Distribution of entactin in the basement membrane of the rat mammary gland

Experimental

Distribution

Cell Research

152 (1984) 240-254

of Entactin in the Basement of the Rat Mammary Gland Evidence

for a Non-epithelial

Membrane

Origin

MICHAEL J. WARBURTON,‘* * PAUL MONAGHAN,’ SHARON A. FERNS,’ PHILIP S. RUDLAND,’ NINA PERUSINGHE’ and ALBERT E. CHUNG’ ‘Ludwig Institute for Cancer Research (London Branch), The Royal Marsden Hospital, Sutton, Surrey SM25PX, UK and ‘Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

Entactin, a sulfated glycoprotein with a molecular weight (MW) of about 150 kD, is present in vascular basement membranes and in the interstitial connective tissue of the mammary glands of virgin rats. It does not appear to be present in the basement membrane surrounding the mammary ductal system. However, in lactating mammary glands entactin is also present in the basement membrane region surrounding the secretory alveoli. Ultrastructural localisation of entactin reveals that it is present on the basal surface of epithelial cells, with patchy staining in the lamina lucida and lamina densa. Entactin also appears to be associated with interstitial collagen tibres. Mammary tibroblastic cells in culture are able to produce entactin, whereas mammary epithelial and myoepithelial cells, which synthesise the basement membrane proteins laminin and type IV collagen, fail to synthesise entactin.

Basement membranes are thin layers of specialised connective tissue that separate epithelia from the surrounding stroma. In addition to being an important factor in the maintenance of tissue architecture [ 11, basement membranes provide a support to which cells can attach and grow 12, 31, and are involved in the processes of morphogenesis and differentiation [4, 51. In some tissues, especially those of the kidney, basement membranes constitute a filtration barrier to the passage of macromolecules [6]. Certain proteins, e.g. laminin and type IV collagen, appear to be restricted to and are present in all basement membranes. In glandular tissues, these proteins are synthesised only by cells in the epithelial compartment [7]. Other proteins, e.g. fibronectin and type V collagen, are present in some basement membranes, especially those of the vascular system [8, 91, and are also major constituents of the interstitial connective tissue [lo]. In vitro, fibronectin is synthesised by both epithelial and mesenchymal cells [ll, 121. Recent evidence suggests that mesenchymal-derived fibronectin can be incorporated into basement membranes [13]. In the rat mammary gland, laminin and type IV collagen are constituents of the basement membrane at all stages in development, whereas tibronectin and type V * To whom offprint requests should be sent. Copyright @ 19134 by Academic Press, Inc. All rights of reproduction in any form resewed 0014~4827/84 $03.00

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Fig. 1. Immunofluorescence localisation of entactin in the rat mammary gland. Sections of mammary gland were placed on glass slides and incubated with anti-entactin serum followed by FITC-goat antirabbit IgG. (a) Virgin rat mammary gland. Note the absence of staining in the basement membrane region (arrow) and the diffuse staining in the surrounding connective tissue (ct). The membranes of fat cells (f) stain strongly. (b) Virgin rat mammary gland stained with non-immune rabbit serum, note the absence of staining. (c) Lactating rat mammary gland. There is strong staining in the basement membrane region surrounding each of the secretory alveoli (a[). (4 Lactating rat mammary gland incubated with non-immune rabbit serum. Note the absence of staining. Bar (a, b) 20 urn; (c-d) 50 pm. Exp

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Fig. 2. An arteriole in a virgin rat mammary gland stained with antiserum to entactin. The inner elastic ~ lamina stains strongly and there is weaker tibrillar staining throughout the tunica media, in the outer elastic lamina, and in the surrounding connective tissue. Bar, 40 urn.

collagen are present only in the alveolar basement membrane region during lactation, although both are constituents of vascular basement membranes and the interstitial connective tissue throughout all developmental phases [ 14, 151. To investigate the contribution of mesenchymal-derived products to the composition of the rat mammary gland basement membrane, we have studied the distribution of entactin during the development of the mammary gland from that of the virgin rat to that of the lactating animal, and its synthesis by mammary cells in vitro. Entactin is suitable for this purpose, since it is a sulfated glycoprotein that is present both in some basement membranes and in the mesenchyme where it is associated with collagen fibres [16].

