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U.S. Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center, Departments of Pediatrics (S.G.B., D.L.H.) and Molecular and Cellular Biology (D.L.H.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. Darryl L. Hadsell, Children’s Nutrition Research Center, 10th floor, 1100 Bates Street, Houston, Texas 77030. E-mail: dhadsell{at}bcm.tmc.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous studies suggest that IGF-I plays an important role in mammary gland development. Firstly, IGF-I is a potent mitogen for normal mammary epithelial cells in culture, and ductal growth can be induced in mammary gland explant cultures by IGF-I in combination with mammogenic hormones (7). Secondly, in vivo local administration of IGF-I induces mammary TEB development (8) and transgenic mice that overexpress IGF-I specifically in the mammary gland during pregnancy and lactation exhibit an increased incidence of mammary hyperplasia and tumorigenesis (9, 10). Lastly, the mRNAs for both IGF-I and the IGF-I receptor (IGF-IR) are expressed in both the mammary stroma (11) and the developing TEB (7), and studies by Ruan and co-workers (12) demonstrated that targeted deletion of IGF-I inhibits normal TEB development.
In cell culture models, IGF-I inhibits apoptosis through IGF-IR-dependent activation of insulin receptor (IR) substrate-1, followed by downstream activation of PI3K and the serine/threonine kinase Akt (13, 14). Much earlier studies in cell culture models have established that IGF-I stimulates cell cycle progression (15). The mechanism through which this occurs is pleiotropic, involving such things as the Ras/Raf/MAPK pathway (16) and a ß-catenin-regulated pathway (17, 18). Transgenic models that overexpress IGF-I in the mammary gland during lactation exhibit delayed mammary involution in conjunction with inhibited apoptosis (9, 19). Delayed postlactational mammary involution and inhibited apoptosis also occur in response to transgenic overexpression of IGF-II (20). This response has been linked with prolonged phosphorylation of Akt. Unfortunately, there has been limited success at overexpressing IGFs in the virgin mammary gland, and analysis of TEB development in the IGF-I ligand knockout has provided little in terms of the intracellular mechanisms of IGF-I action on mammary cells.
In various tumor cell types, blockage of IGF-IR signaling causes extensive cell death (21). In primary cultures of mouse mammary cells, IGF-I or high concentrations of insulin inhibit apoptosis (22). In contrast, IGF-I treatment of mammary gland organ cultures derived from virgin mice stimulates DNA synthesis (23). Because TEB development involves both cellular proliferation and apoptosis (3, 4), and no published data exist on the cellular mechanism through which TEB development is stimulated by IGF-I, an analysis of the relative involvement of apoptosis and cell cycle progression would be a logical first step in defining the mechanism of IGF-I action on the TEB. In addition, because IGF-I can stimulate biological responses through activation of IR:IGF-IR heterodimers (24, 25), the analysis of TEB development in IGF-IR-null mammary tissue would serve as an important means of formally establishing the importance of the IGF-IR to TEB development.
Independent laboratories have previously described mice that carry a targeted mutation of the IGF-IR and IR genes (26, 27). Homozygous IGF-IR-null mice exhibit decreased prenatal growth and diminished bone, skeletal muscle, and skin development. These mice die within minutes of birth. Through the use of a fetal tissue transplantation technique similar to that used to study other knockout models (28), we have been able to directly examine the in vivo function of the IGF-IR in mammary epithelial cell proliferation and death. Preliminary studies conducted in our laboratory with this model suggested that loss of the IGF-IR inhibited mammary ductal development (29). Hence, the goals of this study were 1) to investigate in further detail how loss of the IGF-IR affects mammary TEB development, 2) to determine if the effects of IGF-IR were mediated through cell proliferation or apoptosis, and 3) to determine whether the hormones of pregnancy can affect the development of mammary epithelium that lacks IGF-IR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mammary epithelium transplantation
F9–F11 heterozygous
males and females were bred to generate wild-type
(Igf1r+/+), heterozygous
(Igf1r+/-), and null
(Igf1r-/-) donor embryos for
mammary transplants. On d 16–18 of pregnancy, embryos were harvested
by cesarean section and kept on ice in HBSS until dissection.
