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Department of Pediatrics, U.S. Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center (D.L.H., T.A., Y.K., D.T.); and Breast Center, Department of Medicine (A.V.L.), and Department of Molecular and Cellular Biology (D.L.H., A.V.L.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. Darryl L. Hadsell, Department of Pediatrics, U.S. Department of Agriculture (USDA)/Agrucultural Research Service (ARS) Children’s Nutrition Research Center, Houston, Texas 77030. E-mail: dhadsell{at}bcm.tmc.edu
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Introduction |
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Overexpression of wild-type IGF-I in the mammary glands of transgenic mice has been demonstrated to inhibit apoptosis when analyzed on day 5 of forced involution (14). Studies in our own laboratory have shown that overexpression of the des(1, 2, 3) truncated form of IGF-I under control of the promoter from the rat whey acidic protein gene (WAP-DES) inhibits the apoptosis that occurs during natural mammary involution and increases the frequency of spontaneous mammary tumors (15, 16). However, preliminary studies in our laboratory suggested that WAP-DES was incapable of inhibiting apoptosis during forced involution. The goals of the current study were, therefore, 1) to characterize in greater detail the ability of overexpressed des(1–3])human (h) IGF-I to inhibit apoptosis during forced vs. natural mammary gland involution, and 2) to determine whether expression of IGF signal transducers could account for differential responsiveness of the involuting mammary gland to overexpressed des(1, 2, 3)hIGF-I.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histology
Tissue was fixed and processed for immunohistochemistry
(15). Mammary tissue sections were stained for apoptosis
using a previously described (17) modification of the
terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end
labeling (TUNEL) technique (18). Labeling indexes were
obtained by counting labeled and unlabeled epithelial cells in 8–12
(
1000 cells total) fields at x1000 magnification.
Immunohistochemistry for IRS-1 was conducted on paraffin-embedded
sections of mammary tissue using a rabbit antihuman IRS-1 antibody
(Upstate Biotechnology, Inc., Lake Placid, NY). All
incubations were performed at room temperature, and all washing was
performed with 0.15 M NaCl, 0.01 M Tris-HCl (pH
7.4), and 0.05% Tween 20 unless otherwise stated. Slides were cut at
3–4 µm, baked overnight at 58 C, and deparaffinized using a
Shandon-Lipshaw Varistain (program 2) (Shandon Inc., Pittsburgh,
PA). Heat-induced antigen retrieval was performed in 0.1 M
Tris-HCl (pH 9.0) for 5 min. Endogenous peroxidase activity was blocked
by incubation in 3% hydrogen peroxide solution for 5 min.
Avidin/biotin blocking was performed with the A/B blocking kit
(Vector Laboratories, Inc., Burlingame, CA) by incubation
in solution A for 15 min and then in solution B for 15 min. Slides were
then incubated in IRS-1 antibody (1:800 dilution in TBS plus 1% BSA)
for 1 h, biotin-labeled secondary antibody (1:200) for 30 min, and
horseradish peroxidase-labeled avidin (1:200) for 30 min. For a
negative control, slides were incubated with purified rabbit Ig
(Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA). Detection was then performed by incubation with diaminobenzidine
(DAKO Corp., Carpinteria, CA) for 15 min, followed by
enhancement with 0.2% osmium tetroxide for 30 sec. Slides were
counterstained with 0.05% methylene green for 30 sec, dehydrated, and
mounted using cytoseal. For detection of steroid hormone receptors we
used the same method with antibodies to estrogen receptor 
(6F11,
Vector Laboratories, Inc.) and progesterone receptor
(1294, DAKO Corp.).
RNA analysis
Total RNA was isolated from about 100 mg tissue using Ultraspec
(Biotecx, Houston, TX) as described by the manufacturer. Ribonuclease
protection assays (RPA) were used to analyze the abundance of the
messenger RNAs (mRNAs) for IGF-I receptor (IGF-IR), IRS-1, and IRS-2.
The RPA probe for murine IGF-IR has been previously described
(16). The probes for murine IRS-1 and -2 were produced by
cloning PCR fragments amplified from mouse genomic DNA. For the murine
IRS-1 probe, pDT1-mIRS-1, a 447-bp PCR product, was amplified with the
forward and reverse primers, 5'-CAT CCG AAT TCA CCT GCG CAA GCC CAA GAG
T-3' and 5'-AAA GAG GAT CCT GCC AGA CCT CCT TGA ACG C-3', respectively.
