0022-1514/92/$3.30
Vol. 40, No. 5 , pp. 697-703, 1992 Printed in U X A .
The Journal of Histochemistry and Cytochemistry Copyright 0 1992 by The Histochemical Society, Inc.
Original Article
1 Enhanced Synthesis of Gelatinase and Stromelysin by Myoepithelial Cells During Involution of the Rat Mammary Gland' SANDRA R. DICKSON and MICHAEL J. WARBURTON2 Department of Histopathology, St George's Hospital Medical ScbooL, London SW17 ORE, United Kingdom. Received for publication August 22, 1991 and in revised form November 20, 1991; accepted November 21, 1991 (1A2438)
During the involution of the mammary gland there is destruction of the basement membrane as the secretory alveolar structures degenerate. Immunofluorescencestaining of sections of rat mammary gland with antibodies to 72 gelatinase (MMP2) and stromelysin (MMP3) revealed increased production of these two proteinases during involution. This increased expression was mostly restricted to myo-
Introduction The major phase of mammary growth and differentiation occurs during pregnancy and lactation. As the animal matures, terminal end buds undergo branching to form alveolar buds (1). With the onset of pregnancy, the rate of cell division in the alveolar buds increases dramatically (2). In the fully developed gland there is an enormous increase in cellularity. Under hormonal influences, the epithelial cells begin to synthesize milk proteins and lipids and the myoepithelial cells acquire myofilaments. The entire secretory alveoli are surrounded by a basement membrane ( 3 ) whose synthesis must also increase during pregnancy. After the young are weaned the mammary gland undergoes involution. Several investigators have described the morphological changes occurring during involution (e.g., 4J). Essentially, the alveolar structure collapses. The secretory epithelial cells are degraded by autophagocytosisand phagocytosis by macrophages. About 14 days after weaning, the structure of the involuting gland is similar to that of the resting virgin gland. During involution there are dramatic changes in the structure of the extracellular matrix. The basement membrane around the secretory alveoli becomes discontinuous ( 3 ) ; it also becomes thicker and has a loose structure ( 6 ) . Diffuse staining with antibodies to basement membrane proteins is observed throughout the tissue, suggesting that the basement membrane is being degraded with diffusionof degradationproducts (3,6).Ultrastructural studies could
Supported by a grant from The Medical Research Council. To whom correspondence should be addressed.
epithelial cells. Increased expression during involution was also demonstrated by immunoblotting techniques. Gelatin
zymography indicated that the predominant metalloproteinase present in involuting rat mammary glands was a 66 KD gelatinase. ( J Histochem Cytochem 40:697-703,1992) KEY WORDS: Mammary gland; Metalloproteinase; Myoepithelial cell.
find no evidence for phagocytosis of basement membrane by macrophages or other cells (6),suggesting that dissolution occurs through the activity of secreted enzymes. The scale of the destruction of the basement membrane is unique to the mammary gland. Basement membrane degradation also occurs, but to a much lesser extent, during other developmental processes, e.g., ductal growth in the submandibulargland (7) and angiogenesis (8),and also in certain pathological conditions, e.g., tumor invasion and metastasis (9). Little is known about the enzymes responsible for basement membrane degradation during mammary gland involution, their cellular site of synthesis, and their regulation. We now report the localization of 72 KD gelatinase (MMP-2) and stromelysin (MMP3). two metalloproteinases implicated in basement membrane degradation(lo), during the development of the rat mammary gland through pregnancy, lactation, and involution.
