Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture. RABIH S. TALHOUK1'2', JENNIE R. CHIN2, ELAINE N.
Development 112, 439-449 (1991) Printed in Great Britain © The Company of Biologists Limited 1991
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Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture
RABIH S. TALHOUK1'2', JENNIE R. CHIN2, ELAINE N. UNEMORI 3 , ZENA WERB2 and MINA J. BISSELL1 1
Division of Cell and Molecular Biology, Lawrence Berkeley Laboratory, Berkeley, CA 94720, USA Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, San Francisco, CA 94143, USA 3 Genentech Inc., Department of Developmental Immunology, South San Francisco, CA 94080, USA
2
Summary 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-I mice resolved by zymography contained three major proteinases of 60K (K=10 3 M r ), 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.
Introduction
including cell adhesion, migration, morphology and differentiation (Talhouk et al. 1991; Stoker et al. 1990; Thiery et al. 1988) has led to increasing interest in recent years in factors that modify or remodel the ECM. However, most of the literature so far has concentrated on correlation between growth of cancer cells and metastasis, and the ability of these cells to secrete increased levels of ECM-degrading proteinases (Sloane et al. 1986; Liotta, 1986; Ossowski, 1988; Lyons et al. 1989; Matrisian, 1990). Except for few reports on embryo development (Frisch and Werb, 1989; Brenner et al. 1989), branching morphogenesis in the developing
Two classes of enzymes have been implicated in a proteolytic cascade for extracellular matrix (ECM) degradation: metalloproteinases and plasminogen activators. The metalloproteinases appear to be the ratelimiting enzymes in the degradation of ECM components, while plasminogen activators which are serine proteinases are implicated indirectly in the remodelling of the ECM by participating in the activation of the metalloproteinases (Alexander and Werb, 1989). The effect of ECM on the different aspects of cell function
Key words: gelatinase, caseinase, extracellular matrix (ECM), mammary gland, proteinase.
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salivary gland (Fukuda et al. 1988) and ovulation (Beers et al. 1975), no information is available on the nature and the role of ECM-degrading proteinases in normal developmental processes. The mammary gland is one of the few organs that develops in fetal life and undergoes structural, compositional and functional changes during the lifetime of an individual (Anderson, 1985; Lascelles and Lee, 1978; Russo and Russo, 1987; Forsyth, 1982; Dembinski and Shiu, 1987). Whereas the importance of cell-ECM interaction and deposition of ECM components on the functional differentiation of mammary cells have been well documented (Daniel and DeOme, 1965; Emerman and Pitelka, 1977; Wicha et al. 1980; Lee et al. 1984; Wiens etal. 1987; Blum etal. 1987; Li etal. 1987; Bissell and Barcellos-Hoff, 1987; Chen and Bissell, 1989; Streuli and Bissell, 1990), the nature of ECM-degrading proteinases and their role in the development and involution of the mammary gland has received little attention (Martinez-Hernandez et al. 1976; Ossowski et al. 1979). Given the importance of ECM in the regulation of mammary-specific functions, and the implication from earlier studies (Chin et al. 1985; Unemori and Werb, 1986; Unemori et al. 1987; Matrisian, 1990) that metalloproteinases are important in the remodelling of the ECM, we determined which ECM-degrading proteinases are expressed in vivo during the various stages of mammary growth, differentiation and involution, and in isolated mammary epithelial cells in culture. Materials and methods Reagents Medium 199, Ca2+-free Dulbecco's modified minimum essential medium (DMEM), gentamicin and fetal calf serum were purchased from Gibco Laboratories (Grand Island, NY). Prolactin was obtained from the National Hormone and Pituitary Program (contracted to NIADDK, Baltimore, MD). Gelatin (from porcine skin), 50% by NPGB and PMSF (Table 1). To determine whether this 26K caseinase was mammary specific, we analyzed tissue extracts from kidney, spleen, liver, heart and brain. None of the tissues had a casein-degrading activity at 26K (Fig. 8) while the 92K and 35K caseinases were widely distributed. Thus the