For the murine IRS-2 probe, pDT1-mIRS-2, a 329-bp PCR product, was
amplified with the forward and reverse primers, 5'-GGT AAG AAT TCA GGA
CCT TCC CAG TAA ACG G-3' and 5'-AAT AAG GAT CCT GGT CAT TGT CTC CGC TGC
A-3', respectively. The temperature-time settings for these 30 cycle
amplifications were 95 C for 1 min, 65 C for 2 min, and 72 C for 3 min.
The resulting products were digested with EcoRI and
BamHI and then ligated into the corresponding sites in
pBluescript (Stratagene, La Jolla, CA). The
pTRI-cyclophilin-mouse antisense control template (Ambion, Inc., Austin, TX) was used to measure cyclophilin mRNA as a
loading control. Antisense complementary RNAs were produced using T3
RNA polymerase and resulted in probes of 502, 384, and 138 nucleotides
(nt) for IRS-1 IRS-2, and cyclophilin, respectively. RPAs were
conducted using the RPA III kit (Ambion, Inc.).
Quantitative data were obtained using the Storm PhosphorImager
(Molecular Dynamics, Inc., Sunnyvale, CA.)
IGF-I measurement
Acetic acid extracts (19) of mammary tissue were
assayed for both human IGF-I and mouse IGF-I using species-specific
assay kits (Diagnostics Systems Laboratories, Inc.,
Webster, TX). For the human IGF-I assay, approximately 50 mg frozen
mammary tissue were removed from liquid nitrogen and immediately
homogenized in 2 ml ice-cold 1 M acetic acid. Tissue
extracts prepared from this homogenate were neutralized by diluting 1:6
in a neutralization buffer provided with the assay kit and then diluted
in the assay zero standard. This assay had a working range of 0.15–20
ng/ml and a minimum detection limit of 36 ng/g tissue. The intraassay
coefficient of variation was 6%. The specificity of the assay for the
human IGF-I transgene product was evident by the fact that parallel
analysis of mammary tissue extracts prepared from nontransgenic mice
failed to give a signal above the zero assay standard (data not shown).
For analysis of mouse IGF-I, mammary tissue was homogenized in 5 vol 1
M acetic acid. Extracts were than neutralized as described
above. The rodent-specific RIA had a working range of 5.3–133 ng/ml
and a minimum sensitivity of 114 ng/g tissue. The intraassay
coefficient of variation was 4%.
Western blotting
Mammary tissue extracts were prepared for protein analysis as
previously described (20). For each genotype time point,
equivalent amounts of protein from four individual mammary gland
extracts were pooled. Immunoprecipitations for IRS-1 and IRS-2 were
conducted using 5 µg of each antibody (Upstate Biotechnology, Inc., Lake Placid, NY) and 500 µg of protein from each pool as
previously described (20). These immunoprecipitates were
then resolved by 8% SDS-PAGE and blotted to nitrocellulose
(Schleicher & Schuell, Inc., Keene, NH). The resulting
blots were analyzed for phosphotyrosine as previously described
(20). Equal protein concentrations of the pooled extracts
(10–100 µg depending upon the antigen to be detected) were also
directly electrophoresed on 8% SDS-PAGE gels and used for Western blot
analysis. Equal protein loading was assessed after electrophoretic
transfer by staining the membrane in 0.1% Ponceau S in 5% acetic acid
(Sigma, St. Louis, MO) for 5 min. Blots generated with
these extracts were probed with antibodies specific to IGF-IR (1:1000;
-IR3, Calbiochem, San Diego, CA), IRS-1 (1:1000;
Upstate Biotechnology, Inc.), IRS-2 (1:1000; Upstate Biotechnology, Inc.), p85 subunit of phosphatidylinositol-3
kinase (1:2000; Upstate Biotechnology, Inc.), phospho-Akt
(1:1000; New England Biolabs, Inc., Beverly, MA), total
Akt (1:1000; New England Biolabs, Inc.), phospho-BAD
(1:1000; Upstate Biotechnology, Inc.), and total BAD
(1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
using previously described procedures (20). The blots were
also probed with an antibody to keratin 18 (K18, Progen Biotechnik,
Heidelberg, Germany) to examine epithelial content.