Materials and Methods Electrophoretic Techniques. Gelatin and casein zymography were carried out by previously published procedures (11). Tissue samples were prepared by extracting mammary glands, powdered by pulverizing in liquid nitrogen, with gel sample buffer (2% SDS, 10% sucrose, 0.001% bromophenol blue, 60 mM Tris-HC1, pH 6.8) and removing insoluble matter by centrifugation. For immunoblotting experiments,protein was precipitated from the extract with 90% methanol at - 20°C and redissolved in gel sample buffer. Approximately 250 wg of protein was loaded into each well. Proteins, separated by SDS-polyacrylamide gel electrophoresis, were then transferred to Immobilon PVDF membranes (12) using a Millipore semidry blotting apparatus. The membrane was incubated overnight in 3% marvel/PBS10.2% Tween 20 and then sequentially with primary antibody (diluted l:lOOO), biotin-conjugated anti-rabbit or -sheep IgG (1:2000), and
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I
c
1
Figure 1. Immunofluorescence localization of 72 KD gelatinase during pregnancy, lactation, and involution. Frozen sections of (A) pregnant, (e) lactating, (c) 2-day involuting, (D) Cday involuting, (E) 7-day involuting, and (F) 10-day involuting rat mammary gland were stained with an antibody to 72 KD gelatinase. At all stages staining is largely restricted to the myoepithelial cells (myo) although some epithelial cells (ep) adjacent to the lumen (L) show granular cytoplasmic staining. Original magnifications: A.B,D,E x 360; C,F x 500. Bars: A,B.D.E = 28 pm; C,F = 20 Fm.
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Figure 2. Immunofluorescence localization of stromelysin during pregnancy, lactation, and involution. Frozen sections of (A) pregnant, (B) lactating, (C) 2-day involuting, (D) 4-day involuting, (E)7-day involuting, and (F) 10-day involuting rat mammary gland were stained with an antibody to stromelysin. The myoepithelial cells (myo) demonstrate the strongest stalning, whereas epithelial cells (ep) adjacent to the lumen (L) show granular cytoplasmic staining. Original magnification x 360. Bar = 28 wm.
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Figure 3. Dual localization of 72 KD gelatinase and actin. A frozen section of a 2-day involuting rat mammary gland was stained with an antibody to 72 KD gelatinase. followed by (A) FITC-anti-rabbit IgG and (a)TRITC-phalloidin. (C) A control section was stained with non-immune rabbit serum. Note the similar staining Pattern Of the myoepithelial cells with both 72 K D gelatinase and actin. Original magnification x 360. Bar = 28 wm.
avidin-alkaline phosphatase (1:5000). Each incubation was for 2 hr and the membrane was washed three times with PBS/O.2% Tween 20 between each change of reagent. The membrane was incubated in 0.1 M ethanolamine-HCI, pH 9.6, for 10 min and then with the substrate solution (100 pglml 5-bromo-4-chloro-3-indolyl phosphate. 100 pglml nitroblue tetrazolium, 1 mM MgCI2. 0.1 M ethanolamine-HCI. pH 9.6). The reaction was terminated by rinsing the membrane with water. ExtrAvidin-alkaline phosphatase and all other immunological reagents were purchased from Sigma (Poole. UK). ImmunofluorescenceT&ques. Frozen sections of rat mammary gland were fixed with acetone at - 2O'C for 5 min and immunofluorescence staining was carried out as described previously (13). Actin was visualized by staining with TRITC-phalloidin. Antibodies. Sheep antibodies to human srromelysin (14) were provided by H. Nagase. Antibodies to 72 KD gelatinase were produced by immunizing rabbits with a synthetic peptide (corresponding to residues 472-490 of human 72 KD gelatinase) as described by Sretler-Stevenson et al. (15). The peptide was synthesized with a C-terminal cysteine residue and coupled to keyhole limpet hemocyanin using sulpho-MBS (16). Rabbits were injected every 4 weeks and were bled 1 week after each injection. Antisera positive by ELISA were subjected to affinity purification using a peptide-Sepharose column. Purified antibodies were checked for crossreactivity with a rat 72 KD gelatinase by immunoblorting against medium conditioned by the rat mammary myoepithelial cell line Rama 401 (17). Antibodies producing bands at about 72 KD were used in further studies.
