1 Laboratory of Protein Dynamics and Signaling, Center for Cancer Research,
National Cancer Institute, Frederick, MD 21702-1201, USA
2 Laboratory of Pathology, National Cancer Institute, National Institute of
Health, Bethesda MD 20892, USA
3 Laboratory of Genetics and Physiology, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD
20892, USA
Author for correspondence (e-mail:
sterneck{at}ncifcrf.gov)
Accepted 19 August 2005
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SUMMARY |
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Key words: Mammary gland, Involution, C/EBP, Apoptosis, Mouse
![]() |
Introduction |
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Another gene with reduced expression in both LIF and STAT3-deficient
mammary glands encodes the transcription factor C/EBP (CEBPD, CRP3,
CELF, NF-IL6ß) (Kritikou et al.,
2003
). C/EBP
is a member of the C/EBP family of
transcription factors, which dimerize and bind DNA through a highly homologous
basic region leucine zipper domain. Each of the five C/EBP proteins has unique
properties regulating cell-type-specific growth and differentiation. For
example, within the hematopoietic system, C/EBP
is required for the
development of granulocytes, while lack of C/EBPß affects differentiation
of the B-cell lineage and monocytes
(Takiguchi, 1998
). Expression
of C/EBP
is typically low to undetectable in most cell types and
tissues, but is rapidly induced by a variety of extracellular stimuli, e.g.
growth hormone, insulin, IFN
, IL1, IL6, LPS, TNF
, noradrenalin
and glutamate (Ramji and Foka,
2002
). In-vitro and in-vivo studies have implicated C/EBP
in inflammatory responses (Takiguchi,
1998
), proliferation of osteoblasts
(Umayahara et al., 1997
),
growth arrest in certain cancer cell lines
(Gery et al., 2005
;
Ikezoe et al., 2005
) and
differentiation of lung epithelial cells
(Cassel et al., 2000
). Thus,
C/EBP
appears to have highly diverse functions, depending on cell type
and specific physiological stimuli. However, C/EBP
-deficient mice
display no overt phenotype, are fertile and achieve normal life spans. The
null mutation leads to altered learning and memory functions
(Sterneck et al., 1998
) and
increased mammary ductal branching, specifically in nulliparous outbred mice
(Gigliotti et al., 2003
). It
also exacerbates the differentiation defect of C/EBPß-deficient
adipocytes in vitro (Tanaka et al.,
1997
) and causes genomic instability in embryonic fibroblasts
(Huang et al., 2004
).
In the mammary gland, the C/EBP gene is activated transiently at the
onset of involution (Gigliotti and
DeWille, 1998
), but its function in this process has hitherto not
been elucidated. Several reports analyzing immortalized mammary epithelial
cell lines in vitro demonstrate that expression of C/EBP
is induced
during growth arrest and document that C/EBP
participates specifically
in the growth arrest response of MEC lines in vitro (Dearth and DeWille). One
study also reported reduced cell viability under growth arrest conditions when
MEC lines were stably transfected with a C/EBP
expression construct
(O'Rourke et al., 1999
).
The present study addresses the role of C/EBP in mammary epithelial
cells in vivo and demonstrates that C/EBP
participates in the
initiation of physiological cell death responses in MEC as a crucial target of
STAT3. These data identify a novel role for C/EBP
in the initiation of
cell death, and shed light on the signaling cascade from the activation of
STAT3 to tissue remodeling during the second phase of involution.
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Materials and methods |
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Stat3 fl/fl;WC mice
Mice carrying a Stat3 gene with LoxP-flanked exons 16-21
(Stat3 fl/fl) were bred to mice carrying the Cre transgene under
control of the WAP gene promoter
(Humphreys et al., 2002).
Stat3 fl/fl mice without Cre were used as controls. Litter sizes were
maintained at six to eight pups. All animals were housed and handled according
to approved protocols established by the NIDDK Animal Care and Use Committee
and NIH guidelines.
Cell culture
HC11 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum,
10 ng/ml epidermal growth factor and 10 µg/ml insulin (Invitrogen). For
transient transfections, 0.7 x106 cells were plated per 10-cm
dish, transfected the next day with 10 µg expression constructs plus 3
µg pEGFP-N1 and 20 µl Fugene 6 (Roche). Cells were collected 2 days
later for analysis.
