Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution

Extracellular matrix (ECM) plays an important role in the maintenance of mammary epithelial differentiation in culture. We asked whether changes in mouse mammary specific function in vivo correlate with changes in the ECM. We showed, using expression of beta-casein as a marker, that the temporal expression of ECM-degrading proteinases and their inhibitors during lactation and involution are inversely related to functional differentiation. After a lactation period of 9 d, mammary epithelial cells maintained beta-casein expression up to 5 d of involution. Two metalloproteinases, 72-kD gelatinase (and its 62-kD active form), and stromelysin, and a serine proteinase tissue plasminogen activator were detected by day four of involution, and maintained expression until at least day 10. The expression of their inhibitors, the tissue inhibitor of metalloproteinases (TIMP) and plasminogen activator inhibitor-1, preceded the onset of ECM-degrading proteinase expression and was detected by day two of involution, and showed a sharp peak of expression centered on days 4-6 of involution. When involution was accelerated by decreasing lactation to 2 d, there was an accelerated loss of beta-casein expression evident by day four and a shift in expression of ECM-remodeling proteinases and inhibitors to a focus at 2-4 d of involution. To further extend the correlation between mammary-specific function and ECM remodeling we initiated involution by sealing just one gland in an otherwise hormonally sufficient lactating animal. Alveoli in the sealed gland contained casein for at least 7 d after sealing, and closely resembled those in a lactating gland. The relative expression of TIMP in the sealed gland increased, whereas the expression of stromelysin was much lower than that of a hormone-depleted involuting gland, indicating that the higher the ratio of TIMP to ECM-degrading proteinases the slower the process of involution. To test directly the functional role of ECM-degrading proteinases in the loss of tissue-specific function we artificially perturbed the ECM-degrading proteinase-inhibitor ratio in a normally involuting gland by maintaining high concentrations of TIMP protein with the use of surgically implanted slow-release pellets. In a concentration-dependent fashion, involuting mammary glands that received TIMP implants maintained high levels of casein and delayed alveolar regression. These data suggest that the balance of ECM-degrading proteinases and their inhibitors regulates the organization of the basement membrane and the tissue-specific function of the mammary gland.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture.

The extracellular matrix (ECM) is an important regulator of mammary epithelial cell function both in vivo and in culture. Substantial remodeling of ECM accompanies the structural changes in the mammary gland during gestation, lactation and involution. However, little is known about the nature of the enzymes and the processes involved. We have characterized and studied the regulation of cell-associated and secreted mammary gland proteinases active at neutral pH that may be involved in degradation of the ECM during the different stages of mammary development. Mammary tissue extracts from virgin and pregnant CD-1 mice resolved by zymography contained three major proteinases of 60K (K = 10(3) Mr), 68K and 70K that degraded denatured collagen. These three gelatinases were completely inhibited by the tissue inhibitor of metalloproteinases. Proteolytic activity was lowest during lactation especially for the 60K gelatinase which was shown to be the activated form of the 68K gelatinase. The activated 60K form decreased prior to parturition but increased markedly after the first two days of involution. An additional gelatin-degrading proteinase of 130K was expressed during the first three days of involution and differed from the other gelatinases by its lack of inhibition by the tissue inhibitor of metalloproteinases. The activity of the casein-degrading proteinases was lowest during lactation. Three caseinolytic activities were detected in mammary tissue extracts. A novel 26K cell-associated caseinase--a serine arginine-esterase--was modulated at different stages of mammary development. The other caseinases, at 92K and a larger than 100K, were not developmentally regulated. To find out which cell type produced the proteinases in the mammary gland, we isolated and cultured mouse mammary epithelial cells. Cells cultured on different substrata produced the full spectrum of gelatinases and caseinases seen in the whole gland thus implicating the epithelial cells as a major source of these enzymes. Analysis of proteinases secreted by cells grown on a reconstituted basement membrane showed that gelatinases were secreted preferentially in the direction of the basement membrane. The temporal pattern of expression of these proteinases and the basal secretion of gelatinases by epithelial cells suggest their involvement in the remodelling of the extracellular matrix during the different stages of mammary development and thus modulation of mammary cell function.     