until involution of the rat mammary gland, both 72 KD gelatinase and stromelysin were mostly produced by the basal myoepithelial cells (Figures 1 and 2). Some epithelial cells displayed strongly staining cytoplasmic granules (e.g., Figures 2B and 2C). There was little specific staining of stromal cells. In the glands of pregnant animals (Figures 1A and 2A), the staining of myoepithelial cells varied from one lobule to another. This might reflect differing rates of cell proliferation within individual lobules. The staining of myoepithelial cells of lactating glands (Figures 1B and 2B) was more uniform. During involution (Figures 1C-1F and 2C-2F). the staining of myoepithelial cells became more intense, reaching a maximum around 4-7 days after weaning (Figures 1E and 2E). As the alveolar structures collapse, the epithelial cells degenerate and either dislodge into the lumen andlor are subjected to phagocytosis by macrophages, leaving strings and clumps of positively staining myoepithelial cells (Figures IF and 2F). There appeared to be little difference between the patterns of staining produced by antibodies to the two metalloproteinases. Staining of sections with antibodies to 72 KD gelatinase and TRITC-phalloidin yielded almost identical patterns of staining (Figure 3). Actin is often used as a marker for myoepithelial cells (3,18) and the coincidence of the staining patterns demonstrated the myoepithelial nature of the matrix metalloproteinase-producing cells.
Results
Quantification of 72 KD Gelutinase una' Stromelysin
Immunoloculizution of 72 KD Gelatinuse ana' Stromelysin
The levels of 72 KD gelatinase and stromelysin in rat mammary glands at different stages of development were compared by immunoblotting experiments (Figure 4). The levels of both proteinases reached a maximum at around 4-7 days after weaning and were
At every stage of development investigated, i.e., from pregnancy
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MAMMARY GLAND METALLOPROTEINASES
1
2
3
4
5
6
70 1
7
Figure 5. Gelatinaseactivity in involuting rat mammary gland. An extract of a 7-day involuting rat mammary gland was subjected in gelatin zymography.
A
+66kD
B
I
66 kD
Figure 4. lmmunoblot analysis of 72 K O gelatinase and stromelysin levels during pregnancy, lactation, and involution. Equal amounts of protein (250 Fg) extracted from (1) virgin, (2) pregnant, (3) lactating, (4) 2-day involuting, (5) 4-day involuting, (6) 7-day involuting, and (7)10-day involuting rat mammary glands were subjected to SDS-polyacrylamide gel electrophoresisand transferred to a PVDF membrane. The membrane was stained with antibodies to either (A) 72 KD gelatinase or (6)stromelysin.
four- to fivefold higher than the levels in virgin animals. Ten days after weaning, levels of both enzymes had fallen to about 50% of the maximum levels. Significant staining was observed only for activated forms of 72 KD gelatinase and stromelysin (66 KD and 48 KD, respectively).
Gelatin Zymography Gelatin zymography of an extract of a 7-day involuting mammary gland revealed a major band with an Mr of 66,000 and a minor band with an Mr of 72,000 (Figure 5). Similar patterns were observed regardlessof the developmental state of the gland. No change was observed in these Mr if the mammary gland extracts were incubated with trypsin before electrophoresis. Inclusion of 10 mM EDTA during the incubation period, abolished all activity. Casein zymography failed to reveal any significant bands of proteinase activity.
