- Email: [email protected]
Modulation of α2β1 integrin changes during mammary gland development by β-oestradiol
Biochimica et Biophysica Acta 1499 (2001) 232^241 www.elsevier.com/locate/bba
Modulation of K2L1 integrin changes during mammary gland development by L-oestradiol Tessy Iype a , K. Jayasree b , P.R. Sudhakaran a
a;
*
Department of Biochemistry, University of Kerala, Kariavattom, Trivandrum 695581, India b Department of Cytopathology, Regional Cancer Centre, Trivandrum 695011, India
Received 15 May 2000; received in revised form 16 October 2000; accepted 18 October 2000
Abstract In order to study the role of cell^matrix interactions in mammary gland function, temporal changes in K2 L1 integrin, the major receptor for collagen and the influence of L-oestradiol on its level and distribution in rat mammary gland at different stages of development were studied. The level of K2 L1 integrin determined by ELISA, was found to be high during different days of pregnancy, while in the lactating stage, it was significantly reduced. By immunocytochemical analysis, K2 L1 integrin was found to be localized towards the luminal side of acinar cells, both in the virgin and midpregnant stage, while it was not detected in the lactating stage. The possible role of hormones in modulating the level of integrin was examined in both in vitro and in vivo experiments using L-oestradiol. Supplementing L-oestradiol to isolated mammary epithelial cells from both virgin and lactating glands caused a concentration dependent increase in the incorporation of [35 S]methionine into K2 L1 integrin associated with the cells. Administration of L-oestradiol to virgin and lactating glands caused about 1.4^4-fold increase in the level of K2 integrin, indicating that upregulation of integrin during pregnancy may be due to oestrogen and as the oestrogen level falls during lactating phase, downregulation of K2 L1 integrin occurs. Treatment with L-oestradiol also resulted in the appearance of K2 L1 integrin in the acinar region of the lactating tissue, while in the untreated controls no staining for integrin was seen. These results indicate that oestrogen, apart from directly affecting the cellular activity, can influence mammary tissue function by affecting cell^ECM interactions through the modulation of integrin receptors for matrix proteins. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: L-Estradiol; Extracellular matrix; K2 L1 Integrin upregulation; Mammary gland; Lactation
1. Introduction Interaction of epithelial cells with the extracellular matrix (ECM) is crucial in maintaining proper cellular morphology and tissue-speci¢c gene expression [1^3]. Cells interact with the ECM through a variety of cell surface receptors such as the integrins. The
* Corresponding author. Fax: +91-471-307158; E-mail: [email protected]
integrins are a large family of transmembrane proteins which form heterodimers that mediate cell^ ECM and cell^cell interactions [4,5]. In vitro studies have shown that consequent on ECM^integrin interaction a cascade of events occurs intracellularly, leading to tissue-speci¢c gene expression [6,7]. Mammary gland undergoes proliferation, di¡erentiation and regression during adult life. Functional di¡erentiation in mammary epithelia requires speci¢c hormones and local environmental signals. Various changes occur in the ECM, as well as in cell^ECM
0167-4889 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 8 9 ( 0 0 ) 0 0 1 2 2 - 1
BBAMCR 14706 4-1-01
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
interactions, during this ontogenic process. These changes may be either due to the action of matrix metalloproteinases (MMPs) which alters the cellular microenvironment [8^10] or due to alteration in cell^ matrix interactions because of the altered expression of cell surface receptors such as the integrins. K2 L1 Integrin has been identi¢ed as the major collagen receptor in the mammary gland and appears to play an important role in mammary gland morphogenesis and tissue-speci¢c gene expression [11,12]. The di¡erent phases of mammary epithelial proliferation and di¡erentiation are under stringent hormonal control [13,14]. Recent reports have suggested that hormones in£uence integrin expression [15^17] and matrix remodelling by upregulating MMPs involved in mammary gland involution [18]. The molecular mechanism of alteration in cell^matrix interactions was studied by examining the temporal pattern of expression of K2 L1 integrin in rat mammary gland at di¡erent stages of development. The results presented here indicate that the changes in K2 L1 integrin during mammary gland development were modulated by L-oestradiol. 2. Materials and methods 2.1. Materials Acrylamide, bisacrylamide, L-oestradiol (watersoluble), Eagle's minimal essential medium (MEM), collagenase, trypsin, foetal bovine serum, Hepes, DNase I, EGTA and o-dianisidine were from Sigma Chemical Co. (St. Louis, MO). [125 I]NaI and [35 S]methionine were products of BARC, Mumbai, India. ECL kit was from Boehringer Mannheim. Monoclonal mouse anti-integrin K2 L1 antibody (P1E6, speci¢c for K2 ), biotinylated anti-mouse IgG, streptavidin HRP and diaminobenzidine (DAB) were products of DAKO (Denmark). The anti-L1 antibody was a kind gift from R. Timpl, Max Plank Institute, Martinsreid, Munich. Collagen I^Sepharose (COL I^Sepharose) was prepared by the coupling of collagen I (COL I) to CNBr-activated Sepharose 4B (Pharmacia, Sweden) according to the manufacturer's instructions. Tissue culture plastics were from NUNC (Roskilde, Denmark).
233
2.2. Experimental animals Mammary tissues (from inguinal and abdominal glands) at various stages of ontogeny were isolated from female Sprague^Dawley rats. Virgin tissue from animals at the pro-oestrous stage was used. 2.3. Hormone treatment Both virgin and lactating rats were injected with L-oestradiol (0.6 mg/kg body weight) subcutaneously to the inguinal mammary gland. Saline injected animals served as control. 2.4. Preparation of the plasma membrane extract from rat mammary gland tissue Mammary gland tissue was washed with phosphate-bu¡ered saline (PBS) and homogenized in 20 volumes of hypotonic bu¡er (1 mM NaHCO3 , 0.5 mM CaCl2 , 1 mM phenylmethylsulfonyl £uoride (PMSF)) under ice-cold conditions. It was ¢ltered through cheesecloth and centrifuged at 800Ug. The pellet was repeatedly extracted with the bu¡er and ¢nally washed with 0.025 M Tris^HCl bu¡er (pH 7.4) containing 1 mM PMSF. Pure plasma membrane was prepared by ultracentrifugation of the resuspended pellet over sucrose gradient [19]. The pellet was extracted with detergent bu¡er (0.025 M Tris^HCl, 0.15 M NaCl, 1 mM PMSF, 0.5% deoxycholate, 0.5% NP40) for 12 h, centrifuged at 13 000 rpm for 15 min, and the supernatant was used for a¤nity chromatography. 2.5. Isolation of collagen I binding proteins The plasma membrane extract prepared from the midpregnant rat mammary gland was subjected to a¤nity chromatography on COL I^Sepharose column, and the collagen I binding proteins were eluted with 20 mM EDTA and radioiodinated by chloramine T method [20]. Sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) analysis was done according to the procedure of Laemmli [21] and was visualized by silver staining [22], and was further con¢rmed by immunoblotting using antibodies against integrins. Binding to collagen I and IV
BBAMCR 14706 4-1-01
234
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
was also tested using radiolabelled protein by dot blot assay. 2.6. Enzyme-linked immunosorbent assay (ELISA) for the quantitation of integrin The amount of protein present in the plasma membrane extracts from the mammary gland tissues was estimated according to the method of Lowry et al.[23]. Membrane extract equivalent to 20 Wg of protein from each set was used for coating the multiwells. After incubation at 37³C for 3 h, blocking was done for another 1 h in 0.2% casein/0.05% Tween-20 in PBS. The primary antibody was added and incubated again for 1 h, followed by washing in Tween^PBS and incubation with secondary antibody. Streptavidin^HRP was added and kept for 1 h at room temp. One ml of substrate^chromogen mixture was added to the wells (to 60 ml of 0.1 M citrate-phosphate bu¡er was added 12 Wl of 30% H2 O2 followed by 500 Wl of o-dianisidine (10 mg/ ml)) and the reaction was stopped after 5 min with 50 Wl of 5 N HCl. Yellow-orange colour was developed and the absorbance was measured at 400 nm. 2.7. Immunocytochemical analysis Immunocytochemical analysis was carried out with mammary tissue sections from di¡erent developmental stages. Virgin and lactating glands after oestradiol treatment were also taken. For these studies, tissues from the inguinal glands were used. Cryostat sections were ¢xed in cold acetone for 10 min and blocked with 0.3% H2 O2 in methanol for 30 min to reduce endogenous peroxidase activity. The sections were rinsed with distilled water and incubated with 3% BSA to reduce non-speci¢c antibody binding. The primary antibody, monoclonal anti-integrin K2 _was added and kept overnight at 4³C, washed in PBS and incubated further for 30 min each at room temperature with biotinylated antimouse IgG followed by peroxidase-conjugated streptavidin. After each step the sections were washed in PBS and the reaction was developed by the application of diaminobenzidine, and were lightly counterstained with haematoxylin. In control experiments, the primary antibody was omitted [18].