Autoradiograms were generated by exposing the blots for varying times
from 10 sec to 30 min. The resulting autoradiograms were scanned on the
Personal Densitometer SI (Molecular Dynamics, Inc.,
Sunnyvale, CA) to provide quantitative data. For Quantitative
comparison of IRS and Akt, exposure times were chosen that allowed for
the detection of signals with the least abundance. The data were
expressed as the integrated area under the curve, assuming equivalent
loading based Ponceau S staining and the bicinchoninic acid protein
assay (20).
Statistics
Quantitative data on mammary tissue weight, apoptosis, and
hIGF-I concentration were analyzed using the general linear models
procedure in SPSS for Windows version 10.0.0 (SPSS, Inc.,
Chicago, IL). The effects of involution and genotype on K18,
phospho-Akt and total Akt were determined by one-way ANOVA on data
collapsed for day within each genotype/involution combination. This was
done because there was no apparent effect of day of Akt phosphorylation
and abundance. Decreased apoptosis or increased mammary gland weight in
the WAP-DES mice on individual days was tested for using a one-way
unpaired t test. Differences were considered significant at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In contrast, the
decline in mammary gland weight in the WAP-DES mice was delayed by 2
days, and hence, tissue weight was maintained at 29–30% greater than
that of nontransgenic mice (P < 0.05). Analysis of
mammary tissue apoptosis during natural involution (Fig. 1B) revealed
only 0.2–0.3% apoptosis in nontransgenic and WAP-DES mice on day 16
postpartum. By day 20 postpartum, this value increased 14-fold to
2.8 ± 0.6% in the nontransgenic mice. In contrast, mammary
tissue apoptosis in the WAP-DES mice on day 20 postpartum, although
increased to 1.4 ± 0.3%, was only 51% of that observed in the
nontransgenic animals (P < 0.05). By day 22
postpartum, mammary tissue apoptosis in the WAP-DES mice was still only
61% of that observed in the nontransgenic mice (P <
0.05). Thus, des(1, 2, 3)hIGF-I overexpression inhibited apoptosis during
natural involution of the mammary gland.
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), with an initial,
transient increase due to milk accumulation followed by weight loss
toward the end of the 4-day time period. Mammary cell apoptosis also
underwent similar changes between nontransgenic and WAP-DES mice during
forced involution (Fig. 1D), supporting the conclusion that
overexpression of des(1, 2, 3)hIGF-I does not inhibit the apoptosis that
occurs during the first 4 days of forced involution.
A decreased transgene expression and lower abundance of the transgene
protein during forced involution would explain the inability of WAP-DES
to protect mammary cells from apoptosis. To test this hypothesis,
species-specific assays were used to measure both human and mouse IGF-I
in mammary tissue extracts from WAP-DES and nontransgenic mice during
natural and forced mammary involution (Fig. 2). At 16 days postpartum, the
concentration of des(1, 2, 3)hIGF-I in mammary tissue from the WAP-DES
mice averaged 1090 ± 1038 ng/g tissue (Fig. 2A). During natural
involution, these concentrations gradually increased to a maximum of
4012 ± 233 ng/g tissue on day 22. In contrast, the concentration
of endogenous IGF-I was below the detection limit of 114 ng/g tissue
for both nontransgenic and WAP-DES mice throughout the course of
natural involution. The concentration of des(1, 2, 3)hIGF-I in the mammary
tissue of WAP-DES mice during forced involution ranged from a high of
1599 ± 410 ng/g tissue on day 17 to 969 ± 292 ng/g tissue
on day 20 postpartum (Fig. 2B). During forced involution,
concentrations of endogenous IGF-I ranged from below the limit of
detection on day 17 to as much as 402 ± 37 ng/g tissue on day 18
postpartum. The concentration of endogenous IGF-I during forced
involution was also slightly, but significantly (P <
0.05), lower in mammary tissue from WAP-DES compared with nontransgenic
mice. No significant differences were observed in des(1, 2, 3)hIGF-I
concentrations among the first three time points of natural involution
(days 16, 18, and 20) and forced involution (days 17 18, and 19). This
is the time when significant differences were observed in apoptosis and
wet weight between nontransgenic and WAP-DES mice (see Fig. 1) during
natural, but not forced, involution.
