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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Kidney Int Suppl. Author manuscript; available in PMC 2010 September 10.
Published in final edited form as:
Kidney Int Suppl. 1996 May; 54: S68–S74.
PMCID: PMC2937007
NIHMSID: NIHMS228536

Extracellular matrix remodeling and the regulation of epithelial-stromal interactions during differentiation and involution

Abstract

An intact basement membrane is essential for the proper function, differentiation and morphology of many epithelial cells. The disruption or remodeling of the basement membrane occurs during normal development as well as in the disease state. To examine the importance of basement membrane during development in vivo, we altered the matrix metalloproteinase and tissue inhibitor of metalloproteianses balance in mammary gland. Inhibition of matrix metalloproteinase synthesis by glucocorticoids or implants or transgenic overexpression of tissue inhibitor of metalloproteinases-1 delays matrix degradation and the involution process after weaning. The mammary glands from transgenic mice that inappropriately express auto-activating isoforms of stromelysin-1 are both functionally and morphologically altered throughout development. Transgenic mammary glands have supernumerary branches, and show precocious development of alveoli that express β-casein expression and undergo unscheduled apoptosis during pregnancy. This is accompanied by progressive development of an altered stroma, which becomes fibrotic after postweaning involution, and by development of neoplasias. These data suggest that metalloproteinases and disruption of the basement membrane may play key roles in branching morphogenesis of mammary gland, cell cycle, apoptosis, and stromal fibrosis as well as in induction and progression of breast cancer.

The remodeling of extracellular matrix (ECM) accompanies cell migration, morphogenesis, development and tissue maintenance, as well as in pathological events including inflammation, fibrosis and tumor invasion. The balance between matrix metalloproteinases (MMPs) and their inhibitors [mostly members of the tissue inhibitor of metalloproteinases (TIMP) family] has been implicated in these processes. When active MMPs are higher than TIMPs, net degradation and erosion of ECM take place, whereas when TIMPs are higher than MMPs, net ECM accumulation ensues (Fig. 1).

Fig. 1
ECM remodeling b the net result of ECM degradation mediated by activated MMPs and inhibition of ECM degradation by TIMPs or other proteinase inhibitors such as α2-macroglobulin.

ECM not only provides structural support for cells within a tissue, but also plays a more active role by affecting adhesion, migration, proliferation and differentiation of many cell types. Events that influence the dynamic state of the ECM must therefore play a critical role in both normal and pathological processes. One such event is the remodeling and degradation of the ECM by ECM-degrading proteinases. Many normal physiological processes, such as bone deposition and resorption, angiogenesis, fertilization, trophoblast implantation, and involution of the uterus, prostate, and mammary gland, require ECM remodeling [14]. Likewise, there are pathological processes that have been associated with excessive or insufficient ECM degradation. These conditions include interstitial fibrosis in kidney and liver, rheumatoid arthritis and osteoarthritis, emphysema, periodontal disease, atherosclerosis, wound repair, and tumor invasion and metastasis [46]. MMPs and TIMPs have been implicated in the proteolytic cascade of ECM degradation.

Mammary gland shows significant proteinase activity during virgin gland development, pregnancy and postweaning involution when ECM remodeling is required [710]. To elucidate the molecular mechanisms involved in ECM remodeling, we generated transgenic mice that inappropriately express stromelysin-1. The stromelysins (stromelysin-1 and -2 and matrilysin) have a broad substrate specificity catalyzing the degradation of various components of the ECM, such as proteoglycans, laminin, fibronectin, entactin and the nonhelical globular portions of type IV collagen [2, 5, 6]. The activity of stromelysin-1 can be effectively inhibited by members of the TIMP family, three distinct forms of which have been cloned [5, 11]. Our experimental rationale was to alter the ratio of MMPs to TIMPs in favor of the proteinases in mammary glands, where ECM remodeling is ah essential element of the normal developmental program of pregnancy, lactation and postweaning involution. When the autoactivating stromelysin-1 transgene is maximally expressed from the whey acidic protein WAP promoter, little endogenous proteinase activity is normally present and the gland is functionally differentiated [12]. The phenotypic changes in the transgenic mice show that ECM plays essential roles as a cell survival factor [10,13,14] in addition to being an important regulator of mammary gland function and morphology in vivo, in regulating stromal-epithelial interactions. In the developing virgin gland, transgenic expression of stromelysin-1 has a morphogenetic effect on mammary-epithelial cells, inducing branching morphogenesis, hyperplasia and differentiation. The stromelysin-1 transgenic mice develop a reactive stroma, become fibrotic and are predisposed to the formation of mammary gland tumors [1416]. These results clearly suggest a role of MMPs in vivo in regulating the ECM, which in turn regulates the survival and function of mouse mammary epithelial cells (MMEC), branching morphogenesis, fibrosis, epithelial-stromal interactions, and the multi-step initiation and progression of breast cancer.