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26K caseinase appears to be novel and mammary specific. Discussion The involvement of matrix-degrading proteinases in metastasis and pathological conditions has been postulated and extensively investigated (Liotta, 1986; Sloane et al. 1986; Ballin et al. 1988; Baricos et al. 1988; Ossowski, 1988; Reich et al. 1988; Lyons et al. 1989). Studies documenting the role of matrix-degrading proteinases in the regulation of growth and morphogenesis in normal tissues, in contrast, are few (Dean et al. 1985; Brenner etal. 1989; Werb, 1989; Matrisian, 1990). In this study, we have demonstrated that normal mammary gland contains a number of secreted and cellassociated proteinases and that they are regulated during normal development of the gland in primiparous mice. These proteinases are produced, at least partly, by mammary epithelial cells themselves, and those secreted are routed basally in the direction of the basement membrane. We further show that there is at least one developmentally regulated, 26K cell-associated, proteinase that appears to be unique to the mammary gland. Of the gelatinases found in tissue extracts (60/68K, 70K, and 130K), the one most consistently expressed during all stages of mammary gland development but down-regulated in lactation is a metalloproteinase with an apparent relative molecular mass of 68X103. Recent cloning of this metalloproteinase (Collier et al. 1988) showed that it is the proenzyme form of type IV collagenase with an actual relative molecular mass of 72X103 that migrates under non-reducing conditions at 68K. A similar 68K gelatin-degrading metalloproteinase is known to be secreted by endothelial cells (Herron et al. 1986a,b), and rabbit synovial fibroblasts (Unemori and Werb, 1988). Brenner et al. (1989) reported a 68K gelatinase/type IV collagenase that was implicated in the regulation of peri-implantation development and implantation of mouse embryos and is expressed in embryonal carcinoma cells differentiating to endoderm (Adler et al. 1990). An intact basement membrane is essential to maintain the differentiated function of mammary epithelial cells in culture (Streuli and Bissell, 1990). Our results described here are consistent with this also being the case in vivo: the active form (60K) of type IV collagenase is abundant during pregnancy when mammary growth and ECM remodelling is prominent. The activity decreases three days before parturition and is absent during lactation and early involution when the gland is functionally differentiated (Forsyth, 1982; Knight and Peaker, 1982; Hurley, 1989), and there is minimal restructuring of the gland and remodelling of the ECM. The 60K gelatinase activity reappears 3 to 4 days after initiation of involution when massive restructuring of the ECM occurs. The function of the 130K gelatinase that is expressed during the early stages of involution, before overt degradation of the ECM remains to be determined.
Ossowski et al. (1979) showed a sharp peak of plasminogen activator produced by mammary cells at the fourth day of involution after a 2 day lactation. Similarly, Martinez-Hernandez et al. (1976) demonstrated that involuting mammary tissue extracts from Swiss-Webster mice hydrolyzed 125I-labeled insoluble basement membrane components. The ECM-degrading activity started 2 days postweaning and was maximal by day 4. In a separate study on the involution of the mammary gland, we have determined that stromelysin and tissue plasminogen activator expression reaches a maximum by day 4 of involution. However, the expression of TTMP is maximal during the early stages of involution (days 1 to 4) and decreases thereafter (Talhouk et al. 1990; and manuscript in preparation). These data lead us to postulate that a balance between ECM-degrading proteinases and their inhibitors during mammary gland development and involution may be a critical factor affecting function and morphology of the gland. Of the caseinases detected on substrate gels, the cellassociated 26K activity is novel and most likely mammary specific, since it was not detected in tissue extracts from kidney, spleen, liver, heart and brain. Our inhibitor studies with NPGB and PMSF show that this caseinase is most likely a serine arginine-esterase. NPGB, a potent inhibitor of trypsin, plasminogen activator, plasmin and arginine esterases exerts its effect by complexing with the enzyme to form a stable acyl enzyme (Goldberg et al. 1975), thus making the enzyme unavailable for further activity. It is interesting to note that PMSF was effective against the mammary 26K caseinase, yet the other serine proteinase inhibitors were not. The lack of inhibition of this activity by Trasylol, benzamidine or TLCK suggests that this enzyme is not plasminogen activator, plasmin or one of the tissue kallikreins. The 26K caseinase, like the gelatinases, was lowest in extracts from lactating and early involuting tissues. The relative difference between caseinolytic activity during various developmental stages as detected by [14C]casein degradation (Fig. 3) and those resolved on zymogram gels (Fig. 2) also suggests that proteinase inhibitors modulated the level of proteinase activities in vivo. The high level of casein-degrading activity in the pregnant gland is consistent with the fact that these cells produce substantial amounts of milk proteins which must be degraded before parturition. Studies by Wilde and Knight (1986) and Stewart et al. (1988) demonstrated intracellular degradation of newly synthesized caseins via a non-lysosomal pathway in goat mammary tissue explants. The casein-degrading activity was higher in mammary tissue explants from pregnant goats than from lactating goats. Whether the caseinases observed in our study are involved in ECM degradation or whether they only regulate casein levels in the gland cannot be answered at present. The fact that these caseinases are cell associated argues that they are involved in regulating intercellular casein levels as suggested by Wilde and Knight (1986) and Stewart et al. (1988). On the other hand, the presence of caseinase
