Mammary Derived Signals Activate Programmed Cell Death in the Involuting Gland

References Li, M., Liu, X., Robinson, G., Bar-Paled, U., Wagner, K-U, Young, W.S, Hennighausen, L. and Furth, P.A. (1997) Mammary derived signals activate programmed cell during the involuting mammary gland. Proc. Natl. Acad. Sci. U.S.A., 94, 3425-3430 (Medline Abstract). (PDF reprint)

Background During involution a coordinated process of alveolar programmed cell death (PCD) and lobular-alveolar remodeling restructures the mammary gland. Simple removal of the suckling stimulus triggers this process. With the absence of suckling, milk accumulates within alveolar lumens and levels of systemic lactogenic hormones fall. Mammary gland involution goes through two distinct stages. In the first stage of involution alveolar cells undergo PCD, but there is no remodeling of the lobular-alveolar structure. During the second stage of involution the lobular-alveolar structure of the gland is obliterated as proteinases degrade basement membrane and extra-cellular matrix (ECM). The two stages exhibit characteristic changes in gene expression or activity. First stage changes include up-regulated expression of sulfated glycoprotein-2 (SGP-2), tissue inhibitor of metalloproteinases-1 (TIMP), interleukin -1ß converting enzyme (ICE), and cell cycle control proteins (c-Jun, junB, junD, c-fos, c-myc ) and decreased expression levels of milk protein genes. Second stage changes include increased expression levels of matrix metalloproteinases gelatinase A and stromelysin-1 and serine protease urokinase-type plasminogen activator.

PCD of individual alveolar cells during the first days of involution is correlated with increased expression levels of the death inducers bax and bcl xshort (bcl-xS) as compared to bcl-xlong (bcl-xL), all members of the bcl-2 family. Importantly, this phase of PCD is p53 independent. Changes in activity of two Stat family members accompany mammary gland involution: decreased activity of the prolactin signaling molecules Stat5a and Stat5b and activation of Stat3.

In this study we examined the role of local as compared to systemic factors during the two stages of involution. Specifically, we asked whether local or systemic factors control the onset of PCD, bax induction, and the phosphorylation state of Stat5a, 5b and Stat 3. The role of hormonal stimuli during involution was examined using suckling to maintain physiological levels of lactogenic hormones. Previous studies using exogenous administration of glucocorticoids or other individual lactogenic hormones have yielded some conflicting results. One group reported that glucocorticoids inhibit PCD by interfering with AP-1 function while another found that alveolar cell PCD occurs in the presence of glucocorticoids.

Four mouse models were selected to differentiate effects of local versus systemic factors. In the first model, teat sealing disrupted milk delivery to the pups. In the second model, the use of transplanted glands prevented milk release. These glands develop normally but have no teat connection to deliver milk. In the third model, milk ejection was impaired due to oxytocin deficiency (oxy -/- mice). The oxy -/- mice carry a homozygous targeted disruption of the oxytocin gene and can breed normally but there is no lactation due to failure of milk ejection. Another lactation failure model (WAP-TAg transgenic mice) showed that lactogenic hormones can preserve lobular-alveolar structure even in the presence of extensive PCD. WAP-TAg mice carry a transgene that targets Simian Virus 40 T Antigen (TAg) expression to mammary alveolar cells. There it triggers PCD and inhibition of milk protein synthesis. Finally, removing and replacing pups on non-transgenic dams for different times determined the reversibility of the first stage of involution.

During the first reversible stage of involution, we found that local factors are both necessary and sufficient for induction of PCD, bax and phosphorylation changes in Stat5a, 5b and Stat3. Systemic hormone stimulation prevented progression of the gland into the second irreversible stage of involution, but did not block PCD.

Mammary derived signals were sufficient for induction of alveolar PCD In all models, failure of milk removal from the gland induced alveolar cell PCD in the presence of systemic lactogenic hormone stimulation (Figs. 1,2,3). These results show that local signals are sufficient to induce PCD and the first stage of involution. In the closed teat model, the percentage of cells undergoing PCD (4.2%) was not significantly different from that in a normal d 3 involution gland (4.8%) (Compare Figs. 1E and F)(8). Maintenance of systemic hormone levels by suckling prevented the mice with closed teats from entering the second stage of involution. The critical time for lactation reversal during the first 3 days of involution was found to lie between 48 and 72 h. Lactation was restored to dams whose pups were removed for 48 h, but was not resumed in dams whose pups were removed for 72 h.