Discussion Initial investigations on proteinases involved in mammary gland involution demonstrated the presence of uncharacterized metalloproteinases whose activity was substantially increased during lactation and involution (6). Using immunohistochemical techniques, Monteagudo et al. (19) demonstrated the presence of Type IV collagenase (72 KD gelatinase) in myoepithelial cells of normal human breast and in epithelial cells of breast carcinomas. This implies that 72 KD gelatinase is involved in both normal and pathological basement membrane turnover. More recently, Talhouk et al. (20) have shown that there is an increase in the activity of activated forms of 72 KD gelatinase during involution of the mouse mammary gland. In the mouse, there is a decrease in the activity of this gelatinase during lactation, compared with glands from virgin or pregnant animals. Similar activities were noted for a 26 KD casein-degrading enzyme, and the specific induction of a 130 KD
gelatinase during involution was also noted. In the rat mammary gland, levels of 72 KD gelatinase and stromelysin rise slightly during pregnancy and lactation before increasing four- to fivefold during involution, maximal activity being reached about 4-7 days after weaning. The immunoblotting experiments also indicated that both 72 KD gelatinase and stromelysin are largely present as activated, or partially activated, forms with Mr of 66,000 and 48,000, respectively. We did not find any evidence for the presence of a 130 KD gelatinase (20) or the 92 KD gelatinase (21), even though the latter enzyme is produced by both rat mammary epithelial and myoepithelial cell lines in culture (unpublished observations). Casein zymography of extracts of rat mammary gland at all stages of development did not produce any notable bands, although stromelysin could readily be detected by immunofluorescence and immunoblotting techniques. In the rat mammary gland, 72 KD gelatinase and stromelysin are predominantly produced by the myoepithelial cells. Although the cytoplasm of these cells appears to be diffusely stained, it is unlikely that the two metalloproteinases are present in the cytoplasm. In differentiated myoepithelial cells, cytoplasmic organelles are displaced towards the apical surface by the abundant myofilaments ( 5 ) . The resolution of the light microscope is probably insufficient to provide a detailed staining pattern. Some epithelial cells also display staining with antibodies to 72 KD gelatinase and stromelysin in the form of cytoplasmic granules. Mammary epithelial cells have a well-developed secretory apparatus during lactation, which is responsible for the secretion of milk proteins. Tal-
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houk et al. (20) have demonstrated that metalloproteinases are synthesized by mammary epithelial cells and are secreted via the apical surface. Metalloproteinaseactivity cannot usually be demonstrated in tissue sections by immunocytochemical procedures because of the low levels of enzymes present. However, the extent of involution of the mammary gland after weaning, during which about 90% of the gland is resorbed in 7 days (4), probably necessitates the production of high levels of metalloproteinases for this limited period of time. The integrity of the basement membrane is required for mammary gland morphogenesis (22) and differentiation(23). Most evidence suggests that the mammary gland basement membrane is synthesized by myoepithelial cells (3,17,24)and is under hormonal control (25), although little is known about the precise hormonal requirements. Evidence presented here and by others (19) indicates that the myoepithelial cell is also responsible for basement membrane degradation. Little is yet known about the regulation of metalloproteinase production by myoepithelial cells, but there are several possible regulatory mechanisms. In a number of cell types, metalloproteinase synthesis is regulated by hormones or cytokines (26). Metalloproteinasesare synthesized as inactive precursors. Activation can occur by removal of an NHz-terminal peptide. 72 KD Gelatinase is synthesized as a precursor (Mr 72,000) and is proteolytically cleaved to active forms (Mr 60-68,000) (15). Stromelysin is synthesized as a prwursor with an Mr of 57,000, which is reduced to 48,000 on activation (27). Activation in vivo probably results from proteolytic cleavage. One proteinase capable of activating metalloproteinasesis plasmin (28). Interestingly, plasminogen activator levels have been shown to increase during mammary gland involution (29). The kinetics of its induction are similar to those observed for gelatinases in the mouse and rat involuting mammary gland. Furthermore, plasminogen activator activity in mammary gland organ cultures was repressed by lactogenic hormones and induced by their absence (29). An additional site of regulation is the production of metalloproteinaseinhibitors (TIMP) (30), although little is yet known about the regulation of TIMP during mammary gland development. Therefore, the myoepithelial cell appears to be responsible for both the synthesis and degradation of the mammary gland basement membrane during normal developmentalprocesses. The manner in which these processes are regulated must await the development of suitable myoepithelial cell lines.
Acknowledgments We thank Dr H.Nagase (University of lClnsas Medicd Center) for antiserum to stromelysin and Dr B. Austin (St Georgei Hospitd Medica/ Schooo f i r peptide synthesis.
myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating and involuting rat mammary gland. J Histochem Cytochem 1982;30:667 4. Helminen HJ, Ericsson JLE. Studies on mammary gland involution. J Ultrastruct Res 1968;25:193
-
5 . Radnor CJP. Mvoepitheliumin involuting mammary, elands of the rat. J Anat li72:3
Vol. 40, No. 5 , pp. 697-703, 1992 Printed in U X A .
The Journal of Histochemistry and Cytochemistry Copyright 0 1992 by The Histochemical Society, Inc.