The mammary gland as a model system for studying basement membrane remodeling. In our studies, we have used the mouse mammary gland as a model system to investigate the role of ECM-degrading proteinases in normal growth and development. The mammary gland can undergo repeated cycles of growth and differentiation. It is also easily accessible, and its developmental biology has been extensively studied [17, 18]. The adult gland consists of a fat pad infiltrated by epithelial ducts during puberty with the onset of hormonal stimulation. The mammary gland provides the clearest example to date of the interlocking effects of cell-ECM interactions and ECM remodeling on tissue function. In MMEC, tissue-specific gene expression appears to be exquisitely sensitive to the structure and composition of the ECM. Indeed, in these cells, expression of milk proteins in response to lactogenic hormones appears to require the participation of integrins and to be regulated in vivo by the remodeling of the ECM. This remodeling, in turn, results from changes in the expression of ECM proteinases and their inhibitors. The mammary gland is unusual in that it undergoes dramatic alterations in its ECM during adult life as a result of normal physiological processes, lactation and involution [5]. For this reason, it provides a system for studying the induction of ECM remodeling and the effects of this process on an accessible and abundant cell type, the mammary epithelial cell.

MMEC require a specific morphology and particular interactions with their substrate if they are to express the normal complement of milk proteins in culture [1921]. The regulation of the abundant milk protein β-casein is the best-studied example. Expression of the β-casein gene in cultured MMEC is controlled by cell-ECM interactions, probably in part by protein phosphorylation, and much of this control is exerted at the transcriptional level. A 160 bp promoter element, BCE1, has been identified in the bovine β-casein gene. BCE1 supports the high level expression of a reporter gene in mammary cells when cells are grown in contact with substrates containing laminin and in the presence of lactogenic hormones [22, 23]. Other milk proteins are also regulated by ECM, but there are significant differences in their regulation. For instance, WAP is expressed only in cells that form closed alveoli with tight junctions and polarized morphology, and much of this control appears to be post-transcriptional [18].

As with the expression of milk proteins by MMEC, the expression of ECM components by these cells is sensitive to regulation by ECM contacts. When cells are cultured on plastic or on type I collagen, steady-state levels of laminin and type IV collagen mRNA and protein are dramatically higher than those in cells plated on laminin, suggesting that contact with basement membrane negatively regulates expression of basement membrane components, a novel form of extracellular homeostasis [19]. However, the overexpression of these components does not necessarily suffice to restore a normal substrate for the cells. Cells grown on plastic simply release the material into the medium without depositing it in an insoluble form. Cells grown on a collagen substrate can synthesize their own basement membrane components, but they incorporate them efficiently into a basement membrane only if the substrate is released from the culture dish, thereby allowing cell aggregates to contract. Once formed, the endogenous basement membrane apparently causes cells to down-regulate their synthesis of laminin and other basement membrane components and permits the expression of milk proteins.

