Generation of Recombinant Epimorphin

Recombinant full-length epimorphin (H123) is identical to endogenous epimorphin lacking membrane-spanning region and represents all isoforms (see Fig. ​Fig.33 A). Recombinantly generated epimorphin fragments H2 and H13 represent the cellular recognition domain of epimorphin (amino acids 104–187) and a fusion of NH₂- (amino acids 1–103) and COOH- (amino acids 188–264) terminal coiled-coil domains of epimorphin, respectively. They were tagged with six histidine residues, expressed in Escherichia coli and purified over Ni columns in the presence of urea as described (Oka and Hirai, 1996). Urea was necessary, since all recombinant products precipitated immediately upon removal of urea under the neutral pH. For use in cell culture, recombinant epimorphin was dialyzed against 1.5 mM HCl and filtered under sterile conditions.

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Figure 3

Preparation of recombinant epimorphin. (A) Schematic diagram of epimorphin isoforms and the recombinant epimorphin fragments used in this study. There are three epimorphin isoforms produced by alternative splicing. Isoforms I and II (34-kD products) have a putative membrane-spanning region at the COOH terminus, whereas isoform III (31-kD products) has not. The recombinant epimorphin fragment H123 represents the epimorphin sequence shared by all the isoforms. H13 is a fusion peptide of the NH₂- and COOH-terminal coiled-coil domains of H123, and H2 is a peptide of only the cellular recognition domain inserted between the coiled-coil domains. *, NH₂-terminally tagged histidine residues. (B) Coomassie brilliant blue–stained epimorphin fragments in SDS-PAGE gel.

Antibodies

Affinity-purified antibodies to epimorphin were prepared as follows: rabbit antiserum against untagged recombinant epimorphin (Hirai, 1994) was mixed with an equal volume of PBS, pH 7.4, precipitated with 40% ammonium sulfate, dissolved in PBS, and dialyzed against PBS. Subsequently, antibodies were adsorbed to nitrocellulose membranes (Millipore Corp., So. San Francisco, CA) precoated with 1 mg/ml histidine-tagged epimorphin by incubation for 4 h at ambient temperature. After several washes with PBS, antibodies bound to recombinant epimorphin were eluted with 0.25 M glycine-HCl, pH 2.7. Affinity-purified anti-epimorphin antibody was immediately neutralized with 1 M phosphate buffer, pH 8.0, precipitated with 60% ammonium sulfate and dissolved in DME/F12 (Life Technologies, Ltd.). Epimorphin antibodies were then dialyzed against DME/F12 and filtered under sterile conditions. Purified rabbit IgG (DAKO Corp., Glostrup, Denmark) were dialyzed against DME/F12, sterile filtered, and used as the control.

Mouse anti-vimentin, mouse anti–α-smooth muscle actin, FITC-conjugated sheep anti–mouse IgG, and rhodamine-conjugated goat anti–rabbit IgG antibodies were purchased from Sigma Chemical Co. FITC-conjugated goat anti–rat IgG antibodies were from Caltag Labs (So. San Francisco, CA). HRP-conjugated donkey anti–rabbit Ig antibodies were from Amersham Corp. (Buckinghamshire, U.K.). Function blocking anti-HGF antibodies were from Sigma Chemical Co. Rat monoclonal antibody against E-cadherin, ECCD2 (Shirayoshi et al., 1986), was a generous gift from Dr. Takeichi (Kyoto University, Japan).

