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Department of Physiology, Michigan State University, East Lansing, Michigan 48824
Address all correspondence and requests for reprints to: Sandra Z. Haslam, Ph.D., Department of Physiology, 108 Giltner Hall, Michigan State University, East Lansing, Michigan 48824-1101. E-mail: shaslam{at}pilot.msu.edu
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Abstract |
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We report the novel finding that all ECMs tested, but not a nonspecific attachment factor, poly-L-lysine (PL), promoted a highly synergistic proliferative response to EGF plus IGF-I. EGF receptors were significantly increased with culture time on all ECMs, but not on PL. IGF receptor expression was significantly 2- to 4-fold higher on all ECMs compared with PL. EGF decreased IGF-binding protein-2 (IGFBP-2) and IGFBP-3 by more than 50% in the presence of IGF-I on PL or collagen I. These results indicate that ECM-specific IGF-I/EGF synergism occurs in response to ECM up-regulation of growth factor receptors and EGF down-regulation of inhibitory IGFBPs. Growth factors did not synergize with estrogen and/or R5020. Instead, estrogen plus R5020 decreased EGF- plus IGF-I-induced proliferation in an ECM-dependent manner. These studies demonstrate that proliferation of normal mammary epithelial cells involves complex interactions among steroids, growth factors, binding proteins, and ECMs.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although it has been previously shown that estrogen acts locally in the mouse mammary gland to stimulate cell proliferation and regulate progesterone receptor (PR) levels, it appears that the mitogenic effects of 17ß-estradiol (E) may be mediated indirectly by locally produced growth factors (4, 5, 6). We and others have hypothesized that E produces its mitogenic effect in the mammary gland via the paracrine action of stroma-derived growth factors (7, 8, 9). The observation that growth factors such as epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I), but not E, are mitogenic for mammary epithelial cells in serum-free culture has provided further support for this hypothesis (10). There is also evidence that growth factors can synergize with estrogen or can activate E receptor (ER) in the absence of ligand (11). Thus, the exact mechanisms by which growth factors may mediate and/or modulate estrogenic effects in the mammary gland are not known at present.
In vivo most if not all extracellular matrix (ECM) proteins of the basement membrane in the mouse mammary gland come from the stromal cells (2, 12). ECM proteins have been shown to be important in mediating hormonally regulated expression of milk proteins in mammary epithelial cells both in vivo and in vitro (13, 14, 15). We have reported that stroma-derived ECM molecules can also modulate the proliferative effects of the synthetic progestin, R5020, in mammary epithelial cells in serum-free medium (16). The purpose of the present study was to investigate the effects of the ECM molecules [collagen I and IV (Col I and Col IV), LN, and FN] on the proliferative responses to EGF and IGF-I. As EGF and IGF-I may also mediate or modulate ovarian hormone responses, their interactions with E and R5020 on various ECM proteins were investigated.
We report the novel finding that all ECM proteins tested promoted a highly synergistic effect between EGF and IGF-I through mechanisms that increased growth factor receptors and decreased IGF-I-binding proteins (IGFBPs). In contrast, no additive or synergistic proliferative effect was observed with either EGF or IGF-I and E or R5020. In fact, the opposite was observed; E plus R5020 decreased proliferation in an ECM-dependent manner when added to EGF plus IGF-I.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
Nulliparous (10–12 weeks old) BALB/c mice from our own colony
were the source of mammary tissues. Epithelial cells were obtained by
enzymatic dissociation as previously described (16). Cell
viability was determined by exclusion of trypan blue, and viability was
about 95%. On day 0, freshly dissociated mammary epithelial cells
were plated at 1.4 x 105 cells/well;
cultured in 96-well flat-bottomed plates in serum- and phenol red-free
DMEM/F12 with 0.1 mM nonessential amino acids, 0.3 mg/ml
L-glutamine, 0.1 µg/ml insulin, 100 µg/ml penicillin,
and 50 µg/ml streptomycin; and kept in 5% CO2
at 37 C. E and the synthetic progestin, R5020 (promegestone; NEN Life Science Products, Boston, MA) were used at final
concentrations of 23 and 10 nM, respectively. Growth
factors were used at the concentrations indicated in the text.
Treatment of culture dishes with ECM
Coating concentrations and conditions used for ECM molecules
were previously described (16). One day before cell
plating, ECM molecules were coated onto each culture well as follows:
Col I, Col IV, FN, and LN at 6.25 µg/cm2 and PL
at 125 µg/cm2. After 1-h incubation at 37 C,
the supernatants were removed, and each well was rinsed with HBSS.
