Apoptosis and mammary gland involution: reviewing the process

Apoptosis is a process of programmed cell death. Mammary gland involution is a tissue remodelling process. Mammary epithelial cell apoptosis is an integral component of tissue remodelling but it is only one element. Equally important are the factors which degrade basement membrane and extracellular matrix. Both operations are required for completion of mammary gland involution. The primary apoptotic process occurs first and is temporally distinct from the second stage of involution typified by lobular-alveolar collapse. Local factors related to milk accumulation trigger the first stage, but loss of systemic hormonal stimulation governs the second stage. Changes in the expression patterns of cell cycle control genes and bcl-2 family member genes are found in the first stage. Proteinase gene activation dominates the second stage. These findings support a two stage model of mammary gland involution. Both mammary epithelial cell apoptosis and mammary gland remodelling advance through a process which includes both loss of survival factors and gain of death factors. This review focuses on signalling pathways and genetic controls which are activated and repressed during mammary gland involution.     

IRF6 in development and disease: A mediator of quiescence and differentiation

Post utero development of the mammary gland is a complex developmental process characterized by states of rapid cell proliferation (branching morphogenesis) followed by functional differentiation (lactation) and the consequent apoptosis (involution) of the secretory mammary epithelial cell. This process is cyclical, such that involution returns the mammary gland to a near-virgin-like state capable of responding to morphogenic cues with each consecutive pregnancy. Importantly, many of the regulatory processes which oversee mammary gland development are corrupted or otherwise compromised during the development of breast cancer. For example, Interferon Regulatory Factor 6 (IRF6) is a novel protein with growth inhibitory properties that was initially identified in mammary epithelial cells through its interaction with maspin, a known tumor suppressor in normal breast tissue. Recent findings from our laboratory suggest that IRF6 functions synergistically with maspin to regulate mammary epithelial cell differentiation by acting on the cell cycle. This perspective focuses on the possible involvement of IRF6 in promoting differentiation by regulating exit from the cell cycle and entry into the G(0) phase of cellular quiescence, and how these new findings shed light on normal mammary gland development and the initiation and progression of breast cancer.     

ECM degrading proteases and tissue remodelling in the mammary gland

Matrix degradation and tissue remodelling directed by matrix-degrading proteases are activated in physiological situations such as wound healing and involution of the prostate, ovaries and uterus. Recently, other activities, in addition to the cleavage of matrix proteins, have been attributed to matrix proteases including the release of growth factors from the extracellular matrix and roles in the maturation of adipocytes. This review describes extracellular proteases, including MMPs, plasminogen and cathepsins involved in the tissue remodelling processes that occur in the breast during pubertal mammary development and the mammary cycle of pregnancy, lactation and weaning. It particularly focuses on development and weaning, termed mammary gland involution, when the majority of remodelling occurs. It also brings together recent findings on the exciting new functions of matrix-degrading proteases.     

Knocking Off SOCS Genes in the Mammary Gland

Suppressors of cytokine signaling (SOCS) proteins are critical regulators of cytokinemediatedresponses in diverse tissues. In the mammary gland, signal transductionpathways elicited by cytokines and hormones have been shown to control distinct stagesof development. In vivo evidence points to essential roles for Socs1 and Socs2 as keyphysiological attenuators of prolactin receptor (PRLR) signaling during pregnancy andlactogenesis. Recently, Socs3 has been shown to be a critical regulator of involution, thecoordinated process of programmed cell death and tissue remodelling that is initiatedafter the cessation of lactation. This review will predominantly focus on the antiapoptoticfunction of Socs3 during mammary gland involution in which it acts as a keyattenuator of Stat3-mediated signal transduction. Perturbation of this pathway leads to anincrease in the levels of c-myc and its likely target genes, p53, bax and E2F-1, providingevidence that c-myc is a central effector of apoptosis during involution.     

Method for the generation and cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix

During puberty, pregnancy, lactation and post-lactation, breast tissue undergoes extensive remodelling and the disruption of these events can lead to cancer. In vitro studies of mammary tissue and its malignant transformation regularly employ mammary epithelial cells cultivated on matrigel or floating collagen rafts. In these cultures, mammary epithelial cells assemble into three-dimensional structures resembling in vivo acini. We present a novel technique for generating functional mammary constructs without the use of matrix substitutes.     