2.8. Isolation of cells and metabolic labelling Mammary glands were excised from virgin and lactating rats and the epithelial cells were isolated by the method of Emerman and Bissell [24]. The mammary tissue was dissociated by treating with a medium consisted of 0.1% collagenase, 0.15% trypsin, 1% FCS, 0.12% NaHCO3 , 0.15% Hepes in MEM, for 1 h at 37³C and centrifuged at 20Ug for 1 min. The epithelial and ¢broblast cells were separated by repeated centrifugation and ¢nal pellet consisting of epithelial cells was digested with DNase I (1 ml, 0.4% at 37³C for 15 min) to give a greater number of single cells. Viability of the cells was checked using 0.1% trypan blue. The cells were then seeded onto 35-mm plastic petri dishes at a density of 3^4U106 cells per plate for 30 min at 37³C in MEM, and then methionine-free medium containing 20 WCi/ml of [35 S]methionine was added to the cells. Di¡erent concentrations (1034 M, 1035 M, 1036 M) of oestradiol was supplemented in the medium. After 12 h, the medium and cell layer were separated. The cell layer was extracted with 50 mM Tris^HCl bu¡er (pH 7.5) containing deoxycholate and NP40.The extract was then passed through a COL I^Sepharose column, and the integrin was eluted with 20 mM EGTA. The fractions were subjected to SDS^PAGE analysis, visualized by silver staining, and the bands corresponding to K2 L1 integrin were cut and dissolved in H2 O2 and the radioactivity was measured in a LKB Rack beta liquid scintillation counter. 3. Results 3.1. Isolation and characterization of K2 L1 integrin from rat mammary gland In order to con¢rm the presence of K2 L1 integrin in mammary gland plasma membrane extract of the midpregnant rat mammary gland was subjected to a¤nity chromatography on COL I^Sepharose and the bound proteins were eluted using 20 mM EDTA. The eluted proteins were characterized by electrophoresis. Two bands of molecular size 160 000 and 130 000 appeared on electrophoresis under reducing conditions. Western blotting followed
BBAMCR 14706 4-1-01
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
235
by detection with speci¢c antibodies further con¢rmed the presence of K2 L1 integrin (Fig. 1). Their binding to COL I and COL IV was con¢rmed by dot blot assay using radio iodinated K2 L1 integrin. 3.2. Changes in K2 L1 integrin expression in the mammary gland at di¡erent stages of development Mammary gland undergoes developmental changes during adult life and therefore the changes in the level and/or distribution of the K2 L1 integrin in the gland during di¡erent stages of ontogeny was studied. Mammary glands from varying stages of development, viz. virgin, midpregnant, lactating and involuting stages, were extracted and analysed by ELISA using antibodies against K2 integrin. The virgin tissue was found to express a considerable amount of K2 L1 integrin which increased signi¢cantly in the midpregnant stage. The level of the integrin during pregnancy was found to remain high up to the later stages. A signi¢cant decrease in K2 integrin level was found in the lactating stage when compared with midpregnant or virgin. As involution sets in, the
Fig. 2. Changes in K2 integrin level in mammary gland under di¡erent stages of development. Equal amounts of plasma membrane proteins from virgin (1), midpregnant (2), lactating (3), 2nd day involuting (4) and 6th day involuting (5) stages were coated on multiwell plates and analysed by ELISA. OD units were expressed as percentage of that of virgin tissue. Values given are the average of 5^6 experiments ( þ S.D.). P 6 0.01 when the lactating stage was compared with virgin; P 6 0.05 when the other stages were compared with virgin.
integrin reappears gradually attaining a level similar to that of the virgin tissue (Fig. 2). Thus there is an upregulation of the K2 L1 integrin in pregnancy while it is signi¢cantly downregulated in the lactating phase. 3.3. Localization of the K2 L1 integrin by immunocytochemical analysis
Fig. 1. Immunoblotting of K2 L1 integrin from rat mammary gland. Puri¢ed integrin was subjected to SDS^PAGE followed by Western blotting and detection of integrin using speci¢c antibodies. Immunoblot of collagen binding proteins probed with anti K2 and anti L1 antibodies. Myosin (205 kDa), L-galactosidase (116 kDa) and bovine albumin (66 kDa) were used as markers.
The changes, if any, in the distribution pattern of K2 L1 integrin, as the mammary gland undergoes developmental changes, were also studied by immunocytochemical analysis. Tissue sections from mammary gland at di¡erent stages were stained with antibody against K2 integrin. Positive reaction was indicated by brown staining. K2 Integrin was found to be expressed by virgin, midpregnant and involuting mammary glands, while the lactating gland did not show positive reaction (Fig. 3). For the virgin tissue, expression was moderate and the distribution was found to be mainly towards the luminal area of epithelial cells, along with the ducts and ductules of the gland. In the midpregnant stage also, a similar expression pattern was seen. Although due to the
BBAMCR 14706 4-1-01
236
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
Fig. 3. Immunocytochemical localization of K2 integrin. Virgin (B), midpregnant (C), lactating (D), 2nd day involuting (E) and 6th day involuting (F) mammary tissue sections were stained with antibody against K2 integrin and counterstained with Mayer's hematoxylin as described in Section 2. Midpregnant tissue untreated with primary antibody served as control (A). Arrows indicate regions of positive staining. Bar = 100 Wm.
packing of the acinar structures, the intensity of staining was apparently moderate in the acinar region like the virgin tissue, because of an increase in the number of acini during pregnancy, the total amount of K2 L1 integrin in the gland is increased. It was followed by a downregulation of the integrin in the lactating stage. But as involution sets in, the integrin reappeared and the intensity of staining was mild. No staining was seen in control sections un-
treated with primary antibody, indicating that the reaction observed was not non-speci¢c. 3.4. E¡ect of oestradiol on the production of K2 L1 integrin by isolated mammary epithelial cells in culture In order to study whether the altered expression of K2 L1 integrin in the mammary gland was due to hor-
BBAMCR 14706 4-1-01
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
monal control, the e¡ect of oestradiol on the K2 L1 integrin production by the mammary epithelial cells was studied. Mammary epithelial cells from virgin and lactating tissues in culture were treated with different concentrations of the hormone (1036 ^1034 M) in [35 S]methionine (20 WCi/ml) containing medium. The medium and the cell layer were collected after 12 h. The detergent extract of the cell layer was subjected to a¤nity chromatography on COL I^Sepharose and determined the amount of radioactivity incorporated into K2 L1 integrin. There was an increase in the amount of K2 L1 integrin synthesized both by the virgin tissue and the lactating tissue with increase in the concentration of L-oestradiol, and the maximum e¡ect was observed at a concentration of 10 34 M of the hormone (Fig. 4). The amount of K2 L1 integrin in the cells from virgin stage tissue increased with increase in the amount of hormone. Though the integrin level of the lactating stage was very low compared with the virgin stage, after oestradiol treatment of cells from lactating tissue, there was an increase in the level of integrin.
237
Fig. 5. E¡ect of L-oestradiol on K2 L1 integrin production in vivo. Equal amounts of plasma membrane proteins were prepared from virgin (A) and lactating (B) tissues after oestradiol treatment and ELISA was done using speci¢c antibodies against the integrin. OD units were expressed as percentage of that of virgin tissue. Values given are the average of 3^4 experiments ( þ S.D.). Virgin stage after oestradiol treatment when compared with control, P 6 0.05; lactating stage after treatment when compared with control, P 6 0.01. (E) Untreated; (F) treated.
