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INTRODUCTION |
Among the main regulatory elements that contribute to
transcriptional regulation of extracellular signals are the
cAMP-responsive element
(CRE)1 and activation protein
(AP-1) sequence motifs. It is increasingly accepted that the CRE site
(TGACGTCA) is recognized by a family of basic leucine zipper-containing
proteins known as CRE-binding proteins (CREB) or activating
transcription factors (ATFs), including ATF4. Because ATF binding sites
are present in several growth-regulating gene promoters, ATFs are
believed to be involved in different regulatory circuits, allowing
cells to integrate signals from distinct pathways. The mammalian ATF4
protein has been demonstrated to form heterodimers with members of the
AP-1 and C/EBP family of proteins, including Fos (1), Jun (1-3), JunD
(4), and several C/EBP proteins (C/EBP
, C/EBP
/CRP2, C/EBP, and
C/EBP
/CRP1) (5-8). ATF4 acts both as a transcriptional activator
(7, 9-14) and a transcriptional repressor (4, 15-19), presumably by
sequestering other regulatory factors away from promoters. ATF4 also
interacts with the coactivator CREB-binding protein and components of
the general transcription machinery, such as the TATA-binding protein, TFIIB and the RAP30 subunit of TFIIF (20).
Overexpression of ATF4 in murine NIH3T3 fibroblasts reduces the ability
of the ectopically expressed Ras oncogene to transform cells
as judged by cellular morphology and foci formation and has thus been
proposed to reduce transcription ability of Ras promoter
(18). ATF4-knockout mice display abnormal lens formation (21, 22) that
is at least partially the result of p53-mediated apoptosis because
deletion of the p53 gene in the ATF4-knockout background reduces the
apoptotic phenotype allowing normal lens formation (21). However, the
potential role of ATF4 in mammary gland development remains unknown.
We have shown previously that heregulin, a mesenchymal growth factor in
the mammary gland, up-regulates the expression and transactivation
function of ATF4 in breast cancer cells (14). The present study was
undertaken to analyze the consequences of ATF4 overexpression on
mammary gland development in transgenic animals. We showed that
overexpression of ATF4 in the mammary epithelium of ATF4-transgenic
mice decreased proliferation and impaired differentiation of alveolar
epithelial cells during pregnancy and lactation. In addition, ATF4
overexpression induced apoptosis and accelerated involution of the
mammary gland, suggesting a role for ATF4 in the regulation of normal
mammary gland involution.
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EXPERIMENTAL PROCEDURES |
Generation of Transgenic Mice--
A FLAG-tagged ATF4 cDNA
was digested with SalI, blunt ended, and subcloned into the
EcoRI site of the pBJ41 vector under the control of an ovine
-lactoglobulin promoter (23). Transgenic mice with a B6DF1/J
genetic background were produced by injecting purified DNA fragments
into the pronucleus of fertilized oocytes in B6DFI/J mice as described
previously (23). The founder mice were genotyped using Southern
blotting with 20 µg of tail DNA digested with BamHI. Mice
with exogenous genes were bred with the same strain but recorded as
different lines. Mice from the F5-F6 generation were used for phenotype
analysis. PCR analysis was performed to amplify a 330-bp sequence
between the FLAG tag and the ATF4 cDNA using the forward primer
5'-GACTACAAGGACGACGATGACAAGAT-3' and the reverse primer
5'-AAAGATCACATGTGTCATCCAACG-3'. To determine the pregnancy stage, the
mice were mated and inspected for the presence of vaginal plugs in the
morning. The day of the vaginal plug was counted as day 0 of pregnancy.
Antibodies--
Monoclonal anti-FLAG (M2) was obtained from
Sigma. Anti-ATF4 (C-19), anti-Bax (N-20), and anti-STAT3 (C-20)
were purchased from Santa Cruz Biotechnology. Anti-Bcl-2 (clone 124)
was obtained from Dako, anti-phospho-STAT3-Tyr-705 and anti-ATF2 were
obtained from New England Biolabs, anti-phospho-STAT5a was obtained
from Zymed Laboratories Inc., and anti-keratin 18 KS18.04 was purchased from Progen. Polyclonal STAT5a was generous gift
from the laboratory of Dr. Nicolas J. Donato. Mouse IGFBP-5 polyclonal
antiserum was obtained from Gro Pep. Anti-BrdUrd (Ab-2) was obtained
from Neomarker. Secondary antibodies were peroxidase-labeled anti-mouse
and anti-rabbit, as appropriate (Amersham Biosciences), diluted 1:2,000
or biotinylated goat anti-rabbit antibody diluted 1:100 from Vector
Labs, Inc.