Histological changes of the mouse thymus during involution and regeneration following administration of hydrocortisone

Summary A histological study has been made of the thymus in mice during acute involution and regeneration following administration of hydrocortisone. The cortex undergoes remarkable changes in the microscopic structure during involution and regeneration. During involution the lymphocytes in the cortex rapidly decrease and are removed. Then a rapid replacement of lymphocytes occurs during regeneration. On the basis of formation and repopulation of lymphocytes the regenerative process of the cortex is divided into seven phases. The reconstitution of the cortex proceeds more rapidly in females than in males. Newly formed lymphocytes take origin from the mesenchymal cells in the cortex. Such mesenchymal cells become distinguishable from epithelial reticular cells during involution. They appear to engulf destroyed lymphocytes and debris during involution and then transform into immature lymphoid cells during early regeneration. The findings may support the recent reutilization concept that destroyed lymphocytes are phagocytized and reutilized by reticular cells in heteroplastic differentiation into immature lymphoid cells. In the cortex PAS-positive sudanophilic cells which are derived from the perivascular and subcapsular connective tissue appear with involutionary changes. They become gradually reduced again with progress of the regeneration of the cortex. During involution the medulla are temporarily filled with lymphocytes migrated from the cortex. The epithelial reticular cells in the medulla are found grouped in cords or clumps in the severely involuted thymus. In the medulla there are two types of PAS-positive epithelial reticular cells; one contains a large, colloid-like, PAS-positive inclusion within the cytoplasm and the other has cytoplasm diffusely filled with PAS-positive substance. During involution and early regeneration, the former type increases while the other shows almost no significant changes. Hassall's corpuscles somewhat increase in frequency during involution and early regeneration.     

Inhibitory activity of YKL-40 in mammary epithelial cell differentiation and polarization induced by lactogenic hormones: a role in mammary tissue involution

We previously reported that a secreted glycoprotein YKL-40 acts as an angiogenic factor to promote breast cancer angiogenesis. However, its functional role in normal mammary gland development is poorly understood. Here we investigated its biophysiological activity in mammary epithelial development and mammary tissue morphogenesis. YKL-40 was expressed exclusively by ductal epithelial cells of parous and non-parous mammary tissue, but was dramatically up-regulated at the beginning of involution. To mimic ductal development and explore activity of elevated YKL-40 during mammary tissue regression in vivo, we grew a mammary epithelial cell line 76N MECs in a 3-D Matrigel system in the presence of lactogenic hormones including prolactin, hydrocortisone, and insulin. Treatment of 76N MECs with recombinant YKL-40 significantly inhibited acinar formation, luminal polarization, and secretion. YKL-40 also suppressed expression of E-cadherin but increased MMP-9 and cell motility, the crucial mechanisms that mediate mammary tissue remodeling during involution. In addition, engineering of 76N MECs with YKL-40 gene to express ectopic YKL-40 recapitulated the same activities as recombinant YKL-40 in the inhibition of cell differentiation. These results suggest that YKL-40-mediated inhibition of cell differentiation and polarization in the presence of lactogenic hormones may represent its important function during mammary tissue involution. Identification of this biophysiological property will enhance our understanding of its pathologic role in the later stage of breast cancer that is developed from poorly differentiated and highly invasive cells.     

c-myc as a mediator of accelerated apoptosis and involution in mammary glands lacking Socs3

Suppressor of cytokine signalling (SOCS) proteins are critical attenuators of cytokine-mediated signalling in diverse tissues. To determine the importance of Socs3 in mammary development, we generated mice in which Socs3 was deleted in mammary epithelial cells. No overt phenotype was evident during pregnancy and lactation, indicating that Socs3 is not a key physiological regulator of prolactin signalling. However, Socs3-deficient mammary glands exhibited a profound increase in epithelial apoptosis and tissue remodelling, resulting in precocious involution. This phenotype was accompanied by augmented Stat3 activation and a marked increase in the level of c-myc. Moreover, induction of c-myc before weaning using an inducible transgenic model recapitulated the Socs3 phenotype, and elevated expression of likely c-myc target genes, E2F-1, Bax and p53, was observed. Our data establish Socs3 as a critical attenuator of pro-apoptotic pathways that act in the developing mammary gland and provide evidence that c-myc regulates apoptosis during involution.     