3.5. E¡ect of oestradiol treatment in vivo on K2 L1 integrin in mammary gland In order to study the e¡ect of oestradiol in vivo, virgin and lactating rats were administered oestradiol and the plasma membrane extracts were prepared and ELISA was done for the quantitation of K2 L1 integrin using speci¢c antibody against the integrin. In both cases, after oestradiol administration the level of the integrin was elevated. Though the level of the integrin in the normal lactating gland was very low, after oestradiol treatment it was elevated by about four times the control levels (Fig. 5). 3.6. Immunocytochemical analysis of oestradiol treated tissues
Fig. 4. E¡ect of L-oestradiol on K2 L1 integrin production by mammary epithelial cells. Mammary epithelial cells from virgin (c) and lactating tissues (a) were metabolically labeled with [35 S]methionine (20 WCi/ml) and treated with L-oestradiol (1036 ^1034 M). The cell layer was extracted in detergent bu¡er after 12 h and the K2 L1 integrin was quanti¢ed as described in the text. The radioactivity was measured in an LKB liquid scintillation counter. Cells from virgin and lactating tissues untreated with oestradiol served as control. Values given are the average of 5^6 experiments ( þ S.D.).
The expression of K2 L1 integrin on treatment with L-oestradiol was also tested by immunocytochemical analysis. Sections of both virgin and lactating tissues after oestradiol administration were subjected to immunocytochemical analysis. The amount of K2 L1 integrin in the virgin stage was found to be increased after oestradiol administration. The luminal side of cells in each acini, the ducts and ductules of the gland showed positive staining (Fig. 6). The normal
BBAMCR 14706 4-1-01
238
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
Fig. 6. E¡ect of oestradiol administration on K2 integrin distribution in mammary gland. Animals were administered L-oestradiol and the mammary gland tissues were collected. Tissue sections from virgin (B) and lactating (D) animals administered oestradiol were stained with antibody against the K2 integrin and counterstained with Mayer's hematoxylin. Sections of tissues from untreated, virgin (A) and lactating (C) glands served as control. Arrows indicate regions of positive staining. Bar = 100 Wm.
lactating gland did not show signi¢cant reaction, but after oestradiol treatment the staining was found to be intense. Immune positivity was found to be towards the luminal side of cells in acini; the ducts and myoepithelial cells also showed positive staining. While L-oestradiol administration caused only a slight increase in intensity of staining in virgin tissues, it resulted in intense positive reaction in lactating tissues. 4. Discussion In vitro studies showed that ECM in£uence the expression of tissue-speci¢c functions in mammary epithelial cells. In these studies the crucial role of the extracellular matrix has been demonstrated by showing that mammary cells acquire a glandular morphology, synthesize an organized basement membrane and maintain mammary-speci¢c gene expression when cultured on EHS tumour matrix [25^27].
Evidence in support of a role for ECM in the control of tissue-speci¢c functions in vivo is also accumulating. Alterations in the basement membrane components and their degradation in the mammary gland during development may in£uence cell functions in vivo [28]. The remodelling of the basement membrane by the degradation of ECM components through the coordinated action of matrix degrading metalloproteinases (MMPs) have also been demonstrated [29,30]. The coordinated expression of MMPs and their endogenous regulators, and expression of tissue-speci¢c function such as casein production in mammary gland suggested that the interaction of mammary cells with the basement membrane components in vivo is critically important [31]. The results presented here give further evidence that the molecular mechanisms involved in cell^matrix interactions in intact tissue is altered as the mammary gland undergoes developmental changes. This is evidenced by the changes in K2 L1 integrin which is a receptor for COL I and COL IV. K2 L1 integrin is
BBAMCR 14706 4-1-01
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
the major collagen receptor of the mammary gland and was isolated by a¤nity chromatography on COL I^Sepharose which appeared as 160 000/130 000 bands in SDS^PAGE analysis. In the virgin mammary tissue, the K2 integrin was seen towards the luminal side of the epithelial cells in the acini, and also in the duct system. During gestation stage, when proliferation of epithelial cells occurs, there is a signi¢cant increase in K2 L1 integrin level, which appeared at the cell^matrix contact sites. This may cause alterations in cell^matrix interaction and contribute to tissue remodelling during mammary gland development. Keely et al. [12] have shown that the K2 L1 integrin was present on the basal, lateral and apical surfaces of the mammary epithelium throughout post natal development and pregnancy. A high level of expression of the K2 L1 integrin is associated with orderly and regulated proliferation of epithelial cells, including the ducts and ductules of normal breast [11]. Our results show that in the lactating tissue, where epithelia showed a structural change and tissue-speci¢c gene expression such as milk production is maintained, K2 integrin level is very low. It appears that there is a down regulation of K2 integrin, during lactating stage. Earlier reports have suggested that K2 L1 integrin might mediate some mammary cell response to collagen [11]. A decrease in K2 L1 integrin levels disrupted the ability of mammary cells to organize in three-dimensional collagen gels, indicating that K2 L1 integrin plays a critical role in collagen induced morphogenesis [32]. From our results, it is evident that a high level of K2 L1 integrin is maintained during gestation, when epithelial proliferation and glandular structure formation occur in the mammary gland. The downregulation of the integrin in the lactating stage may be a regulatory mechanism to arrest the morphogenetic event. The normal development and ductal morphogenesis of the mammary gland depend on functional L1 integrins, which permit contacts with the ECM and with laminin in particular [33]. It is also suggested that cellular growth, survival and morphogenesis of acinar structures by normal cells are integrin dependent and loss of proper integrin mediated cell^ECM interactions may be critical to breast tumour formation [34]. The results presented here provide further evidence in support of a critical role for the interaction of cells with ECM in vivo in
239
mammary epithelial function. Changes in the level of K2 L1 integrin not only a¡ect epithelial-basement membrane interactions, but also may in£uence intracellular events. The increase in K2 L1 integrin during pregnancy may be proliferation associated, but during lactation there is a down regulation of the integrin, though the epithelial mass remains the same. This may also be due to the action of hormones, since the development of the mammary epithelium and milk production during the later stages of pregnancy and in lactation are under hormonal control. The normal development of the mammary gland was found to be partly under control of interaction between gonadotropic hormone and oestrogen [14]. Results on the production and distribution of K2 L1 integrin presented here suggest that hormones in£uence cell^matrix interactions as well. It appears that the production and distribution of K2 L1 integrin is modulated by oestrogen. This conclusion is based on the following observations. (a) In mammary glands from midpregnant rats, which is under oestrogen stress, the level of the K2 L1 integrin is elevated. (b) In lactating tissue, which is relieved from oestrogen stress, there is decrease in K2 L1 integrin level. (c) Administration of L-oestradiol caused an increase in the level of the integrin. Both in the virgin and in the lactating stage, the level of the integrin was signi¢cantly enhanced after oestradiol treatment. The distribution pattern of the K2 L1 integrin in lactating glands treated with oestradiol was also similar to that of virgin and pregnant stages. (d) In vitro experiments by supplementing oestradiol to primary cultures of mammary epithelial cells also demonstrated that the K2 L1 integrin production by cells from both the virgin and lactating tissues is increased in the presence of oestradiol. Although these experiments do not indicate how oestrogen a¡ects the production and distribution of K2 L1 integrin in mammary gland, its enhanced production by primary cultures of mammary epithelial cells on treatment with oestradiol indicates that oestradiol e¡ect is not a systemic e¡ect. The in£uence of hormones on the cell surface receptors and changes in tissue-speci¢c functions have been reported in different systems [15^17]. The addition of oestradiol and progesterone to cultured stromal cells of the human endometrium in the early proliferative phase increased the expression of L1 integrins in vitro
BBAMCR 14706 4-1-01
240
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
[15]. It has been suggested that oestrogen enhances Kv _L3 integrin expression by avian osteoclast precursors through stabilization of L 1 integrin mRNA. It appears that steroid hormones, particularly oestrogen, has an important role in regulating cell^matrix interactions in mammary gland. It has been reported that steroid hormones apart from their intracellular e¡ects in£uence composition of the extracellular matrix [35]. A role for L-oestradiol in mediating matrix remodelling by regulation of MMPs involved in mammary gland involution has also been demonstrated [18]. The present data give evidence for the hormonal modulation of cell^ECM interactions as a result of the changes occurring in the cell surface receptors for matrix proteins as shown in the case of K2 L1 integrin expression in mammary gland. As indicated before, in vitro and in vivo studies have shown that cell^matrix interactions modulate expression of tissue-speci¢c function in the mammary gland. Oestrogen plays an important role in the control of the biochemical activity of mammary epithelial cells and the expression of di¡erentiated functions. Results presented above indicate that oestrogen, apart from directly a¡ecting the cellular activity, can in£uence mammary tissue function by a¡ecting cell^matrix contact through the modulation of integrin receptor for matrix proteins. Further investigations are under way to elucidate the mechanism of action of L-oestradiol in modulating K2 L1 integrin expression.