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). The receptor protein, however, was
not diminished by forced mammary involution. The variation observed in
the abundance of the receptor also showed little apparent relationship
to genotype. Western blotting for K18 demonstrated variation unrelated
to genotype or involution. This was repeatedly found with all of the
blots in which K18 was measured (see also Figs. 4 and 7). Furthermore, statistical
analysis of the densitometry data from all three K18 blots failed to
detect a significant change in response to either genotype or
involution (data not shown). Ribonuclease protection analysis
demonstrated that the abundance of the IGF-IR mRNA was not diminished
by forced involution and was little affected by the WAP-DES transgene
(Fig. 3B).
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). Western blot
analysis of immunoprecipitates prepared from pooled mammary tissue
extracts demonstrated abundant tyrosine phosphorylation of IRS-1
(Py-IRS-1) during natural involution (Fig. 4A, lanes 3–12). The degree
of IRS-1 phosphorylation appeared to be slightly higher in pooled
extracts from the WAP-DES mice on days 20 and 22 than in the
nontransgenic (compare lanes 6 and 7 with 11 and 12 in Fig. 4A). This
effect, however, was not dramatic. During forced involution,
phosphorylation of IRS-1 was detected only in the day 17 nontransgenic
pool (Fig. 4A, lane 15). Phosphorylation of IRS-2 (Py-IRS-2) was not
detected during natural involution, but was present at detectable, but
low, levels in some of the pools obtained from mammary tissue during
forced involution (Fig. 4A, lanes 15–22). Western blot analysis
demonstrated abundant IRS-1 and -2 protein during natural involution in
mammary gland extracts from both nontransgenic (Fig. 4A, lanes 3–7)
and WAP-DES (Fig. 4A, lanes 8–12) mice. The abundance of these two
proteins was similar between nontransgenic and WAP-DES mice on days
8–18 postpartum. Both IRS-1 and -2 decreased on days 20 and 22
postpartum (Fig. 4A, lanes 6, 7, 11, and 12). For IRS-1, this decrease
was slightly greater for the nontransgenic mice than for the WAP-DES
mice. For IRS-2, the decrease appeared to be delayed 1 day in WAP-DES
compared with nontransgenic mice. For both proteins, however, the
apparent effect of the transgene was relatively small compared with the
effect of forced involution. Consequently, the densitometric data from
the IRS-1 and -2 blot were pooled across genotype, as presented in Fig. 4, B and C.
Abundance of IRS-1 and -2 decreased over the course of natural
involution by 70% and 93%, respectively (Fig. 4B). For IRS-1, the
decrease occurred gradually throughout the course of natural
involution. For IRS-2, the decrease was only apparent on days 20 and 22
when apoptosis was increased. During forced mammary involution, a
dramatic reduction in IRS-1 and -2 protein abundance preceded the onset
of apoptosis in both the nontransgenic and WAP-DES mice (Fig. 4A, lanes
15–22). On day 17 postpartum, after only 24 h of forced
involution, mammary gland IRS-1 and -2 abundance was reduced by 54%
and 96% compared with that on day 16 of lactation (Fig. 4, B and C).
By day 18 postpartum, IRS-1 was reduced by 97%, and IRS-2 was
undetectable. Reprobing the same blots with antibodies to the p85
regulatory subunit of phosphatidylinositol 3-kinase (PI3 kinase)
and K18 demonstrated little effect of genotype or mammary gland
involution (Fig. 4A) on the abundance of these two proteins. The
decrease in IRS-1 protein abundance during forced involution was
confirmed by immunohistochemical staining of mammary tissue sections
(Fig. 5). Intense cytoplasmic IRS-1
staining was observed in the mammary epithelial cells of nontransgenic
mice undergoing natural involution (Fig. 5A). In the absence of primary
antibody, only a low level of background staining was detected (Fig. 5, B and D). In mammary epithelial cells of mice undergoing forced
involution, the intensity of IRS-1 staining was reduced to near
background levels (Fig. 5C). Loss of IRS-1 and -2 proteins during
forced involution has also been observed in a separate series of
studies on developmental regulation of these proteins during a complete
lactational cycle (Lee, A.V., and D. L. Hadsell, unpublished
observations). These data support the hypothesis that decreased IRS-1
and -2 expression in the epithelium is associated with an inability of
overexpressed des (1, 2, 3)hIGF-I to inhibit mammary gland apoptosis
during forced involution.