Constructs
To construct wild-type C/EBP fused to EGFP, the full-length mouse
C/EBP
coding region was amplified by PCR from a pMEX expression
construct (Colangelo et al.,
1998
) with specific primers
(5'-CAAGCTAGCATGAGCGCCGCGCTTTTC-3';
5'-CAAGGATCCACCGGCAGTCGGCGCCGG-3') and the GC-rich PCR system
(Roche), inserted into the TOPO TA vector (Invitrogen), sequenced and cloned
into pEGFP-N1 (Clontech) by NheI and BamHI
(`filled-in').
Cell-cycle analysis
Cells were fixed in 50% ethanol, lysed with 0.1% sodium citrate, 1 mg/ml
RNase A, 50 µg/ml propidium iodide, and subjected to FACS (Becton Dickinson
FacsCaliber) analysis. At least 20,000 gated events per sample were quantified
(Cellquest software). Cells with DNA content below G1 were scored as
apoptotic. For transiently transfected cells, data represent the total
unsorted population (transfection efficiencies averaged at 60% by EGFP).
Histological analysis
One abdominal mammary gland per mouse was fixed in 10% neutral buffered
formalin and sections were stained with haemotoxylin and eosin. Photographs
were taken with an AxioHOME microscope (Zeiss). For quantifications, 10-12
pictures per gland were taken at random across the whole gland at 200-fold
magnification. The areas occupied by fat cells were identified manually and
quantified by the public domain NIH1.62 Image program
(http://rsb.info.nih.gov/nihimage/).
Immunohistochemical staining was performed with the aid of an automated immunostainer (DakoCytomation Inc., Carpinteria, CA). Deparaffinized tissue sections on glass slides were subjected to heat-induced antigen retrieval by placing the slides into target retrieval solution, high pH (DakoCytomation), and steaming them in a commercial vegetable steamer at full temperature for 30 minutes. Following the antigen retrieval procedure the slides were incubated with a C/EBP delta rabbit polyclonal antibody (Santa Cruz Biotechnology, M17; dilution 1:500) overnight at 4°C, and the detection was carried out on the automated system using an HRP/DAB polymer based rabbit detection system (Envision+ DakoCytomation) according to the manufacturer's recommendations.
RNA analysis
Total RNA was isolated using TRIZOL reagent (Life Technologies, Ind.). Ten
micrograms of each sample was fractionated and analyzed by standard Northern
blotting techniques. Expression of beta-casein mRNA was analyzed using
specific oligonucleotides as previously described
(Robinson et al., 1996). To
generate a DNA probe for BFL1, a fragment was amplified from C57BL/6 mouse
genomic DNA (primers: 5'-TTTGCAGTCTTTGCCTCCTT-3';
5'-TTCTGCCGTATCCATTCTCC-3'), sub-cloned and sequenced. All other
radiolabeled DNA probes were prepared from isolated cDNA clones for the
indicated genes. The multiprobe ribonuclease protection assay using 10 µg
total RNA per sample was performed as described
(Hodge et al., 2002
), using
the mAPO2 multiprobe template set (PharMingen). The signals were visualized by
phosphorimaging and densitometry analysis performed by ImageQuant software
(Molecular Dynamics).
|
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Results |
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STAT3 is required for C/EBP expression in mammary epithelial cells
The C/EBP promoter can be activated by STAT3 in human hepatoma and
mouse MEC lines (Cantwell et al.,
1998
; Hutt et al.,
2000
). Furthermore, Oncostatin M and STAT3 can activate
C/EBP
in mouse and human MEC lines
(Hutt and DeWille, 2002
;
Zhang et al., 2003
),
identifying C/EBP
as a target of STAT3 in vitro. However, mice with a
conditional null allele of Stat3 revealed that STAT3 is not necessary
for induced C/EBP
expression in the liver
(Alonzi et al., 2001
). By
contrast, STAT3- or LIF-deficient mammary glands exhibited approximately
2.5-fold and 4.5-fold reduced C/EBP
levels on Inv d2 compared with
controls (Kritikou et al.,
2003
). To define the exact extent to which C/EBP
gene
expression is dependent on STAT3 in MEC, mice with a conditional
Stat3 null allele transgenic for Cre recombinase driven by the MEC
specific whey acidic protein (WAP) gene promoter
(Humphreys et al., 2002
) were
analyzed on Inv d0-3. In the absence of STAT3, C/EBP
levels were
greatly reduced even in the lactating gland
(Fig. 2). On Inv d1,
C/EBP
expression was highly induced in wild-type mice, as shown
previously (Gigliotti and DeWille,
1998
; Thangaraju et al.,
2004
). However, induction of C/EBP
was dramatically
impaired in the absence of STAT3. The low level induction of C/EBP
still detectable in involuting mutant glands may stem from luminal cells with
inefficient deletion of the Stat3 gene, or could be due to stromal
cells. Expression of C/EBP
was reduced by Inv d3 in wild-type glands
and absent in mutant tissue. IGFBP5, an inhibitor of the pro-survival signal
IGF, was induced at early stages of involution by the STAT3 pathway in
wild-type tissue but not in mutant tissue, as shown previously
(Kritikou et al., 2003
).