Three-week-old FVB recipient females were prepared by clearing the inguinal fat pad of endogenous host epithelium as described by DeOme et al. (30). After clearing, embryonic mammary buds were dissected by cutting around the teat of the embryo and transplanting the mammary bud along with the overlying skin into the cleared inguinal gland of the recipient. The transplanted epithelium was allowed to develop for 4 or 8 wk in virgin hosts and then harvested for analysis. To test the effects of pregnancy on the grafted epithelium, hosts were bred 3.5 wk after grafting, and the glands were harvested 10 d postbreeding. Pregnancy was confirmed by observation of embryos in the host uteri.
Embryo genotyping
Embryo genotype was established by Southern analysis as
previously described (27). The sex of the embryo was
established by PCR on embryo DNA. Amplification of the sex-determining
region of the Y chromosome (SRY) gene was used to identify male
embryos. The forward SRY primer was 5'-cgccccatgaatgcatttatg-3', and
the reverse primer was 5'-cctccgatgaggctgatat-3'. PCR cycling was for 1
min at 94 C, 2 min at 55 C, and 2 min at 72 C for 30 cycles.
Mammary gland whole mounts and morphometric analysis
The transplanted outgrowths were harvested and fixed in 10%
neutral buffered formalin overnight and prepared as whole mounts as
described by Medina et al. (31). The whole
mounts were analyzed for growth by measuring the percentage of the fat
pad filled (PFPF) by the ductal system of the outgrowth. PFPF was
estimated by measuring the distance the ductal outgrowth
extended across the fat pad divided by the total length of the fat pad.
Whole mounts were also analyzed for branch point and TEB number and
area. These analyses were performed using Adobe Photoshop (Adobe
Systems, San Jose, CA) and/or Scion Image (Scion Corp., Frederick, MD)
image processing and analysis software. Gray scale tagged-image file
format (TIFF) images of whole mounts were captured with a Dage black
and white CCD72 video camera (Dage-MTI, Inc., Michigan City, IN) and
Scion LG3 frame grabber (Scion Corp.) at a resolution of 72
pixels/inch. To quantitate branch points, images of whole mounts (x10
magnification) were viewed in Adobe Photoshop. Nodes (branch points) in
the ductal tree were marked on an overlying layer using the paintbrush
tool. The layer containing the marked nodes was saved in a TIFF
format, and the marks representing nodes or branches were counted by
Scion Image software. To measure TEB number and area, a similar method
was used. Briefly, x10 images of whole mounts, captured as described,
were displayed in Adobe Photoshop. TEBs were traced or outlined and
then filled. The layer containing only the traced images of the TEBs
was saved in a TIFF format and quantitated using Scion Image software.
The analyze particles function of the Scion Image software was used to
quantitate the area in square millimeters and the total number of the
traced TEBs. The total number of terminal structures possessing an area
0.03 mm2 or more and with morphology distinct to
a TEB structure were recorded following the paradigm of Ball
(32). The area of the two largest TEBs was used to
determine the mean TEB area, because the minimal average number of TEBs
in all groups was 1.8. One
Igf1r-/- outgrowth included
in the mean TEB area calculation possessed only one TEB.
Cell proliferation and cell death assays
Anti-bromodeoxyuridine (anti-BrdU) immunohistochemistry was used
to measure cell proliferation in the mammary outgrowths, whereas the
terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick-end
labeling (TUNEL) assay was used to measure cell death. These procedures
were conducted as previously described (10, 33). Counting
at least 1000 cells/section quantitated the percentage of cells
proliferating or dying. In some cases, sections of the
Igf1r-/- outgrowths did not
contain 1000 cells because of the limited growth of the
Igf1r-/- epithelium. In
these instances, all cells in the entire section of multiple
(2, 3, 4, 5, 6) sections were counted.