Histological and Morphological Analysis--
For histological
analysis, mammary gland tissue was fixed in 10% neutral buffered
formaldehyde and embedded in paraffin by standard methods. Sections,
each 4 µm thick, were stained with hematoxylin and eosin. For whole
mount analysis, mammary glands were stained with carmine and aluminum
as described previously (23). Briefly, the glands were fixed with
acetic acid/ethanol (1:3) for 2 h and stained with 0.5% carmine
and 0.2% aluminum potassium sulfate for 16 h. After briefly being
rinsed with distilled water, the mammary glands were dehydrated using
graded ethanol, and lipids were removed with two changes of acetone.
Finally, the glands were preserved in methyl salicylate.
Immunohistochemical Methods--
Immunohistochemical detection
of STAT5a phosphorylated at Tyr-694 was performed as described
previously (36). Briefly, deparaffinized tissue sections were treated
with 1 µg/ml proteinase K (Sigma) for 15 min at 37 °C to expose
phosphorylated STAT5a epitopes. Endogenous peroxidase was inactivated
by incubating the sections in 0.3% H2O2 in
phosphate-buffered saline for 20 min at room temperature. Anti-phospho-STAT5 antibody was diluted to 10 µg/ml and detected by
biotinylated goat anti-rabbit antibody diluted to 15 µg/ml. Phosphate-buffered saline containing 10% goat serum was used to block
sections and dilute antibodies. Immunostained sections were lightly
counterstained in hematoxylin according to the manufacturer's instructions, dehydrated in graded ethanol, cleared in xylene, and
mounted on a coverslip with Permount (Fisher Scientific Co.). For
immunostaining of STAT3 phosphorylated at Tyr-705, deparaffinized sections were subjected to antigen retrieval by heating in 10 mM citric acid buffer, pH 6.0, and antibody was diluted
1:50. For immunostaining of the FLAG tag and IGFBP-5, sections were treated with 1 µg/ml proteinase K, and the remainder of the procedure was performed as described previously (24). FLAG tag monoclonal antibody was diluted 1:50, and IGFBP-5 was diluted 1:250.
BrdUrd Labeling and TUNEL Assays--
A sterile solution of 20 mg/ml BrdUrd (Sigma) in phosphate-buffered saline, pH 7.4, was
administered to mice by intraperitoneal injection (50 mg/kg). Mammary
glands were harvested after 3 h, embedded in paraffin, and
sectioned. BrdUrd incorporation was detected by immunohistochemistry
using a mouse anti-BrdUrd monoclonal antibody as described previously
(25). Apoptosis was detected in paraffin sections by TUNEL analysis
with terminal deoxynucleotidyl transferase (Roche Diagnostics) as
described previously (26). Ten random fields/section were
documented by photomicroscopy, and the percentage of TUNEL-positive
epithelial cell nuclei relative to the total number of epithelial cell
nuclei was calculated. Mean values were determined from results
from at least six different mice.
Caspase-3 Assay--
The caspase-3 assay (R&D Systems Europe
Ltd., Abington, UK) was based on the hydrolysis of the peptide
substrate acetyl-Asp-Val-Asp p-nitroanilide resulting in the
release of the p-nitroaniline moiety. This was quantified
spectrophotometrically at 405 nM. Tissues were homogenized
in the lysis buffer provided (250 mM Hepes, pH 7.4, 1%
CHAPS, 50 mM dithiothreitol, 20 mM EDTA).
RT-PCR and Northern Blot Analysis--
RT-PCR was performed
using the Access Quick reverse transcription RT-PCR system (Promega) as
described previously (27). RNA was extracted from frozen tissue using
TriZol reagent (Invitrogen), denatured, analyzed on a 1% agarose gel
containing 6% formaldehyde, and transferred to a nylon membrane.
Probes for the Northern blots were radiolabeled using random primers
(Invitrogen) and [32P]dCTP (PerkinElmer Life Sciences).
Immunoblot Analysis--
Protein was extracted from frozen
tissue by homogenization in 1% Nonidet P-40 buffer (20 mM
Tris, pH 7.4, 100 mM NaCl, 10 mM NaF, 10%
glycerol, 5 mM sodium orthovanadate, and protease inhibitor
mixture (Sigma)). Total protein (20-200 µg) was separated on a 10%
SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, and
immunoblotted with various antibodies. Bound antibodies were detected
using horseradish peroxidase-conjugated secondary antibody and the ECL
system (Amersham Biosciences) according to the manufacturer's
recommendations. Densitometry analysis was performed using a computing
densitometry, and proteins were quantitated from the images using Sigma
gel software.
Statistical Analysis--
Results are expressed as the mean ± S.E. Statistical analysis of the data was performed using Student's
t test.