[3H]Thymidine incorporation and DNA
content
[3H]Thymidine incorporation was used to
measure DNA synthesis of cell cultures with or without hormone and/or
growth factor treatments as previously described (17).
Briefly, cells were incubated with 0.1 µCi/well
[3H]thymidine (SA, 50 Ci/mmol; ICN Pharmaceuticals, Inc., Irvine, CA) at 37 C for 6 h. This
was followed by washing with HBSS, ice-cold 90% ethanol, 10% (wt/vol)
trichloroacetic acid, and HBSS. After air-drying, the cells were
dissolved with 1 M NH4OH/NaOH (pH
12.3) for 4–5 h and neutralized with 1 M
KH2PO4. Radioactivity was
determined by liquid scintillation counting. DNA content for each well
was determined by a fluorometric assay using Hoescht 33258 by the
method of West et al. (18). Calf thymus DNA was
used as a standard.
Steroid hormone binding assays
Ligand binding assays were used to determine ER and PR contents
as previously described (19). DNA content was determined
as described above.
EGF receptor (EGF-R) binding assay
EGF-R on cultured cells were determined by the receptor binding
assay described by Krane et al. (20). First,
the saturated concentration of [125I]EGF (SA,
107 µCi/µg; Amersham Pharmacia Biotech, Arlington
Heights, IL) was determined in the cultured mammary epithelial cells by
Scatchard analysis with a range of 1–18 ng/ml
[125I]EGF. It was found that 10 ng/ml EGF
saturated EGF-R binding. In the saturated ligand binding assay, cells
were rinsed twice with warm HBSS and incubated with 50 µl/well of a
10 ng/ml solution of [125I]EGF with or without
a 1000-fold excess of cold EGF at 4 C for 6 h. Specific binding
was calculated by subtracting nonspecific binding obtained in the
presence of unlabeled EGF from total binding obtained in the absence of
unlabeled EGF. DNA content was determined as described above.
IGF receptor (IGF-R) binding assay
IGF-R on cultured cells were determined by the ligand binding
method described by Stewart et al. (21) and
Yamasaki et al. (22). Initially, the saturating
concentration of [125I]IGF-I (SA, 327
µCi/µg; NEN Life Science Products) was determined in
the cultured mammary epithelial cells by Scatchard analysis with
0.1–1.6 nM/ml
[125I]IGF-I. It was found that 0.4–0.6
nM IGF-I saturated IGF-R binding. In the
saturated ligand binding assay, cells were rinsed twice with warm HBSS
and incubated with 50 µl/well of a 0.6-nM
solution of [125I]IGF-I with or without a
1000-fold excess of cold IGF-I at 4 C for 2.5 h. Specific binding
was calculated by subtracting nonspecific binding obtained in the
presence of unlabeled IGF-I from total binding obtained in the absence
of unlabeled IGF-I. DNA content was determined as described above.
IGFBP analysis
Freshly isolated murine mammary epithelial cells were plated in
serum-free medium (SFM) in 24-well dishes at 1 x
106 cells/well on PL (12.5
µg/cm2), Col I (12.5
µg/cm2), or FN (12.5
µg/cm2) for 48 h, and conditioned medium
was collected. Medium was prepared for Western blotting as previously
described (23). Briefly, the following protease inhibitors
were immediately added upon medium collection:
phenylmethylsulfonylfluoride (2 mM), leupeptin (3
µM), aprotinin (16 µM), sodium
orthovanadate (500 µM), and EDTA (2 mM). The
conditioned medium was centrifuged (1000 x g, 4 C, 10
min) to remove any cells, concentrated 10-fold with Micron low protein
binding 10,000 mol wt cut-off filters (Amicon, Inc., Beverly, MA), and
immediately frozen in aliquots at -80 C until Western blot analysis.
Samples, in the presence of protease inhibitors (listed above), were
separated on 12% acrylamide SDS-PAGE gels. Samples were
electrotransferred onto nitrocellulose filters and probed with
[125I]IGF-I (SA, 2000 Ci/mmol; Amersham Pharmacia Biotech) as previously described (23).
Filters were exposed overnight to 2 days in a PhosphorImager cassette
and subsequently scanned for band intensity using a PhosphorImager
(Molecular Probes, Inc., San Francisco, CA). To confirm
the identity of IGFBPs, Western immunoblots were performed, using
rabbit antimouse polyclonal antiserum to either IGFBP-2 or IGFBP-3
(GroPep Pty. Ltd.) under nonreducing conditions according
to the manufacturer’s instructions. Briefly, for Western immunoblot,
sample buffer for SDS-PAGE contained only 0.2% SDS, because higher
concentrations of SDS resulted in no binding or reduced antibody
binding. After SDS-PAGE, Western immunoblots were incubated in 5% milk
and 0.2% Tween-20 for 2 h (before antibody incubation) at room
temperature to allow renaturation of IGFBPs, which was required for
optimal antibody binding.