Expression of M-N#1, a histo-blood group B-like antigen, is strongly up-regulated in nonapoptosing mammary epithelial cells during rat mammary gland involution.

Antibodies against the histo-blood group B-like antigen M-N#1 efficiently block the growth in vivo of rat mammary carcinoma cells that bear the antigen (Sleeman et al., 1999, Oncogene 18, 4485--4494). To try to understand the function of the M-N#1 antigen, we investigated when and where the antigen is expressed during the normal function of the rat mammary gland. Expression was virtually only seen during mammary gland involution. Here, strong expression of the antigen was observed in mammary epithelial cells, beginning around 2 days postweaning and lasting throughout the involution process. Dexamethasone treatment of animals postlactation inhibited alveolar collapse and remodeling in the mammary gland but inhibited neither the apoptosis of mammary epithelial cells nor the expression of the M-N#1 antigen. We show that up-regulation of carbohydrate antigens is not a general phenomenon during mammary gland involution, and thus that M-N#1 antigen expression is specifically regulated. Up-regulation of alpha(1,2)fucosyltransferase A, an enzyme required for M-N#1 antigen synthesis, is at least partly responsible for regulated M-N#1 antigen expression postlactation. Most significantly, we observed that the M-N#1 antigen is virtually exclusively expressed on nonapoptosing epithelial cells in the involuting mammary gland. These data suggest that M-N#1 antigen expression might either provide a survival function and/or be expressed in epithelial cells that are destined to grow and remodel mammary duct structures.     

Modulation of neutral matrix metalloproteinases of involuting rat mammary gland by different cations and glycosaminoglycans

The synthesis and regulation of the matrix metalloproteinases (MMPs) are important factors contributing to the involution of mammary gland. In order to understand the role of these MMPs in involution and in remodeling of the mammary gland, the different MMPs (130K, 68K, and 60K gelatinases) were partially purified by gel filtration and affinity chromatography over gelatin Sepharose and subjected to kinetic analysis. Comparative analysis of the different gelatinases showed that the 130K that appears at the early involuntary phase and the constitutive 68K enzyme are more specific for Col IV of the basement membrane, while the inducible 60K that appeared at the later phase of involution degraded Col I more efficiently. These neutral proteinases required Ca2+/Zn2+ for their activity and the analysis of cation dependence revealed that Ca2+ at 10 mM concentration and above completely inhibited the enzyme. The 60K was active at very low concentration of Zn2+ (5 microM); but at higher concentration of Zn2+ (2 mM), where the 68K and 130K were active, the 60K gelatinase was inhibited, indicating a difference in the cation dependence of these enzymes. Chondroitin sulfate A and chondroitin sulfate C caused inhibition of the 130K, 68K, and 60K, while hyaluronic acid and heparin did not show any effect, suggesting that the chondroitin sulfate proteoglycan that decorates collagen in the ECM can modu late the activity of the collagenases in vivo. These results suggest that the 130K gelatinase expressed during the early phase of involution degraded Col IV of the basement membrane, making the 60K gelatinase formed at a later stage of involution more accessible to its preferred substrate (Col I of the underlying stroma), highlighting the role of these MMPs in mammary gland involution.     

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.     

Milk-protein mRNAs and poly(A) in the involuting rat mammary gland return to the levels found during late pregnancy

Analyses of the rat mammary gland show that the increase in the milk-protein mRNAs during the development of lactation and the rapid disappearance of these sequences during involution are not accompanied by similar changes in the poly(A) content. During the development of lactation the casein mRNA is initially in great excess to the whey-protein mRNA and this differential expression of the genes for the two types of milk proteins is again observed during early involution. Since the amounts of poly(A) and of both milk-protein mRNAs are also similar to the amounts found in the gland during late pregnancy, these results indicate that during early involution the mammary gland has reverted to the pattern of mRNA metabolism that occurs during late pregnancy.     