Furthermore, this analysis revealed that expression of matrix
metalloproteinase 3 (MMP3, stromelysin 1), a marker for the second phase of
involution (Lund et al., 1996
;
Strange et al., 1992
), was not
induced in the absence of STAT3 (Fig.
2).
|
Involution is delayed in the absence of C/EBP
Next, the role of C/EBP in involution was analyzed by use of
C/EBP
-deficient mice. On Inv d1, alveoli are expanded because of
accumulating milk, and the contribution of fat to tissue morphology is low. As
in lactating glands (data not shown), there was no difference between mutants
and controls (Fig. 3A). On Inv
d2, wild-type alveoli began to regress and fill with debris while fat cells
started to re-fill. This process was significantly blunted in
C/EBP
-deficient glands compared with controls. On Inv d3, we found
heterogeneity in tissue morphology between mice of the same genotype. The
sections shown in Fig. 3 are
representative of the most and least involuted appearance per group. However,
in general wild-type alveoli are almost completely collapsed, while in
C/EBP
null glands significantly expanded alveoli can still be detected.
Because of the aforementioned tissue heterogeneity, the proportional
contribution of fat to tissue morphology was quantified from several
independent glands per time point (Fig.
3B). Mutant and wild-type mice had similar amounts of fat on Inv
d1. However, the proportional increase in adipose tissue was significantly
delayed on Inv d2-4 in mutant mice, in agreement with delayed regression of
the epithelial compartment. These data show that in the absence of
C/EBP
, the kinetics of post-lactational mammary gland involution is
delayed at the morphological level.
C/EBP is required for the timing of pro-apoptotic events
Analyses of STAT3-deficient glands had demonstrated a defect in the
initiation of apoptosis (Chapman et al.,
1999; Humphreys et al.,
2002
). Therefore, we first addressed apoptotic markers in
C/EBP
-deficient involuting mammary glands. Apoptosis is initiated
through regulated proteolytic activation events of the caspase system
(Marti et al., 2001
). By
Western analysis, wild-type mice displayed significant levels of activated,
cleaved caspase 3 on Inv d3 and 4 (Fig.
4). By contrast, cleaved caspase 3 was not detected before Inv d4
in mutant mice and pro-caspase 3 remains high through Inv d4. In wild-type
mice, cleavage of the protease substrate PARP
(Marti et al., 2001
) was
detected from day 1 to 4 of involution, but only by Inv d4 in C/EBP
null tissue. In wild-type mice, significant cleavage of PARP was detected
earlier than caspase 3 activation. This could be because PARP cleavage is a
particularly sensitive measure of apoptosis
(Duriez and Shah, 1997
), or
because other caspases or protease activities
(Soldani and Scovassi, 2002
)
may be more important at early stages of involution. Importantly, by the
measure of both caspase 3 and PARP cleavage, protease activation is delayed in
the absence of C/EBP
.
The first stage of involution is dependent on the proapoptotic tumor
suppressor p53 in some mouse strains
(Blackburn and Jerry, 2002;
Li et al., 1996
). p53 protein
levels were elevated in wild-type mice on Inv d1-4. In mutant glands, p53
protein failed to accumulate through the first 3 days of involution.