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). The
results of this analysis demonstrated that the changes in IRS protein
levels during involution occurred independently of ER and PR status,
and that the abundance of the IRS-1 and -2 mRNAs was similar between
nontransgenic and WAP-DES mice and did not change in response to
natural or forced involution.
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, lanes 3–11) and forced (Fig. 7A, lanes 12–19) involution. The abundance of phospho-Akt was dramatically
reduced with forced involution. In addition, there appeared to be
moderate enhancement of phospho-Akt in extracts prepared from WAP-DES
mice during forced involution compared with that in nontransgenic mice.
These results were obtained in two separate sets of gels run with two
independently prepared sets of extracts. In both sets of extracts there
was no apparent effect of day of involution on the abundance of Akt.
Therefore, the densitometric data for day of involution were collapsed
within each genotype/involution combination to allow statistical
analysis of treatment effects. This analysis demonstrated that the
overall abundance of phospho-Akt was decreased (P <
0.05) in forced involution to only 12% of that observed during natural
involution (Fig. 7B). Overall abundance of total Akt during forced
involution was decreased (P < 0.05) to 33% of that
observed during natural involution (Fig. 7C). The enhancement
(P < 0.05) of phospho-Akt in WAP-DES mice during
forced involution, although statistically significant, was relatively
small compared to the overall reduction that was observed with forced
involution. In addition, this small, but statistically significant,
increase in phospho-Akt was not accompanied by a similarly significant
increase in total Akt. Like IRS-1 and -2, the decrease in Akt preceded
the onset of apoptosis during forced involution. Reprobing of the blots
demonstrated little effect of genotype or involution on K18
abundance. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The direct measurement of des(1, 2, 3)hIGF-I concentrations demonstrate that the tissue concentrations present in the mammary glands of the WAP-DES mice during forced involution are essentially the same as those observed during natural involution, with the exception of elevated concentrations on day 22. Although this elevation on day 22 is counterintuitive and currently unexplained, it also probably has little relevance to the apoptotic processes that began on days 18–20 postpartum. The concentrations of des(1, 2, 3)hIGF-I are much higher than those of endogenous IGF-I and are probably more than adequate to stimulate a responsive epithelium. Because the des(1, 2, 3)IGF-I analog expressed by the WAP-DES transgene has reduced affinity for all IGFBPs (24, 25, 26), reduced bioavailability through interaction with IGFBPs seems unlikely. The present studies cannot, however, rule out the possibility that the differential ability of WAP-DES to inhibit apoptosis is due to decreased bioactivity during forced involution. Despite this caveat, we believe that the data strongly implicate the third possibility, that mammary epithelial cells lose their responsiveness to IGF signals during the early stages of forced involution.
The IRS proteins have been shown to mediate proliferative and antiapoptotic responses in a number of different insulin/IGF-I-responsive cell types and tissues, including breast tumor cells (20, 27, 28). Analyses of the tyrosine phosphorylation state of IGF-IR or signaling molecules such as IRS-1 would presumably serve as a meaningful indicator of IGF-I bioactivity based on cell culture models (29). Although the procedures used in these studies were not sensitive enough to detect phosphorylated IGF-IR in involuting mammary tissue extracts, phosphorylation of IRS-1, IRS-2, and Akt was detectable. The fact that the phosphorylation state of these molecules showed only modest increases in the pooled extracts from WAP-DES mice was probably due to high background phosphorylation in those derived from the nontransgenics. In addition to phosphorylation by insulin or IGF-I receptors, IRS-1 and -2 could serve as substrates for JAK2-dependent phosphorylation in response to PRL or GH stimulation (30). Importantly, the level of tyrosine phosphorylation of IRS-1 declined significantly with the onset of forced involution. This decline was associated not with decreased IGF-IR abundance, but with a decrease in the abundance of IRS-1 protein. The ability to detect IRS-2 phosphorylation during forced, but not natural, involution is somewhat puzzling and remains unexplained. However, like IRS-1, a dramatic loss of IRS-2 protein occurred with the onset of forced involution.