Statistical analysis
All data were expressed as the mean ± SEM, and
statistical significance was determined using Student’s t
test or ANOVA followed by the Tukey test, as appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) showed that at 48 h posttreatment
a maximal proliferative response was obtained with 10–100 ng/ml EGF
and 200–800 ng/ml IGF-I, respectively. Both growth factors caused a 2-
to 3-fold increase in proliferation on all ECM proteins tested and on
the nonspecific attachment factor, PL. Time-course studies using high
doses of either EGF (50 ng/ml) or IGF-I (300 ng/ml) revealed that
maximum [3H]thymidine incorporation was
obtained 24 h after growth factor addition regardless of whether
growth factors were added at the time of plating or after 1 day of
culture (data not shown). Thus, all subsequent thymidine incorporation
studies were carried out at 24 h posttreatment.
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). A highly
synergistic, 6- to 11-fold increase in proliferation was observed in
response to the combined growth factors on all ECM proteins. In
contrast, a significantly smaller (4-fold), additive response to the
combined growth factors was obtained on PL. The synergistic stimulation
observed on ECM proteins was probably not due to differences in cell
densities, because we have previously shown that cell attachments on
PL, LN, and Col IV are very similar (16).
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). After 48 h in culture
(Control-48 h), there was a 2-fold increase in EGF-R levels in cells
cultured on Col I, Col IV, and LN and a 3-fold increase in cells
cultured on FN. There was no significant increase in EGF-R in cells
cultured on PL. These results suggest that the cells cultured on ECM
proteins, but not on PL, are able to up-regulate EGF-R levels. Addition
of IGF-I at 24 h did not further increase EGF-R levels on any ECM
proteins (Fig. 3).
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). Furthermore, the level of IGF-R on LN
was 2-fold higher than that on the other ECM proteins. Addition of EGF
at 24 h produced no additional increase in IGF-R levels on any ECM
protein or PL. The pattern of increased proliferation observed with EGF
plus IGF-I on each ECM (Fig. 2
) was most closely correlated with the
level of EGF-R on each ECM (Fig. 3). However, the greater proliferative
responses to EGF plus IGF-I on all ECM proteins compared with PL are
probably due to higher levels of both EGF-R and IGF-R.
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). Des(1, 2, 3)-IGF-I stimulated
proliferation more than IGF-I when comparing the means of ECM groups
treated with des(1, 2, 3)-IGF-I to the means of ECM groups treated with
IGF-I (P < 0.01), indicating the possible presence of
inhibitory IGFBPs. However, there were no significant differences
between the proliferative responses obtained with EGF plus IGF-I or EGF
plus des(1, 2, 3)-IGF-I, suggesting that an inhibitory effect of IGFBPs
did not occur when EGF was also present.
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). Western
ligand blots (as opposed to Western immunoblots) were chosen to
quantitate IGFBPs, because 1) ligand blots use the native ligand,
IGF-I, so Western ligand blots not only indicate quantity of protein,
but also functional binding; and 2) Western ligand blots can more
accurately compare quantities of IGFBP2 to IGFBP3 because they use a
single detection protein (IGF-I) for both proteins; the different
antibodies needed for IGFBP2 and for IGFBP3 used in immunoblots would
have different affinities and avidities (with the potential for each
polyclonal antibody to recognize one or several sites on each protein).
Two IGFBPs, 34 and 42–46 kDa, were detected on the ECM proteins and PL
(Fig. 6A). These two IGFBPs migrated at the same speed and had the same
banding patterns as proteins that have previously been identified in
mammary epithelial cell lines as IGFBP-2 and IGFBP-3, respectively
(25, 26). We confirmed the identify of these binding
proteins by Western immunoblotting with IGFBP-2- and IGFBP-3-specific
rabbit antimouse polyclonal antibodies (Fig. 6B). Treatment with EGF
alone did not increase or decrease IGFBP-2 or -3 secretion on either
ECM or PL (Fig. 6C) In contrast, treatment with des(1, 2, 3)-IGF-I
increased the levels of IGFBP-3 6-, 10-, and 5- fold on PL, Col I, and
FN, respectively. IGFBP-2 was increased by des(1, 2, 3)-IGF-I 4-fold on PL
and FN and 8-fold on Col I. Addition of EGF with des(1, 2, 3)-IGF-I
reduced the IGFBP-3 level by more than half on PL and Col I compared
with des(1, 2, 3)-IGF-I alone; no decrease was observed on FN. Addition of
EGF with des(1, 2, 3)-IGF-I reduced IGFBP-2 by 54% on Col I and PL and by
17% on FN compared with IGF-I alone. However, overall levels of
IGFBP-3 and -2 on FN were lower and similar to the reduced levels
obtained on Col I after treatment with EGF plus des(1, 2, 3)-IGF-I. These
results suggest that IGFBP-2 and -3 inhibit IGF-I action on Col I and
PL and that down-regulation of these IGFBPs by EGF reduces this
inhibition.