Mammary-associated proteinases 447 activity in tissue extracts from virgin and involuting mammary gland (Fig. 2), where lactogenesis does not occur, implies that caseinases could be involved in other processes such as matrix remodelling. The presence of proteinases and proteinase inhibitors in milk has been recognized by others (reviewed by Lonnerdal, 1985), but it was hypothesized that this activity was derived from leukocytes in milk and not secreted by the mammary cells. However, mouse neutrophils and macrophages do not express the 68K gelatinase (Werb and Coworkers, unpublished observation) and the expression of gelatinases and caseinases in MMEC in culture is evidence that these proteinases can be synthesized by mammary epithelial cells. Furthermore the presence of the latent form of type IV collagenase in the lumenal fraction in cells on EHSmatrix indicate that there is some apical secretion explaining its existence in milk. Nevertheless, the fact that the majority of gelatinases are secreted in the direction of the basement membrane argues further that such gelatinases are involved in the degradation and remodelling of the stroma and basement membrane. Both gelatinases and caseinases found in mammary gland extracts in vivo were also expressed by MMEC in culture. An analysis of the pattern of expression in culture and a comparison to in vivo situation unravelled two additionally interesting facts. First, MMEC under our culture conditions resemble the lactating tissue morphologically, ultrastructurally and in their ability to synthesize milk proteins and fat (Barcellos-Hoff et al. 1989; Chen and Bissell, 1989). However, the abundance of high relative molecular mass gelatinases (>70-200K) and the low levels of the 60K gelatinase relative to the 68K gelatinase by cells on EHS-matrix and floating collagen gel indicate that the differentiated epithelial cells in culture resemble those in early involution tissue, at a time when milk protein synthesis is still quite high (Knight and Peaker, 1982; Hurley, 1989). Second, the absence of discernable modulation of ECM-degrading proteinases by substrata indicate another interesting phenomenon. Streuli and Bissell (1990) have shown that deposition of a basement membrane by MMEC in culture positively correlated with the expression of differentiated functions, and Wicha et al. (1980) reported that inhibition of collagen deposition induced an involution-like process in the mammary gland. These data suggest an intimate relationship between basement membrane and differentiated function of mammary cells. In view of this, the separation of the differentiation events that encompass organization of a basement membrane (Streuli and Bissell, 1990) leading to a lactation-competent phenotype in vitro from the regulation of secretion of ECM-degrading proteinases was surprising. It appears from the data presented here and the fact that MMEC on plastic do not differentiate but express higher levels of TIMP mRNA than do differentiated cells on EHS-matrix orfloatingcollagen gels (Bissell et al. 1990) that the major regulation of ECM remodelling may be due to the regulated production of proteinase inhibitors, which could affect
both matrix deposition and remodelling. This hypothesis is being tested by introducing the TIMP gene into mammary cells. In conclusion, we have denned the nature and regulation of ECM-degrading proteinases during the normal development of the mammary gland. Of these proteinases, a novel, most likely mammaryspecific, cell-associated 26K proteinase is characterized. Furthermore, we have demonstrated that ECMdegrading proteinases are produced by mammary epithelial cells and secreted basally. Studies are currently in progress to determine how these ECMdegrading enzymes and their inhibitors are involved in regulating the growth, differentiation and involution of the mammary gland. This work was supported by the US Department of Energy, Office of Health and Environmental Research (contracts DEAC03-76-SF00098 [for M.J.B.] and DE-AC03-76-SF01012 [for Z.W.]), a research grant from the National Institutes of Health (HD 23539), and a National Research Service Award (T32ES07106) from the National Institute of Environmental Health Sciences. A brief report of part of these data has been presented in abstract form (Unemori et al. 1987). We thank Drs A. Howlett, M. Martins-Green, R. Schwarz and C. Streuli, for critical reading of this manuscript. References ADLER, R. R., BRENNER, C. A. AND WERB, Z. (1990). Expression
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(Accepted 25 February 1991)