Down-regulation of protein tyrosine phosphatase gene expression in lactating mouse mammary gland

Detailed analysis of protein tyrosine phosphatase (PTP) expression in mouse mammary gland and mammary epithelial cells using a set of degenerate primers corresponding to the PTP core domain sequence revealed the presence of 16 different receptor-type and intracellular PTPs. Northern blot and RT-PCR analyses revealed that some PTPs were up-regulated during gestation, suggesting that these enzymes are involved in development of mammary gland. However, expression of most PTPs dramatically decreased during lactation, whereas the beta-casein gene expression was increased and remained at a high level. At the involution stage after weaning, most PTPs were up-regulated and their expression returned almost to the virgin level. Such up-regulation was also induced by forced weaning in lactating mother mice. These results suggest the possible contribution of PTPs to the development, involution, and remodeling of mammary gland and their possible inhibitory action on maintaining high expression of milk genes during lactation.     

The 24p3 gene is induced during involution of the mammary gland and induces apoptosis of mammary epithelial cells

To understand the molecular mechanism of mammary gland involution we identified involution-induced clones by differential screening of a mouse mammary gland cDNA library. Characterization of clones by sequencing and Northern analysis showed that expression of 24p3 was induced during involution of the mammary gland. RNA in situ hybridization showed that it was mainly expressed in the secretory epithelial cells surrounding the lumen of the mammary gland alveoli. Induction of 24p3 was also observed in apoptotic HC11 mammary epithelial cells under serum starvation. In these cells, dexamethasone increased 24p3 gene expression four-fold. Transient expression of 24p3 increased the percentage of apoptotic cells 3- to 4-fold over a period of 3 days after transfection. This study provides evidence that overexpression of 24p3 gene can induce apoptosis of mammary epithelial cells.     

Retinoids induce MMP-9 expression through RARalpha during mammary gland remodeling

Retinoic acid (RA) is a signaling molecule in the morphogenesis of the mammary gland, modulating the expression of matrix metalloproteinases (MMPs). The aim of this paper was to study the role of RA during weaning, which consists of three events: apoptosis of the secretory cells, degradation of the extracellular matrix, and adipogenesis. CRABP II and CRBP-1 carrier proteins increased significantly during weaning compared with lactating glands but reverted to control values after the litter resuckled. The effects of RA are mediated by the nuclear receptors RARalpha, RARbeta, RARgamma, and RXRalpha, which underwent an increase in protein levels during weaning. In an attempt to elucidate the RARalpha-dependent signaling pathway, ChIP assays were performed. The results showed the binding of RARalpha to the MMP-9 promoter after 24- and 72-h weaning together with its coactivator p300; this fact could be responsible for the increase found in MMP-9 mRNA and protein levels in these conditions. Expression of related MMPs (MMP-2 and MMP-3) was also increased during weaning. Using gelatine zymography, we observed a time-dependent increase in active forms of MMP-9 and MMP-2. On the other hand, the inhibitor of MMPs, TIMP-1, was almost undetectable at 24- and 72-h weaning by Western blot. The role of retinoids in matrix remodeling is reinforced by the fact that administration of an acute dose of retinol palmitate to control lactating rats also induces MMP-9 expression. This emphasizes the importance of retinoids in vivo to regulate mammary gland involution.     

Differential expression of TGF-β isoforms during postlactational mammary gland involution

After cessation of lactation, the mammary gland undergoes involution, which is characterized by a massive epithelial cell death and proteolytic degradation of the extracellular matrix. Whereas the expression patterns and also the function of TGF-beta isoforms during mammary gland branching morphogenesis and lactation are well understood, their expression during postlactational involution and therefore a possible role in this process is poorly known. In this study we show that TGF-beta3 expression is dramatically induced (>fivefold) during mouse mammary gland involution when compared to that of virgin mouse, reaching a maximal expression level at day 4 after weaning. In contrast, other TGF-beta isoforms do not display significant increase in expression during involution (TGF-beta1, 1.3-fold and TGF-beta2, <1.5-fold) when compared to that of virgin or lactating mice. During mammary gland involution, TGF-beta3 is expressed in the epithelial layer and particularly in myoepithelial cells. A comparison of the kinetics of TGF-beta3 expression to that of programmed cell death and degradation of the basement membrane suggests that TGF-beta3 functions in the remodeling events of the extracellular matrix during the second stage of involution.     