To test the role of laminin-integrin interactions in MMEC, Streuli and co-workers [1921] examined cells plated within matrices of different compositions. Within Engelbreth-Holm-Swarm, an artificial matrix containing laminin, even unpolarized single cells expressed β-casein, and an antibody to β1 integrin reduced β-casein expression by 80 to 100%. Within a type I collagen matrix, on the other hand, few cells expressed β-casein unless they formed clusters, and such clusters generally contained endogenously synthesized laminin [20, 21]. The nature of the β1 integrin that responds to laminin is still not known, but a recent experiment suggests that the E3 domain of laminin is involved in the interaction [21]. Interestingly, two potential receptors, α3β1 integrin [24] and dystroglycan [25] interact with this fragment of laminin. These results suggest that a specific cell-ECM interaction mediates this effect and that a signal related to the composition of the ECM is transduced through this integrin to affect the pattern of gene expression in these cells. Furthermore, the regulation by integrins of β-casein synthesis appears to be a direct result of contact with laminin, rather than a secondary effect resulting from the induction of cell-cell interactions or cell polarity [26]. In contrast, the synthesis and deposition of laminin seems to require cell-cell interactions and may be subject to more complex regulation [13].

The indications that expression of milk proteins in vitro requires integrin-mediated contacts with the ECM raise the possibility that changes in these contacts during the course of developmentally regulated ECM remodeling could affect milk synthesis. This issue has been addressed by first characterizing the proteinases and proteinase inhibitors expressed in the mammary gland before, during, and after lactation. It was then possible to interfere with the expression and activity of ECM-remodeling proteinases in otherwise normal mice and to examine their synthesis of milk proteins during lactation and involution. Remarkably, these experiments indicate that changes in the secretory phenotype of mammary epithelial cells during these physiological processes are regulated by the remodeling of the ECM.

During pregnancy, mammary epithelial cells proliferate, and by parturition the gland is packed with secretory alveoli, consisting of polarized epithelial cells linked by tight junctions, contacting a uniform basement membrane. During this period, milk proteins such as β-casein are induced. This structure, as well as the expression of milk-specific genes, is maintained during lactation. After weaning, however, with milk stasis and normal alteration the alveoli regress, milk protein genes are repressed, and the gland assumes a structure similar to that found in virgin animals [7, 8,10, 27].

ECM-degrading proteinases in the involuting mammary gland. MMPs are expressed throughout mammary gland development [7, 8, 10]. Two peaks of expression are seen for stromelysin-1: one between days 6 and 10 of pregnancy and a second major induction between days 4 and 10 of involution. The first expression of stromelysin-1 is associated with a gain-of-function phenotype as the mammary gland branches and begins to acquire alveolar differentiation during early pregnancy, whereas the peak of expression in involution is associated with a loss-of-function phenotype during which major tissue remodeling is characterized by basement membrane (basement membrane) degradation, alveolar regression, apoptosis of epithelial cells, as well as a biosynthetic phase of stromal ECM synthesis, angiogenesis and adipocyte differentiation.

A variety of secreted proteinases and proteinase inhibitors are expressed during this cycle. Among the proteinases are stromelysin-1, stromelysin-3 and the 72 kDa gelatinase A [7,8,10,27]. The MMPs are suppressed during lactation, compared with pregnancy, but are highly expressed during involution. The time course of induction of proteins involved in ECM remodeling correlates with loss of expression of β-casein during involution. During lactation, β-casein mRNA is abundantly expressed, whereas mRNAs for proteinases and inhibitors are hardly detectable. During involution, the basement membrane is degraded and the mammary epithelial cells lose the capacity to express milk protein genes and are shed into the lumina, where they die. The degradation of the ECM appears to involve cooperation between epithelial and mesenchymal cells [10, 2730].

When extracts from involuting mammary glands are analyzed for expression of ECM-degrading proteinases, gelatinase A (72 kDa and its 62 kDa active form) and a 130 kDa gelatinase, which is not inhibited by TIMP-1, account for the major gelatinolytic species [7, 8,10]. These gelatinases are vectorially secreted in the direction of the basement membrane by MMEC in culture, suggesting their involvement in the ECM-remodeling events of involution in vivo. Expression of stromelysin-1 is prominent in the involuting gland, whereas very little is present when the gland is fully functional during lactation. Stromelysin-1 is expressed in periductal and perioalveolar stromal cells in virgin, pregnant and involuting mammary glands [10,14]. The major casein-degrading enzymes with apparent molecular weights of 26 kDa, 35 kDa, 92 kDa and > 100 kDa are not secreted but instead are cell-associated. Tissue-type and urokinase-type plasminogen activator (tPA and uPA, respectively) are also found during involution of the mammary gland. The expression such ECM-degrading proteinases suggests extensive remodeling of the basement membrane during mammary gland involution.