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RESULTS |
ATF4 Expression and Activity during Mammary Gland
Development--
To investigate whether ATF4 has any role in normal
mammary gland development, we first examined the level of ATF4 and ATF2 protein in wild type mammary glands using Western blot analysis. As
shown in Fig. 1A, top
panel, ATF4 expression levels were high in virgin and pregnant
glands but were dramatically lower during lactation and higher after
weaning. ATF2 was expressed in pregnant glands (Fig. 1A,
middle panel). Paxillin was used as a control (Fig.
1A, bottom panel).

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Fig. 1.
ATF4 expression during murine mammary
gland development. A, total tissue lysates were
immunoblotted with anti-ATF4, and the membranes were stripped
and reprobed with anti-ATF2 antibody. Paxillin was used as a control.
The abbreviations are: Vir, virgin; P, pregnancy;
L, lactation; and PW, postweaning. B,
schematic showing the ATF4 transgene construct. The transgene contains
the -lactoglobulin promoter and FLAG epitope-tagged ATF4 cDNA
followed by a poly(A) tail from the -lactoglobulin gene.
C, Southern blot detection of the ATF4 transgene in the tail
genomic DNA of transgenic and wild type (WT) littermates of
F2 from lines 23, 24, and 25 as indicated. D, ATF4
expression of three transgenic lines was analyzed by Western blotting
FLAG antibody. E, the time course of ATF4 transgene
expression in line 23 transgenic mice and the ratio of transgene ATF4
expression to endogenous ATF4 levels were analyzed by RT-PCR followed
by Southern blotting. F, expression of the ATF4 transgene in
the mammary gland from 12 day lactation, as analyzed by
immunohistochemistry using anti-FLAG antibody. Note that the transgene
is located in the nucleus of the luminal epithelial cells.
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Generation and Analysis of ATF4-transgenic Lines--
A
FLAG-tagged ATF4 mouse cDNA was cloned into the pBJ41 vector under
the control of an ovine
-lactoglobulin promoter, which has been
shown to confer tissue-specific, hormonally regulated expression of
heterologous genes in the mammary epithelials cells of transgenic mice
during pregnancy and lactation (28) (Fig. 1B). Six founders
with transgene integration were identified by PCR, and results were
confirmed by Southern blot. These founders were then bred with the wild
type mice to generate females to test expression of ATF4. A
representative Southern blot derived from BamHI-digested
genomic DNA are shown for the three founders (TG23, TG24, TG24) in Fig.
1C. The mice from founders TG23, TG24, and TG25 expressed
the transgene protein product, as detected by Western blotting of
mammary extracts with FLAG antibody, which specifically recognizes the
FLAG epitope tag present in the transgene product and does not react
with endogenous ATF4 (Fig. 1D). These lines of mice varied
in their levels of ATF4 expression, thus permitting dose responsiveness
of the phenotype to be assessed. One line, TG23, was selected for
further analysis because it had the highest levels of protein expression.
The level of ATF4 expression in the various stages of mammary gland
development was determined using semiquantitative RT-PCR. RNA
expression was compared between aged-matched wild type and transgenic
glands (TG23) at days 10 and 15 of pregnancy, days 2 and 12 of
lactation, and days 1, 3, and 7 of involution (Fig. 1E). The
ratio of transgene ATF4 to endogenous ATF4 was 2-fold at day 10 of
pregnancy, 1.6-fold at day 15 of pregnancy, increased to 8.2-fold at
day 2 of lactation and 5.2-fold at day 12 of lactation, and reduced at
day 7 of involution (Fig. 1E).
To establish the cellular localization of ATF4, we performed
immunohistochemistry on sections of mammary glands from wild type and
ATF4-transgenic animals using anti-FLAG antibody to detect the
expression of FLAG-tagged ATF4 transgene. Samples from day 2 of
lactation showed ATF4 expression in both the nucleus and the cytoplasm
of epithelial cells lining the alveoli (Fig. 1F).
ATF4 Regulates Proliferation and Differentiation of the Mammary
Alveolar Epithelium--
To evaluate whether ATF4 affected alveolar
development, we studied mammary gland structures in pregnant wild type
and transgenic mice. Analysis of both whole mounts and hematoxylin and
eosin-stained sections during pregnancy revealed that ductal branching
and alveolar development were already impaired by day 10 of pregnancy
(compare Fig. 2, A and
C with B and D). The wild type mammary
glands had sprouted many new side branches of alveoli (Fig.