[3]
[4] [5]
[6] [7]
[8]
[9]
[10]
[11]
[12]
[13]
Acknowledgements Financial assistance received from UGC, New Delhi, to T.I. in the form of SRF is gratefully acknowledged.
[14]
[15]
References [1] M. Bern¢eld, S.D. Banerjee, J.E. Koda, A.C. Rapreager, Remodeling of the basement membrane as a mechanism of morphogenetic tissue interaction, in: R. Trelstad (Ed.), The Role of Extracellular Matrix in Development, Alan R. Liss, New York, 1984, pp. 545^572. [2] M.J. Bissell, J. Aggeler, Dynamic reciprocity: How do extracellular matrix and hormones direct gene expression?, in: M.C. Cabot, W.L. McKeehan (Eds.), Mechanisms of Signal
[16]
[17]
Transduction by Hormones and Growth Factors, Alan R. Liss, New York, 1987, pp. 251^262. M.H. Barcellos-Ho¡, J. Aggeler, T.G. Ram, M.J. Bissell, Functional di¡erentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane, Development 105 (1989) 223^235. R.O. Hynes, Integrins: A family of cell surface receptors, Cell 48 (1987) 549^554. C.A. Buck, A. F Horwitz, Cell surface receptors for extracellular matrix molecules, Annu. Rev. Cell Biol. 3 (1987) 179^205. R.O. Hynes, Integrins: Versatility, modulation and signaling in cell adhesion, Cell 69 (1992) 11^25. K. Burridge, C. Turner, L. Romer, Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly, J. Cell Biol. 119 (1992) 893^904. R.S. Talhouk, Z. Werb, M.J. Bissell, Functional interplay between extracellular matrix degrading proteinases in mammary gland: a coordinate system for regulating mammary epithelial function, in: T.P. Fleming (Ed.), Epithelial Organization and Development, Chapman and Hall, London, 1992, pp. 329^349. R.S. Talhouk, M.J. Bissell, Z. Werb, Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution, J. Cell Biol. 118 (1992) 1271^1282. M. Ambili, R. Pillai, P.R. Sudhakaran, Characteristics of a 60K gelatinase involved in rat mammary gland involution, Ind. J. Biochem. Biophys. 34 (1997) 347^353. M.M. Zutter, A.S. Santoro, Widespread histologic distribution of the K2 L1 integrin cell surface collagen receptor, Am. J. Pathol. 137 (1990) 113^120. P.J. Keely, J.E. Wu, A.S Santoro, The spatial and temporal expression of the K2 L1 integrin and its ligands collagen I, collagen IV and laminin suggest important roles in mouse mammary morphogenesis, Di¡erentiation 59 (1995) 1^13. Y.J. Topper, C.S. Freeman, Multiple hormone interactions in the developmental biology of the mammary gland, Physiol. Rev. 60 (1980) 1049^1060. M. Feldman, W.F. Ruan, B.C. Cunningham, J.A. Wells, D.L. Kleinberg, Evidence that the growth hormone receptor mediates di¡erentiation and development of mammary gland, Endocrinology 133 (1993) 1602^1608. Y. Yoshimura, K. Miyakoshi, T. Hamatani, K. Iwahashi, J. Takahashi, N. Kobayashi, K. Sueoka, T. Miyazaki, N. Kuji, M. Tanaka, Role of L1 integrins in human endometrium and decidua during implantation, Horm. Res. 2 (1998) 46^55. M. Sillem, S. Prifti, M. Schmidt, T. Rabe, B. Runnebaum, Endometrial integrin expression is independent of oestrogen or progestin treatment in vitro, Fertil. Steril. 67 (1997) 877^ 882. S. Sonohara, R. Mira-y-Lopez, M.M. Brentani, Laminin and oestradiol regulation of the plasminogen activator system in MCF-7 breast carcinoma cells, Int. J. Cancer 76 (1998) 77^ 85.
BBAMCR 14706 4-1-01
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241 [18] M. Ambili, K. Jayasree, P.R. Sudhakaran, 60K gelatinase involved in mammary gland involution is regulated by Loestradiol, Biochim. Biophys. Acta 1403 (1998) 219^231. [19] D.M. Neville, Isolation of an organ speci¢c antigen from cell surface membrane of rat liver, Biochim. Biophys. Acta 154 (1968) 540^552. [20] F.C. Greenwood, W.M. Hunter, J.S. Clover, The preparation of a 131 I-labelled human growth hormone of high speci¢c radioactivity, Biochem. J. 89 (1963) 114^123. [21] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage-T4, Nature 227 (1970) 680^685. [22] B.R. Oakely, D.R. Kirsch, N.R. Morris, A simpli¢ed ultra sensitive silver staining for detecting proteins in polyacrylamide gels, Anal. Biochem. 105 (1980) 361^363. [23] O.H. Lowry, N.J. Rosebrough, A. Farr, R.J. Randall, Protein measurement with the Folin-phenol reagent, J. Biol. Chem. 193 (1951) 265^275. [24] J.T. Emerman, M.J. Bissell, Cultures of mammary epithelial cells : extracellular matrix and functional di¡erentiation, Adv. Cell culture 6 (1988) 137^159. [25] E.Y.H. Lee, G. Parry, M.J. Bissell, Modulation of secreted proteins of mouse mammary epithelial cells by the extracellular matrix, J. Cell Biol. 98 (1984) 146^155. [26] M.J. Bissell, H.G. Hall, Form and function in the mammary gland: The role of extracellular matrix, in: M. Neville, C. Daniel (Eds.), The Mammary Gland: Development, Regulation and Function, Plenum, New York, 1987, pp. 97^ 146. [27] J. Aggeler, J. Ward, L. Mackenzie Blackie, M.H. BarcellosHo¡, C.H. Streuli, M.J. Bissell, Cytodi¡erentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to development in vivo, J. Cell Sci. 99 (1991) 407^417.
241
[28] M.L. Li, J. Aggeler, D.A. Farson, C. Hatier, J. Hassell, M.J. Bissell, In£uence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells, Proc. Natl. Acad. Sci. USA 84 (1987) 136^140. [29] H. Birkedel-Hansen, W.G.I. Moore, M.K. Bodden, L.J. Windsor, B. Birkedel-Hansen, A. DeCarlo, J.A. Engler, Matrix metalloproteinases: A review, Crit. Rev. Oral Biol. Med. 4 (1993) 197^250. [30] P. Mignatti, D.B. Rifkin, H.G. Welgus, W.C. Parks, Proteinases and remodeling, in: R.A.F. Clark (Ed.), The Molecular and Cellular Biology of Wound Repair, Plenum, New York, 1996, pp. 426^473. [31] C.H. Streuli, N. Bailey, M.J. Bissell, Control of mammary epithelial di¡erentiation : Basement membrane induces tissue speci¢c gene expression in the absence of cell-cell interaction and morphological polarity, J. Cell Biol. 115 (1991) 1383^ 1395. [32] P.J. Keely, A.M. Fong, M.M. Zutter, S.A. Santoro, Alteration of collagen dependent adhesion, motility and morphogenesis by the expression of antisense K2 integrin mRNA in mammary cells, J. Cell Sci. 108 (1995) 595^607. [33] T.C.M. Klinowska, J.V. Soriano, G.M. Edwards, J.M. Oliver, A.J. Valentijn, R. Montesano, C.H. Streuli, Laminin and L1 integrins are crucial for normal mammary gland development in the mouse, Dev. Biol. 215 (1999) 13^32. [34] A.R. Howlett, N. Bailey, C. Damsky, O.W. Petersen, M.J. Bissell, Cellular growth and survival are mediated by L1 integrins in normal human breast epithelium but not in breast carcinoma, J. Cell Sci. 108 (1995) 1945^1957. [35] Z. Feng, A. Marti, B. Jehn, H.J. Altematt, G. Chicaiza, R. Jaggi, Glucocorticoid and progesterone inhibit involution and programmed cell death in mouse mammary gland, J. Cell Biol. 131 (1995) 1095^1103.