The dramatic decrease in phospho-Akt levels during forced involution correlated with the decline in IRS-1 and -2 phosphorylation and expression and was only partially explained by a decrease in the abundance of Akt protein. This decrease is consistent with previous reports demonstrating IRS dependence of Akt phosphorylation (12, 22, 23). The presence of detectable phospho-Akt in mammary extracts during forced involution in the absence of detectable IRS phosphorylation, however, supports the possibility that other factors can mediate Akt phosphorylation in the absence of IRS proteins. The observation that phospho-Akt was higher in extracts prepared from WAP-DES mice during forced involution suggests that these other factors may be capable of mediating an IGF signal. The integrin-linked kinase has been shown to phosphorylate and activate Akt in an insulin-dependent, PI3 kinase-dependent, fashion (31). These potential IRS-independent mechanisms, however, must serve a relatively minor role, because the most dramatic changes in Akt phosphorylation correlated with the loss of IRS-1. During the course of these studies an attempt was also made to measure the abundance of both BAD and phospo-BAD as a means of determining whether the apoptotic pathway downstream of Akt was also decreased during force involution. These attempts were unsuccessful due to sensitivity limitations of the available antibodies.
The interpretation of the differences between forced and natural
involution with respect to IRS-1, IRS-2, and Akt abundance must include
two important considerations. Firstly, these decreases occurred during
a time when the mammary gland undergoes significant epithelial cell
loss. The wet weight data in Fig. 1 illustrates this loss and agrees
with previously published morphometric data that suggest that over the
course of 6 days of forced involution the mammary gland loses about
half of its epithelial cell population (10). On this
basis, the abundance of epithelial markers such as K18 would be
expected to change by 2-fold at most. The small changes in K18
abundance after 4 days of involution in the present study agree with
previously published results (10), which showed only
minimal changes in K18 abundance over 6 days of forced involution. This
is probably due to the fact that despite losses over 4–6 days of
involution the epithelium remain the predominant contributor on a mass
basis to the protein that would be obtained from a whole gland lysate.
Hence the loss of epithelium that occurs during forced involution is
far too small to account for the 10-fold loss in IRS-1, IRS-2, and
phospho-Akt. The second consideration in the comparison of natural with
forced involution is the potential for differences to exist in the rate
of epithelial cell loss between the two paradigms. Although direct
comparisons between forced and natural involution in the mouse are
limited (1), studies of lactation in a variety of species
support the suggestion that the main difference between the two
paradigms is the occurrence of transient alveolar, distension, and
diminished secretion of galactopoietic hormones (32). Data
from the present study demonstrate that overall mammary gland weight
loss during the 4 days of forced involution was similar to that which
occurred over 6 days of natural involution. Hence, the daily rate of
epithelial loss during natural involution could be estimated to be
about 66% of that observed with forced involution. This observation
coupled with the finding that K18 abundance showed little systematic
change between natural and forced involution support the conclusion
that epithelial cell loss does not account for the dramatic differences
in the abundance of IRS-1, IRS-2, and phospho-Akt. The
immunohistochemical staining for IRS-1 (Fig. 5) supports the conclusion
that the decreased IRS-1 detected by Western blotting is predominantly
due to decreased expression of IRS-1 in the epithelium. This conclusion
does not imply that IRS-1 is not expressed in adipocytes of the mammary
gland. In fact, immunohistochemical staining suggests that it is
expressed in mammary adipocytes (data not shown). However, as greater
than 97% of a typical adipocyte consists of either lipid or water, the
actual contribution of mammary adipocytes to the total protein present
in the whole gland lysates analyzed in these studies is probably small
(33). In addition, the increase in adipocyte number or
volume during involution is not associated with an increase in IRS-1
expression, suggesting that adipocytes are probably not the predominant
cell type responsible for the loss in IRS-1 expression in the present
studies.