Epithelial cells remove apoptotic epithelial cells during post-lactation involution of the mouse mammary gland

Following the cessation of lactation, the mammary gland undergoes a physiologic process of tissue remodeling called involution in which glandular structures are lost, leaving an adipose tissue compartment that takes up a much larger proportion of the tissue. A quantitative morphometric analysis was undertaken to determine the mechanisms for clearance of the epithelial cells during this process. The involution process was set in motion by removal of pups from 14-day lactating C57BL/6 mice. Within hours, milk-secreting epithelial cells were shed into the glandular lumen. These cells became apoptotic, exhibiting exposure of phosphatidylserine residues on their surfaces, activation of effector caspase-3, staining for caspase-cleaved keratin 18, loss of internal organellar structure, and nuclear breakdown, but minimal blebbing or generation of apoptotic bodies. Clearance of residual milk and the shed epithelial cells was rapid, with most of the removal occurring in the first 72 h. Intact apoptotic epithelial cells were engulfed in large numbers by residual viable epithelial cells into spacious efferosomes. This process led to essentially complete involution within 4 days, at which point estrous cycling recommenced. Macrophages and other inflammatory cells did not contribute to the clearance of either residual milk or apoptotic cells, which appeared to be due entirely to the epithelium itself.     

Differential expression of claudin 1, 3, and 4 during normal mammary gland development in the mouse

The claudins are a family of tight junction proteins that display varied tissue distribution and preferential specificity. We recently identified by microarray analysis, members of this family, particularly claudin 1 (cldn1), as highly upregulated genes in the mouse mammary gland during early involution. Gene expression was confirmed by immunohistochemistry and real-time PCR. We then examined gene and protein expression throughout normal mammary gland development. The cldn3 gene showed a steady increase in expression from pregnancy to involution, while cldn1 and cldn4 gene expression increased during pregnancy, but decreased sharply by D10 of lactation, and once again was significantly increased by D1 of involution (P < 0.001 for both genes). The different patterns of gene expression observed between cldn3, and cldn1, and 4 suggest that different family members may be functionally important at different times during mouse mammary gland development. All three genes exhibited a high level of expression at day 1 (D1) of involution, followed by a dramatic decrease in gene expression to day 10 of involution. Immunostaining with the cldn3 antibody showed intense staining of epithelial cells; however, a lesser degree of staining was evident with the cldn1 antibody. In addition to the lateral staining of epithelial cells, basal staining was evident at D1 and D2 of involution and cytoplasmic staining was evident during lactation. Since claudins are known to play a role as tight junction proteins, lateral and basal staining may suggest a role in other functions such as vesicle trafficking or remodeling of tight junctions at different stages of mammary gland development. Cldn1 and 3 antibodies also stained epithelial cells in mouse mammary tumors. In summary, cldn1, 3, and 4 are differentially expressed in the mammary gland during pregnancy, lactation, and involution, suggesting different roles for these proteins at different stages of mammary gland function. In addition, cldn1 and cldn3 are detected in mammary tumors and the wide distribution of cldn3 in particular, suggest specific roles for these proteins in mammary tumorigenesis.     

Calmodulin content of rat mammary tissue and isolated cells during pregnancy and lactation.

The calmodulin content of heat-treated extracts of rat mammary tissue and isolated cells was measured by using stimulation of cyclic nucleotide phosphodiesterase (PDE) activity and radioimmunoassay (r.i.a.) procedures. The calmodulin content of mammary tissue increased 2.5-fold near the time of parturition, remained at the elevated level during lactation, then, after the onset of involution, decreased to values similar to those measured from mammary tissue of pregnant rats. When tissue from 15 animals in different stages of pregnancy, lactation and involution were compared, the r.i.a. gave 2.6-fold higher results than the PDE assay. To investigate further the increase in calmodulin content of mammary tissue, secretory and myoepithelial cells were enzymically dissociated from rat mammary tissue during different stages of pregnancy, lactation and involution. Protein, DNA, lactose, glucose-6-phosphate dehydrogenase and alkaline phosphatase were assayed to characterize the cell fractions. By using r.i.a., the calmodulin content per mg of protein in isolated secretory-cell fractions was high near parturition, then decreased and remained relatively constant during lactation. The amount of calmodulin expressed per mg of DNA in secretory cells did not show a marked change near parturition, suggesting a constant amount of calmodulin per cell. The calmodulin content of myoepithelial cells dissociated from mammary tissue and measured by using r.i.a. was 6-fold lower than in secretory cells and remained relatively constant during the course of lactation. The changing levels of calmodulin in rat mammary tissue during development are suggested to be related to proliferation and destruction of secretory epithelial cells, events that occur near parturition and involution respectively.     