2A). In contrast, the mammary glands from ATF4-transgenic
mice had developed few new lateral branches with little alveolar
development (Fig. 2B). The morphological differences between
wild type and transgenic animals were increased at days 15 and 18 of
pregnancy and consistently showed decreased lobuloalveolar development
in ATF4-transgenic mice (compare Fig. 2, E and I
with F and J). In addition to their size, alveoli
in wild type at day 18 of pregnancy contained copious amount of lipid,
whereas those of ATF4 mice did not (Fig. 2, K and
L).

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Fig. 2.
Impaired mammary gland development in
ATF4-transgenic mice during pregnancy and lactation. Whole mount
analysis of mammary glands by carmine aluminum staining in wild type
mice (WT) and ATF4-transgenic mice (TG23) at day 10 (A and B), day 15 (E and
F), and day 18 (I and J) of pregnancy
(P) and lactation (Lac) day 2 (M and
N) is shown. Hematoxylin and eosin staining of wild type and
ATF4-transgenic mice is shown at day 10 (C and
D), day 15 (G and H), and day 18 (K and L) of pregnancy. A-L
magnification, ×200.
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The alveolar density in the fat pads of transgenic mice from three
different founder lines was severely impaired (Fig.
3A). A significantly greater
area was occupied by adipocytes in glands from ATF4-TG23 (24 ± 4%) compared with the wild type mice (4 ± 1%) during pregnancy
day 15 (Fig. 3B).

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Fig. 3.
Defective proliferation of pregnant ATF4
mammary epithelial cells. A, whole mount staining of
mammary gland in wild type (WT) and three ATF4-transgenic
lines at day 15 of pregnancy. B, quantification of adipocyte
area of mammary gland. Each bar represents the mean of data
collected from three mice. C, BrdUrd staining of mammary
glands from wild type and three ATF4-transgenic lines at day 10 of
pregnancy. D, BrdUrd labeling indices of mammary tissue
sections from 10- and 15-day pregnant (P) wild type mice and
three independent line of ATF4-transgenic mice. 300-400 epithelial
cell nuclei were examined from each section of wild type and
ATF4-transgenic glands. The values represent the average fraction of
BrdUrd-positive epithelial cells/total number of epithelial cells of
three different mice.
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In addition to the abnormalities observed at day 18 of pregnancy,
decreased lobuloalveolar development was observed in ATF4 females at
day 2 of lactation (compare Fig. 2, M and N).
To explore the mechanism of impairment of mammary gland development we
examined rates of BrdUrd incorporation on days 10 and 15 of pregnancy,
when proliferation is high. Wild type and ATF4-transgenic mice from
three independent founder lines at different developmental stages were
pulse labeled with BrdUrd and then sacrificed. The percentage of
BrdUrd-positive epithelial cells was determined by quantitative
analysis of anti-BrdUrd-stained sections (Fig. 3D). Cell
proliferation was reduced in ATF4-transgenic mice throughout pregnancy
(Fig. 3C). At day 10 of pregnancy, it was reduced
significantly up to 85% of the rate observed in wild type mice, and it
remained low until day 15 (Fig. 3D).
ATF4 Overexpression Up-regulates Cyclin-dependent
Kinase Inhibitors--
To investigate the molecular events underlying
this proliferation defect in ATF4 mammary epithelium, we analyzed the
expression levels of cell cycle regulators in early and midpregnancy.
The cyclin-dependent kinase inhibitor p21WAF1
and p27Kip1, which play important roles in cell cycle
regulation, were chosen (29). The p21WAF1 protein was
expressed at 2-8-fold higher levels in ATF4-transgenic mice than in
wild type mice (Fig. 4, A and
B). The p27Kip1 protein, which is regulated by
translational and post-translational mechanisms (30-32), was
up-regulated 3-7-fold in ATF4-transgenic mammary tissues compared with
that of wild type gland 3-6-fold (Fig. 4, A and
B). Thus, the proliferation defect of ATF4-transgenic mammary epithelial cells was accompanied by up-regulation of
p21WAF1 and p27Kip1.

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Fig. 4.
Up-regulation of p21WAF1 and
p27Kip1 in ATF4-transgenic mice. A, Western
blot analysis of p27Kip1, p21WAF1, and keratin
18 (K18) at day 15 of pregnancy. WT, wild type.
B, densitometry for four independent mice; values ± S.E. were calculated as the percentage of highest value for each blot.
In the graph on the right, values relative to keratin 18 were calculated as a proportion of the mean (hence, no error bars are
included).
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ATF4 Overexpression Impairs Lactation--
At parturition, litters
were adjusted to eight in number. Pups were weighed daily to monitor
growth; if losses occurred, replacement pups were added to maintain
litter size. Littermates from ATF4-transgenic mice exhibited a 40-50%
lower body weight than did wild type mice (Fig. 5A). At day
12, the mean weight of transgenic mice was 1.7 ± 0.3 g, and
that of wild type mice was 5.0 ± 0.63 g. At 3 weeks, mean
weights were 4.88 ± 0.25 g for ATF4-transgenic mice and
9.76 ± 0.8 g for wild type mice.