Statistical analysis
All data were analyzed by one-way ANOVA using Minitab
statistical software (State College, PA). Mean separation was
accomplished using Fisher’s pairwise comparison. Differences were
considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A–C, show that transplanted embryonic
mammary buds of all three genotypes were able to grow in the host fat
pad to form a mammary ductal tree. When transplanting wild-type mammary
epithelium, the rates of successful grafts for
Igf1r+/+ and
Igf1r+/- epithelium were 78% and 71%,
respectively, whereas the rate of successful grafts of
Igf1r-/- mammary epithelium
was only 36% (Fig. 1D). Therefore, although mammary epithelium from
all three genotypes can be successfully transplanted to study postnatal
mammary development, the
Igf1r-/- epithelium does not
possess the same growth potential as its wild-type or heterozygous
counterpart.
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). The ductal morphology of the Igf1r+/+
(Fig. 1A) and Igf1r+/- (Fig. 1B)
outgrowths appeared normal. In contrast, the
Igf1r-/- (Fig. 1C) ducts
appeared unorganized and displayed irregular branching. Although growth
and morphogenesis appeared abnormal in
Igf1r-/- outgrowths, studies
of hematoxylin- and eosin-stained paraffin sections did not reveal
abnormalities at the cellular level (data not shown).
Four-week outgrowths exhibit limited ductal development that is
restored by pregnancy
The initial studies of grafts harvested 8 wk posttransplantation
established that Igf1r-/-
epithelium gave rise to a limited mammary ductal system. To further
explore the defect in ductal growth, we performed an analysis of grafts
harvested 4 wk posttransplantation. Study of the outgrowths at this
time point would allow an analysis of the highly proliferative
compartment of the developing ductal system, the TEB, which is absent
at the 8-wk point. Representative whole mounts from each genotype are
shown (Fig. 2, A–C).
Igf1r+/+ and
Igf1r+/- outgrowths exhibited a greater
level of development than
Igf1r-/- outgrowths (Fig. 2C). Quantitative analysis of the outgrowths showed that after 4 wk,
the Igf1r-/- epithelium
filled only 17.5 ± 2.4% of the fat pad vs. 66.9
± 7.4% and 65.9 ± 4.1% for
Igf1r+/+ and
Igf1r+/- outgrowths, respectively (Fig. 2G).
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, D–F), the
extent of ductal elongation in
Igf1r-/- grafts (Fig. 2F)
was restored to levels observed in
Igf1r+/+ and
Igf1r+/- grafts grown in virgin hosts
(Fig. 2, A and B). This development was more representative of what
would be observed in early pregnant mammary gland development
characterized by increased branching and alveolar budding. The extent
of growth in Ig1fr+/+ and
Igf1r+/- grafts from pregnant hosts was
also increased above levels observed in virgin hosts (Fig. 2, D and E).
Growth in Igf1r-/- grafts
from pregnant hosts was 3.5 times greater than growth of
Igf1r-/- grafts that were
allowed to develop for a similar amount of time in virgin hosts (Fig. 2G). Although the extent of growth in
Igf1r-/- grafts from
pregnant hosts was still significantly less than
Igf1r+/+ and
Igf1r+/- growth in pregnant hosts, the
magnitude of difference was smaller than that observed in grafts from
virgin hosts (Fig. 2G). Therefore, although
Igf1r-/- ductal development
was growth inhibited in virgin hosts, the hormonal environment provided
by pregnancy was able to partially restore ductal growth.
TEBs in Igf1r-/- outgrowths are smaller
A morphometric study of TEB structures in the 4-wk ductal
outgrowths was also performed. Images of whole mounts were used to
quantitate TEB number and size. The mean number of TEBs found in
Igf1r+/+ and
Igf1r+/- outgrowths was 3.6- and 4.0-fold
greater, respectively, than that observed in
Igf1r-/- outgrowths (Table 1). Additionally, the mean TEB area of
Igf1r+/+ and
Igf1r+/- outgrowths was 31% greater than
the mean of Igf1r-/-
outgrowths (Table 1). These findings indicate that reduced ductal
growth in the IGF-IR null tissue is due to reduced TEB development.