METHODS Antibody

to Entactin

Entactin was purified from the endodermal cell line M1536-B3 1171. Antibodies to entactin were raised by immunizing rabbits with purified entactin. Any contaminating antibodies to laminin were removed by passing the serum through a column of laminin-Sepharose. The specificity of this antibody was determined by immuno-staining the proteins of M1536-B3 cell membrane sacs which had been electrophoretically transferred to nitrocellulose after their separation on sodium dodecyl sulfate (SDS) polyacrylamide gels. Only one band of staining that co-migrated with purified entactin, was observed [16].

Electron Microscopy Lobuloalveolar structures were dissected from the mammary glands of rats which had been lactating for 7 days. Care was taken to minimise the amount of adhering stroma. Small cubes (1 mm3) of tissue were incubated with 100 ul of antibody to entactin (diluted 1 : 100 with 0.5 % bovine serum albumin in PBS) at room temperature for 2 h, washed for I5 min with four changes of PBS, and then incubated with 100 pl of sheep anti-rabbit IgG conjugated to horseradish peroxidase (diluted 1 : 50) for a further 2 h at room temperature. The cubes of tissue were again rinsed with PBS and incubated with 0.05 % (w/v) diaminobenzidine and 0.05 % (v/v) hydrogen peroxide in PBS for 5 min. The tissues were then fixed in 2 % (v/v) glutaraldehyde for 2 h, and post-fixed in 1% (w/v) osmium tetroxide for 16 h at 4°C. Both fixatives were sodium phosphate buffered (50 mM, pH 7.4), and the osmotic pressure was Exp CellRes

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Fig. 3. Ultrastructural localisation of entactin in the lactating rat mammary gland. Cubes of gland were incubated with either (A) anti-entactin serum or (B) non-immune serum followed by peroxidaseconjugated goat anti-rabbit IgG. (A) The basal surface of the secreting epithelial cell (cp) is stained in a semi-periodic manner (arrows). Weaker, diffuse staining is observed over the lamina lucida and lamina densa. Collagen tibres (c), external to the basement membrane are also stained. (8) The section incubated with non-immune rabbit serum is devoid of staining. Section (R) was counterstained with lead citrate and uranyl acetate. Bar (A) 0.22 pm; (8) 0.19 pm.

adjusted to 350 mosmol by addition of sucrose. Tissues were then processed for electron microscopy without further staining, as described previously [18]. Cubes of tissue treated with non-immune serum were used as controls.

Cell Lines The epithelial cell line Rama 25 and one of its elongated cell converts (Rama 29) were derived from a dimethylbenzanthracene-induced rat mammary tumor and have been described previously [19]. Exp

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Fig. 4. Ultrastructural localisation of entactin in lactating rat mammary gland. (A) In some regions, staining of the lamina densa is more pronounced (arrows). Staining of semi-periodic structures which extend from the basal cell surface (orror.&eads) through the lamina lucida is observed, regardless of whether an epithelial (ep) or myoepithelial (myo) cells is adjacent to the basement membrane. (B) At higher magnification the staining of the semi-periodic structures (arrowheads) and lamina densa (arrow) are clearly observed. Bar, (A) 0.17 urn; (B) 0.13 w.

Epithelial (Rama 704) and myoepithelial-like cell lines (Rama 401 and 712) from normal, neonatal rat mammary glands were obtained by a previously published procedure [17, 201. The Bbroblastic cell line, Rama 27, was obtained from a normal rat mammary gland [19]. All cell lines were grown in Dulbecco’s modified Eagles’ medium containing 5% fetal calf serum (FCS), insulin (50 &ml) and hydrocortisone (50 &ml). Primary cultures from the mammary glands of a lactating rat were established and grown as described previously [21].