Orchestrated expression of ECM-degrading proteinases and their inhibitors during involution. The transition from a fully functional lactating gland to an involuting gland is characterized by three major events. First, during lactation and the early stages of involution, ECM-degrading proteinases are expressed at low levels. However, three to four days after weaning, stromelysin-1, tPA, uPA, gelatinase A, and the 26 kDa caseinase are up-regulated [7, 8, 10]. This expression reaches a maximum around day 5 to 6 and remains high for at least 10 days into involution. Second, the expression of the proteinase inhibitors, TIMP-1 and plasminogen activator inhibitor-1, precedes the expression of the ECM-degrading proteinases. TIMP-1 mRNA is detected on day 2 of involution, peaks, then declines very rapidly. Third, the expression of β-casein, a mammary gland marker of intact cell-ECM interactions, is dramatically reduced when the balance of ECM-degrading proteinases and their inhibitors favors the proteinases, Between days 4 and 8 of involution, the epithelial cells of the mammary gland undergo massive programmed dell death or apoptosis [10] by a mechanism involving interleukin-1β converting enzyme (ICE) [10, 13] and are phagocytosed by neighboring epithelial cell or macrophages. Also during this time, the adipocytes differentiate to fill the gland, returning it to a state similar to that of the virgin gland. These events strongly suggest that the high levels of ECM-degrading proteinases during involution result in the disruption of cell-ECM interactions that modulate MMEC morphology and function.

Significantly, the expression of β-casein mRNA drops dramatically around day 5, when proteinase expression increases and inhibitor expression begins to decline [8,10]. These results suggest that the balance between proteinases and their inhibitors, rather than the level of expression of either class of molecule, modulate the differentiated state of mammary cells.

The correlation between excess proteinase expression and loss of β-casein holds under various physiological conditions, although the balance between proteinases and inhibitors is tipped with different time courses and with different absolute levels of proteinases and inhibitors. Thus, after brief periods of lactation, involution proceeds more rapidly, but with the same order of events, and β-casein expression ceases when proteinases begin to predominate over inhibitors, after only two days of involution, Conversely, sealing a mammary gland to prevent suckling induces relatively high levels of both TIMP-1 and stromelysin-1, but with an excess of the inhibitor. Under these conditions, loss of β-casein expression is significantly retarded [8].

Modified development and involution in mammary glands of transgenic mice with altered matrix metalloproteinase function. To probe the correlation between excess degradative activity and repression of β-casein expression, Talhouk, Bissell and Werb [8] perturbed the balance directly. When slow-release pellets containing TIMP-1 were implanted into mammary glands in vivo two days after the cessation of suckling, TIMP-1 delayed both the onset of involution and the decline of β-casein expression in a dose-dependent manner. The effect of TIMP-1 is seen not only in the stability of the basement membranes in treated mammary glands, but also in the failure of MMEC in these glands to be lost by apoptosis [9], suggesting that TIMPs can delay or regulate apoptosis by maintaining the integrity of the basement membrane. Notably, stromelysin-3, an MMP associated with apoptosis, is expressed during involution but not in pregnancy, when ECM remodeling occurs as part of mammary growth [27], suggesting that an altered balance between this enzyme and TIMPs may trigger apoptosis. Inhibition of MMP synthesis by glucocorticoid treatment in vivo completely inhibits ECM remodeling of MMEC apoptosis [10]. Direct evidence for the role of MMPs and basement membrane degradation in regulating apoptosis has been found by using MMEC in culture [13]. Similarly, apoptosis of other cell types, including endothelial cells [31] and keratinocytes, has been found to be regulated by interactions with the ECM. Loss of contact with the ECM regulates the differentiation of keratinocytes and the apoptosis of these cells [reviewed in 32]. Since proliferating basal cell keratinocytes can secrete MMPs [33], it may be that these enzymes alter integrin-ECM interactions to promote differentiation in this system. In MMEC in culture, the apoptotic program involves loss of ECM, triggering the expression and activation of ICE [13]. Taken together, these data indicate that the balance between proteinases and their inhibitors regulates integrin-ECM contacts by controlling turnover of the basement membrane. Signaling through integrins appears to be necessary for control of tissue-specific gene expression and cell viability.