Induction of bax gene expression was solely dependent upon local factors The teat closure model was used to evaluate bax mRNA and protein expression in the presence of systemic lactogenic hormones. Normal first stage induction of both bax mRNA and protein were observed (Fig. 3A and B). Local factors triggered loss of phosphorylation and heterodimerization of Stat5a and Stat5b. The local mechanism underlying loss of Stat 5 activity during involution (13,14)was determined using the closed teat model. Local factors induced loss of 5a and 5b phosphorylation resulting the absence of heterodimerization (Fig. 3C). In contrast, both phosphorylation and heterodimerization were maintained in ipsilateral open glands. The levels of Stat 5a and 5b proteins remained stable. Steady state levels of WAP (Fig. 3B), a milk protein whose in vivo expression is partially dependent on Stat5 activity (17), were also reduced in the closed glands.

Mammary derived factors stimulated Stat3 phosphorylation The teat closure model was used to examine activation of Stat3. Strong phosphorylation of Stat3 appeared in the closed gland by 12 h and persisted, although at slightly lower levels, through d 3 (Fig. 3D) indicating that local factors alone stimulate Stat3 activation during involution (14). Maintenance of systemic lactogenic hormones by suckling preserved lobuloalveolar structure without blocking PCD. Normally, mammary glands are remodelled by day 6 of involution (2). Typical histologic changes include obliteration of the alveolar luminae and collapsed lobules (Fig. 4A). Maintenance of systemic hormone stimulation by suckling blocked progression into the second stage of involution. In the 6 d closed glands, the alveolar lumens remained open and the lobules were intact (Fig. 4B). Systemic hormone stimulation did not, however, block PCD (Fig. 4C). The time of suckling was extended to 10 d with the same results (data not shown).

A model of lactation failure, WAP-TAg transgenic mice, was used as a second approach to evaluate the role of suckling and systemic hormone stimulation on PCD and lobuloalveolar structure. WAP-TAg mice exhibit alveolar cell PCD and early involution due to lactation failure (8,11). At 3 d post-partum, WAP-TAg glands normally exhibit collapsed and compressed alveoli (Fig. 5A), and there is no mRNA expression of the milk protein b-casein (Fig. 5E, lane 6). Serial litter replacement was used to test if maintenance of systemic lactogenic hormones through suckling would preserve lobuloalveolar structure or block PCD. Continuous suckling protected lobuloalveolar structure (Fig. 5B). The alveolar lumens remained open with well distributed lobules (Fig. 5B). b-casein mRNA expression persisted in the suckled glands (Fig. 5E, lanes 2-5). Consistent with the results from the closed teat model, hormonal stimulation did not block PCD (Fig. 5C).

In a third experimental approach, glucocorticoid was administered to WAP TAg mice to test if this single hormone had the same effect as suckling (1,2). Similar to physiological stimulation, cells undergoing PCD persisted in concert with preservation of alveolar structure (Fig. 5D). Significantly, the alveolar diameters of the glucocorticoid treated mice were larger and contained more cells than the alveoli of the suckled mice. This suggested that high levels of exogenous glucocorticoids produce different cellular effects than physiologically elevated levels of endogenous glucocorticoid. Two pathways provided by suckling, milk removal and systemic lactogenic hormone stimulation, are both necessary and sufficient to maintain lactation. Serial litter replacement using normal mice was used to test if lobuloalveolar architecture and lactation could be maintained indefinitely by continuous suckling. During a normal lactation cycle, involution is triggered at approximately three weeks of age as the pups begin to wean themselves. By day 25 post-partum, the gland is in the process of being remodelled (Fig. 6A). In contrast, if continuous suckling is supplied by serial litter replacement, the alveoli remain widely open, the lobules remain intact, and milk production continues (Fig. 6B).

Keywords involution, apoptosis, bax, bcl-x

Contributed By

Priscilla A. Furth
Institute of Human Virology
University of Maryland Medical School
Phone: 410-706-4606
Fax: 410-706-4619
e-mail: furth@nih.gov

More info about the Furth lab ("http://mammary.nih.gov/Groups/Furth/Furth_group.html")

last update: June 1998