Original Article
1 Enhanced Synthesis of Gelatinase and Stromelysin by Myoepithelial Cells During Involution of the Rat Mammary Gland' SANDRA R. DICKSON and MICHAEL J. WARBURTON2 Department of Histopathology, St George's Hospital Medical ScbooL, London SW17 ORE, United Kingdom. Received for publication August 22, 1991 and in revised form November 20, 1991; accepted November 21, 1991 (1A2438)
During the involution of the mammary gland there is destruction of the basement membrane as the secretory alveolar structures degenerate. Immunofluorescencestaining of sections of rat mammary gland with antibodies to 72 gelatinase (MMP2) and stromelysin (MMP3) revealed increased production of these two proteinases during involution. This increased expression was mostly restricted to myo-
Introduction The major phase of mammary growth and differentiation occurs during pregnancy and lactation. As the animal matures, terminal end buds undergo branching to form alveolar buds (1). With the onset of pregnancy, the rate of cell division in the alveolar buds increases dramatically (2). In the fully developed gland there is an enormous increase in cellularity. Under hormonal influences, the epithelial cells begin to synthesize milk proteins and lipids and the myoepithelial cells acquire myofilaments. The entire secretory alveoli are surrounded by a basement membrane ( 3 ) whose synthesis must also increase during pregnancy. After the young are weaned the mammary gland undergoes involution. Several investigators have described the morphological changes occurring during involution (e.g., 4J). Essentially, the alveolar structure collapses. The secretory epithelial cells are degraded by autophagocytosisand phagocytosis by macrophages. About 14 days after weaning, the structure of the involuting gland is similar to that of the resting virgin gland. During involution there are dramatic changes in the structure of the extracellular matrix. The basement membrane around the secretory alveoli becomes discontinuous ( 3 ) ; it also becomes thicker and has a loose structure ( 6 ) . Diffuse staining with antibodies to basement membrane proteins is observed throughout the tissue, suggesting that the basement membrane is being degraded with diffusionof degradationproducts (3,6).Ultrastructural studies could
Supported by a grant from The Medical Research Council. To whom correspondence should be addressed.
epithelial cells. Increased expression during involution was also demonstrated by immunoblotting techniques. Gelatin
zymography indicated that the predominant metalloproteinase present in involuting rat mammary glands was a 66 KD gelatinase. ( J Histochem Cytochem 40:697-703,1992) KEY WORDS: Mammary gland; Metalloproteinase; Myoepithelial cell.
find no evidence for phagocytosis of basement membrane by macrophages or other cells (6),suggesting that dissolution occurs through the activity of secreted enzymes. The scale of the destruction of the basement membrane is unique to the mammary gland. Basement membrane degradation also occurs, but to a much lesser extent, during other developmental processes, e.g., ductal growth in the submandibulargland (7) and angiogenesis (8),and also in certain pathological conditions, e.g., tumor invasion and metastasis (9). Little is known about the enzymes responsible for basement membrane degradation during mammary gland involution, their cellular site of synthesis, and their regulation. We now report the localization of 72 KD gelatinase (MMP-2) and stromelysin (MMP3). two metalloproteinases implicated in basement membrane degradation(lo), during the development of the rat mammary gland through pregnancy, lactation, and involution.