Zmmunojluorescence

Staining

Cells cultured on polystyrene coverslips (Lux Scientific Corp., Newbury Park, Calif.) were washed with PBS and fixed in methanol for 16 h at 4°C. After washing with PBS, the cells were- incubated with anti-entactin serum (1 : 2.5 dilution) at room temperature for 1 h followed by seven washes with PBS. Antibodies to laminin, vimentin and keratin were used at a dilution of 1 : 50. The characterisation of these antibodies has been described previously [14, 181. Incubation was then continued with fluorescein-conjugated goat anti-rabbit IgG (1: 20 dilution) at room temperature for 1 h. The coverslips were then washed seven times with PBS and mounted in polyvinyl alcohol. Fluorescencp was observed with a Reichert Polyvar microscope and photographs were taken on Ilford XPl film. Exp Cell Res 152 (1984)

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Frozen sections of rat mammary gland, which had been fixed in 1% (v/v) formaldehyde: 0.2 % (v/v) ghrtaraldehyde for 10 min at 4”C, were placed on glass slides and stained as described above. Tissue from the mammary glands of 50-day-old virgin rats or rats which had been lactating for 7 days were used.

Zmmunoprecipitation

of Entactin

Confluent cultures of Rama 29 cells were washed with PBS and incubated with 6 U/ml lactoperoxidase, 6 U/ml ghrcose oxidase, 5 mM ghrcose and 250 gCi of Na 12’1 (carrier-free) in 2 ml of PBS. After 5 min, the cells were washed three times with phosphate-buffered sodium iodide, scraped into PBS containing 2 mM phenylmethylsulfonyl fluoride and collected by centrifugation. Alternatively, cells were labelled with [35S]methionine (10 &i/ml) for 24 h in medium containing 5% of the normal methionine concentration. Labelled cells were extracted with 1 M urea: 0.5 % Triton X-100 : PBS : 1 mM phenylmethylsulfonyl fluoride for 30 min at 4°C and insoluble material was then removed by centrifugation at 10000 g for 5 min. To the supernatant (500 pl) was added either anti-entactin serum or, as controls, anti-fibronectin serum or non-immune serum (2.5 pl). The mixture was incubated at 4°C for 1 h, and then protein A-Sepharose (20 pl) was added and the incubation continued at 4°C for a further 1 h with constant mixing. The Sepharose beads were collected by centrifugation, washed three times with 1 M urea: 0.5 % ‘Riton X-100 : PBS and once with PBS. Immune complexes were eluted from the protein A-Sepharose by boiling in 1% sodium dodecyl sulfate (SDS) : 5 % 2-mercaptoethan01 : 10% sucrose : 10 mM sodium phosphate, pH 7.0 for 5 min, and analysed by electrophoresis on 7 % polyacrylamide/SDS gels.

RESULTS Distribution

of Entactin

in the Rat Mammary

Gland

In the mammary glands of virgin rats, entactin is distributed diffusely throughout the interstitial connective tissue, but does not appear to be present in the basement membrane that surrounds the ducts (fig. 1a). In the lactating mammary gland, entactin, in addition to being present in blood vessels and interstitial connective tissues also appears to be a constituent of the basement membrane region that surrounds each of the secretory alveoli (fig. 1c). Cytoplasmic staining with antiserum to entactin, which is indicative of active synthesis, was not observed in any cells in either the virgin or lactating glands. However, this result does not preclude that entactin is being synthesised at a low rate. Entactin is also present in the basement membrane region surrounding blood vessels and vascular smooth muscle cells and around fat cells (fig. 2). Ultrastructural Localisation Rat Mammary Gland

of Entactin

in the Lactating

The ultrastructural localisation of entactin was studied by incubating small cubes of lactating mammary gland tissue in anti-entactin serum followed by localisation of the antibody by an indirect peroxidase technique. Entactin occurs in a semi-periodic manner along the basal surface of the secretary epithelial cells (fig. 3A). The lamina lucida and densa also exhibit patchy staining. In some regions, the semi-periodic staining of the cell surface extends into the lamina lucida and there is stronger staining of the lamina densa (fig. 4). Peroxidase reaction product was also seen on some collagen tibres located external to the Exp Cell

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et al.

