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Department of Neuroscience and Anatomy, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: Teresa L. Wood, Ph.D., Department of Neuroscience and Anatomy, Pennsylvania State University College of Medicine, P.O. Box 850, 500 University Drive, Hershey, Pennsylvania 17033. E-mail: twood{at}psu.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ovarian and pituitary hormones mediate postnatal growth of the mammary gland, at least in part, through local induction of peptide growth factors (1). The insulin-like growth factors (IGFs) are among the growth factors implicated in postnatal growth of the mammary gland. IGF-I and IGF-II are mitogenic peptides that have essential growth promoting actions during embryonic development (15, 16). The evidence that the IGF peptides have a role in postnatal development of mammary tissue comes from in vitro as well as in vivo studies. In vitro, both IGF-I and IGF-II are potent mitogens for normal and tumorigenic mammary epithelial cells (17, 18, 19, 20). Moreover, conditions for optimal growth of mammary gland explant cultures use micromolar concentrations of insulin (13, 21, 22, 23). At these superphysiological concentrations, insulin stimulates the IGF type I receptor (IGF-IR), the primary signaling receptor for both IGF-I and IGF-II (24). In vivo, IGF-I (implanted into mammary fat pads of hypophysectomized, estrogen-treated male rats) induces epithelial growth (25).
Endogenous production of IGF-I is likely an important component of GH, as well as estrogen-mediated mammary development. Administration of GH to hypophysectomized rats induces ductal and alveolar growth, as well as a dose-dependent increase in mammary expression of IGF-I messenger RNA (mRNA) (26, 27, 28). IGF-I also synergizes with estrogen to stimulate ductal growth when coimplanted into glands of hypophysectomized, ovariectomized female rats (27, 29). The mechanism for this synergism is unknown; however, evidence that IGF-I induces transcription of the estrogen receptor in breast cancer cell lines suggests that IGF-I may enhance estrogen responsiveness of mammary tissue (30). These studies support a model for IGF-I action in postnatal mammary growth as a downstream mediator of GH that, once induced, amplifies the mammogenic actions of estrogen.
In addition to its role as a mitogen for mammary epithelium, exogenous IGF-I promotes survival of mammary epithelial cells in vitro (31), and mammary overexpression of IGF-I in transgenic mice results in impaired involution caused by disruption of the normal process of cell death in mammary epithelium (32, 33). These results suggest that a reduction in IGF-I levels may be important for normal involution of the mammary gland.
Although in vivo and in vitro manipulations with
exogenously added IGFs support the hypothesis that these factors have
important endogenous roles in mammary epithelial growth, defining the
spatial and temporal patterns of IGF and IGF-IR expression is critical
for determining the precise roles for these factors in normal
development of the mammary gland. Endogenous expression patterns for
other growth factors and receptors implicated in mammary development
have been well documented, including those for EGF/TGF-
(34, 35, 36),
the TGF-ßs (37, 38), and FGFs (39). In contrast, very little is known
about the endogenous expression of the IGFs and the IGF-IR during
postnatal stages of mammary growth.
To determine the spatial and temporal expression of the IGFs and the IGF-IR during normal postnatal growth of the mammary gland, we have used in situ hybridization (ISH) to identify cellular sites of the mRNAs for these genes in the mouse mammary gland during the rapid growth phases of puberty and pregnancy. Results of these studies are presented here and demonstrate that IGF-I, IGF-II, and IGF-IR mRNA expression patterns are consistent with a role for the IGFs in TEB proliferation during pubertal growth. Moreover, using a whole-organ culture system, we also demonstrate that IGF-I induces ductal growth in mammary glands stimulated with mammogenic hormones in the presence of nanomolar levels of insulin. In contrast, EGF (under identical conditions) failed to stimulate ductal growth. Finally, a surprising finding from these studies is the demonstration that IGF-II mRNA is a major component of ductal epithelium during both pubertal and pregnancy-induced growth and is expressed in a pattern that correlates with the pattern of highly proliferative cells along the ducts. In contrast, IGF-I mRNA expression is restricted to the stroma, except for the TEBs during puberty and the differentiated alveoli in late pregnancy. These results suggest distinct roles for IGF-I and IGF-II during postnatal development of the mammary gland.