BBAMCR 14706 4-1-01
Modulation of K2L1 integrin changes during mammary gland development by L-oestradiol Tessy Iype a , K. Jayasree b , P.R. Sudhakaran a
a;
*
Department of Biochemistry, University of Kerala, Kariavattom, Trivandrum 695581, India b Department of Cytopathology, Regional Cancer Centre, Trivandrum 695011, India
Received 15 May 2000; received in revised form 16 October 2000; accepted 18 October 2000
Abstract In order to study the role of cell^matrix interactions in mammary gland function, temporal changes in K2 L1 integrin, the major receptor for collagen and the influence of L-oestradiol on its level and distribution in rat mammary gland at different stages of development were studied. The level of K2 L1 integrin determined by ELISA, was found to be high during different days of pregnancy, while in the lactating stage, it was significantly reduced. By immunocytochemical analysis, K2 L1 integrin was found to be localized towards the luminal side of acinar cells, both in the virgin and midpregnant stage, while it was not detected in the lactating stage. The possible role of hormones in modulating the level of integrin was examined in both in vitro and in vivo experiments using L-oestradiol. Supplementing L-oestradiol to isolated mammary epithelial cells from both virgin and lactating glands caused a concentration dependent increase in the incorporation of [35 S]methionine into K2 L1 integrin associated with the cells. Administration of L-oestradiol to virgin and lactating glands caused about 1.4^4-fold increase in the level of K2 integrin, indicating that upregulation of integrin during pregnancy may be due to oestrogen and as the oestrogen level falls during lactating phase, downregulation of K2 L1 integrin occurs. Treatment with L-oestradiol also resulted in the appearance of K2 L1 integrin in the acinar region of the lactating tissue, while in the untreated controls no staining for integrin was seen. These results indicate that oestrogen, apart from directly affecting the cellular activity, can influence mammary tissue function by affecting cell^ECM interactions through the modulation of integrin receptors for matrix proteins. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: L-Estradiol; Extracellular matrix; K2 L1 Integrin upregulation; Mammary gland; Lactation
1. Introduction Interaction of epithelial cells with the extracellular matrix (ECM) is crucial in maintaining proper cellular morphology and tissue-speci¢c gene expression [1^3]. Cells interact with the ECM through a variety of cell surface receptors such as the integrins. The
* Corresponding author. Fax: +91-471-307158; E-mail: [email protected]
integrins are a large family of transmembrane proteins which form heterodimers that mediate cell^ ECM and cell^cell interactions [4,5]. In vitro studies have shown that consequent on ECM^integrin interaction a cascade of events occurs intracellularly, leading to tissue-speci¢c gene expression [6,7]. Mammary gland undergoes proliferation, di¡erentiation and regression during adult life. Functional di¡erentiation in mammary epithelia requires speci¢c hormones and local environmental signals. Various changes occur in the ECM, as well as in cell^ECM
0167-4889 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 8 9 ( 0 0 ) 0 0 1 2 2 - 1
BBAMCR 14706 4-1-01
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
interactions, during this ontogenic process. These changes may be either due to the action of matrix metalloproteinases (MMPs) which alters the cellular microenvironment [8^10] or due to alteration in cell^ matrix interactions because of the altered expression of cell surface receptors such as the integrins. K2 L1 Integrin has been identi¢ed as the major collagen receptor in the mammary gland and appears to play an important role in mammary gland morphogenesis and tissue-speci¢c gene expression [11,12]. The di¡erent phases of mammary epithelial proliferation and di¡erentiation are under stringent hormonal control [13,14]. Recent reports have suggested that hormones in£uence integrin expression [15^17] and matrix remodelling by upregulating MMPs involved in mammary gland involution [18]. The molecular mechanism of alteration in cell^matrix interactions was studied by examining the temporal pattern of expression of K2 L1 integrin in rat mammary gland at di¡erent stages of development. The results presented here indicate that the changes in K2 L1 integrin during mammary gland development were modulated by L-oestradiol. 2. Materials and methods 2.1. Materials Acrylamide, bisacrylamide, L-oestradiol (watersoluble), Eagle's minimal essential medium (MEM), collagenase, trypsin, foetal bovine serum, Hepes, DNase I, EGTA and o-dianisidine were from Sigma Chemical Co. (St. Louis, MO). [125 I]NaI and [35 S]methionine were products of BARC, Mumbai, India. ECL kit was from Boehringer Mannheim. Monoclonal mouse anti-integrin K2 L1 antibody (P1E6, speci¢c for K2 ), biotinylated anti-mouse IgG, streptavidin HRP and diaminobenzidine (DAB) were products of DAKO (Denmark). The anti-L1 antibody was a kind gift from R. Timpl, Max Plank Institute, Martinsreid, Munich. Collagen I^Sepharose (COL I^Sepharose) was prepared by the coupling of collagen I (COL I) to CNBr-activated Sepharose 4B (Pharmacia, Sweden) according to the manufacturer's instructions. Tissue culture plastics were from NUNC (Roskilde, Denmark).
233
2.2. Experimental animals Mammary tissues (from inguinal and abdominal glands) at various stages of ontogeny were isolated from female Sprague^Dawley rats. Virgin tissue from animals at the pro-oestrous stage was used. 2.3. Hormone treatment Both virgin and lactating rats were injected with L-oestradiol (0.6 mg/kg body weight) subcutaneously to the inguinal mammary gland. Saline injected animals served as control. 2.4. Preparation of the plasma membrane extract from rat mammary gland tissue Mammary gland tissue was washed with phosphate-bu¡ered saline (PBS) and homogenized in 20 volumes of hypotonic bu¡er (1 mM NaHCO3 , 0.5 mM CaCl2 , 1 mM phenylmethylsulfonyl £uoride (PMSF)) under ice-cold conditions. It was ¢ltered through cheesecloth and centrifuged at 800Ug. The pellet was repeatedly extracted with the bu¡er and ¢nally washed with 0.025 M Tris^HCl bu¡er (pH 7.4) containing 1 mM PMSF. Pure plasma membrane was prepared by ultracentrifugation of the resuspended pellet over sucrose gradient [19]. The pellet was extracted with detergent bu¡er (0.025 M Tris^HCl, 0.15 M NaCl, 1 mM PMSF, 0.5% deoxycholate, 0.5% NP40) for 12 h, centrifuged at 13 000 rpm for 15 min, and the supernatant was used for a¤nity chromatography. 2.5. Isolation of collagen I binding proteins The plasma membrane extract prepared from the midpregnant rat mammary gland was subjected to a¤nity chromatography on COL I^Sepharose column, and the collagen I binding proteins were eluted with 20 mM EDTA and radioiodinated by chloramine T method [20]. Sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) analysis was done according to the procedure of Laemmli [21] and was visualized by silver staining [22], and was further con¢rmed by immunoblotting using antibodies against integrins. Binding to collagen I and IV
BBAMCR 14706 4-1-01
234
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
was also tested using radiolabelled protein by dot blot assay. 2.6. Enzyme-linked immunosorbent assay (ELISA) for the quantitation of integrin The amount of protein present in the plasma membrane extracts from the mammary gland tissues was estimated according to the method of Lowry et al.[23]. Membrane extract equivalent to 20 Wg of protein from each set was used for coating the multiwells. After incubation at 37³C for 3 h, blocking was done for another 1 h in 0.2% casein/0.05% Tween-20 in PBS. The primary antibody was added and incubated again for 1 h, followed by washing in Tween^PBS and incubation with secondary antibody. Streptavidin^HRP was added and kept for 1 h at room temp. One ml of substrate^chromogen mixture was added to the wells (to 60 ml of 0.1 M citrate-phosphate bu¡er was added 12 Wl of 30% H2 O2 followed by 500 Wl of o-dianisidine (10 mg/ ml)) and the reaction was stopped after 5 min with 50 Wl of 5 N HCl. Yellow-orange colour was developed and the absorbance was measured at 400 nm. 2.7. Immunocytochemical analysis Immunocytochemical analysis was carried out with mammary tissue sections from di¡erent developmental stages. Virgin and lactating glands after oestradiol treatment were also taken. For these studies, tissues from the inguinal glands were used. Cryostat sections were ¢xed in cold acetone for 10 min and blocked with 0.3% H2 O2 in methanol for 30 min to reduce endogenous peroxidase activity. The sections were rinsed with distilled water and incubated with 3% BSA to reduce non-speci¢c antibody binding. The primary antibody, monoclonal anti-integrin K2 _was added and kept overnight at 4³C, washed in PBS and incubated further for 30 min each at room temperature with biotinylated antimouse IgG followed by peroxidase-conjugated streptavidin. After each step the sections were washed in PBS and the reaction was developed by the application of diaminobenzidine, and were lightly counterstained with haematoxylin. In control experiments, the primary antibody was omitted [18].