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). Higher magnifications
of the outgrowths are shown in Fig. 3, A–C, to allow closer
examination of the TEB structures in gland whole mounts. As described
in the TEB morphometric analyses, TEBs in
Igf1r-/- outgrowths appeared
smaller, but exhibited normal morphology (Fig. 3C). Hematoxylin and
eosin-stained sections of the whole mounted outgrowths revealed that
ductal cell organization was normal in
Igf1r-/- outgrowths (Fig. 3F). In all three genotypes, a single layer of ductal epithelium lined
the lumen (Fig. 3, D–F). TEB anatomy also appeared normal (Fig. 3, G–I). A layer of cap cells was present in the leading edge of
Igf1r-/- end bud structures
along with a normal body cell component and lumen (Fig. 3, G and I).
Therefore, although TEB number and size are reduced in
Igf1r-/- outgrowths, the
cellular structure of the TEB is normal.
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). BrdU- or TUNEL-positive cells were
counted separately in the TEBs or ductal epithelium of the outgrowths.
The number of BrdU-labeled cells was significantly lower in
Igf1r-/- TEBs, whereas the
ductal epithelium showed no difference in the number of labeled cells
among the three genotypes (Fig. 5A). The
percentage of BrdU-labeled cells in
Igf1r-/- TEBs was 40% less
than that observed in Igf1r+/+ and
Igf1r+/- TEBs (Fig. 5A). Closer analysis
of the TEBs shown in Fig. 4, A and C, suggests that as much as a 2-fold
difference in proliferation exists between the cap cells found in
Igf1r+/+ TEBs and those found in
Igf1r-/- TEBs. The majority
(86%) of cells in the cap cell layer of the
Igf1r+/+ TEB were BrdU labeled (Fig. 4A),
in contrast to the minimal number (38%) seen in an
Igf1r-/- TEB (Fig. 4C). This
finding coupled with the observation that
Igf1r-/- TEBs are smaller
support the conclusion that IGF-IR-dependent proliferation in this very
small, but important, population of cells mediates TEB growth and the
subsequent development of the mammary ductal system.
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, B and D). Surprisingly,
Igf1r-/- TEBs or ductal
epithelium did not show an increased percentage of TUNEL-positive cells
over Igf1r+/+ epithelium (Fig. 5B).
However, the Igf1r+/- ductal epithelium
exhibited a significantly higher number of apoptotic cells than either
the Igf1r+/+ or the
Igf1r-/- ducts (Fig. 5B).
Igf1r+/- ductal epithelium exhibited
2.4-fold more apoptotic cells than
Igf1r+/+ ducts and 2.6-fold more than
Igf1r-/- ducts. Although a
statistically significant difference between
Igf1r+/- and
Igf1r+/+ TEB apoptosis was not observed,
Igf1r+/- mammary epithelium exhibited
significantly more cell death than either
Igf1r+/+ or
Igf1r-/- epithelial cells
when the percentage of apoptosis among ducts and TEBs was considered as
a whole (data not shown). These results suggest that IGF-IR
haploinsufficiency results in increased mammary cell apoptosis.
Although this increased apoptosis was not large enough to influence the
percentage of the fat pad filled by the outgrowth (Fig. 2G) or TEB
development (Table 1), analysis of ductal branching in 4-wk outgrowths
revealed that Igf1r+/- ductal systems
exhibited a reduction in branches compared with
Igf1r+/+ grafts. The ducts in
Igf1r+/- grafts had only 66% the number
of branches observed in Igf1r+/+ ducts
(Fig. 6). By contrast, branching in
Igf1r-/- outgrowths was only
14% of that observed in Igf1r+/+ and 22%
of that observed in Igf1r+/- outgrowths.