The dramatic changes in epithelial differentiation which occur in the
mammary gland during lobuloalveolar development are reflected at the
molecular level by tightly regulated and temporally ordered milk
protein expression (33). To determine whether ATF4-transgenic mice
manifested a defect in epithelial differentiation, we determined mRNA expression levels for a panel of markers of mammary gland differentiation (lactoalbumin, WAP, and
-casein) in
mammary glands from ATF4-transgenic and wild type mice during
lactation. Northern blot analysis showed significantly lower levels of
-lactoalbumin, WAP, and
-casein mRNAs in the mammary
glands from the transgenic than in those from the wild type mice at day
12 of lactation (Fig. 5B). In
contrast, expression levels of the epithelial cell marker keratin
18 did not differ significantly between mammary glands from wild type
and ATF4-transgenic mice.

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Fig. 5.
Impaired lactation in ATF4-transgenic
mice. A, size of transgenic mice that overexpress ATF4
and nontransgenic littermates at day 12 and 21. WT, wild
type. B, Northern analysis of gene expression for epithelial
differentiation markers (lactoalbumin, WAP, -casein) in the mammary
gland of wild type or ATF4-transgenic animals at day 12 of lactation.
C, immnohistochemical localization of phospho-STAT5a in
mammary glands obtained from wild type and ATF4-transgenic mice on
lactation day 12. D, quantitation of phospho-STAT5a
staining. E, immunoblot analysis of phospho-STAT5a and
vinculin in wild type and ATF4-transgenic mice at day 12 of
lactation.
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Phosphorylation of STAT5a also has been shown to be tightly linked to
mammary gland differentiation and milk protein expression (34,
35). To determine whether the inability to transcribe milk
protein genes correlated with reduced phosphorylation of STAT5a, we
examined immunohistochemical localization of phospho-STAT5a in the
lactating mammary gland in transgenic and wild type mice (36). As shown
in Fig. 5, C and D, the STAT5a tyrosine
phosphorylation was significantly lower in mammary glands from
ATF4-transgenic mice than those from matched wild type mice at day 12 of lactation. The similar results were obtained by Western blotting
using phospho-STAT5a antibody (Fig. 5E).
Accelerated Mammary Gland Involution in ATF4-transgenic
Mice--
In addition to the anomalies observed during pregnancy of
ATF4-transgenic mice, decreased lobuloalveolar development was observed at lactation. Fig. 6 shows hematoxylin
and eosin-stained sections of ATF4 and wild type mammary glands during
lactation and involution. At days 2, 12, and 18 of lactation, the
majority of the wild type gland was composed of alveoli lined by
epithelial cells that secrete milk components into the alveolar lumina
(Fig. 6, A, C, and E). The well
organized, secretory lobuloalveolar structures characteristic of
lactating animals remained intact for the 1st day after weaning (Fig.
6G). By days 2 and 12 of lactation, the mammary glands of ATF4-transgenic mice had fewer large alveolar distanced structures compared with those of wild type mice, and some of the structure was in
a in collapsing state and had smaller luminal spaces (Fig. 6,
B and D). By day 18 of lactation and the 1st day
of involution, the glands from transgenic mice had extensive tissue
remodeling; some of the lobuloalveolar structure had collapsed, and the
adipocytes reappeared (Fig. 6F).

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Fig. 6.
Involution is accelerated in mammary glands
of ATF4-transgenic mice. Shown is hematoxylin and eosin staining
of mammary glands of wild type (WT) and ATF4-transgenic mice
at day 2 (A and B), day 12 (C and
D), and day 18 (E and F) of lactation
(Lac) and at day 1 (G and H), day 3 (I and J), and day 7 (K and
L) of involution (postweaning; PW).
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At day 3 of involution, most of the lobuloalveolar structures in the
mammary glands of wild type mice had also collapsed, leaving mainly
ducts, vessels, and clusters of epithelials cords, some with small
lumina. Adipocytes, which constitute most of the tissue in a resting
gland, reappeared (Fig. 6I). In contrast, the alveolar
structures in ATF4-transgenic mice were remodeled with occasional
epithelial cords and ducts remaining, and surrounding stroma and
adipocytes (Fig. 6J). Taken together, our results suggest that the mammary glands from ATF-transgenic mice at day 18 of lactation
were phenotypically similar to those of wild type mice at day 3 of
involution (compare Fig. 6, I and F). Thus,
involution is accelerated in ATF4-transgenic mice.