Cell death by apoptosis during involution of the lactating breast in mice and rats

The role of cell death in involution of lactating breast was investigated in mice and rats by light and electron microscopy. Apoptosis, recognized by sharply demarcated compaction of chromatin against the nuclear envelope and by shrinkage and budding of the whole cell to form membrane-bounded apoptotic bodies, was responsible for major loss of cells in both species. In the mouse, rapid involution during the first 2 days was associated with shedding of large numbers of apoptotic bodies derived from alveolar epithelial cells into alveolar lumens. This was followed by more gradual regression, during which the bodies were mostly phagocytosed by macrophages within the epithelium. In the rat, glandular involution was a more gradual and uniform process, with shedding of apoptotic epithelial cells into alveolar lumens being much less conspicuous. Apoptosis of myoepithelial cells was observed in mice, the resulting apoptotic bodies being phagocytosed by intraepithelial macrophages, but was not detected in rats. Apoptosis of capillary endothelial cells caused rapid regression of the capillary beds in both mice and rats. Intraepithelial macrophages increased in number during involution, developed cytoplasmic lipofuscin pigment, and either remained within the epithelium or migrated to the interstitium and regional nodes. Cell loss by apoptosis has been demonstrated during involution and atrophy of a variety of other glands. It characteristically results in shrinkage of a tissue without disruption of its basic architecture.     

Annexin A8 Identifies a Subpopulation of Transiently Quiescent c-Kit Positive Luminal Progenitor Cells of the Ductal Mammary Epithelium

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G₀/G₁ arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin.     

The rat as an animal model for fetoplacental development: a reappraisal of the post-implantation period

Following implantation in rodents, the uterine stromal fibroblasts differentiate into densely packed decidual cells. This process, called decidualization, is well-orchestrated and progresses both antimesometrially and mesometrially, creating two regions with distinctive cellular morphologies. In addition, subsequent placental development is dependent on the invasion of the trophoblast, the process intimately linked to the endometrial tissue remodelling and depending largely on the environment created by the decidua; this phenomenon is crucial for the establishment and maintenance of pregnancy. The key mechanisms underlying the maternal tissue remodelling and trophoblast invasion remain poorly understood. The rat, just like human beings, exhibits a highly invasive type of placental development, the haemochorial placentation. For obvious ethical reasons, the studies of endometrial tissue remodelling throughout pregnancy in humans are greatly limited. Although the rat differs somewhat from humans with regards to the implantation process, it is an appropriate model for studying the mechanisms of decidualization as well as subsequent remodelling of the uterine tissues and fetoplacental development. As decidual remodelling is very closely linked to placentation and the maternal-fetal interactions in the rat show several important similarities to human placentation, the morphological alterations occurring during the post-implantation period in the rat have been addressed in the present review.     

Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2

The prognosis of breast cancer in young women is influenced by reproductive history. Women diagnosed within 5 years postpartum have worse prognosis than nulliparous women or women diagnosed during pregnancy. Here we describe a mouse model of postpartum breast cancer that identifies mammary gland involution as a driving force of tumor progression. In this model, human breast cancer cells exposed to the involuting mammary microenvironment form large tumors that are characterized by abundant fibrillar collagen, high cyclooxygenase-2 (COX-2) expression and an invasive phenotype. In culture, tumor cells are invasive in a fibrillar collagen and COX-2-dependent manner. In the involuting mammary gland, inhibition of COX-2 reduces the collagen fibrillogenesis associated with involution, as well as tumor growth and tumor cell infiltration to the lung. These data support further research to determine whether women at high risk for postpartum breast cancer would benefit from treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) during postpartum involution.