To demonstrate the importance of the ECM in promoting and maintaining tissue-specific morphology and function in vivo, we generated transgenic mice that ectopically express autoactivating isoforms of stromelysin-1 at a time when an intact basement membrane is essential for normal growth and differentiation of MMEC [12]. We targeted the transgene to the mammary gland by using the promoter from the WAP gene, which encodes a milk protein that begins to accumulate in midpregnancy. Stromelysin-1 was chosen because it has been implicated as a key player in mammary gland involution and neoplasias, it digests a wide variety of ECM molecules, and autoactivating rat mutant cDNAs have been generated and well characterized. The WAP promoter was chosen because it has been used extensively to target transgenes to the mammary gland during midpregnancy and lactation.

The small amount of stromelysin-1 transgene expression in the adult virgin gland also produced a gain-of-function phenotype. The mammary gland of prepubertal female mice is essentially quiescent. However, with the onset of ovarian activity (between 20 to 25 days), primary ducts, once confined to the immediate area of the teat, form large, club-shaped terminal end buds and infiltrate the fat pad [12,16]. These ducts usually arrive at the end of the fat pad at around day 70 (mature adult virgin), and the gland remains dormant until pregnancy. Examination of glands taken from normal 70-day virgin females revealed the typical branching pattern. However, the mammary glands from stromelysin-1 transgenic mice showed precocious alveolar development and maturation, with primary and secondary ducts that filled the fat pad. This phenotype is both morphologically and functionally (as measured by their ability to express β-casein) very similar to that of a normal 9- to 12-day pregnant gland, in which endogenous stromelysin-1 is normally expressed. These results suggest that the morphology of the developing gland is exquisitely sensitive to pericellular proteolysis. Therefore, ectopic expression of stromelysin-1 appears to stimulate epithelial cell growth and differentiation in the virgin gland, producing a gain-of-function phenotype that resembles early pregnancy. This suggests that stromelysin-1 may play a role in branching morphogenesis in early pregnancy. This hypothesis is currently being investigated in stromelysin-1 null mice.

Histological examination of the midpregnant glands from several lines of these transgenic mice revealed that unscheduled involution of the mammary gland had been induced. The degradation of the basement membrane results in the collapse of alveolar structures. The glands were also impaired functionally, because the level of tissue-specific gene expression was drastically reduced [12]. In situ DNA fragmentation, a hallmark of apoptosis, is induced in the transgenic but not the normal CD-1 midpregnant glands [13, 34, 35]. Approximately 15% of the epithelial cells in the transgenic gland are in the process of apoptosis at 15 days of pregnancy, whereas no apoptosis or DNA fragmentation can be detected in the normal CD-1 glands. Furthermore, the CID-9 mammary epithelial cell line is transfected with the autoactivating mutant of stromelysin-1 driven by an inducible promoter, the basement membrane is disrupted, and apoptosis is triggered in the cells by a mechanism involving the induction of ICE [13].

Expression of the stromelysin-1 transgene increased slightly as the animals progressed through lactation. Unlike the normal CD-1 glands, which had large distended alveoli, the transgenic glands contained smaller collapsed alveoli (Fig. 2) that had reduced amounts of milk proteins, consistent with an early involution phenotype [12]. Surprisingly, the transgenic mice were still capable of lactating even though the basement membrane was severely disrupted. Despite gross abnormalities during the growth and differentiation phases of the transgenic glands, the involution process appeared to proceed normally.