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Materials and Methods |
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Probes. [35S]-labeled RNA transcripts for IGF-I (40), IGF-II (41, 42), the IGF-IR (43), and the milk protein gene WDNM-1 (6) were synthesized from linearized plasmids containing partial complementary DNA (cDNA) inserts to rat sequences. Linearized DNAs were incubated with either Sp6 (IGF-IR), T7 (IGF-I, IGF-II), or T3 (WDNM-1) RNA polymerase in the presence of CTP, GTP, ATP, and [35S]-uridine 5'-triphosphate ([35S]-UTP), according to standard RNA transcription protocols (Promega Corp., Madison, WI). The resulting RNA transcripts were purified on Sephadex G-50 (Boehringer Mannheim, Indianapolis, IN) and used without hydrolysis.
For ISH analyzes, frozen cryostat sections were removed from -80 C and immediately postfixed for 10 min in 4% paraformaldehyde/PBS, rinsed in PBS, and dehydrated through ethanols. The sections were then acetylated in 0.25% acetic anhydride (vol/vol) in 0.05 M triethanolamine (pH 8.0), washed in 0.2 x SSC (1 x SSC contains 0.15 M NaCl and 0.015 M sodium citrate), and dehydrated through ethanols. Sections were prehybridized for 2–3 h at room temperature (RT) in a solution containing 50% (vol/vol) deionized formamide, 0.6 M sodium chloride, 10 mM Tris (pH7.5), 1 mM EDTA, 0.02% Ficoll, 0.02% BSA (fraction V), 0.02% polyvinylpyrrolidone, 0.5 mg/ml sheared herring sperm DNA, 0.5 mg/ml yeast total RNA, and 0.05 mg/ml yeast transfer RNA. Hybridization was carried out at 50 C overnight in prehybridization buffer containing 0.1 mg/ml herring sperm DNA with the addition of 10% dextran sulfate, 10 mM dithiothreitol, 0.1% SDS, and 35S-labeled complementary RNA probe (4 x 107 cpm/ml). After hybridization, the sections were rinsed in 2 x SSC and washed for 30 min at 45 C in 50% deionized formamide/1 x SSC/10 mM dithiothreitol. Unhybridized probe was removed by treatment with ribonuclease (RNase) A (100 mg/ml in 0.5 M NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA) for 30 min at RT, followed by a 2-h wash in 0.2 x SSC at 55–60 C. After the high stringency wash, the sections were dehydrated through ethanols and coated with autoradiographic emulsion (NTB2, Eastman Kodak Co., Rochester, NY), diluted 1:1 with deionized-distilled water. Slides were stored at 4 C in a desiccated light-proof box for 4–8 weeks. After exposure, the slides were developed (Kodak D19, 3.5 min, 15 C), rinsed in H2O, and fixed (15 C in Kodak fixer for 5–6 min). Sections were washed extensively in H2O, counterstained with hematoxylin, dehydrated through graded ethanols and xylene, and coverslipped with Permount mounting media (Fisher). All ISH analyzes were performed on at least 2–3 glands from each age. Hybridization with control (sense) RNA yielded very low background in all cases.
Nonradioactive ISH
Nonradioactive ISH was performed using Digoxigenin RNA probes
(Boehringer Mannheim). Digoxigenin-labeled RNA probe for IGF-II was
prepared according to standard protocols (Genius Kit, Boehringer
Mannheim) using Digoxigenin-UTP undiluted with cold UTP. The procedure
for nonradioactive ISH was as described above for ISH with radioactive
probes, except for the following modifications: the hybridization
temperature was 72 C, the concentration of the Digoxigenin-labeled
probe was 0.4 mg/ml, and the slides were washed in 0.2 x SSC at 72 C
for 1 h after the hybridization followed by 0.2 x SSC at RT for 5 min.
Sections were then treated with ribonuclease (RNase) A (as described
above), rinsed in RNase buffer without RNase A, and taken immediately
into immunodetection for Digoxigenin. Immunodetection of Digoxigenin
was performed according to Boehringer Genius Kit instructions
(Boehringer Mannheim).