Cell Attachment and Proliferation Assays

Cell attachment assays were carried out as previously described (Oka and Hirai, 1996), with minor modifications. In brief, each well of 24-well plates (nontreated for cell culture; Falcon, Becton Dickinson Labware, Franklin Lakes, NJ), was coated with 5 μg/cm² of recombinant epimorphin (H123), epimorphin fragments H2 or H13 in 1.5 mM HCl, and subsequently air dried. Cells (2 × 10⁴) were plated in DME/F12 medium containing 4 mg/ ml of BSA onto epimorphin-coated substrata and incubated at 37°C. Anti-epimorphin antibodies and control antibodies, at a total concentration of 20 μg/ml, were added to medium. After an 8-h incubation, plates were tapped sharply and washed twice with PBS. Cells bound to the plates were then detached after trypsinization and counted. Wells uncoated and coated with 5 μg/cm² type I collagen (Cellagen™ ; ICN, Irvine, CA) in 1.5 mM HCl were used as negative and positive controls. All experiments were conducted in duplicate and repeated three times.

For proliferation assays, cells (1 × 10⁵) were seeded into each well of 12-well plates coated with epimorphin fragments, as described above. After 8 h of incubation, replicate cultures of attached cells were washed, detached, and counted (N₀). Cells attached in similar numbers regardless of whether wells were precoated with H123, H2, or collagen type I. Growth medium was added to remaining wells and incubated for an additional 2 d. After such time, cell number was determined (N₂) and growth ratio (N₂/ N₀) was calculated. The proliferation assay was also carried out in the presence of 50 ng/ml recombinant HGF (Becton Dickinson, Bedford, MA) or 50 ng/ml EGF (Sigma Chemical Co.). The proliferation assays were performed in duplicate and repeated three times.

To measure the effects of HGF, EGF, KGF, and FGF on proliferation of cell clusters in collagen gels (see three-dimensional cultures, below), the diameters of >50 cell clusters cultured for 8 d with one of these growth factors (50 ng/ml) were measured and compared to those of control.

Preparation of Cell Clusters

The cell clusters were prepared as follows: agarose was heated in PBS (final 2%) and 250 μl of the solution was added to each well of 24-well plates. After the agarose gelled, 1.5 ml of growth medium was added to each well and incubated for 30 min at 37°C in CO₂ incubator. This medium was then discarded and 5 × 10⁴ cells, suspended in 500 μl of growth medium containing 1,000 U of desoxyribonuclease I (DNase I; Sigma Chemical Co.), were seeded on top of the agarose gel and incubated with gentle rotation (100 rpm) for 24 h at 37°C. After incubation, cells formed smoothly rounded and well packed clusters of 150 to 250 μm diam. The remaining single cells were removed by centrifugation for 30 s at 200 rpm. To obtain clusters of primary mammary cells, a cell suspension of glands from 12 to 15 d pregnant mice was prepared as follows: the glands were minced with a sharp razor blade and incubated with 0.2% trypsin (Life Technologies, Ltd.) and 0.2% collagenase I (Life Technologies, Ltd.) in DME/F12 with agitation (100 rpm) for 30 min at 37°C, followed by centrifugation at 1,000 rpm for 3 min. After discarding the fat-containing supernatant, cells in the pellet were resuspended in growth medium supplemented with 1,000 U of DNase I and incubated for 2 min. Cells were then washed three times with growth medium.

Three-Dimensional Cultures

For three-dimensional cell cultures, cell clusters were embedded in type I collagen gels. The embedding process into collagen gels was similar to that described for single cells by Montesano et al. (1991a). Acid-soluble collagen (7.5 vol of a 0.5% solution; Cellagen™ AC-5; ICN) was mixed gently on ice with 1 vol of 1.5 mM HCl (blank solution) and 1 vol of 10× DME/ F12, followed by mixing with 0.5 vol of alkaline solution to neutralize the pH. After adding 1:20 vol of FCS, 250 μl of the collagen solution was poured into each well of a 24-well dish, and the dish was incubated at 37°C to allow gelation of the basal collagen layer. The cell clusters were suspended in growth medium at a concentration of 2,500 to 4,000 clusters/ml, and 10 μl of the suspension (25–40 clusters) was mixed with 250 μl of ice-cold collagen solution, poured onto the basal collagen layer, and placed immediately at 37°C. To prepare collagen substrate containing recombinant epimorphin or control peptides, one vol of a 1 mg/ml stock solution of H123 or its fragments (H2 or H13) in 1.5 mM HCl was used instead of the blank solution. To prepare cell clusters in which recombinant epimorphin were present around each cell, cells were precultured on H123- or H2-coated plates for 4 d, as described in Cell Attachment and Proliferation Assay before making clusters (see Fig. ​Fig.77 B). After gelation of collagen, 500 μl of growth medium was added to each well. Medium was exchanged every 5 d. HGF, EGF, KGF (Sigma Chemical Co.), or bFGF (Sigma Chemical Co.) were added to replicate wells at a concentration of 50 ng/ml. Blocking antibodies to epimorphin, HGF (Sigma Chemical Co.) or control antibodies were added to replicate wells at a concentration of 100 μg/ml.