The observation that IRS-1 and -2 mRNA abundance does not change with
forced involution supports the conclusion that these decreases in IRS-1
and -2 are due to changes in protein turnover and that the regulation
of IRS proteins in the mammary gland in vivo differs from
the steroid hormone-dependent regulation that has been documented in
cell culture models (20, 21). Immunohistochemistry
conducted on mammary tissue from mice undergoing either natural or
forced involution failed to detect the expression of ER and PR. This
observation is consistent with the findings of previous studies of
estrogen and progesterone responsiveness of the mammary gland
(34) and supports the suggestion that the
steroid-dependent regulation of IRS mRNA abundance in not involved with
the loss of IRS during mammary involution. Instead, altered IRS protein
turnover analogous to a proteasome-dependent mechanism that has
recently been described in several cell types (35, 36) is
a more likely mechanism. Investigation of this possibility is currently
in progress.
In summary, these studies highlight the existence of mechanistic differences between the regulation of natural and forced mammary gland involution and suggest an important role for key members of the IGF signaling pathway in the maintenance of IGF-I-dependent mammary cell survival. Further studies using animal models with targeted alterations in these signaling molecules will determine the mechanistic importance of IRS expression to mammary gland involution.
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Acknowledgments |
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, and PR. Thanks to Drs. Doug Burrin, Jeff Rosen, and Dan
Medina for critical review of the manuscript. |
Footnotes |
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2 Recipient of a Susan G. Komen Foundation Research Award.
Received September 8, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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 A. V. Lee, R. Schiff, X. Cui, D. Sachdev, D. Yee, A. P. Gilmore, C. H. Streuli, S. Oesterreich, and D. L. Hadsell New Mechanisms of Signal Transduction Inhibitor Action: Receptor Tyrosine Kinase Down-Regulation and Blockade of Signal Transactivation Clin. Cancer Res., January 1, 2003; 9(1): 516s - 523s. [Abstract] [Full Text]
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S. L. Grimm, T. N. Seagroves, E. B. Kabotyanski, R. C. Hovey, B. K. Vonderhaar, J. P. Lydon, K. Miyoshi, L. Hennighausen, C. J. Ormandy, A. V. Lee, et al. Disruption of Steroid and Prolactin Receptor Patterning in the Mammary Gland Correlates with a Block in Lobuloalveolar Development Mol. Endocrinol., December 1, 2002; 16(12): 2675 - 2691. [Abstract] [Full Text] [PDF]
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R. C. Humphreys, B. Bierie, L. Zhao, R. Raz, D. Levy, and L. Hennighausen Deletion of Stat3 Blocks Mammary Gland Involution and Extends Functional Competence of the Secretory Epithelium in the Absence of Lactogenic Stimuli Endocrinology, September 1, 2002; 143(9): 3641 - 3650. [Abstract] [Full Text] [PDF]
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 E. Tonner, M. C. Barber, G. J. Allan, J. Beattie, J. Webster, C. B. A. Whitelaw, and D. J. Flint Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice Development, January 10, 2002; 129(19): 4547 - 4557. [Abstract] [Full Text] [PDF]
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S. G. Bonnette and D. L. Hadsell Targeted Disruption of the IGF-I Receptor Gene Decreases Cellular Proliferation in Mammary Terminal End Buds Endocrinology, November 1, 2001; 142(11): 4937 - 4945. [Abstract] [Full Text] [PDF]
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) mice were
placed with litters of 10 pups each at 2 days postpartum. At days 16,
18, 20, and 22 postpartum, mammary glands were collected for analysis
of natural involution. Forced involution was induced at day 16
postpartum. Mammary tissue samples were then collected at days 17, 18,
19, and 20 postpartum. Wet weight was measured on one of the number 4
mammary glands. Apoptosis was detected by TUNEL staining and was
enumerated by counting positive and negative cells in 10 randomly
chosen x100 fields. *, P < 0.05.
) or WAP-DES (
) mice during either natural (A) or forced (B)
involution. The tissue concentration of des(
) was
measured using a species-specific immunoradiometric assay on mammary
tissue extracts from WAP-DES mice during natural (A) or forced (B)
involution. Each bar represents the mean ±
SEM for three or four mice. The dashed line
indicates the detection limit for the measurement of endogenous IGF-I.
Arrows show the day of peak apoptosis. The
asterisk in A indicates a statistically significant
increase in des(
) in
mammary tissue extracts during either natural (B) or forced (C) mammary
involution. Because the effect of genotype was small, the data were
pooled across genotype. Each bar represents the
mean ± SD for the two observations.
Arrows indicate the day of peak apoptosis.