2.8. Isolation of cells and metabolic labelling Mammary glands were excised from virgin and lactating rats and the epithelial cells were isolated by the method of Emerman and Bissell [24]. The mammary tissue was dissociated by treating with a medium consisted of 0.1% collagenase, 0.15% trypsin, 1% FCS, 0.12% NaHCO3 , 0.15% Hepes in MEM, for 1 h at 37³C and centrifuged at 20Ug for 1 min. The epithelial and ¢broblast cells were separated by repeated centrifugation and ¢nal pellet consisting of epithelial cells was digested with DNase I (1 ml, 0.4% at 37³C for 15 min) to give a greater number of single cells. Viability of the cells was checked using 0.1% trypan blue. The cells were then seeded onto 35-mm plastic petri dishes at a density of 3^4U106 cells per plate for 30 min at 37³C in MEM, and then methionine-free medium containing 20 WCi/ml of [35 S]methionine was added to the cells. Di¡erent concentrations (1034 M, 1035 M, 1036 M) of oestradiol was supplemented in the medium. After 12 h, the medium and cell layer were separated. The cell layer was extracted with 50 mM Tris^HCl bu¡er (pH 7.5) containing deoxycholate and NP40.The extract was then passed through a COL I^Sepharose column, and the integrin was eluted with 20 mM EGTA. The fractions were subjected to SDS^PAGE analysis, visualized by silver staining, and the bands corresponding to K2 L1 integrin were cut and dissolved in H2 O2 and the radioactivity was measured in a LKB Rack beta liquid scintillation counter. 3. Results 3.1. Isolation and characterization of K2 L1 integrin from rat mammary gland In order to con¢rm the presence of K2 L1 integrin in mammary gland plasma membrane extract of the midpregnant rat mammary gland was subjected to a¤nity chromatography on COL I^Sepharose and the bound proteins were eluted using 20 mM EDTA. The eluted proteins were characterized by electrophoresis. Two bands of molecular size 160 000 and 130 000 appeared on electrophoresis under reducing conditions. Western blotting followed
BBAMCR 14706 4-1-01
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
235
by detection with speci¢c antibodies further con¢rmed the presence of K2 L1 integrin (Fig. 1). Their binding to COL I and COL IV was con¢rmed by dot blot assay using radio iodinated K2 L1 integrin. 3.2. Changes in K2 L1 integrin expression in the mammary gland at di¡erent stages of development Mammary gland undergoes developmental changes during adult life and therefore the changes in the level and/or distribution of the K2 L1 integrin in the gland during di¡erent stages of ontogeny was studied. Mammary glands from varying stages of development, viz. virgin, midpregnant, lactating and involuting stages, were extracted and analysed by ELISA using antibodies against K2 integrin. The virgin tissue was found to express a considerable amount of K2 L1 integrin which increased signi¢cantly in the midpregnant stage. The level of the integrin during pregnancy was found to remain high up to the later stages. A signi¢cant decrease in K2 integrin level was found in the lactating stage when compared with midpregnant or virgin. As involution sets in, the
Fig. 2. Changes in K2 integrin level in mammary gland under di¡erent stages of development. Equal amounts of plasma membrane proteins from virgin (1), midpregnant (2), lactating (3), 2nd day involuting (4) and 6th day involuting (5) stages were coated on multiwell plates and analysed by ELISA. OD units were expressed as percentage of that of virgin tissue. Values given are the average of 5^6 experiments ( þ S.D.). P 6 0.01 when the lactating stage was compared with virgin; P 6 0.05 when the other stages were compared with virgin.
integrin reappears gradually attaining a level similar to that of the virgin tissue (Fig. 2). Thus there is an upregulation of the K2 L1 integrin in pregnancy while it is signi¢cantly downregulated in the lactating phase. 3.3. Localization of the K2 L1 integrin by immunocytochemical analysis
Fig. 1. Immunoblotting of K2 L1 integrin from rat mammary gland. Puri¢ed integrin was subjected to SDS^PAGE followed by Western blotting and detection of integrin using speci¢c antibodies. Immunoblot of collagen binding proteins probed with anti K2 and anti L1 antibodies. Myosin (205 kDa), L-galactosidase (116 kDa) and bovine albumin (66 kDa) were used as markers.
The changes, if any, in the distribution pattern of K2 L1 integrin, as the mammary gland undergoes developmental changes, were also studied by immunocytochemical analysis. Tissue sections from mammary gland at di¡erent stages were stained with antibody against K2 integrin. Positive reaction was indicated by brown staining. K2 Integrin was found to be expressed by virgin, midpregnant and involuting mammary glands, while the lactating gland did not show positive reaction (Fig. 3). For the virgin tissue, expression was moderate and the distribution was found to be mainly towards the luminal area of epithelial cells, along with the ducts and ductules of the gland. In the midpregnant stage also, a similar expression pattern was seen. Although due to the
BBAMCR 14706 4-1-01
236
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
Fig. 3. Immunocytochemical localization of K2 integrin. Virgin (B), midpregnant (C), lactating (D), 2nd day involuting (E) and 6th day involuting (F) mammary tissue sections were stained with antibody against K2 integrin and counterstained with Mayer's hematoxylin as described in Section 2. Midpregnant tissue untreated with primary antibody served as control (A). Arrows indicate regions of positive staining. Bar = 100 Wm.
packing of the acinar structures, the intensity of staining was apparently moderate in the acinar region like the virgin tissue, because of an increase in the number of acini during pregnancy, the total amount of K2 L1 integrin in the gland is increased. It was followed by a downregulation of the integrin in the lactating stage. But as involution sets in, the integrin reappeared and the intensity of staining was mild. No staining was seen in control sections un-
treated with primary antibody, indicating that the reaction observed was not non-speci¢c. 3.4. E¡ect of oestradiol on the production of K2 L1 integrin by isolated mammary epithelial cells in culture In order to study whether the altered expression of K2 L1 integrin in the mammary gland was due to hor-
BBAMCR 14706 4-1-01
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
monal control, the e¡ect of oestradiol on the K2 L1 integrin production by the mammary epithelial cells was studied. Mammary epithelial cells from virgin and lactating tissues in culture were treated with different concentrations of the hormone (1036 ^1034 M) in [35 S]methionine (20 WCi/ml) containing medium. The medium and the cell layer were collected after 12 h. The detergent extract of the cell layer was subjected to a¤nity chromatography on COL I^Sepharose and determined the amount of radioactivity incorporated into K2 L1 integrin. There was an increase in the amount of K2 L1 integrin synthesized both by the virgin tissue and the lactating tissue with increase in the concentration of L-oestradiol, and the maximum e¡ect was observed at a concentration of 10 34 M of the hormone (Fig. 4). The amount of K2 L1 integrin in the cells from virgin stage tissue increased with increase in the amount of hormone. Though the integrin level of the lactating stage was very low compared with the virgin stage, after oestradiol treatment of cells from lactating tissue, there was an increase in the level of integrin.
237
Fig. 5. E¡ect of L-oestradiol on K2 L1 integrin production in vivo. Equal amounts of plasma membrane proteins were prepared from virgin (A) and lactating (B) tissues after oestradiol treatment and ELISA was done using speci¢c antibodies against the integrin. OD units were expressed as percentage of that of virgin tissue. Values given are the average of 3^4 experiments ( þ S.D.). Virgin stage after oestradiol treatment when compared with control, P 6 0.05; lactating stage after treatment when compared with control, P 6 0.01. (E) Untreated; (F) treated.
