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

TLDR: The 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, characterize by extrace cellular matrix remodeling and altered mesenchymal-epithelial interactions, followed by apoptotic cells that are losing differentiated functions.

Abstract: 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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Frequently asked questions

Q1. What is the C-terminal region of membrane type matrix metalloproteinase?

The C-terminal region of membrane type matrix metalloproteinase is a functional transmembrane domain required for pro-gelatinase A activation. 

Q2. What is the effect of hydrocorticoid treatment on the ventral prostate?

Glucocorticoid treatment has earlier been shown to inhibit the involution of the ventral prostate gland after castration and to affect apoptosis-related gene expression (Rennie et al., 1989; Freeman et al., 1990). 

Q3. What is the RNA level after hydrocortisone treatment?

After normalization to the level obtained for the 28S RNA, the value obtained at day 5 of involution without treatment was set equal to 1 for each probe, and the values after hydrocortisone treatment are indicated as -fold induction or reduction. 

Q4. What is the effect of a transforming growth factor 1 transgene on the mamm?

Targeting expression of a transforming growth factor 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation. 

Q5. How much did uPA decrease in the hydrocortisone-treated gland?

in the hydrocortisone-treated gland, -casein decreased only to approximately 35% of its lactating level, similar to that of a normal day 2 involuting gland, whereas in the saline-treated involuting mice it decreased to 5% (Figs 2C,D, 9D). 

Q6. how long did hydrocortisone delay the growth of the mammary gland?

The start of hydrocortisone treatment could be delayed up to 3 days after weaning and still delay the regression of the gland considerably, as judged by morphological and biochemical criteria (data not shown). 

Q7. How did the authors find connective tissue collagens around major ducts and vessels?

By trichrome staining, the authors found connective tissue collagens around major ducts and vessels, whereas only very faint staining or none was observed around the intact lobules and the interstitial septae (Fig. 1D). 

Q8. how many mice are used to reduce the effect of variability?

The results shown are from a typical single experiment, which included pooled tissue extracts from at least 3 mice to reduce the effect of variability between individual mice. 

Q9. What is the role of stromal cells in the regulation of progelatinase?

In a recent study MT-MMP and gelatinase A, but not stromelysin-1 and interstitial collagenase, were shown to be co-expressed by stromal cells in human colon, breast and head and neck cancer (Okada et al., 1995). 

Q10. What is the role of MT-MMP in the mammary gland?

Future studies are required to determine the cells responsible for the synthesis of MT-MMP in the mammary gland and the biochemical effects of this potential activator of progelatinase A in vivo.