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Figure 7

Nonpolar presence of recombinant epimorphin around each cell of the cluster leads to “lumen” formation. (A) Immunoblot detection of recombinant epimorphin in the clusters of SCp2 cells. (Right lane) Clusters of SCp2 cells precultured on H123 for 4 d (SCp2H123IN). (Left lane) Purified H123 (5 ng). (B) Detection of recombinant epimorphin (H123) in the cell clusters. Sections of the clusters were stained with anti-epimorphin antibodies (a) and with DAPI (b). (C) Appearance of the clusters of H123-containing SCp2 cells (SCp2 H123IN) in collagen gels, cultured for 8 d in the absence (a) or presence (b) of 50 ng/ml HGF. (D) Quantification of the branching and “lumenal” phenotype of the clusters of SCp2. Clusters of cells precultured on H123, H2, or H13 are shown as H123IN, H2IN, or H13IN, respectively. Note that H123 but not H2 and H13 fragments in the cluster induced “lumen” formation. EGF as well as HGF dramatically enhanced the effect of H123. Bars: (B) 120 μm; (C) 200 μm.

Analysis of the Morphogenic Phenotypes of Cell Clusters

The phenotype of cell clusters embedded in collagen gels was determined after cultivation for 8 d. A cluster was defined as a “branching” phenotype if it had at least three independent extending cell processes that were longer than the diameter of the cell cluster. A cluster was defined as a “lumenal” phenotype if it had a clearly visible lumen occupying >50% of the volume of cluster. There were few clusters that had the double phenotype; these were scored in each category. The quantification of the phenotypic appearance was carried out by counting the percentage of a particular phenotype from at least 50 randomly selected clusters in each experiment. The experiments were repeated at least three times.

Effect of Epimorphin on Attachment and Proliferation of Mammary Epithelial Cells

Given that mammary lumenal epithelial cells in vivo do not express epimorphin but are surrounded by epimorphin-positive cell populations, one could postulate the existence of epimorphin receptors (not yet characterized) if epimorphin were to act directly on epithelium. We asked whether mammary epithelial cells in culture can interact with epimorphin. Since SCp2 cells were largely devoid of endogenous epimorphin, as are mammary epithelial cells in vivo (Fig. ​(Fig.2,2, g–i), these cells were tested for their ability to bind to purified recombinant epimorphin (Fig. ​(Fig.3).3). About 80% of plated SCp2 cells attached to substratum-bound recombinant epimorphin within 8 h (Fig. ​(Fig.4,4, A and B). Attachment of cells to epimorphin was as efficient as their attachment to type I collagen. As was shown previously for endothelial cells (Oka and Hirai, 1996), cells attached to the H2 cell-binding domain of epimorphin and to full-length epimorphin H123 but not to the coiled-coil domain H13 (Fig. ​(Fig.44 B). Binding of cells to epimorphin-coated plates could be inhibited with anti-epimorphin antibodies in a dose-dependent fashion, whereas binding to type I collagen could not. These observations suggest that SCp2 cells can interact with extracellularly presented epimorphin.