Detection of proliferating cells
Mice at days 12–13 of pregnancy were injected with 0.01 ml/g BW
of a solution of 5-Bromo-2'-deoxyuridine and 5-Fluoro-2'-deoxyuridine
(10:1 ratio, Amersham, Arlington Heights, IL). After 2.5 h,
the tissues were perfusion-fixed with 4% paraformaldehyde, and
abdominal mammary glands were removed, embedded in paraffin, and
sectioned at 4–5 microns. The sections were deparafinized through
xylenes and ethanols, treated with 0.1% trypsin in 0.1 M
Tris buffered saline plus 0.1% CaCl2 for 20 min at 37 C,
rinsed in PBS, and treated with 0.3% hydrogen peroxide in PBS for 10
min at RT to block endogenous peroxidase activity. Sections were then
rinsed in PBS, treated with 2 N HCl for 1 h at 37 C,
rinsed twice in 0.1 M borate buffer (pH 8.5), rinsed in
PBS, and washed in 0.2% Triton X-100 for 30 min at RT. After
additional rinses in PBS, sections were blocked for 1 h at RT in
PGB superblock (10% BSA, 0.05% NaN3, 10% normal goat
serum in PBS) and incubated for 2 h at 37 C with
peroxidase-conjugated antibromodeoxyuridine (BrdU) antibody (1:15,
Boehringer Mannheim) in PGB diluent (PGB superblock diluted 1:5 with
0.5 M phosphate buffer, pH 7.6). The sections were rinsed
in PBS and incubated with inactive DAB (2.5 mg 3'-3' diaminobenzadine,
450 µl NiCl2 in PBS, pH 7.6) for 5 min, followed by
activated DAB (inactive DAB solution, containing 7%
H2O2) for 10 min. Finally, the sections were
washed in distilled H2O and coverslipped with Aqua-Mount
(Lerner Laboratories, Pittsburgh, PA).
Organ culture
Four-week-old C57Bl6/J female mice were estrogen- and
progesterone-primed by implantation of 9-day time-release
pellets of 3 mg progesterone and 3 µg estrogen
(Innovative Research of America, Sarasota, FL).
After 9 days, the mice were killed by cervical dislocation, and two
abdominal mammary glands were removed from each mouse, under sterile
conditions. The glands were placed on Gelfoam (Upjohn, Kalamazoo,
MI) in 6-well culture dishes, maintained at 37 C in 5%
CO2 in defined media, and fed every other day for 5 days.
Defined media contained: Waymouth’s medium (752/1 Gibco BRL, Gaithersburg, MD) plus 100 U/ml penicillin (Gibco BRL); 100 µg/ml streptomycin (Gibco BRL); 5
µg/ml PRL (ovine PRL NIH-P-S-12, obtained through the National
Hormone Pituitary Program, NIDDK, NICHHD, USDA); 5
µg/ml hydrocortisone (Sigma Chemical Co., St. Louis, MO); 1 µg/ml aldosterone (Sigma Chemical Co.);
and either 50 ng/ml insulin alone (Sigma Chemical Co.), 50
ng/ml insulin plus 100 ng/ml IGF-I (recombinant rat IGF-I; Gro-Pep,
Adalaide, Australia), or 50 ng/ml insulin plus 60 ng/ml EGF
(recombinant human EGF; Collaborative Research, Bedford, MA).
Whole-mount staining of mammary glands
Whole-mount staining of mammary glands was performed basically
according to previously published procedures (44). Briefly, mammary
glands were removed from culture and placed in tissue cassettes between
Whatman filter paper and sponges and were fixed overnight
in 10% neutral buffered formalin. The glands then were washed 3 x
1 h in acetone, followed by 30 min each in 100% and 95% ethanol, and
stained for 1.5 h in hematoxylin (0.13 g FeCl3, 13.5
ml H2O, 1.74 ml stock hematoxylin (10% hematoxylin in 95%
ethanol), 200 ml 95% ethanol, pH 1.25). After staining, glands were
soaked overnight in H2O, dehydrated in acidic 50% ethanol,
and dehydrated through graded ethanols to xylene and photographed.