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, A and B) or any
other ECM protein (data not shown). Hormones plus growth factors were
also added 24 h after plating, and cell proliferation was measured
24 or 48 h later according to our previous scheme in which a
proliferative response to R5020 was observed (16). Again,
we did not observe differences in proliferation with EGF plus E, EGF
plus R5020, IGF-I plus E, or IGF-I plus R5020. Culturing cells in the
presence of E or R5020 using maximal doses of EGF or IGF-I also failed
to produce an increased proliferative response (data not shown).
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).
Proliferation was significantly decreased by the addition of EGF,
IGF-I, and R5020 on LN and by EGF, IGF-I, E, and R5020 on both Col I
and LN. Thus, the progestin R5020, alone or in combination with E,
decreased the EGF- plus IGF-I-induced proliferative response in an
ECM-dependent manner.
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As E regulation of PR requires the presence of ER, we analyzed the effects of growth factors and ECM composition on ER levels at 24 or 48 h after treatment. Similar levels of ER were maintained on all ECM proteins, and neither EGF nor IGF-I had a significant effect on ER levels (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synergistic effects of EGF and IGF-I have previously been observed on keratinocyte proliferation in vitro in serum-free medium (20, 29). It was concluded from that study that the synergism was due to a 4-fold increase in EGF-R expression by IGF-I. Synergism between EGF and IGF-I has also been investigated in the IEC-6 rat intestinal epithelial cell line. It was found that IGF-I decreases the expression of IGF-R messenger RNA and that EGF attenuated this effect (30). We also examined the regulation of EGF-R and IGF-R as a possible mechanism for EGF/IGF synergy. EGF-R were significantly increased on Col I and FN. However, on Col IV or LN, EGF-R levels were similar to those observed on PL. Addition of IGF-I did not have any effect on EGF-R levels. We found that IGF-R levels were increased on all ECM proteins compared with those on PL. In particular, there was a 2-fold greater increase on LN than on any other ECM. EGF had no additional effect on IGF-R binding levels. Thus, it appears that ECM proteins had differential effects on growth factor receptor concentrations. Col I and FN were the most effective in increasing EGF-R levels, whereas LN was the most effective ECM protein for maintaining or increasing IGF-R levels; PL had the least effect in both cases.
EGF/IGF synergism in the IEC-6 rat intestinal epithelial cell line has also been attributed to the additive decrease in expression of IGFBP-2 by EGF and IGF-I (30). Additionally, it has previously been reported that EGF and IGF-I differentially regulate IGFBP secretion in a mouse mammary epithelial cell line (COMMA-D/MME) in serum-free medium (26). We also analyzed the influence of growth factors and ECM molecules on secreted IGFBPs. When we compared cell proliferation obtained with IGF-I to that obtained with des(1, 2, 3)-IGF-I, which has very low affinity for IGFBPs, we found a slight increase in proliferation in the presence of des(1, 2, 3)-IGF-I alone. However, the proliferative responses to EGF and des(1, 2, 3)-IGF-I were not significantly different from those observed with EGF and IGF-I. This suggested that IGFBPs were increased in cells cultured in IGF-I alone, but decreased in the presence of EGF and IGF-I. This hypothesis was supported by the observation that IGFBP-2 and -3 levels were increased by IGF-I and were consistently reduced when cells were treated with EGF and IGF-I. The exception to this was cells cultured on FN, where IGFBPs were not reduced by EGF plus IGF-I. However, overall levels of IGFBPs were significantly lower on FN regardless of growth factor treatment. Thus, the combined effect of ECM proteins to increase EGF-R and IGF-R levels and of IGF-I plus EGF to decrease IGFBP levels appear to be major contributing factors to the synergistic proliferative response obtained upon treatment with EGF and IGF-I on ECM proteins. This suggests that the balance between growth factor receptor levels and IGFBP levels is a critical determinant of the overall proliferative response. The presence of IGFBPs combined with lower levels of EGF-R and IGF-R present on cells cultured on PL are probably responsible for the lower proliferative response observed in cells cultured on this nonspecific attachment factor.