Materials and Methods Electrophoretic Techniques. Gelatin and casein zymography were carried out by previously published procedures (11). Tissue samples were prepared by extracting mammary glands, powdered by pulverizing in liquid nitrogen, with gel sample buffer (2% SDS, 10% sucrose, 0.001% bromophenol blue, 60 mM Tris-HC1, pH 6.8) and removing insoluble matter by centrifugation. For immunoblotting experiments,protein was precipitated from the extract with 90% methanol at - 20°C and redissolved in gel sample buffer. Approximately 250 wg of protein was loaded into each well. Proteins, separated by SDS-polyacrylamide gel electrophoresis, were then transferred to Immobilon PVDF membranes (12) using a Millipore semidry blotting apparatus. The membrane was incubated overnight in 3% marvel/PBS10.2% Tween 20 and then sequentially with primary antibody (diluted l:lOOO), biotin-conjugated anti-rabbit or -sheep IgG (1:2000), and
697 Downloaded from jhc.sagepub.com at UNIV OF GEORGIA on June 28, 2015
698
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I
c
1
Figure 1. Immunofluorescence localization of 72 KD gelatinase during pregnancy, lactation, and involution. Frozen sections of (A) pregnant, (e) lactating, (c) 2-day involuting, (D) Cday involuting, (E) 7-day involuting, and (F) 10-day involuting rat mammary gland were stained with an antibody to 72 KD gelatinase. At all stages staining is largely restricted to the myoepithelial cells (myo) although some epithelial cells (ep) adjacent to the lumen (L) show granular cytoplasmic staining. Original magnifications: A.B,D,E x 360; C,F x 500. Bars: A,B.D.E = 28 pm; C,F = 20 Fm.
Downloaded from jhc.sagepub.com at UNIV OF GEORGIA on June 28, 2015
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Figure 2. Immunofluorescence localization of stromelysin during pregnancy, lactation, and involution. Frozen sections of (A) pregnant, (B) lactating, (C) 2-day involuting, (D) 4-day involuting, (E)7-day involuting, and (F) 10-day involuting rat mammary gland were stained with an antibody to stromelysin. The myoepithelial cells (myo) demonstrate the strongest stalning, whereas epithelial cells (ep) adjacent to the lumen (L) show granular cytoplasmic staining. Original magnification x 360. Bar = 28 wm.
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Figure 3. Dual localization of 72 KD gelatinase and actin. A frozen section of a 2-day involuting rat mammary gland was stained with an antibody to 72 KD gelatinase. followed by (A) FITC-anti-rabbit IgG and (a)TRITC-phalloidin. (C) A control section was stained with non-immune rabbit serum. Note the similar staining Pattern Of the myoepithelial cells with both 72 K D gelatinase and actin. Original magnification x 360. Bar = 28 wm.
avidin-alkaline phosphatase (1:5000). Each incubation was for 2 hr and the membrane was washed three times with PBS/O.2% Tween 20 between each change of reagent. The membrane was incubated in 0.1 M ethanolamine-HCI, pH 9.6, for 10 min and then with the substrate solution (100 pglml 5-bromo-4-chloro-3-indolyl phosphate. 100 pglml nitroblue tetrazolium, 1 mM MgCI2. 0.1 M ethanolamine-HCI. pH 9.6). The reaction was terminated by rinsing the membrane with water. ExtrAvidin-alkaline phosphatase and all other immunological reagents were purchased from Sigma (Poole. UK). ImmunofluorescenceT&ques. Frozen sections of rat mammary gland were fixed with acetone at - 2O'C for 5 min and immunofluorescence staining was carried out as described previously (13). Actin was visualized by staining with TRITC-phalloidin. Antibodies. Sheep antibodies to human srromelysin (14) were provided by H. Nagase. Antibodies to 72 KD gelatinase were produced by immunizing rabbits with a synthetic peptide (corresponding to residues 472-490 of human 72 KD gelatinase) as described by Sretler-Stevenson et al. (15). The peptide was synthesized with a C-terminal cysteine residue and coupled to keyhole limpet hemocyanin using sulpho-MBS (16). Rabbits were injected every 4 weeks and were bled 1 week after each injection. Antisera positive by ELISA were subjected to affinity purification using a peptide-Sepharose column. Purified antibodies were checked for crossreactivity with a rat 72 KD gelatinase by immunoblorting against medium conditioned by the rat mammary myoepithelial cell line Rama 401 (17). Antibodies producing bands at about 72 KD were used in further studies.