These data demonstrate links between branching, and proliferation and
apoptosis and support the conclusion that IGF-IR haploinsufficiency may
inhibit some aspects of mammary gland development. More importantly,
these results also clearly show that reduced TEB development in
response to the complete loss of IGF-IR signaling is not due to
increased apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to demonstrating a requirement for IGF-IR within the context of an intact animal, these studies make several novel observations on the mechanism of IGF-I action in the mammary gland. These are as follows. 1) The most dramatic effect of loss of the IGF-IR is decreased proliferation in a very small, but important, population of cells, the TEB cells. 2) Complete loss of the IGF-IR does not affect apoptosis in the TEB. 3) Pregnancy partially restores the development of Igf1r-/- mammary glands. 4) IGF-IR haploinsufficiency increases mammary cell apoptosis and decreases ductal branching. These observations, although limited in the degree to which they define the molecular mechanisms of IGF-I action, serve to identify an important target cell for IGF-I in the mammary gland and provide a basis for more technically challenging studies, which could eventually define IGF-IR-dependent signaling pathways of importance to TEB development.
The cap cell was originally defined by its physical presence in the outer layer of cells on the TEB (1). Ultrastructural and immunohistochemical analyses of TEBs demonstrated that these cells are myoepithelial cell precursors (1, 35). In addition, previously published analysis of cell proliferation within the TEB suggests that proliferation is often highest in the cap cell layer (3, 4). These observations coupled with the finding that proliferation in the Igf1r-/- TEB was inhibited most dramatically in the cap cell layer support the idea that continued study of IGF-I action on this particular cell population within the TEB will yield valuable insights into the role of the IGF-IR in mammary gland development.
The fact that total loss of the IGF-IR reduced proliferation without increasing apoptosis supports the suggestion that stimulation of cell survival pathways by IGF-IR may not be as important to TEB development as stimulation of cell cycle pathways. In this respect, analysis of the phosphorylation state of proteins involved in cell survival, such as Akt (14), or proteins involved in proliferation, such as MAPK (16) or ß-catenin (17, 18), would be valuable. However, because the amount of epithelium in the grafts generated by these studies varies tremendously between Igf1r+/+ and Igf1r-/- tissue, and because the cells of the TEB represent only a fraction of the total cellular mass of the grafts, an immunohistochemical approach would theoretically be the only method by which these signaling events could be detected. Unfortunately, the in situ analysis of the phosphorylation of these proteins appears to only be possible in models in which the proteins are artificially activated above normal levels (36). Current efforts in our laboratory to detect localized phosphorylation of IGF-IR signaling molecules in the TEB have had limited success. However, even if such techniques become possible, the most convincing test of involvement for a specific pathway would rely on genetic crosses of the Igf1r-/- mice with transgenic mice in which specific signaling pathways are constitutively activated. These studies are currently in the planning phase.
The relief of Igf1r-/- mammary cell dependence on IGF-IR signaling during early pregnancy may result from increased levels of E and progesterone that cause secondary changes in local growth factor expression and subsequent activation of alternate growth factor receptors. In other knockout models where mammary development is compromised, multiple cycles of pregnancy and lactation are able to restore mammary gland function (37, 38), indicating that exposure to signals from pregnancy is able to compensate for the loss of otherwise important mammary signaling pathways. The restoration of Igf1r-/- ductal growth observed during pregnancy may also result from changes in mammary cell sensitivity to insulin-like signals mediated by the IR. There is genetic evidence that the IR can mediate the growth-promoting function of IGF-II (39). Additionally, IGF-IR null fibroblasts that overexpress IR are able to grow in serum-free medium supplemented solely with insulin or IGF-II, but not IGF-I (40). Furthermore, changes in insulin responsiveness during the development of rodent mammary epithelium have been documented (41, 42). Carrascosa et al. (41) have shown that increased insulin sensitivity during pregnancy is not due to changes in the level of IR binding, but to up-regulation of IR tyrosine kinase activity. In addition, our own preliminary studies (Lee, A. V., and D. L. Hadsell, unpublished data) suggest that the IGF signaling molecules, IR substrate-1 and –2, are also up-regulated by pregnancy. Again, the ultimate demonstration of these as underlying mechanisms for pregnancy-dependent growth compensation will require genetic crosses of the Igf1r-/- mice with gain of function transgenic mice, as described above.