Involution is characterized by apoptosis of epithelial cells that can
be distinctly identified by condensed chromatin (37). Apoptotic cells
are shed into the lumina and the lobuloalveolar structure, where they
usually decrease in size and with their condensed chromatin becoming
encapsulated into apoptotic bodies that are phagocytosed by neighboring
cells. To assess whether the observed acceleration of involution in the
mammary gland of ATF4-transgenic mice was caused by apoptotic cell
death, we performed TUNEL assays on lactating and involuting mammary
epithelia from wild type and ATF4-transgenic mice. Apoptotic cells were
nearly absent on days 2, 12, and 18 of the lactating mammary gland of wild type mice (Fig. 7, A,
C, and E). The mean percentage of apoptotic cells
in ATF4-transgenic mice by day 2 of lactation (5.74 ± 2.7) was
similar to that of wild type mice at day 2 of involution (5.68 ± 1.2) (Fig. 7G). Apoptosis peaked at day 3 of involution in
the mammary gland of wild type mice (6.45 ± 2) and at day 2 of
involution in the mammary gland of ATF4-transgenic mice (Fig.
7G). In wild type mice the number of apoptotic cells
decreased at day 7 of involution when most of the gland had been
remodeled; in transgenic mice, apoptosis was decreased by day 3 of
involution (Fig. 7G).

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Fig. 7.
Increased apoptosis of mammary epithelial
cells during lactation in ATF4-transgenic mice. Shown is TUNEL
staining of apoptotic cells in the mammary glands of wild type
(WT) and ATF4-transgenic at day 2 (A and
B), day 12 (C and D), and day 18 (E and F) of lactation (Lac). The
percentage of TUNEL-positive nuclei was calculated as the number of
TUNEL-positive nuclei/number of total nuclei (G). Values
presented are the mean of a total of 10 fields from each section
analyzed. Data are representative of six mice. Error bars
represent ±S.D. from the mean.
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The increased apoptosis in ATF4-transgenic mice prompted us to
investigate the levels of apoptosis-regulating proteins. Bax, an
inducer of apoptosis (38), is up-regulated at the start of involution
and is thought to act as an apoptotic signal for epithelial cells
(57). Bax levels were increased in mammary glands from ATF4-transgenic mice compared with wild type mice at lactation days 12 and 18 (Fig. 8A). Compared
with relative levels of keratin 18, the expression level of Bax at day
12 of lactation in ATF4-transgenic mice was 100-200-fold higher in
ATF4-transgenic mice than in age-matched wild type mice. Bcl-2 levels
promotes cell survival and tumor formation in transgenic mice (38). The
levels of Bcl-2 were lower during lactation and involution in mammary
glands from ATF4-transgenic compared with the levels present in
age-matched mammary glands from wild type mice (Fig. 8B). In
addition, during lactation, when milk production was most strongly
impaired in ATF4-transgenic mice, caspase-3 activity was increased
significantly (Fig. 9).

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Fig. 8.
Regulation of apoptosis-related proteins
during lactation and involution. Western blot analysis of Bax
(A), keratin 18 and Bcl2 (B) at days 12 and 18 of
lactation (Lac) and days 1, 3, and 7 of postweaning
(PW). The bottom graph shows results of
densitometry for three independent mice; the value ± S.E. was
calculated as the percentage of the highest value for each blot. Values
relative to keratin 18 were calculated as a proportion of the mean
(hence; no error bars are included). Solid bars, wild type
(WT) mice; open bars, ATF4-transgenic
(ATF4-TG23) mice.
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Fig. 9.
Caspase-3 activity in mammary homogenates of
wild type or transgenic females on day 2, 12, or 18 of lactation
(Lac). Values are the mean ± S.E. of three
to seven mice.
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Activation of STAT3 in ATF4-transgenic Mice--
Earlier studies
have shown that STAT3 has a role in the normal programming of apoptosis
and involution in the mammary gland and that it targets IGFBP-5 during
induction of involution (39). Once phosphorylated on the specific
tyrosine residue (Tyr-705), STAT3 translocates to the nucleus and
interacts with consensus promoter sequences to regulate transcription
from target genes (40). STAT3 is activated at the start of involution
(41), which is characterized by removal of epithelial cells by
apoptosis (37, 42). On the basis of this information, we next
sought to determine the status of STAT3 phosphorylation during
lactation in ATF4-transgenic mice. Mammary glands from ATF4-transgenic
mice showed elevated levels of phosphorylated STAT3 at day 18 of
lactation compared with the wild type mice (Fig.