until involution of the rat mammary gland, both 72 KD gelatinase and stromelysin were mostly produced by the basal myoepithelial cells (Figures 1 and 2). Some epithelial cells displayed strongly staining cytoplasmic granules (e.g., Figures 2B and 2C). There was little specific staining of stromal cells. In the glands of pregnant animals (Figures 1A and 2A), the staining of myoepithelial cells varied from one lobule to another. This might reflect differing rates of cell proliferation within individual lobules. The staining of myoepithelial cells of lactating glands (Figures 1B and 2B) was more uniform. During involution (Figures 1C-1F and 2C-2F). the staining of myoepithelial cells became more intense, reaching a maximum around 4-7 days after weaning (Figures 1E and 2E). As the alveolar structures collapse, the epithelial cells degenerate and either dislodge into the lumen andlor are subjected to phagocytosis by macrophages, leaving strings and clumps of positively staining myoepithelial cells (Figures IF and 2F). There appeared to be little difference between the patterns of staining produced by antibodies to the two metalloproteinases. Staining of sections with antibodies to 72 KD gelatinase and TRITC-phalloidin yielded almost identical patterns of staining (Figure 3). Actin is often used as a marker for myoepithelial cells (3,18) and the coincidence of the staining patterns demonstrated the myoepithelial nature of the matrix metalloproteinase-producing cells.
Results
Quantification of 72 KD Gelutinase una' Stromelysin
Immunoloculizution of 72 KD Gelatinuse ana' Stromelysin
The levels of 72 KD gelatinase and stromelysin in rat mammary glands at different stages of development were compared by immunoblotting experiments (Figure 4). The levels of both proteinases reached a maximum at around 4-7 days after weaning and were
At every stage of development investigated, i.e., from pregnancy
Downloaded from jhc.sagepub.com at UNIV OF GEORGIA on June 28, 2015
MAMMARY GLAND METALLOPROTEINASES
1
2
3
4
5
6
70 1
7
Figure 5. Gelatinaseactivity in involuting rat mammary gland. An extract of a 7-day involuting rat mammary gland was subjected in gelatin zymography.
A
+66kD
B
I
66 kD
Figure 4. lmmunoblot analysis of 72 K O gelatinase and stromelysin levels during pregnancy, lactation, and involution. Equal amounts of protein (250 Fg) extracted from (1) virgin, (2) pregnant, (3) lactating, (4) 2-day involuting, (5) 4-day involuting, (6) 7-day involuting, and (7)10-day involuting rat mammary glands were subjected to SDS-polyacrylamide gel electrophoresisand transferred to a PVDF membrane. The membrane was stained with antibodies to either (A) 72 KD gelatinase or (6)stromelysin.
four- to fivefold higher than the levels in virgin animals. Ten days after weaning, levels of both enzymes had fallen to about 50% of the maximum levels. Significant staining was observed only for activated forms of 72 KD gelatinase and stromelysin (66 KD and 48 KD, respectively).
Gelatin Zymography Gelatin zymography of an extract of a 7-day involuting mammary gland revealed a major band with an Mr of 66,000 and a minor band with an Mr of 72,000 (Figure 5). Similar patterns were observed regardlessof the developmental state of the gland. No change was observed in these Mr if the mammary gland extracts were incubated with trypsin before electrophoresis. Inclusion of 10 mM EDTA during the incubation period, abolished all activity. Casein zymography failed to reveal any significant bands of proteinase activity.