Consequently, downregulation of the p53-repressed target survivin (BIRC5
Mouse Genome Informatics), which inhibits p53-mediated apoptosis
(Hoffman et al., 2002
;
Mirza et al., 2002
), was
delayed and reduced in mutant glands (Fig.
4).
As seen in Stat3 null mammary tissue
(Fig. 2)
(Chapman et al., 1999), IGFBP5
expression was significantly impaired even in the absence of C/EBP
alone. These data raised the question of whether STAT3 activation itself was
affected by the C/EBP
mutation. However, both the expression levels and
kinetics of STAT3 activation as judged by its tyrosine phosphorylation were
completely normal in mutant glands. On Inv d8, all results were comparable
between wild-type and mutant glands (Fig.
4). These data demonstrate that C/EBP
is downstream of
STAT3 and upstream of several cell death regulators, such as protease
activation, and p53 and IGFBP5 protein expression.
C/EBP is essential for regulation of pro-apoptotic gene expression
As C/EBP is a transcription factor, we next assessed gene expression
patterns at the level of RNA. Because cell death was delayed in the absence of
C/EBP
, we first analyzed expression of some members of the BCL2 family
of apoptosis regulators, known to be modulated during involution
(Furth, 1999
). Activation of
the pro-apoptotic genes Bak on Inv d1 and Bax on Inv d2 was
impaired in mutant glands (Fig.
5A). Expression of Bcl-x-S/L
(Walton et al., 2001
) was
slightly elevated in mutants on Inv d2 (data not shown). However, the
anti-apoptotic genes Bfl1 (Bcl2a1) and Bcl-w
(Bcl2l2) were downregulated in wild-type glands by Inv d1, but
remained expressed in the absence of C/EBP
(Fig. 5A). These data are in
agreement with a delay in cell death initiation in C/EBP
-deficient
glands.
|
In the absence of C/EBP, regulation of most of these genes was
significantly perturbed. The proliferation-associated genes Cyclin D1,
Brca1 and Brca2 were not repressed
(Fig. 5B), while IGFBP5 and
SGP2 induction was delayed and significantly blunted
(Fig. 5B,C). Consistent with
the delayed tissue regression, CK18 levels continued to be elevated through
Inv d2, and induction of the pro-apoptotic p53 gene was delayed to Inv d4
(Fig. 5B,C). As seen in
conditionally Stat3 null mammary glands
(Fig. 2), expression of MMP3 on
Inv d2-4 was completely eliminated in the C/EBP
null glands. However,
the C/EBP family member C/EBPß was first activated normally on Inv d2,
although this induced expression was not maintained through days 3 and 4 of
involution (Fig. 5B).
Because of the cascade of cellular events during involution and the
heterogeneity of the tissue after the onset of epithelial cell apoptosis,
interpretation of differences in gene expression at later stages of involution
are complicated in the absence of cellular localization. Furthermore, the most
direct consequences of C/EBP-deficiency take place during the early
stages of involution. Therefore, we have focused our attention on day 1 of
involution, when C/EBP
levels are highest in wild-type glands,
C/EBP
-expressing luminal epithelial cells represent the majority of the
tissue, and no histological differences are detected between mutant and
wild-type glands. Four to five mice per genotype and time point were
quantified for expression of some of the genes shown in
Fig. 5A-B. This approach
confirmed the complete block in p53 and IGFBP5 induction in the absence of
C/EBP
. Furthermore, not only was Cyclin D1 expression maintained in
C/EBP
null glands, but BFL1 expression was in fact induced rather than
suppressed compared with wild-type glands
(Fig. 5D).
|
C/EBP is sufficient to initiate involution-specific events in HC11 cells
To investigate whether C/EBP was sufficient to trigger some of the
genetic events impaired in the mutant glands, we transfected the
non-transformed mouse MEC line HC11 (Hynes
et al., 1990
) with various expression vectors. As shown previously
(O'Rourke et al., 1999
),
C/EBP
elicited a growth arrest response in HC11 cells with concomitant
upregulation of Gas1 mRNA levels (Fig.