10A). Immunostaining of mammary glands at day 18 of lactation also revealed a significant elevated level of phosphorylated STAT3 in the nucleus of epithelial cells lining the alveoli of ATF4-transgenic mice compared with the
alveoli of wild type mice (Fig. 10, B and C).

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Fig. 10.
Activation of STAT3 in ATF4-transgenic
mice. A, immunoblot analysis of phospho-STAT3, STAT3,
and vinculin at day 18 of lactation in mammary glands of wild type
(WT) and ATF4-transgenic mice. B, quantitation of
staining. Each bar represents the mean of data collected
from four mice. Error bars represent ± S.E. of the
mean. C, immunohistochemistry for phospho-STAT3 at day
18 of lactation in wild type and ATF4-transgenic mice.
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Up-regulation of IGFBP-5 during Lactation in ATF4-transgenic
Mice--
During mammary involution, when serum prolactin levels
decline, IGFBP-5 expression is dramatically up-regulated, and IGFBP-5 binds with high affinity to IGF-I, preventing IGF-I from interacting with the IGF-I receptor and acting as a survival factor (43). Recent
study demonstrated that IGFBP-5 induces premature cell death in the
mammary gland of transgenic mice (25). To establish whether IGFBP-5
levels were altered in ATF4-transgenic mice, we determined IGFBP-5
mRNA levels by Northern blotting in aged-matched mammary glands
from wild type and transgenic mice during day 12 of lactation. The
level of IGFBP-5 mRNA was significantly up-regulated (3-5-fold) in
ATF4-transgenic mice compared with wild type mice (Fig.
11A). Consistent with the
up-regulation of IGFBP-5 mRNA in ATF4-transgenic mice, the
transgenic also had higher levels of IGFBP-5 expression in epithelials
cells during lactation than did wild type mice as assessed by
immunostaining with an anti-IGFBP-5 monoclonal antibody (Fig.
11B).

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Fig. 11.
Up-regulation of IGFBP-5 during lactation in
ATF4-transgenic mice. A, Northern blot analysis of
IGFBP-5 at day 12 of lactation in wild type (WT) and
ATF4-transgenic mice. Results were normalized to GAPDH (lower
panel). B, immunohistochemical detection of IGFBP-5 in
mammary glands of wild type and ATF4-transgenic mice at day 12 of
lactation.
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DISCUSSION |
The present study describes the phenotypes of transgenic mice with
deregulated expression of ATF4 in the mammary gland during pregnancy
and lactation. The data presented suggest that 1) expression of ATF4 in
the mammary gland is regulated during development with the highest
expression during virgin and pregnancy and the lowest expression during
lactation; 2) ATF4 overexpression disrupts normal lobuloalveolar
development during pregnancy and lactation, specifically, decreasing
epithelial cell proliferation during pregnancy and impairing alveolar
cell differentiation throughout pregnancy and lactation; and 3)
deregulated expression of ATF4 accelerates involution accompanied by an
increase in apoptosis of epithelial cells during lactation.
Results from BrdUrd incorporation experiments indicated that ATF4
overexpression in the mammary gland resulted decreased cell proliferation at early and late phases of pregnancy. Proliferation of
mammary epithelial cells during pregnancy reaches its maximum in the
early phase, coinciding with high concentrations of serum hormones such
progesterone (44). The high levels of p21WAF1 and
p27Kip1 expression suggested that activation of
cyclin-dependent kinase inhibitors might be responsible for
the attenuated cell proliferation in ATF4-transgenic mice during
pregnancy. Thus the defect identified in ATF4-transgenic mice suggests
that the function of ATF4 in pregnancy involves inhibiting the cell
cycle progression of mammary epithelial cells. During the early stages
of pregnancy the mammary tissue of ATF4 mice exhibits a decrease in the
number of alveolar epithelial cells compared with that of their WT
littermates. This reduction is accompanied by a significant defect in
lactation such that ATF4 mice exhibit growth retardation. Furthermore,
the ATF4-expressing lobuloalveoli are less differentiated than wild type glands, as evidenced by reduced expression of differentiation markers (
-casein,
-, and WAP) in ATF4-transgenic animals during lactation.
Several genes and signaling pathway that control alveolar development
have been identified, including prolactin receptor, STAT5 (45), ErbB2
(46) and ErbB4 (36), cyclinD1 (47, 48), C/EBP
(49, 50), the
osteoclast differentiation factor RANKL, and its receptor RANK (51).
Data from these mouse models suggest that alveologenesis is a complex
process requiring the functional cooperation of numerous molecules.