Fig. 2
Induction of apoptosis is initiated by active stromelysin-1 (curved arrow), which then mediates basement membrane degradation under the epithelial cell

We have also observed a dramatic alteration in the mesenchymal-epithelial interaction in stromelysin-1 transgenic mice. In particular, there is a discordance in the phenotypes of these two compartments, with the stromal phenotype resembling a wound site or an involuting gland [12]. Tissue injury is often associated with tumor development. An up-regulation of MMP expression in the reactive stroma surrounding malignant tumors is a key feature of tumor progression [26]. Indeed, stromelysin-1 has been cloned several times in screens for tumor specific genes. Thus, it is interesting to note that we have observed a marked increase in the incidence of tumors in four independent lines of the stromelysin-1 transgenic mice [12]. All transgenic mice, thus far, display a reactive stroma [14] and phenotypic abnormalities ranging from severe epithelial hyperplasia, resembling a lactational phenotype, to adenocarcinomas [15, 16]. The hormonal stimulation of pregnancy does not appear to be absolutely necessary for tumor formation, because tumors have developed in glands of virgin mice as young as four months old.

To test the hypothesis that proteolysis regulates mammary gland morphogenesis, we have generated animals that ectopically express TIMP-1 [34, 35]. Preliminary results suggest that crossing TIMP-1 transgenic mice with stromelysin-1 transgenic mice can quench the premature involution phenotype during pregnancy that is induced by the ectopic expression of stromelysin-1 [35]. This system should be amenable for testing the function of MMP inhibitors in vivo. Lactating stromelysin-1 transgenic animals produce less (although qualitatively normal) milk than do normal mice, and their mammary alveoli are smaller, with degraded, discontinuous basement membranes. The precocious apoptotic phenotype can be completely reversed by cloning the stromelysin-1 transgenic mice with transgenic mice overexpressing human TIMP-1 [34,35].

Discussion

These studies clearly demonstrate that coordinated expression and a critical balance between ECM-degrading proteinases and their inhibitors regulate epithelial cell function and morphology during involution of the mammary gland. As the ratio of ECM-degrading proteinases to inhibitors increases, basement membrane is degraded, mammary epithelial cells lose their ability to express β-casein and undergo apoptosis, and alveolar regression becomes evident. Our in vivo studies of the mouse mammary gland have led us to propose a model for the series of events that occur during, and regulate the process of involution. After cessation of milk removal from the gland, milk continues to be secreted and accumulates in the alveolar lumina for one to two days. At this time, local factors induce the expression of ECM-degrading proteinases and their inhibitors in a coordinated and temporal pattern. We believe that events that regulate cell-ECM interactions occur in a microenvironment at the individual cell level within an alveolus. Each cell or group of cells within an alveolus is part of a milieu with various amounts of either the ECM-degrading proteinases or the inhibitors. The ECM-degrading proteinases and their inhibitors are secreted either by the epithelia themselves or by neighboring fibroblasts in the underlying stroma. The postulated events that occur at the level of an individual alveolus are as follows. As the lumen of a particular alveolus is engorged owing to cessation of milk removal, a local signal instructs certain cells in the alveolus or in the immediate microenvironment (myoepithelial cells or fibroblasts) to secrete ECM-degrading proteinases. Local concentrations of ECM-degrading proteinases then degrade the basement membrane in the immediate vicinity and alter cell-ECM interactions. This leads to the generation of bioactive ECM fragments, and eventually to detachment of a cell from its degraded basement membrane and from neighboring cells within the alveolus. At some time during this process the cells lose their ability to express milk proteins. The detachment of the cells from the basement membrane could trigger signals for programmed cell death or apoptosis, including the induction of ICE gene expression. Apoptotic events commence as the ratio of ECM-degrading proteinases to inhibitors increases during involution of the mammary gland, or in an unscheduled way in the glands from midpregnant WAP-stromelysin-1 transgenic mice. After the apoptotic cell detaches from its underlying degraded basement membrane and from neighboring cells in the alveolus, the remaining cells, which rest on an intact basement membrane, join together (Fig. 2). As involution proceeds, the net result is a smaller alveolus with a continuous basement membrane [36]. It is not clear what regulates these events at the individual alveolus, and why certain cells within an alveolus are destined to die before others. It is possible that only cells that are progressing through the cell cycle are able to die. We have postulated that the local concentrations of inhibitors bound to the basement membrane protect surviving cells within an alveolus and temporally regulate programmed cell death and alveolar regression.