6). In addition, C/EBP
alone was able to induce expression
of SGP2 and IGFBP5, and inhibited expression of Cyclin D1. By contrast, p53
mRNA (Fig. 6) and protein (data
not shown) levels were not induced by C/EBP
. This result suggests that
the activation of p53 expression downstream of C/EBP
in the mammary
gland is indirect and/or requires additional signals. Expression of
Brca1 and Brca2, two genes associated with proliferation and
differentiation (Kubista et al.,
2002
), was suppressed by p53 but not by C/EBP
, suggesting
that the decline of BRCA1 and BRCA2 on day 1 of mammary gland involution is
mediated by p53. This data is in agreement with the ability of p53 to repress
the Brca1 promoter (MacLachlan et
al., 2000
), and the frequent co-regulation of these two genes
(Rajan et al., 1997
).
Interestingly, p53 activated expression of the endogenous C/EBP
gene in
HC11 cells. Thus, in the mammary gland, C/EBP
and p53 pathways may
engage in a positive feedback loop on expression. HC11 cells harbor p53
alleles with two independent mis-sense mutations
(Merlo et al., 1994
).
Overexpression of wild-type p53 itself triggered apoptosis in HC11 cells
(Fig. 6C). Co-transfection of
C/EBP
significantly enhanced this apoptotic response, indicating that
p53 and C/EBP
cooperate in the initiation of MEC apoptosis.
Furthermore, this result raises the possibility that growth arrest rather than
apoptosis is the predominant response of cell lines to C/EBP
in vitro
(Dearth and DeWille, 2003
, and
references therein), because most cell lines harbor mutant p53 genes.
Taken together, these data show that C/EBP alone is able to initiate
involution-specific genetic responses in an in-vitro model and cooperates with
p53 to elicit apoptosis, further supporting its active role in regulation of
mammary epithelial cell death during involution.
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Discussion |
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|
In Stat3 mutant MEC, IGFBP5 activation was initially impaired, but
recovered by day 3. However, C/EBP levels remained low. Thus,
activation of C/EBP
expression is very specific for the signaling
pathways that elicit the physiological first phase of apoptosis in MEC. Many
extracellular signals can potentially activate C/EBP
expression
(Ramji and Foka, 2002
).
Nevertheless, STAT3 alone is essential for high levels of expression in MEC
during involution. However, STAT3 alone may not be sufficient, because
C/EBP
expression declines already on Inv d2 before STAT3 activation
ceases on Inv d4. Interestingly, STAT3 often exhibits constitutive activation
in breast tumor cells (Bromberg,
2002
), while C/EBP
expression is significantly reduced in
mammary and breast tumors compared with normal tissue
(Kuramoto et al., 2002
;
Porter et al., 2003
). Thus,
one can speculate that lack of C/EBP
as an apoptosis mediator may in
part explain why tumor cells with constitutive STAT3 activity do not undergo
apoptosis.
C/EBP is also inducible in macrophages, fibroblasts and adipocytes
(Ramji and Foka, 2002
), cell
types that participate in mammary gland involution subsequent to initiation of
epithelial cell apoptosis (Monks et al.,
2002
; Werb et al.,
1996
). Our data indicate transient induction of C/EBP
expression in a subset of stromal cells in parallel with its massive
expression in the luminal epithelial cell layer. Thus, we cannot rule out that
some phenotypes in the C/EBP
null glands may be caused in part by
defective responses of adipocytes in the early phase of involution. However,
C/EBP
null glands and mammary glands with Stat3 deletion
specifically in secretory epithelial cells
(Clarkson et al., 2004
;
Stein et al., 2004
) exhibit
several overlapping phenotypes, including impaired expression of IGFBP5, MMP3,
BAX and SGP2, demonstrating that several significant effects are caused by the
role of C/EBP
as a STAT3 target in secretory epithelial cells.
|
Despite the dramatic effects of C/EBP-deficiency on gene expression
patterns, the mammary gland still involutes, albeit with delayed kinetics. In
fact, to our knowledge, no reported genetically engineered mouse model has led
to a complete block of involution. This is most probably due to the fact that
a cell in distress can find many ways to die
(Lockshin and Zakeri, 2004
).
In the conditional Stat3 null model, elevated levels of p53 mediate
in part the compensatory apoptotic response
(Matthews and Clarke, 2005
).