Mammary gland involution is delayed by activated Akt in transgenic mice

Activation of the antiapoptotic protein kinase Akt is induced by a number of growth factors that regulate mammary gland development. Akt is expressed during mammary gland development, and expression decreases at the onset of involution. To address Akt actions in mammary gland development, transgenic mice were generated expressing constitutively active Akt in the mammary gland under the control of the mouse mammary tumor virus (MMTV) promoter. Analysis of mammary glands from these mice reveals a delay in both involution and the onset of apoptosis. Expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of matrix metalloproteinases (MMPs), is prolonged and increased in the transgenic mice, suggesting that disruption of the MMP:TIMP ratio may contribute to the delayed mammary gland involution observed in the transgenic mice.     

Lactose synthetase activities in rat and mouse mammary glands

The activities of lactose synthetase (in the presence and absence of α-lactalbumin), galactosyltransferase and α-lactalbumin levels were determined in rat and mouse mammary glands during pregnancy, lactation and involution. In the rat, essentially none of the above activities were detected prior to parturition. This was followed by a sharp increase with the maximum occurring during late lactation and then by a rapid drop during involution. In the mouse, detectable levels of all the activities occurred at 15 days of pregnancy and these levels increased during pregnancy and reached a maximum level during early lactation and again sharp decreases occurred during involution. The study shows that mammary gland development occurs at an earlier date in the mouse than in the rat.     

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.     

Acute involution in the tammar wallaby: identification of genes and putative novel milk proteins implicated in mammary gland function

Marsupials provide a suitable alternative model to studying mammary gland involution. They have evolved a different reproductive strategy from eutherians, giving birth to an altricial young and secreting milk that changes in composition during lactation. In this study, we used a marsupial-specific EST microarray to identify 47 up-regulated genes during mammary gland involution in the tammar wallaby (Macropus eugenii). These include the pro-apoptotic tumour necrosis factor receptor superfamily 21 (TNFRSF21) gene, whose expression in the mammary gland has not previously been reported. Genes encoding putative novel milk proteins which may protect the mammary gland from infection were also found to be up-regulated, such as amiloride binding protein 1 (ABP1), complement component 1QB (C1QB), complement component 4A (C4A) and colony stimulating factor 2 receptor β (CSF2Rβ). Our results show that the marsupial reproductive strategy was successfully exploited to identify genes and putative novel milk proteins implicated in mammary gland involution.     

The metastasis-promoting protein S100A4 regulates mammary branching morphogenesis

High levels of the S100 calcium binding protein S100A4 also called fibroblast specific protein 1 (FSP1) have been established as an inducer of metastasis and indicator of poor prognosis in breast cancer. The mechanism by which S100A4 leads to increased cancer aggressiveness has yet to be established; moreover, the function of this protein in normal mammary gland biology has not been investigated. To address the role of S100A4 in normal mammary gland, its spatial and temporal expression patterns and possible function in branching morphogenesis were investigated. We show that the protein is expressed mainly in cells of the stromal compartment of adult humans, and during active ductal development, in pregnancy and in involution of mouse mammary gland. In 3D culture models, topical addition of S100A4 induced a significant increase in the TGFα mediated branching phenotype and a concomitant increase in expression of a previously identified branching morphogen, metalloproteinase-3 (MMP-3). These events were found to be dependent on MEK activation. Downregulation of S100A4 using shRNA significantly reduced TGFα induced branching and altered E-cadherin localization. These findings provide evidence that S100A4 is developmentally regulated and that it plays a functional role in mammary gland development, in concert with TGFα by activating MMP-3, and increasing invasion into the fat pad during branching. We suggest that S100A4-mediated effects during branching morphogenesis provide a plausible mechanism for how it may function in breast cancer progression.