Author(s):

Mijoung LEE ◽

Sanghyun NA ◽

Deahyun JEON ◽

Hyungha KIM ◽

Yunjaie CHOI ◽

...

Keyword(s):

Gene Expression ◽

Mammary Gland ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Mammary Gland Involution

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Laminin mediates tissue-specific gene expression in mammary epithelia.

The Journal of Cell Biology ◽

10.1083/jcb.129.3.591 ◽

1995 ◽

Vol 129 (3) ◽

pp. 591-603 ◽

Cited By ~ 235

Author(s):

C H Streuli ◽

C Schmidhauser ◽

N Bailey ◽

P Yurchenco ◽

A P Skubitz ◽

...

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Epithelial Cells ◽

Basement Membrane ◽

Specific Gene ◽

Mammary Cells ◽

Tissue Specific ◽

Tissue Specific Gene ◽

Specific Gene Expression ◽

Beta Casein

Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

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