However, these alternative pathways would not represent the physiological
sequence of events. With respect to the morphological regression and
remodeling of the gland, the C/EBP
null mutation causes a more subtle
delay than mutations in Stat3 or its upstream activators gp130 and
LIF (Chapman et al., 1999
;
Humphreys et al., 2002
;
Kritikou et al., 2003
;
Zhao et al., 2004
). This is
not surprising, as C/EBP
is surely only one of many target genes of
STAT3. Similarly, no single target of C/EBP
can be responsible for the
phenotype of C/EBP
null mammary glands. Rather, the combined actions of
the multiple pro- and anti-apoptotic proteins whose regulation of expression
is perturbed in the absence of C/EBP
underlies the delay in apoptosis
and involution.
|
C/EBP also induced SGP2/clusterin, which is mostly known as a
secreted protein whose expression is activated in many examples of
developmental tissue regression. Its role in cell survival has been
controversial, but recent reports show that nuclear isoforms can elicit growth
arrest and apoptosis (Scaltriti et al.,
2004
). Thus, lack of SGP2 expression in C/EBP
mutant mice
may further contribute to delayed apoptosis.
Cyclin D1 and Bfl1, two genes that are normally
suppressed at the onset of involution, continued to be expressed in
C/EBP null glands. Cyclin D1 promotes G1/S progression through
regulation of cyclin-dependent kinases but can also affect histone acetylation
and chromatin remodeling (Fu et al.,
2004
). BFL1, whose promoter also contains a functional C/EBP site
(Edelstein et al., 2003
),
inhibits the pro-apoptotic activity of other BCL2 members
(Werner et al., 2002
) and of
p53 (D'Sa-Eipper et al.,
1996
). Thus, continued expression of these two genes in the
absence of C/EBP
can contribute to delayed apoptosis and
involution.
Remarkably, loss of C/EBP had profound effects on the later stages
of involution, despite its transient peak expression on Inv d1. A null
mutation in Stat3 null epithelial cells causes delayed activation of
MMP9, while MMP2 activity is not affected
(Humphreys et al., 2002
). The
present study shows that MMP3 expression during involution is completely
dependent on C/EBP
as well as its upstream activator STAT3. Expression
of the C/EBPß gene during the second phase of involution is also affected
by lack of C/EBP
. C/EBPß is essential for proliferation and
differentiation of epithelial cells during mammary gland development
(Robinson et al., 1998
;
Seagroves et al., 1998
).
However, C/EBPß is also a crucial player during adipocyte differentiation
in vitro (Tanaka et al.,
1997
). While it is known that MMP3 is expressed in stromal cells
during mammary development and involution
(Lund et al., 1996
), it
remains to be determined which cell types express C/EBPß during
involution. However, the differences in expression kinetics between
C/EBP
, C/EBPß and MMP3 suggest that C/EBP
does not regulate
either of them directly.
C/EBP expression alone may not be sufficient for activation of its
target genes. The C/EBP
protein requires phosphorylation for efficient
transactivation (Ji et al.,
2003
) and DNA binding, such as by CaseinKinase II
(Osada et al., 1996
), IKKi
(Kravchenko et al., 2003
) or
IL1 receptor signaling (Lacorte et al.,
1997
). Expression of IKKi as well as the IL1 ligand receptor
system is activated at the onset of involution
(Clarkson et al., 2004
), and
may contribute to C/EBP
activation. Furthermore, C/EBP
interacts
with many other transcription factors, including Smad3
(Choy and Derynck, 2003
), which
promotes involution as a mediator of TGFß3 receptor activation
(Nguyen and Pollard, 2000
;
Yang et al., 2002
). Thus,
C/EBP
potentially serves to integrate the pathways from several
extracellular signals such as LIF, TGFß and interleukins to initiate
apoptosis of mammary epithelial cells.
Clearly, many genes are dynamically regulated in unique spatial and
temporal patterns during the mammary involution process. Data derived from
individual gene deletions in mice allow us to place genes into signaling
cascades and derive their crucial functions. The present study places
C/EBP between STAT3 and several pro- and anti-apoptotic genes as an
inducer of a physiological cell death response in epithelial cells at the
onset of mammary gland involution.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/21/4675/DC1
* Present address: Department of Biochemistry and Molecular Biology, Medical
College of Georgia, Augusta, GA 30912-0006, USA
Present address: Department of Cancer Biology, Vanderbilt University
Medical Center, Nashville, TN 37232, USA
Present address: Department of Biochemistry, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
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