Stained glands were stored at 4 C in methyl salicylate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; A, C, and E). IGF-I mRNAs also were
highly expressed in stroma surrounding the TEBs during pubertal growth
(Fig. 1A). In glands from both pubertal and postpubertal mice (15 weeks
of age), IGF-II and IGF-IR mRNAs also were expressed in ductal
epithelial cells (Fig. 1, D and F). Expression of IGF-II mRNA in the
postpubertal glands was nonuniform along the ductal epithelium (Fig. 1D), whereas IGF-IR mRNAs were expressed uniformly along the ducts
(Fig. 1F). IGF-I mRNAs were low-to-undetectable along the ductal
epithelium in postpubertal glands (Fig. 1B) when analyzed after longer
exposure times (6 weeks) than those used to detect IGF-I mRNA in the
TEBs (4 weeks).
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) was
detected in a punctate pattern along the ducts and in isolated alveolar
cells. IGF-I mRNA expression (Fig. 2A) was detectable in the epithelium
at this age and in isolated stromal cells immediately surrounding the
ducts. Expression of mRNAs for the milk proteins WDNM-1 (Fig. 2E) and
ß-casein (not shown) were readily detected in the developing alveoli.
By day 18 of pregnancy, both IGF-I (Fig. 2B) and IGF-IR (Fig. 2F) mRNAs
were widely expressed in ductal and alveolar epithelium, whereas
expression of IGF-II (Fig. 2D) was retained in a nonuniform pattern
along the ducts, identical to the expression pattern observed during
postpubertal (Fig. 1D
) and midpregnancy (Fig. 2C) stages.
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). To determine
whether the expression pattern of IGF-II correlated with proliferating
cells along the ductal epithelium, we used a 2.5-h pulse of BrdU to
label rapidly proliferating cells in midpregnant mammary glands (Fig. 3B). Detection of cells with an antibody to BrdU revealed individual
cells along the ducts that had incorporated BrdU in a pattern similar
to that of IGF-II expression. This is in contrast to the expression
pattern for the milk protein genes (see Fig. 2E), which were expressed
predominantly in developing alveoli throughout the second half of
pregnancy.
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) was sufficient to promote extensive
ductal growth, compared with 50 ng/ml of insulin alone (Fig. 4A). In
contrast, addition of EGF to 50 ng/ml of insulin (Fig. 4C) did not
promote growth of epithelial structures greater than that observed in
glands cultured in nanomolar levels of insulin alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is interesting that, in contrast to IGF-I, EGF was unable to promote
ductal growth in our culture experiments. These results were surprising
for several reasons: 1) the EGF receptor is expressed in TEBs during
pubertal growth (36); 2) EGF implanted into regressed pubertal mammary
glands of ovariectomized mice induces the formation of TEBs and ductal
growth (34, 45); and 3) previous studies, using a similar whole-organ
culture system, used identical concentrations of EGF, in the presence
of micromolar levels of insulin, to promote alveolar development (13).
Thus, the failure of EGF to induce ductal growth in our culture system
suggests that EGF (or TGF-) may synergize with the IGFs in mediating
ductal growth during puberty. In contrast to the in vivo EGF
implant experiments, which were conducted in pituitary-intact animals,
the glands in whole-organ culture were cultured in the absence of GH,
which can stimulate endogenous expression of IGF-I in mammary glands
(28).
Because signaling through the IGF-IR results in antiapoptotic as well as mitogenic effects in a variety of cell types (46, 47, 48, 49, 50, 51, 52), it is possible that the predominant action of the IGFs in the TEBs is to promote survival of TEB cells. TEB cells are known to have low rates of cell death, particularly in the outer cap cell layer (2, 53, 54). Similarly, the failure of EGF to induce ductal growth in the absence of IGF-IR stimulation could be caused by decreased survival of mammary epithelial cells in the absence of either high levels of insulin or IGF-I. A role for IGF-I in survival of mammary epithelium during late pregnancy and lactational stages is suggested by the IGF-I expression data presented here, taken together with the previously published data on transgenic mice that overexpress IGF-I from a milk protein promoter (32, 33). After the disappearance of the TEBs in postpubertal ages, IGF-I mRNA expression was detected only at low levels in mammary epithelium and isolated stromal cells. IGF-I mRNA expression increased in mammary epithelium in the differentiated alveoli at the end of pregnancy, coincident with high expression of IGF-IR mRNAs. These results are consistent with results showing that the major phenotype of IGF-I overexpression in pregnant mammary glands of transgenic mice was inhibition of cell death during involution (32, 33).