Interestingly, comparable phenotypes were observed in some of these
mice, lack of alveolar development. ErbB2 resulted in condensed alveoli
and reduced luminal secretion at parturition (46). ErbB4 dominant
negative epithelium formed condensed alveoli and failed to expand at
midlactation, which correlated with reduced expression of
-lactoalbumin and WAP and loss of STAT5 activity. The ErbB3 and
ErbB4 ligand heregulin up-regulates ATF4 mRNA levels in breast
cancer cells (14). Furthermore, targeted expression of a heregulin
transgene causes persistence of terminal end bud and late development
of mammary adenocarcinomas, suggesting that heregulin inhibits signals
that normally lead to terminal differentiation (52). Because
ATF4-overexpressing mammary epithelia remain poorly differentiated, it
is possible that ATF4 acts downstream of the ErbB receptor signaling
pathway to control proliferation and differentiation of the mammary
alveolar epithelium. ATF4 also inhibited STAT5a phosphorylation at
Tyr-694, suggesting that STAT5a is an important downstream mediator of ATF4.
Similar to the models described here, C/EBPb null mice possess
undifferentiated alveolar epithelium; in contrast, branching morphogenesis was also impaired. In C/EBPb mutant mice at term expression of the WAP, WDNM1, or
-casein was virtually undetectable. In ATF4-overexpressing mice, milk protein expression were
down-regulated during midlactation. It has been shown that ATF4 can
form heterodimers with C/EBP
proteins (53). It is likely that the
heterodimers are unable to bind the C/EBP consensus site and
consequently inhibit C/EBP-mediated transcriptional activation. Thus,
down-regulation of ATF4 during lactation may be essential to induce the
expression of milk protein.
ATF4-overexpressing mice exhibited increased levels of apoptosis during
lactation and accelerated involution compared with the wild type mice,
suggesting that down-regulation of ATF4 during lactation acts an
essential survival signal for the gland. Further support for a role of
ATF4-mediated apoptosis in the mammary gland is the increased
expression of Bax and decreased expression of Bcl-2 during late stages
of lactation in ATF4-transgenic mice. Caspase-3 activity was increased
significantly during lactation in transgenic mice, providing strong
evidence that expression of ATF4 was able to promote inappropriate
apoptosis and extracellular remodeling at this time similar to that
which occurs during normal mammary involution. In addition, STAT3 is
activated at the end of lactation in ATF4-transgenic mice, which raises
the possibility that ATF4 might act as an upstream effector of STAT3.
Studies of the conditional knockout of STAT3 demonstrated that
activation of STAT3 at the start of involution acts as an essential
death signal for the gland, and IGFBP-5 is a direct or indirect target of STAT3 (39).
IGF-I is a potent mitogen for epithelial cells and has been shown to be
a survival factor in vitro. Transgenic animal studies have
shown that overexpression of IGF-I in the mammary gland delays involution (54, 55), thus IGF-I could act as an important survival
factor for mammary epithelial cells. During involution, epithelial
cells synthesize and secrete high levels of IGFBP-5 (43). IGFBP-5 has
been proposed to induce apoptosis by sequestering IGF-I to casein
micelles, thus preventing it from binding to its receptor (25).
Prolactin may act by suppressing the production of IGFBP-5 from the
mammary epithelium and inhibit apoptosis (56). Recently, Tonner
et al. (25) provided evidence for a causal relationship
between IGFBP-5 and apoptosis by producing transgenic mice
expressing IGFBP-5 from a mammary-specific promoter
-lactoglobulin. Overexpression of IGFBP-5 can lead to impaired mammary development, increased expression of proapoptotic molecule caspase-3, increased plasmin generation, and decreased expression of prosurvival molecules of Bcl-2 family. In addition, IGFBP-5 expression is reduced and involution delayed in STAT3 knockout mice, whereas in interferon regulatory factor-1 knockout mice involution and IGFBP-5
expression are both accelerated (39). We showed that IGFBP-5 is
up-regulated during midlactation (day 12) in ATF4-transgenic animals.
Morphologically, the mammary glands of ATF4-transgenic mice showed an
accelerated involution that corresponded to acceleration in the onset
of apoptosis. ATF4 overexpression in transgenic mice also increased
apoptosis rates during lactation.
In light of these findings, we hypothesized that high levels of IGFBP-5
in the lactating mammary glands of ATF4-transgenic mice would decrease
the biological potency of IGF-1, which in turn would induce apoptosis
and accelerate involution. In this study we provide the first evidence
that ATF4 inhibits cellular proliferation and induces cell death when
expressed in the mammary gland of transgenic mice and may play a role
in the normal program of apoptosis and involution in the mammary gland.
We propose that one of the targets of ATF4 in the induction of
involution could be IGFBP-5. Further studies will focus on the in
vivo and in vitro models to unravel further the
mechanism of action and targets of ATF4 in this process.