The most surprising and perplexing result from these studies is the fact that increased apoptosis was observed in the epithelium of Igf1r+/-, but not Igf1r-/-, grafts. The observation that branchpoint number was also reduced in Igf1r+/- supports the surprising conclusion that IGF-IR haploinsufficiency may have biological effects on the mammary gland that would not have been expected based on previously published studies with the Igf1r-/- mice (43). These results also support the suggestion that discreet IGF-IR expression thresholds exist for maintaining cell cycle progression as opposed to cell survival. Studies performed in tumor cells have shown that inhibition of IGF-IR signaling induces apoptosis (21). In contrast, studies performed in normal fibroblast that lack an IGF-IR have shown that these cells are not more susceptible to apoptosis (44). The increased sensitivity of tumor cells to apoptotic signals may be related to the fact that they are rapidly dividing. There is evidence that suggests that cell cycle progression and apoptosis are linked (45). Therefore, the ability to proliferate normally may make the grafted Igf1r+/- epithelium sensitive to a halving of the potential survival signals that would normally come from a full genetic complement of the IGF-IR gene.
In conclusion, we found the IGF-IR to be involved in the processes of cell proliferation and cell death in the developing mouse mammary gland. Moreover, we found the IGF-IR to be required for TEB cell proliferation during virgin morphogenesis and to be less important for pregnancy-dependent growth processes. Future studies involving genetic crosses of the Igf1r-/- mice with transgenic mice that exhibit activation of specific signaling pathways should allow for more definitive analysis of the molecular mechanisms of IGF-IR action on TEB development and mammary tumorigenesis.
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Acknowledgments |
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Footnotes |
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Abbreviations: BrdU, Bromodeoxyuridine; IGF-IR, IGF-I receptor; IR, insulin receptor; PFPF, percentage of the fat pad filled; TEB, terminal end bud; TIFF, tagged-image file format; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick-end labeling.
Received April 13, 2001.
Accepted for publication July 30, 2001.
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 S. Yeh, Y.-C. Hu, P.-H. Wang, C. Xie, Q. Xu, M.-Y. Tsai, Z. Dong, R.-S. Wang, T.-H. Lee, and C. Chang Abnormal Mammary Gland Development and Growth Retardation in Female Mice and MCF7 Breast Cancer Cells Lacking Androgen Receptor J. Exp. Med., December 15, 2003; 198(12): 1899 - 1908. [Abstract] [Full Text] [PDF]
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A. V. Lee, P. Zhang, M. Ivanova, S. Bonnette, S. Oesterreich, J. M. Rosen, S. Grimm, R. C. Hovey, B. K. Vonderhaar, C. R. Kahn, et al. Developmental and Hormonal Signals Dramatically Alter the Localization and Abundance of Insulin Receptor Substrate Proteins in the Mammary Gland Endocrinology, June 1, 2003; 144(6): 2683 - 2694. [Abstract] [Full Text] [PDF]
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G. Chakravarty, D. Hadsell, W. Buitrago, J. Settleman, and J. M. Rosen p190-B RhoGAP Regulates Mammary Ductal Morphogenesis Mol. Endocrinol., June 1, 2003; 17(6): 1054 - 1065. [Abstract] [Full Text] [PDF]
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 B. S. Wiseman and Z. Werb Stromal Effects on Mammary Gland Development and Breast Cancer Science, May 10, 2002; 296(5570): 1046 - 1049. [Abstract] [Full Text] [PDF]
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M. A. Stull, M. M. Richert, A. V. Loladze, and T. L. Wood Requirement for IGF-I in Epidermal Growth Factor-Mediated Cell Cycle Progression of Mammary Epithelial Cells Endocrinology, May 1, 2002; 143(5): 1872 - 1879. [Abstract] [Full Text] [PDF]
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; D). The proportion of successful grafts was
determined by dividing the number of outgrowths observed by the total
number of embryonic mammary buds transplanted (
; D). Data are
expressed as the mean ± SEM. Numbers in
parentheses indicate the number of outgrowths analyzed,
and the asterisk indicates a statistical difference
(P < 0.05).