A major and unexpected finding of this study was the high epithelial expression of IGF-II in the mammary gland throughout all stages of postnatal development. Though both IGF-I and IGF-II are potent mitogens for mammary epithelial cells in vitro, previously published results, supporting a role for the IGFs in postnatal mammary development, have focused on IGF-I as the primary mediator of mammary epithelial growth. IGF-II was highly expressed in individual cells along the ductal epithelium by late pubertal stages and through pregnancy-induced growth. It is of potential interest that immunodetection of EGF in pubertal mammary glands in mice showed a similar nonuniform pattern of EGF in luminal epithelial cells along the ducts (34). Comparison of the single cell expression of IGF-II at midpregnancy with the pattern of cells identified by a short pulse of BrdU showed a similar pattern of isolated epithelial cells along the ductal structure. Because data presented here, as well as previously published data (6), have demonstrated mRNA expression of the milk protein genes in developing alveoli, and not ductal cells at this stage, these data support a potential autocrine or paracrine role for IGF-II as a mitogen for specific cells along the ducts. This hypothesis is further supported by the results from IGF-II overexpression in mammary glands of transgenic mice that demonstrate hyperproliferation of the mammary epithelium (55). However, because the IGF-IR is expressed throughout the ductal epithelium, and because IGF-I is expressed in the stroma at these stages and is present at high levels in the circulation, we cannot rule out the possibility that stromal or circulating IGF-I mediates epithelial growth during late puberty and in pregnancy. Analyzes of mammary glands from IGF-II null mutant mice are in progress in our laboratory and will provide a direct test of an essential function for IGF-II in mammary development. Though it is known that these mice are lactationally competent (56), it is possible that they might show mild deficits in mammary epithelial growth or differentiation that do not compromise the ability to support litters.
Finally, our results demonstrating high levels of mRNA expression for both IGF ligands and the IGF-IR in mammary epithelium and in the TEBs in particular suggest the possibility that locally produced IGFs could be involved in the proposed role of IGFs and the IGF-IR in estrogen-mediated TEB growth and in breast cancer. Estrogen receptor null mutant mice lack TEBs and demonstrate severely reduced epithelial growth during puberty (3). As discussed previously, IGF-I can synergize with estrogen in promoting ductal growth in pubertal glands (29) and can induce transcription of the estrogen receptor in breast cancer cell lines in vitro (30). That interactions between estrogen and the IGF system may be an important component of breast tumorigenesis is suggested by the following studies: 1) estrogen induces IGF-IR expression in breast tumor epithelial cell lines (57); and 2) high levels of both the estrogen receptor and the IGF-IR are correlated with susceptibility to breast cancer in humans (58, 59). The structure that is most susceptible to tumor formation in breast tissue is the TEB in rodents and the equivalent structure in the terminal ductal lobule unit in humans (2, 53, 54, 60). This is likely because of the high proliferative rates and low percentage of cell death in the TEBs, particularly in the outer, cap cell layer. Therefore, changes in the genome of these cells are more likely to be transmitted to daughter cells, resulting in transformation and tumors. In addition to the fact that IGF-I and IGF-II are among the most potent mitogens for breast cancer cell lines and for primary breast tumors in both humans and rodents, IGF-II and the IGF-IR specifically have been connected to malignancy and breast cancer. Overexpression of IGF-II in breast epithelial cells in vitro results in a malignant phenotype (61), and genetic overexpression of IGF-II in mouse mammary glands results in a high incidence of breast tumors (55). Consistent with these results, recent studies in breast cancer patients have confirmed abnormally high levels of IGF-II in the stroma of invasive breast tumors, closely associated with malignant epithelial cells (62). Moreover, numerous studies have demonstrated that blockade of the IGF-IR inhibits growth of breast tumor cells both in vitro and in vivo (20, 58, 63, 64, 65, 66). Taken together, these studies, along with the data presented here, support a role for endogenous IGFs in proliferation and survival of mammary epithelial cells and suggest possible mechanisms for IGF function in estrogen-responsive breast cancer.
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Acknowledgments |
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Footnotes |
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Received May 29, 1998.
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and epidermal growth factor during mouse mammary gland ductal
morphogenesis. Dev Biol 88:276–280
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