Other studies have also reported IGF/EGF synergism in serum-free medium (20, 30). However, it should be noted that cells in these studies were initially plated in serum-containing medium. As serum contains growth-stimulating factors and ECM molecules, particularly FN, it is not possible to rule out their contributions to the synergism. The cells cultured in our studies were plated on defined ECM proteins in SFM and cultured in SFM. As all ECM proteins tested in this study, namely Col I, Col IV, FN, and LN, have been shown to be produced by mammary stroma in vivo (2, 12), our results provide novel information about how mammary stroma may influence the proliferative response of the epithelium to growth factors.
Previously, we have shown in the same culture system, that R5020 stimulates cell proliferation on FN and Col IV (16). However, in the present study no added stimulatory effects on proliferation were obtained by adding EGF or IGF-I alone or in combination with E and/or R5020. In fact, the opposite was observed; R5020 and E plus R5020 decreased EGF- plus IGF-I-induced proliferation. Although there was a trend toward reduced proliferation on all ECM proteins with the combination of E, R5020, EGF, and IGF-I, statistical significance was reached only on Col I and LN. It has recently been reported that both IGF-I and IGF-R messenger RNAs are expressed in mouse mammary gland at high levels in the terminal end buds of the pubertal gland, are undetectable in the adult gland, and are reexpressed at late pregnancy and during lactation (31). In the pubertal gland IGF-I is associated with ductal elongation, and during lactation with cell survival and involution (31). Previous studies from our laboratory have demonstrated that PR levels are very low at puberty, are highest in the adult gland and during early pregnancy, and are reduced again at late pregnancy and during lactation (32). Furthermore, we found that pubertal and lactating mammary glands are nonresponsive to the proliferative effect of progestins (33). A major proliferative response to progestins is ductal side-branching and alveolar morphogenesis. Thus, the expression of mammary IGF-I and IGF-R, and PR and progestin responsiveness are inversely related and appear to be important in two distinct stages of proliferation: IGF-I during ductal elongation, and progestins during alveolar development. Further evidence for a critical role of IGF-I in ductal development has recently been shown in IGF-I knockout mice (34). In this context the present findings that R5020 and E plus R5020 reduce the proliferative response obtained with EGF plus IGF-I in vitro also suggest that progestins may have an inhibitory effect on IGF-Iinduced proliferation in vivo.
E did not increase PR levels either alone or in combination with EGF and/or IGF-I. Previously, it has been shown that IGF-I can increase PR levels in the absence of E in MCF-7 breast cancer cells in the presence of low levels of serum (0.1%), and this effect could be completely abrogated by antiestrogen, suggesting that IGF-I stimulation of PR was mediated through the ER (28). Recently, we have also observed that EGF implants into the mammary gland of ovariectomized adult mice can induce mammary PR, and this response is blocked by antiestrogen (35). The reason for the discrepancy between the EGF effects on PR regulation in vivo and in vitro is not known. From in vivo studies we have also shown that there is an important stromal component to estrogenic regulation of PR (36). Thus, it is possible that there are additional factors that are provided by mammary stromal cells in vivo that are required for PR regulation.
In summary, in primary, serum-free cultures, normal mammary epithelial cells grown on various ECM proteins exhibit a significant synergistic proliferative response to EGF and IGF-I. It is likely that a number of different mechanisms operate to produce this synergistic response, including increased EGF-R and IGF-R levels and reduced IGFBPs levels. The novel observation from these studies is that various stroma-derived ECM proteins regulate specific aspects of the response and are necessary for IGF-I/EGF synergism. Furthermore, the synergism between EGF and IGF-I may be reduced when a progestin, R5020, is added. Deciphering the complex interactions involved in growth factor and ovarian steroid-dependent proliferation in the normal mammary gland has specific relevance to understanding the alterations in the growth control that occur in breast cancer. Furthermore, determining the mechanistic bases of epithelial-stromal interactions in normal development and during mammary carcinogenesis may provide a conceptual basis for novel approaches to the prevention and treatment of breast cancer that also focus on mammary stroma.
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Footnotes |
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Received January 11, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2ß1 integrin and its
ligands, collagen I, collagen IV, and laminin, suggests important roles
in mouse mammary morphogenesis. Differentiation 59:1–13[CrossRef][Medline]
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