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Figure 4

Attachment and proliferation of an epithelial cell line on recombinant epimorphin. (A and B) Attachment of SCp2 cells to substrate-coated epimorphin fragments. Cells bound to each epimorphin fragment in 8 h were photographed (A) and quantified (B). Uncoated (−) and collagen type I–coated wells were used as negative and positive controls, respectively. Medium contained mock antibodies and anti-epimorphin antibodies at a total concentration of 20 μg/ml as indicated. P values between − and H123 (mock Ab20), and H123 (mock Ab20) and H123 (anti-EPM20) were <0.0001. (C) The growth ratios (cell number in 2 d plus 8 h divided by cell numbers 8 h after plating) of SCp2 cells cultured on type I collagen, H123, and H2 fragments. HGF and EGF were tested at 50 ng/ml. Significant inhibition of cell growth was observed in cells cultured on H123 in the absence of additional growth factors. *, P values versus H2 (−) and collagen (−) were <0.001.

When cells were maintained in the presence of serum-containing medium, substratum-bound epimorphin H123, but not its cell-binding H2 domain alone, slightly inhibited proliferation of SCp2 cells (Fig. ​(Fig.44 C), as was seen previously in endothelial cells (Oka and Hirai, 1996). When HGF or EGF were added to the culture medium, cells overcame the inhibitory effects of epimorphin and continued to grow (Fig. ​(Fig.44 C).

Effect of Epimorphin on Branching Morphogenesis of Mammary Epithelial Cells

To analyze the effect of epimorphin on morphogenesis of mammary epithelial cells, clustered SCp2 cells were embedded in collagen gels containing recombinant epimorphin. Cell clusters rather than individual cells were used in this assay because of epimorphin's growth inhibitory effect (Fig. ​(Fig.44 C).

As shown in Fig. ​Fig.5,5, A and B, ~20% of cell clusters of SCp2 cells exhibited branching morphogenesis in the presence of epimorphin (H123), compared to ~5% in the absence of epimorphin. HGF, in the absence of epimorphin, accelerated growth but had no effect on morphogenesis of SCp2 cells (Fig. ​(Fig.55 A, d, and B). However, the percentage of cell clusters undergoing visible branching morphogenesis was markedly increased to 80% when cells were stimulated by both epimorphin and HGF. The H2 cell-binding domain of epimorphin alone was insufficient to induce branching morphogenesis (Fig. ​(Fig.55 B). We asked whether or not HGF was necessary or whether another growth factor could replace its branching function. EGF, KGF, and FGF also promoted growth of SCp2 cell, and morphogenesis was stimulated only when epimorphin was also present (Fig. ​(Fig.5,5, C and D). The branch length was roughly proportional to the ability of the factor to induce growth (Fig. ​(Fig.5,5, C and D). Anti-HGF function-blocking antibodies did not prevent branching if HGF was replaced by EGF and if epimorphin was present. These observations demonstrate that epimorphin positions the cells to undergo branching morphogenesis in response to several growth factors.

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Figure 5

Effect of recombinant epimorphin and growth factors on branching morphogenesis of SCp2 cells. (A) Morphology of SCp2 cell clusters cultured for 8 d in collagen gels mixed with (a and b) or without (c and d) recombinant epimorphin H123. Presence of H123 in substrate and outside the clusters is indicated as H123EX. HGF (50 ng/ml) was added to the medium in b and d. b′ and d′ are sections of b and d stained with hematoxylin, respectively. (B) Quantification of the branching phenotype of SCp2 cells depicted in A. Cell clusters were surrounded by H123, H2, or H13 in collagen (indicated as H123EX, H2EX, or H13EX, respectively). Note that H123, but not H2 and H13 fragments induced branching morphogenesis in collagen gels. HGF dramatically enhanced the effect of H123 and functional blocking antibodies to HGF (100 μg/ml) completely neutralized HGF. (C) Effect of other growth factors on morphology of SCp2 cell clusters. EGF (a, d, and g), KGF (b and e), and FGF (c and f) can also induce branching morphogenesis in the presence (a–c and g) but not in the absence (a–c) of epimorphin H123 (H123EX). All of the factors were added to medium at 50 ng/ml. Note that EGF induced epimorphin-dependent branching morphogenesis even in the presence of function-blocking anti-HGF antibodies. (D) Summary of above data. For growth in collagen, when the average diameter of >50 clusters was 1–2× the control (size of cluster without the factor addition) and P value versus control was <0.1, it was denoted +; 2–3× the control is ++, and 3× or larger, +++. Branching was scored as described in Materials and Methods. Bars: (A and C) 200 μm.

