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First published online 28 September 2005
doi: 10.1242/dev.02050
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is a crucial regulator of pro-apoptotic gene expression during mammary gland involution
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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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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) is a crucial mediator of
pro-apoptotic gene expression events in mammary epithelial cells. In the
absence of C/EBP, involution is delayed, the pro-apoptotic genes
encoding p53, BAK, IGFBP5 and SGP2/clusterin are not activated, while the
anti-apoptotic genes coding for BFL1 and Cyclin D1 are not repressed.
Consequently, p53 targets such as survivin, BRCA1, BRCA2 and BAX are not
regulated appropriately and protease activation is delayed. Furthermore,
expression of MMP3 and C/EBP during the second phase of involution is
perturbed in the absence of C/EBP. In HC11 cells, C/EBP alone is
sufficient to induce IGFBP5 and SGP2. It also suppresses Cyclin D1 expression
and cooperates with p53 to elicit apoptosis. This study places C/EBP
between STAT3 and several pro- and anti-apoptotic genes promoting the
physiological cell death response in epithelial cells at the onset of mammary
gland involution.
Key words: Mammary gland, Involution, C/EBP, Apoptosis, Mouse
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). Our current knowledge on these processes has been
summarized in a recent, comprehensive review
(Green and Streuli, 2004).
Involution can be divided into two morphologically recognizable stages. The
first reversible phase constitutes the initiation of mammary epithelial cell
(MEC) apoptosis and the second phase includes extensive tissue remodeling
(Lund et al., 1996).
Furthermore, adipocytes refill during the process of involution. Microarray
expression analyses lead to the characterization of several distinct clusters
of gene expression patterns, suggesting a number of sub-stages with intricate
regulation and communication of multiple signaling pathways occurring in
several cell types that fine-tune the tissue remodeling process
(Clarkson et al., 2004;
Stein et al., 2004). Several
hundred genes are dynamically regulated during involution. However, a specific
role has been established by gene targeting approaches in mice for only a few.
According to such studies, the signals contributing to initiation of
epithelial cell apoptosis include death-receptor activation and
TGFß3/SMAD3 signaling (Green and
Streuli, 2004). Well characterized is the crucial role of the
signal transducer and activator of transcription 3 (STAT3), which is activated
by the gp130 cytokine receptor with leukemia inhibitory factor (LIF) as a
critical activating ligand (Kritikou et
al., 2003; Zhao et al.,
2004). Stat3 null mice are embryonic lethal
(Takeda et al., 1997).
However, conditional deletion of the Stat3 gene in secretory MEC
leads to a severe delay in involution with expanded, lactation-competent
alveoli present for several days. Stat3 null epithelial cells exhibit
significantly less apoptosis than controls and protease activity in the second
phase of involution is delayed (Chapman et
al., 1999; Humphreys et al.,
2002). STAT3 mediates MEC apoptosis in part by inducing expression
of regulatory PI(3) kinase subunits and IGFBP5, which lead to inhibition of
the pro-survival signals from AKT and IGF1
(Abell et al., 2005;
Chapman et al., 1999).
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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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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mutant mice+/– mice
(Sterneck et al., 1998)
backcrossed for at least 11 generations into C57BL/6 were intercrossed to
generate subjects. Females were mated at approximately 7 weeks of age and
males were removed postcoitum. Litters were standardized to six pups at birth.
Pups were removed on day 7 of lactation
(Strange et al., 1995;
Thangaraju et al., 2004).
Females were euthanized by CO2 and the #4 glands removed for
analysis. All animals were housed and handled according to approved protocols
established by the NCI Animal Care and Use Committee and NIH guidelines.
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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). Briefly, tissue was homogenized in lysis buffer,
incubated at 4°C overnight and cleared by centrifugation. HC11 cells were
lysed by sonication (3 x10 pulses) in lysis buffer and extracts cleared
by centrifugation. Fifty micrograms of protein were fractionated on
tris-glycine gels and transferred to Protran nitrocellulose membrane
(Schleicher & Schull). Membranes were blocked with bovine serum albumin
(BSA) in 10 mmol/l Tris-HCl, pH 7.5, 150 mmol/l NaCl and 0.5% Tween 20 (TBST)
exposed to primary antibody (1:15,000 in TBST) at 4°C overnight, followed
by the secondary antibody (1:50,000 in TBST) at room temperature for 1 hour.
Proteins were visualized using the ECL super signal western system (Pierce).