3.5. E¡ect of oestradiol treatment in vivo on K2 L1 integrin in mammary gland In order to study the e¡ect of oestradiol in vivo, virgin and lactating rats were administered oestradiol and the plasma membrane extracts were prepared and ELISA was done for the quantitation of K2 L1 integrin using speci¢c antibody against the integrin. In both cases, after oestradiol administration the level of the integrin was elevated. Though the level of the integrin in the normal lactating gland was very low, after oestradiol treatment it was elevated by about four times the control levels (Fig. 5). 3.6. Immunocytochemical analysis of oestradiol treated tissues
Fig. 4. E¡ect of L-oestradiol on K2 L1 integrin production by mammary epithelial cells. Mammary epithelial cells from virgin (c) and lactating tissues (a) were metabolically labeled with [35 S]methionine (20 WCi/ml) and treated with L-oestradiol (1036 ^1034 M). The cell layer was extracted in detergent bu¡er after 12 h and the K2 L1 integrin was quanti¢ed as described in the text. The radioactivity was measured in an LKB liquid scintillation counter. Cells from virgin and lactating tissues untreated with oestradiol served as control. Values given are the average of 5^6 experiments ( þ S.D.).
The expression of K2 L1 integrin on treatment with L-oestradiol was also tested by immunocytochemical analysis. Sections of both virgin and lactating tissues after oestradiol administration were subjected to immunocytochemical analysis. The amount of K2 L1 integrin in the virgin stage was found to be increased after oestradiol administration. The luminal side of cells in each acini, the ducts and ductules of the gland showed positive staining (Fig. 6). The normal
BBAMCR 14706 4-1-01
238
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
Fig. 6. E¡ect of oestradiol administration on K2 integrin distribution in mammary gland. Animals were administered L-oestradiol and the mammary gland tissues were collected. Tissue sections from virgin (B) and lactating (D) animals administered oestradiol were stained with antibody against the K2 integrin and counterstained with Mayer's hematoxylin. Sections of tissues from untreated, virgin (A) and lactating (C) glands served as control. Arrows indicate regions of positive staining. Bar = 100 Wm.
lactating gland did not show signi¢cant reaction, but after oestradiol treatment the staining was found to be intense. Immune positivity was found to be towards the luminal side of cells in acini; the ducts and myoepithelial cells also showed positive staining. While L-oestradiol administration caused only a slight increase in intensity of staining in virgin tissues, it resulted in intense positive reaction in lactating tissues. 4. Discussion In vitro studies showed that ECM in£uence the expression of tissue-speci¢c functions in mammary epithelial cells. In these studies the crucial role of the extracellular matrix has been demonstrated by showing that mammary cells acquire a glandular morphology, synthesize an organized basement membrane and maintain mammary-speci¢c gene expression when cultured on EHS tumour matrix [25^27].
Evidence in support of a role for ECM in the control of tissue-speci¢c functions in vivo is also accumulating. Alterations in the basement membrane components and their degradation in the mammary gland during development may in£uence cell functions in vivo [28]. The remodelling of the basement membrane by the degradation of ECM components through the coordinated action of matrix degrading metalloproteinases (MMPs) have also been demonstrated [29,30]. The coordinated expression of MMPs and their endogenous regulators, and expression of tissue-speci¢c function such as casein production in mammary gland suggested that the interaction of mammary cells with the basement membrane components in vivo is critically important [31]. The results presented here give further evidence that the molecular mechanisms involved in cell^matrix interactions in intact tissue is altered as the mammary gland undergoes developmental changes. This is evidenced by the changes in K2 L1 integrin which is a receptor for COL I and COL IV. K2 L1 integrin is
BBAMCR 14706 4-1-01
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
the major collagen receptor of the mammary gland and was isolated by a¤nity chromatography on COL I^Sepharose which appeared as 160 000/130 000 bands in SDS^PAGE analysis. In the virgin mammary tissue, the K2 integrin was seen towards the luminal side of the epithelial cells in the acini, and also in the duct system. During gestation stage, when proliferation of epithelial cells occurs, there is a signi¢cant increase in K2 L1 integrin level, which appeared at the cell^matrix contact sites. This may cause alterations in cell^matrix interaction and contribute to tissue remodelling during mammary gland development. Keely et al. [12] have shown that the K2 L1 integrin was present on the basal, lateral and apical surfaces of the mammary epithelium throughout post natal development and pregnancy. A high level of expression of the K2 L1 integrin is associated with orderly and regulated proliferation of epithelial cells, including the ducts and ductules of normal breast [11]. Our results show that in the lactating tissue, where epithelia showed a structural change and tissue-speci¢c gene expression such as milk production is maintained, K2 integrin level is very low. It appears that there is a down regulation of K2 integrin, during lactating stage. Earlier reports have suggested that K2 L1 integrin might mediate some mammary cell response to collagen [11]. A decrease in K2 L1 integrin levels disrupted the ability of mammary cells to organize in three-dimensional collagen gels, indicating that K2 L1 integrin plays a critical role in collagen induced morphogenesis [32]. From our results, it is evident that a high level of K2 L1 integrin is maintained during gestation, when epithelial proliferation and glandular structure formation occur in the mammary gland. The downregulation of the integrin in the lactating stage may be a regulatory mechanism to arrest the morphogenetic event. The normal development and ductal morphogenesis of the mammary gland depend on functional L1 integrins, which permit contacts with the ECM and with laminin in particular [33]. It is also suggested that cellular growth, survival and morphogenesis of acinar structures by normal cells are integrin dependent and loss of proper integrin mediated cell^ECM interactions may be critical to breast tumour formation [34]. The results presented here provide further evidence in support of a critical role for the interaction of cells with ECM in vivo in
239
mammary epithelial function. Changes in the level of K2 L1 integrin not only a¡ect epithelial-basement membrane interactions, but also may in£uence intracellular events. The increase in K2 L1 integrin during pregnancy may be proliferation associated, but during lactation there is a down regulation of the integrin, though the epithelial mass remains the same. This may also be due to the action of hormones, since the development of the mammary epithelium and milk production during the later stages of pregnancy and in lactation are under hormonal control. The normal development of the mammary gland was found to be partly under control of interaction between gonadotropic hormone and oestrogen [14]. Results on the production and distribution of K2 L1 integrin presented here suggest that hormones in£uence cell^matrix interactions as well. It appears that the production and distribution of K2 L1 integrin is modulated by oestrogen. This conclusion is based on the following observations. (a) In mammary glands from midpregnant rats, which is under oestrogen stress, the level of the K2 L1 integrin is elevated. (b) In lactating tissue, which is relieved from oestrogen stress, there is decrease in K2 L1 integrin level. (c) Administration of L-oestradiol caused an increase in the level of the integrin. Both in the virgin and in the lactating stage, the level of the integrin was signi¢cantly enhanced after oestradiol treatment. The distribution pattern of the K2 L1 integrin in lactating glands treated with oestradiol was also similar to that of virgin and pregnant stages. (d) In vitro experiments by supplementing oestradiol to primary cultures of mammary epithelial cells also demonstrated that the K2 L1 integrin production by cells from both the virgin and lactating tissues is increased in the presence of oestradiol. Although these experiments do not indicate how oestrogen a¡ects the production and distribution of K2 L1 integrin in mammary gland, its enhanced production by primary cultures of mammary epithelial cells on treatment with oestradiol indicates that oestradiol e¡ect is not a systemic e¡ect. The in£uence of hormones on the cell surface receptors and changes in tissue-speci¢c functions have been reported in different systems [15^17]. The addition of oestradiol and progesterone to cultured stromal cells of the human endometrium in the early proliferative phase increased the expression of L1 integrins in vitro
BBAMCR 14706 4-1-01
240
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241
[15]. It has been suggested that oestrogen enhances Kv _L3 integrin expression by avian osteoclast precursors through stabilization of L 1 integrin mRNA. It appears that steroid hormones, particularly oestrogen, has an important role in regulating cell^matrix interactions in mammary gland. It has been reported that steroid hormones apart from their intracellular e¡ects in£uence composition of the extracellular matrix [35]. A role for L-oestradiol in mediating matrix remodelling by regulation of MMPs involved in mammary gland involution has also been demonstrated [18]. The present data give evidence for the hormonal modulation of cell^ECM interactions as a result of the changes occurring in the cell surface receptors for matrix proteins as shown in the case of K2 L1 integrin expression in mammary gland. As indicated before, in vitro and in vivo studies have shown that cell^matrix interactions modulate expression of tissue-speci¢c function in the mammary gland. Oestrogen plays an important role in the control of the biochemical activity of mammary epithelial cells and the expression of di¡erentiated functions. Results presented above indicate that oestrogen, apart from directly a¡ecting the cellular activity, can in£uence mammary tissue function by a¡ecting cell^matrix contact through the modulation of integrin receptor for matrix proteins. Further investigations are under way to elucidate the mechanism of action of L-oestradiol in modulating K2 L1 integrin expression.