To prove that epimorphin was indeed essential for branching morphogenesis of SCp2 cells, we isolated D6 and I6 cell lines, two clonal derivatives of the epimorphin-positive subpopulation of SCp2 cells. These cells expressed both epimorphin monomers comprised of 31 (isoform III) and 34 kD (isoform I and II; Hirai, 1993) and epimorphin dimers and tetramers comprised of ~70 and 150 kD (Hirai, 1994), respectively (Fig. ​(Fig.66 A). When preclustered, these cells expressed epimorphin surprisingly only at the periphery of cell aggregates (Fig. ​(Fig.66 B), reminiscent of the epimorphin staining pattern in the mammary gland around ducts and alveoli (Fig. ​(Fig.11 A). Consistent with what was observed in the mixed populations such as CID-9, this cell type displayed branching morphogenesis in the presence of growth factors without exogenously added epimorphin. However, the extent of the branching could be increased further by addition of epimorphin to the collagen gels (Fig. ​(Fig.66 C). The branching phenotype of D6 and I6 cells in the presence of HGF was blocked by anti-epimorphin antibodies (Fig. ​(Fig.6,6, C and D). On the other hand, cell clusters isolated from the epimorphin-negative subpopulations could not branch in the absence of exogenous epimorphin (Fig. ​(Fig.66 D). We therefore conclude that epimorphin is essential for branching morphogenesis of mammary epithelial cells in culture.

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Figure 6

Branching morphogenesis of clonal derivatives of SCp2 cells. (A) The level of endogenous epimorphin in parental SCp2 and an epimorphin-positive clonal population (D6). Note that only very faint bands of endogenous epimorphin were detected in the SCp2 cells, as would be expected from the immunofluorescence studies (only ~5% express epimorphin). (B) Detection of endogenous epimorphin in the clusters of SCp2 and D6 cells. The sections of SCp2 (a and c) and D6 (b and d) cells were stained for epimorphin (a and b) and DAPI (c and d). The expression of epimorphin was still heterogenous in D6 cells and only the peripheral portion of the clusters were epimorphin-positive. (C) Appearance of cell clusters of D6 cultured for 8 d in collagen gels with (c) or without (a and b) additional recombinant epimorphin H123, in the presence of 50 ng/ml HGF. Mock antibodies (a and c) or anti-epimorphin antibodies (b) were added to medium at 100 μg/ ml. c′ is a section of c stained with hematoxilin. Note that D6 demonstrated branching morphogenesis in collagen, but exogenous epimorphin (H123EX) enhanced, and anti-epimorphin antibodies perturbed the phenotypic appearance. (D) Effect of H123 and anti-epimorphin antibodies on the appearance of branching phenotype of several subclones derived from SCp2. About 60% of D6 and 40% of I6 cells were epimorphin positive, whereas PTSEa and PTSEb (refer to Fig. ​Fig.8)8) were epimorphin negative. The values indicate mean ± SD of three separate experiments. Bars: (B) 120 μm; (C) 200 μm.

The authors thank Drs. E.R. Reichmann and M. Takeichi for EpH4 cells and ECCD-2 antibody, respectively. We are also grateful to Dr. R. Schwarz for his computer analysis and B. Johansen for his administrative support.

This work was supported by the Office of Health and Environmental Research of the U.S. Department of Energy (contract DE-AC03-76SF00098) with additional support from the National Institutes of Health (contract CA57621) and the Science and Technology Agency of Japan.