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5. Immunofluorescence localisation of entactin and laminin in cultured rat mammary cells. Cells were fixed in methanol, and incubated with either (a, c, e, g, i) anti-entactin or (6, d, f, h, J) antilaminin serum followed by FITC-conjugated goat anti-rabbit IgG. (a. b) Rama 25; (c, 6) ridge of elongated cells in a late passage Rama 25 culture; (e, JI Rama 29; (g. h) Rama 401; and (i, j) Rama 27 ceils. Bar, 30 urn. Fig.

basement membrane. Control tissue treated with non-immune serum did not stain (fig. 3 B). The staining pattern observed with anti-entactin serum is unlikely to be artefactual for reasons discussed previously [251. Synthesis of Entactin

by Cultured

Mammary

Cells

We have investigated the distribution of entactin in cultures of mammary cell lines by immunofluorescence techniques. Epithelial (Rama 704) and myoepithelial-like (Rama 401 and 712) cells derived from neonatal rat mammary glands and epithelial (Rama 25) cells derived from a dimethyl-benzanthracene-induced rat Exp Cell

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Fig. 6. Immunofluorescence locafisation of intermediate filament proteins in cultured rat mammary cells. Cells were fixed in methanol and incubated with either (a, c, e, g) anti-vimentin or (6, d, f, h) anti-keratin serum. (a, 6) Rama 25; (c, CT)Rama 29; (e,j) Rama 401; and (g, h) Rama 27 cells. Bar, 25 Pm. &p

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Entactin in rat mammary gland 249

Fig. 7. Immunofluorescence localisation of entactin in primary cultures. Primary cultures were established from the mammary glands of a lactating rat, fixed in methanol, and incubated with antientactin serum followed by FITC-conjugated goat anti-rabbit IgG. (a) An epithelial cell colony; (b) a fibroblastic cell colony. Bar, SO pm.

mammary tumor failed to stain with anti-entactin serum (fig, 5a, g, data not shown for Rama 704 and 712 cells). However, the ridges of elongated cells which formed in later passagesof Rama 25 cells [23] stained strongly in a fibrillar pattern (fig. 5 c). Similarly, staining of Rama 29 cells, which were derived from the elongated converts of Rama 25 cells [ 191, revealed a network of fine fibrils (fig. 5 e). Fibroblastic cells, e.g. Rama 27, stained in a similar manner to Rama 29 cells (fig. 5 i). In each case, a similar distribution of entactin was observed if the cells were first fixed with formaldehyde, and then made permeable to the antiserum by treatment with ‘I’Mon X-100. The distribution of the basement membrane protein laminin was also investigated in these cell lines. Staining of epithelial cells, e.g. Rama 25, reveals punctate structures that are located beneath the cells (fig. 5 b). The ridges of elongated cells stain strongly for laminin (fig. 54; however, Rama 29 cells stained only very weakly (fig. 5 e). The myoepithelial cell line, Rama 401, stains strongly with antibodies to laminin (fig, 5/z), whereas the fibroblastic cell line, Rama 27, was negative (fig. 5~). Cells incubated with non-immune rabbit serum failed to stain (data not shown). The epithelial origin of Rama 25 and 401 cell lines was demonstrated by their staining with antiserum to keratin (fig. 6). All the above cell lines stained with antiserum to vimentin, but Rama 27 and 29 cells were negative for keratin. Treatment of primary cultures from lactating rat E.xp Cell Res 152 (1984)