[3]
[4] [5]
[6] [7]
[8]
[9]
[10]
[11]
[12]
[13]
Acknowledgements Financial assistance received from UGC, New Delhi, to T.I. in the form of SRF is gratefully acknowledged.
[14]
[15]
References [1] M. Bern¢eld, S.D. Banerjee, J.E. Koda, A.C. Rapreager, Remodeling of the basement membrane as a mechanism of morphogenetic tissue interaction, in: R. Trelstad (Ed.), The Role of Extracellular Matrix in Development, Alan R. Liss, New York, 1984, pp. 545^572. [2] M.J. Bissell, J. Aggeler, Dynamic reciprocity: How do extracellular matrix and hormones direct gene expression?, in: M.C. Cabot, W.L. McKeehan (Eds.), Mechanisms of Signal
[16]
[17]
Transduction by Hormones and Growth Factors, Alan R. Liss, New York, 1987, pp. 251^262. M.H. Barcellos-Ho¡, J. Aggeler, T.G. Ram, M.J. Bissell, Functional di¡erentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane, Development 105 (1989) 223^235. R.O. Hynes, Integrins: A family of cell surface receptors, Cell 48 (1987) 549^554. C.A. Buck, A. F Horwitz, Cell surface receptors for extracellular matrix molecules, Annu. Rev. Cell Biol. 3 (1987) 179^205. R.O. Hynes, Integrins: Versatility, modulation and signaling in cell adhesion, Cell 69 (1992) 11^25. K. Burridge, C. Turner, L. Romer, Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly, J. Cell Biol. 119 (1992) 893^904. R.S. Talhouk, Z. Werb, M.J. Bissell, Functional interplay between extracellular matrix degrading proteinases in mammary gland: a coordinate system for regulating mammary epithelial function, in: T.P. Fleming (Ed.), Epithelial Organization and Development, Chapman and Hall, London, 1992, pp. 329^349. R.S. Talhouk, M.J. Bissell, Z. Werb, Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution, J. Cell Biol. 118 (1992) 1271^1282. M. Ambili, R. Pillai, P.R. Sudhakaran, Characteristics of a 60K gelatinase involved in rat mammary gland involution, Ind. J. Biochem. Biophys. 34 (1997) 347^353. M.M. Zutter, A.S. Santoro, Widespread histologic distribution of the K2 L1 integrin cell surface collagen receptor, Am. J. Pathol. 137 (1990) 113^120. P.J. Keely, J.E. Wu, A.S Santoro, The spatial and temporal expression of the K2 L1 integrin and its ligands collagen I, collagen IV and laminin suggest important roles in mouse mammary morphogenesis, Di¡erentiation 59 (1995) 1^13. Y.J. Topper, C.S. Freeman, Multiple hormone interactions in the developmental biology of the mammary gland, Physiol. Rev. 60 (1980) 1049^1060. M. Feldman, W.F. Ruan, B.C. Cunningham, J.A. Wells, D.L. Kleinberg, Evidence that the growth hormone receptor mediates di¡erentiation and development of mammary gland, Endocrinology 133 (1993) 1602^1608. Y. Yoshimura, K. Miyakoshi, T. Hamatani, K. Iwahashi, J. Takahashi, N. Kobayashi, K. Sueoka, T. Miyazaki, N. Kuji, M. Tanaka, Role of L1 integrins in human endometrium and decidua during implantation, Horm. Res. 2 (1998) 46^55. M. Sillem, S. Prifti, M. Schmidt, T. Rabe, B. Runnebaum, Endometrial integrin expression is independent of oestrogen or progestin treatment in vitro, Fertil. Steril. 67 (1997) 877^ 882. S. Sonohara, R. Mira-y-Lopez, M.M. Brentani, Laminin and oestradiol regulation of the plasminogen activator system in MCF-7 breast carcinoma cells, Int. J. Cancer 76 (1998) 77^ 85.
BBAMCR 14706 4-1-01
T. Iype et al. / Biochimica et Biophysica Acta 1499 (2001) 232^241 [18] M. Ambili, K. Jayasree, P.R. Sudhakaran, 60K gelatinase involved in mammary gland involution is regulated by Loestradiol, Biochim. Biophys. Acta 1403 (1998) 219^231. [19] D.M. Neville, Isolation of an organ speci¢c antigen from cell surface membrane of rat liver, Biochim. Biophys. Acta 154 (1968) 540^552. [20] F.C. Greenwood, W.M. Hunter, J.S. Clover, The preparation of a 131 I-labelled human growth hormone of high speci¢c radioactivity, Biochem. J. 89 (1963) 114^123. [21] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage-T4, Nature 227 (1970) 680^685. [22] B.R. Oakely, D.R. Kirsch, N.R. Morris, A simpli¢ed ultra sensitive silver staining for detecting proteins in polyacrylamide gels, Anal. Biochem. 105 (1980) 361^363. [23] O.H. Lowry, N.J. Rosebrough, A. Farr, R.J. Randall, Protein measurement with the Folin-phenol reagent, J. Biol. Chem. 193 (1951) 265^275. [24] J.T. Emerman, M.J. Bissell, Cultures of mammary epithelial cells : extracellular matrix and functional di¡erentiation, Adv. Cell culture 6 (1988) 137^159. [25] E.Y.H. Lee, G. Parry, M.J. Bissell, Modulation of secreted proteins of mouse mammary epithelial cells by the extracellular matrix, J. Cell Biol. 98 (1984) 146^155. [26] M.J. Bissell, H.G. Hall, Form and function in the mammary gland: The role of extracellular matrix, in: M. Neville, C. Daniel (Eds.), The Mammary Gland: Development, Regulation and Function, Plenum, New York, 1987, pp. 97^ 146. [27] J. Aggeler, J. Ward, L. Mackenzie Blackie, M.H. BarcellosHo¡, C.H. Streuli, M.J. Bissell, Cytodi¡erentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to development in vivo, J. Cell Sci. 99 (1991) 407^417.
241
[28] M.L. Li, J. Aggeler, D.A. Farson, C. Hatier, J. Hassell, M.J. Bissell, In£uence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells, Proc. Natl. Acad. Sci. USA 84 (1987) 136^140. [29] H. Birkedel-Hansen, W.G.I. Moore, M.K. Bodden, L.J. Windsor, B. Birkedel-Hansen, A. DeCarlo, J.A. Engler, Matrix metalloproteinases: A review, Crit. Rev. Oral Biol. Med. 4 (1993) 197^250. [30] P. Mignatti, D.B. Rifkin, H.G. Welgus, W.C. Parks, Proteinases and remodeling, in: R.A.F. Clark (Ed.), The Molecular and Cellular Biology of Wound Repair, Plenum, New York, 1996, pp. 426^473. [31] C.H. Streuli, N. Bailey, M.J. Bissell, Control of mammary epithelial di¡erentiation : Basement membrane induces tissue speci¢c gene expression in the absence of cell-cell interaction and morphological polarity, J. Cell Biol. 115 (1991) 1383^ 1395. [32] P.J. Keely, A.M. Fong, M.M. Zutter, S.A. Santoro, Alteration of collagen dependent adhesion, motility and morphogenesis by the expression of antisense K2 integrin mRNA in mammary cells, J. Cell Sci. 108 (1995) 595^607. [33] T.C.M. Klinowska, J.V. Soriano, G.M. Edwards, J.M. Oliver, A.J. Valentijn, R. Montesano, C.H. Streuli, Laminin and L1 integrins are crucial for normal mammary gland development in the mouse, Dev. Biol. 215 (1999) 13^32. [34] A.R. Howlett, N. Bailey, C. Damsky, O.W. Petersen, M.J. Bissell, Cellular growth and survival are mediated by L1 integrins in normal human breast epithelium but not in breast carcinoma, J. Cell Sci. 108 (1995) 1945^1957. [35] Z. Feng, A. Marti, B. Jehn, H.J. Altematt, G. Chicaiza, R. Jaggi, Glucocorticoid and progesterone inhibit involution and programmed cell death in mouse mammary gland, J. Cell Biol. 131 (1995) 1095^1103.
BBAMCR 14706 4-1-01