2 DISTINCT PHASES OF APOPTOSIS IN MAMMARY-GLAND INVOLUTION - PROTEINASE-INDEPENDENT AND PROTEINASE-DEPENDENT PATHWAYS

Postlactational involution of the mammary gland is characterized by tw o distinct physiological events: apoptosis of the secretory, epithelia l cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and te mporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was al most absent during lactation but became evident at day 2 of involution , when p-casein gene expression was still high. Apoptotic cells were t hen seen at least up to day 8 of involution, when beta-casein gene exp ression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1 beta converting enzyme (ICE) and tissue inhibit or of metalloproteinases-1 was upregulated at day 2, when apoptotic ce lls were seen initially. Expression of the matrix metalloproteinases g elatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly u pregulated in parallel starting at day 4 after weaning, coinciding wit h start of the collapse of the lobulo-alveolar structures and the inte nsive tissue remodeling in involution. The major sites of mRNA synthes is 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 mese nchymal-epithelial interaction. To elucidate the functional role of th ese proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is kno wn to inhibit mammary gland involution. Although the initial wave of a poptotic cells appeared in the lumina of the gland, the dramatic regre ssion 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 al l, 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 gel atinase A. Concomitantly, the gland filled with an overabundance of mi lk. Our data support the hypothesis that there are at least two distin ct 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 o f the extracellular matrix, and a second phase, characterized by extra cellular matrix remodeling and altered mesenchymal-epithelial interact ions, followed by apoptosis of cells that are losing differentiated fu nctions.