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8. Immunoprecipitation of entactin from Rama 29 cultures labelled with [35S]methionine. Confluent cultures of Rama 29 cells were labelled with [35S]methionine (10 uCi/ml) for 24 h. The Iabelled cells were extracted with 1 M urea : 0.5 % Triton X-100 : PBS : 1 mM phenylmethylsulfonyl fluoride, and the extract incubated with either (A) anti-entactin serum, or (B) non-immune rabbit serum (2.5 ~1). Immune complexes were collected on protein A-Sepharose and analysed on a SDSpolyacrylamide gel (7 %). MW are expressed in kD. Fig. 9. Immunoprecipitation of entactin Rama 29 cultures labelled with [‘Z51]iodine. Confluent cultures of Rama 29 cells were subjected to lactoperoxidase-catalysed iodination. After washing, the cells were extracted with 1 M urea: 0.5 % Triton X-100 : PBS : 1 mM phenylmethylsulfonyl fluoride, and the extract incubated with either (B) anti-entactin serum; (C) non-immune serum, or (D) antitibronectin serum (2.5 ~1). The total extract, approx. 100 ug of protein, was applied in (A). Immune complexes were collected on protein A-Sepharose and analysed on a SDS-polyacrylamide gel (7 %). MW are expressed in kD. Fig.

mammary gland with anti-entactin serum (fig. 7) revealed a tibrillar network associated with the fibroblast cells, but not with the epithelial colonies [24]. Immunoprecipitation

of Entactin

Entactin synthesised by rat mammary cells was further characterised by immunoprecipitation techniques. Incubation of a urea: Triton X-100 extract of [3SS]methionine-labelled Rama 29 cells with anti-entactin serum specifically precipitated a major protein with a molecular weight (MW) of 145 kD and, consistently, a minor protein with a MW of 140 kD (fig. 8). Non-immune rabbit serum failed to precipitate either of these proteins. The 145 kD MW component was also precipitated when an extract of iodinated Rama 29 cells was incubated with antientactin serum (fig. 9). In addition, some minor, higher MW proteins were also immunoprecipitated, the major one corresponded to that of tibronectin. Since small amounts of tibronectin were also immunoprecipitated with non-immune serum this probably represented non-specific precipitation. Entactin could not be immunoprecipitated from medium conditioned by [35S]methionine-labelled Rama 25 or Rama 401 cells (data not shown). Exp Cell Res 152 (1984)

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DISCUSSION The protein components of the basement membrane of the adult rat mammary gland appear to belong to two classes. The first class, which includes laminin and type IV collagen, is present in the basement membrane at all stages of post-natal development. The second class, which includes fibronectin and type V collagen, is only present in the basement membrane region in lactating glands [14, 151. We have now demonstrated that entactin belongs to this second class of proteins, in that it is absent from the mammary basement membrane of virgin animals but present in that of lactating rats. At the ultrastructural level, the above two classes of proteins can be distinguished by their topographical arrangement within the basement membrane. In the lactating rat mammary gland, both laminin and type IV collagen are predominantly located in the lamina densa, with smaller amounts occurring in semiperiodic clusters which extend from the cell surface through the lamina lucida [25]. Fibronectin and type V collagen, however, are not present in the lamina densa, but are located on the basal surface of epithelial cells which abut onto the basement membrane and also, in the case of fibronectin, in semi-periodic clusters that are present in the lamina lucida [15, 251. However, the topographical arrangement of entactin shows similarities to both that of fibronectin and that of laminin, in that it is present on the basal cell’s surface and is also partly associated with regions of the lamina lucida and densa. The only glandular basement membrane in which the ultrastructural localisation of entacin has been investigated is the mouse kidney [ 16, 261. Here, entactin is predominantly located on the basal surface of the tubular cells which are in contact with the basement membrane. The basement membrane was weakly and diffusely stained, although interstitial collagen fibres were quite strongly stained In a recent study of the distribution of entactin in human tissues, Jaffe et al. [27] have found that entactin is present in all vascular basement membranes, but is absent from all the epithelial-derived basement membranes examined including breast. In Reichert’s membrane, entactin is distributed uniformly throughout its entire thickness [28]. In vitro, entactin is synthesised by mammary fibroblastic cell lines and fibroblastic cells in primary culture. It is also synthesised by elongated cell lines (e.g., Rama 29) which spontaneously accumulate in cultures of cuboidal epithelial cells [19, 231. Rama 29 cells more closely resemble mesenchymal rather than epithelial cells in that they synthesise type I collagen and vimentin-containing intermediate filaments rather than type IV collagen and keratin-containing intermediate filaments [29, 301. Myoepithelial cells (e.g., Rama 401) which synthesise relatively large amounts of the basement membrane proteins, laminin and type IV collagen [18] and epithelial cells (e.g., Rama 25) which synthesise these proteins in small amounts [31], both fail to synthesise entactin. Similarly, entactin can not be detected in epithelial colonies in primary cultures. The ability of the epithelial and myoepithelial cell lines to continue laminin and type IV collagen synthesis in vitro Exp