Discussion Initial investigations on proteinases involved in mammary gland involution demonstrated the presence of uncharacterized metalloproteinases whose activity was substantially increased during lactation and involution (6). Using immunohistochemical techniques, Monteagudo et al. (19) demonstrated the presence of Type IV collagenase (72 KD gelatinase) in myoepithelial cells of normal human breast and in epithelial cells of breast carcinomas. This implies that 72 KD gelatinase is involved in both normal and pathological basement membrane turnover. More recently, Talhouk et al. (20) have shown that there is an increase in the activity of activated forms of 72 KD gelatinase during involution of the mouse mammary gland. In the mouse, there is a decrease in the activity of this gelatinase during lactation, compared with glands from virgin or pregnant animals. Similar activities were noted for a 26 KD casein-degrading enzyme, and the specific induction of a 130 KD
gelatinase during involution was also noted. In the rat mammary gland, levels of 72 KD gelatinase and stromelysin rise slightly during pregnancy and lactation before increasing four- to fivefold during involution, maximal activity being reached about 4-7 days after weaning. The immunoblotting experiments also indicated that both 72 KD gelatinase and stromelysin are largely present as activated, or partially activated, forms with Mr of 66,000 and 48,000, respectively. We did not find any evidence for the presence of a 130 KD gelatinase (20) or the 92 KD gelatinase (21), even though the latter enzyme is produced by both rat mammary epithelial and myoepithelial cell lines in culture (unpublished observations). Casein zymography of extracts of rat mammary gland at all stages of development did not produce any notable bands, although stromelysin could readily be detected by immunofluorescence and immunoblotting techniques. In the rat mammary gland, 72 KD gelatinase and stromelysin are predominantly produced by the myoepithelial cells. Although the cytoplasm of these cells appears to be diffusely stained, it is unlikely that the two metalloproteinases are present in the cytoplasm. In differentiated myoepithelial cells, cytoplasmic organelles are displaced towards the apical surface by the abundant myofilaments ( 5 ) . The resolution of the light microscope is probably insufficient to provide a detailed staining pattern. Some epithelial cells also display staining with antibodies to 72 KD gelatinase and stromelysin in the form of cytoplasmic granules. Mammary epithelial cells have a well-developed secretory apparatus during lactation, which is responsible for the secretion of milk proteins. Tal-
Downloaded from jhc.sagepub.com at UNIV OF GEORGIA on June 28, 2015
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houk et al. (20) have demonstrated that metalloproteinases are synthesized by mammary epithelial cells and are secreted via the apical surface. Metalloproteinaseactivity cannot usually be demonstrated in tissue sections by immunocytochemical procedures because of the low levels of enzymes present. However, the extent of involution of the mammary gland after weaning, during which about 90% of the gland is resorbed in 7 days (4), probably necessitates the production of high levels of metalloproteinases for this limited period of time. The integrity of the basement membrane is required for mammary gland morphogenesis (22) and differentiation(23). Most evidence suggests that the mammary gland basement membrane is synthesized by myoepithelial cells (3,17,24)and is under hormonal control (25), although little is known about the precise hormonal requirements. Evidence presented here and by others (19) indicates that the myoepithelial cell is also responsible for basement membrane degradation. Little is yet known about the regulation of metalloproteinase production by myoepithelial cells, but there are several possible regulatory mechanisms. In a number of cell types, metalloproteinase synthesis is regulated by hormones or cytokines (26). Metalloproteinasesare synthesized as inactive precursors. Activation can occur by removal of an NHz-terminal peptide. 72 KD Gelatinase is synthesized as a precursor (Mr 72,000) and is proteolytically cleaved to active forms (Mr 60-68,000) (15). Stromelysin is synthesized as a prwursor with an Mr of 57,000, which is reduced to 48,000 on activation (27). Activation in vivo probably results from proteolytic cleavage. One proteinase capable of activating metalloproteinasesis plasmin (28). Interestingly, plasminogen activator levels have been shown to increase during mammary gland involution (29). The kinetics of its induction are similar to those observed for gelatinases in the mouse and rat involuting mammary gland. Furthermore, plasminogen activator activity in mammary gland organ cultures was repressed by lactogenic hormones and induced by their absence (29). An additional site of regulation is the production of metalloproteinaseinhibitors (TIMP) (30), although little is yet known about the regulation of TIMP during mammary gland development. Therefore, the myoepithelial cell appears to be responsible for both the synthesis and degradation of the mammary gland basement membrane during normal developmentalprocesses. The manner in which these processes are regulated must await the development of suitable myoepithelial cell lines.
Acknowledgments We thank Dr H.Nagase (University of lClnsas Medicd Center) for antiserum to stromelysin and Dr B. Austin (St Georgei Hospitd Medica/ Schooo f i r peptide synthesis.
myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating and involuting rat mammary gland. J Histochem Cytochem 1982;30:667 4. Helminen HJ, Ericsson JLE. Studies on mammary gland involution. J Ultrastruct Res 1968;25:193
-
5 . Radnor CJP. Mvoepitheliumin involuting mammary, elands of the rat. J Anat li72:3