Many recent studies suggest a role for apoptosis in maintaining homeostasis in normal development, such as embryonic morphogenesis, endometrial cycling, and immune cell selection [37]. In these and other systems, apoptosis offers a physiological way to limit the number of cells, remove cells that have completed a given function, or eliminate premalignant cells. It is clear from our studies that the degradation of ECM by proteinases plays an important role in the apoptosis of MMEC of the involuting mouse mammary gland. However, in the midpregnant glands from transgenic mice, it is not clear if the epithelial cells undergoing apoptosis are indeed normal, leaving premalignant cells that escape apoptosis.

The initiation of apoptosis has been correlated in several cell types with aberrant regulation of the cell cycle and proliferation. Analysis of a variety of cells in tissue culture has shown that at least one of the cyclins is induced in the absence of the rest of the normal regulatory pathway for proliferation with the timing of induction that associates it closely with commitment to apoptosis [3844]. Inhibiting the induction of the cyclin or the activation of the associated kinase suppresses apoptosis [38, 39]. Suppression of apoptosis by stable overexpression of bcl-2 prevents the inappropriate expression or activation of the cell cycle regulatory molecules [42,43], in one case by allowing induction of the normal complement of cyclins [43]. The specific cell cycle regulatory molecules that are induced during apoptosis differ between cell types; specific examples are cyclin D1 in cultured neurons [44], cyclin A in HeLa cells [42] and embryonic fibroblasts [43] and B-type cyclins in hematopoietic cells [38, 39, 41]. It is not yet clear whether misregulation of the cell cycle is a necessary component of apoptosis in all cases, as there are several examples in which there arc as yet no obvious aberrations in cell cycle regulation [45,46]. The observations that link apoptosis and misregulation of the cell cycle have not yet been extended into intact tissues. It is interesting to note that in the involuting mammary gland, apoptosis is induced at four days postweaning and is largely confined to the epithelial cells that line the alveoli (A. MacAuley, L. Lund and Z. Werb, unpublished observation). Bromodeoxyuridine labeling during this period has demonstrated that there is a contemporaneous increase in the number of cells entering S phase that is also largely confined to the epithelial cells of the alveoli. In addition, the precocious apoptosis that occurs in the stromelysin-1 transgenic mice during pregnancy, when the epithelial cells are actively proliferating, ceases during lactation, when these cells exit the cell cycle [35]. It seems possible that induction of proliferation during the radical changes in the cellular environment that accompany involution may result in misexpression of cell cycle regulatory components in many of the epithelial cells, leading to apoptosis.

Taken together, the observations on mammary glands in which the balance of MMPs and inhibitors has been perturbed indicate that in the context of a normal gland, ECM remodeling is both necessary and sufficient to alter the lactational phenotype of mammary epithelial cells. The experiments on cultured cells described above suggest one mechanism for this, namely, laminin-based signaling through a β1 integrin. However, it remains an outstanding challenge in this system to link the in vivo results with observations on cultured mammary cells. Meeting this challenge will require a more detailed understanding of integrin-mediated signaling in mammary cells. It may also entail designing a cell culture model of mammary involution and/or genetically manipulating the mammary gland in live animals to alter signal transduction systems or cell-ECM contacts.

The current body of evidence suggests that a complex pattern of mutual regulation between integrin-mediated signaling and matrix proteolysis has profound influences on a variety of cell behaviors. Thus, ECM regulates cellular function by signaling through integrins, which in turn regulate the turnover of ECM, and these intersecting events may alter both cellular proliferation and programmed cell death, as well as cell migration and invasion, differentiation, and the maintenance of tissue-specific functions. This mechanism operates in the mammary gland, and it is likely that ECM turnover in other tissues is the critical event regulating morphogenesis and differentiation both in developmental tissue regeneration and in pathogenic situations. Because ECM, in turn, signals to the cells through integrins and results in the regulation of MMPs and TIMPs, this interplay serves to choreograph migratory, invasive and differentiative events in multicellular organisms.

Acknowledgments

This work was supported by the U.S. Department of Energy, Office of Health and Environmental Research (contracts DE-AC03-76-SF01012 and DE-AC03-76-SF00098), and grants from the National Cancer Institute (CA 57621 and CA 58207) and from the Women’s Health Initiative, Office of the Director, NIH (CA 57621S1).

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