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suggests that the failure to synthesise entactin is not a tissue culture artefact. Thus, the synthesis of entactin by rat mammary cells in vitro appears to be restricted to cells with mesenchymal characteristics. Entactin was first recognised as a product of the mouse endodermal cell line M1536-B3 [16, 171 and has since been found to be synthesised by F9 embryonal carcinoma cells [32, 331, PYS parietal endoderm cells [34], and by Swiss 3T3 and ST0 cell lines which are of presumptive mesenchymal origin [34]. Entactin generally appears to be synthesised as a glycosylated polypeptide with a MW in the range 145-158 kD [16, 32-351. F9 cells synthesise a fully processed entactin with a MW of 150 kD, but minor, partially processed forms with MW of 145 and 140 kD can be detected [32]. The 145 kD MW entactin synthesised by Rama 29 cells probably represents the fully processed molecule, since it can be immunprecipitated from cell surface-labelled cultures. The minor 140 kD MW entactin which is only seen in [35S]methionine-metabolically labelled cells, may represent a partially processed intracellular molecule. The function of entactin is unknown, but its localisation on the basal surface of epithelial cells suggests that it may be involved in interactions between cells and their adjacent basement membrane [ 16, 261. Immunoprecipitation of entactin from cells which also synthesise laminin often results in the co-precipitation of laminin [33, 34, 361. This co-precipitation, which can be abolished by 0.5 % SDS [33], suggest that laminin and entactin may form a non-covalent complex in vivo. The occurrence of entactin in the lactating gland, but not in the mammary glands of virgin rats, may be related to the attachmentof mammary epithelial cells to the basement membrane in lactating glands, whereas they are separated from it by a layer of myoepithelial cells in the mammary glands of virgin rats [ 141. Studies on the attachment of mammary epithelial cells to entactin-coated substrata may help to resolve this problem. If the entactin, type V collagen and possibly fibronectin that are present in the basement membrane region of the lactating rat mammary gland do not originate from the epithelium, then by what mechanism are they transported to the alveoli? It has recently been demonstrated that both plasma- and mesenchymal-derived tibronectin can be incorporated into basement membranes [13,37]. Amenta et al. [381 have also suggested that fibronectin may be considered as an “extrinsic” component of basement membranes in that it is derived from plasma, and is only present in basement membranes having a filtering function. Entactin, fibronectin and type V collagen could be deposited in the basement membrane of the lactating rat mammary gland by shedding from either tibroblastic cells, or from blood vessels that are closely apposed to the basement membrane surrounding the alveoli and which are more numerous in lactating glands [14, 251. Entactin, fibronectin and type V collagen are major components of vascular basement membranes [8, 9, 271. Milk contains several serum proteins [39], and evidence has been obtained for the transport of the serum proteins across the alveolar basement membrane, through the secretory alveolar eels and into the luminal Exp Cell Res 152 (1984)

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spaces [40]. Thus the basement membrane of the lactating mammary gland qualifies as one having a filtration function, and the presence of entactin, fibronectin and type V collagen within it may be a consequence of the close proximity of blood vessels. We thank Dr R. 0. Hynes for the gift of anti-vimentin serum, Christine Hughes and John Ellis for technical assistance and Linda Adlam for typing the manuscript.

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Exp Cell

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