Antisera were from Santa Cruz Biotechnology (STAT3, sc-7179; p53, sc-100;
PARP, sc-7150; anti-goat, sc-2094), Upstate Cell Signaling (phospho-STAT3
Y705, # 07-391; IGFBP5 # 06-110), R&D Systems (Survivin, AF886), RDI
Biotechnology (Caspase 3, RDI-CPP32 Nabg), Promega (anti-rabbit WA401B,
#17330901) and Pierce (anti-mouse #31432, Pierce). |
Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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is expressed in luminal mammary epithelial cells expression in the mammary gland were
based on whole organ RNA levels. Although MEC lines in vitro clearly express
inducible C/EBP, the gene can also be expressed in many other cell
types, including adipocytes, smooth muscle cells and endothelial cells. Thus,
we first applied immunohistochemistry to assess the range of cell types in
which C/EBP is expressed at different stages. By this approach we were
unable to detect C/EBP protein in any cell type of lactating glands
(data not shown) or at 12 hours after removal of the pups
(Fig. 1). However, on day 1 of
involution nuclear C/EBP protein expression was evident in most of the
luminal epithelial cells specifically in wild-type mice. Parallel staining for
smooth muscle actin suggested that myoepithelial cells do not express
C/EBP (see Fig. S1 in the supplementary material), similar to
endothelial cells (data not shown). However, a subset of stromal adipocytes
also expressed nuclear C/EBP (Fig.
1). No specific staining for C/EBP was detectable in the
mammary gland from day 3 of involution (Inv d3) (data not shown), in agreement
with the significantly reduced mRNA levels at this stage reported for the same
C57BL/6 strain (Thangaraju et al.,
2004) as well as FVB/N mice
(Gigliotti and DeWille, 1998).
The present data confirm massive induction of nuclear C/EBP protein in
the luminal epithelial layer of the mammary gland specifically in the early
phase of involution. Furthermore, a subset of stromal cells expressed
C/EBP protein on Inv d1.
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).
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expression during mammary involution in vivo.
Furthermore, the expanded kinetic analysis revealed that this requirement
extends to the lactation period and Inv d1-3, with no compensatory mechanism
developing by Inv d3. Furthermore, STAT3 was found to be required for MMP3
expression during the later phase of involution.
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.
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was induced in
wild-type glands by Inv d1. In the C57BL/6 strain used here, this activation
faded already on Inv d2 (see Discussion). Other genes induced in wild-type
glands at the RNA level 24 hours after removal of the pups were p53, SGP2
(clusterin), IGFBP5 and STAT3, while Cyclin D1, BRCA1 and BRCA2 were
repressed. The elevated levels of Stat3 mRNA at the onset of
involution are by contrast to unchanged protein levels
(Fig. 4), which suggests
additional regulation of STAT3 expression at the protein level. On Inv d2,
C/EBPß was induced, and MMP3 was highly expressed on Inv d3 and 4. With
the disappearance of epithelial cells, expression of epithelial specific
keratin 18 (CK18) was significantly reduced by Inv d2. Surprisingly,
ß-casein (Fig. 5B) as well
as WAP (data not shown) mRNA levels continued to be expressed, decreasing only
in parallel with the epithelial marker CK18. This pattern was also observed by
microarray analysis of involution in the same C57BL/6 background
(Clarkson et al., 2004), and
indicates that metabolic de-differentiation may not be required for
involution. As described previously (Allar
and Wood, 2004), IGFBP3 mRNA levels did not change during the
involution process.
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).
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null mammary glands. With the exception of Stat3 mRNA expression and
protein phosphorylation, every gene we have analyzed and that showed specific
regulation on day 1 of involution in wild-type mice was significantly affected
by lack of C/EBP. Thus, impaired regulation of genes whose products
participate in the regulation and execution of the involution process
underlies the morphologically delayed tissue regression in C/EBP null
mice.
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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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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as a master regulator of
pro-apoptotic gene regulation during initiation of physiological cell death,
specifically at the onset of mammary gland involution. Based on the phenotype
of involuting C/EBP null mammary glands, the genetic response of HC11
cells to C/EBP expression, and known targets of p53, we propose the
following model for the signaling cascade in involution of C57BL/6 mice
(Fig. 7). C/EBP, induced
by STAT3, is upstream of and required for induced expression of p53, IGFBP5,
SGP2 and BAK. Furthermore, C/EBP activity leads to suppression of
Cyclin D1 and BFL1 expression. C/EBP is necessary but not sufficient to
induce expression of p53, which leads to activation of BAX, and inhibition of
survivin, BRCA1 and BRCA2 expression. However, p53 and other factors may
cooperate also in the regulation of genes downstream of C/EBP as well
as C/EBP itself. In addition, C/EBP activity is required for
expression of MMP3 during the later phase of involution and for maintenance of
C/EBPß expression. Furthermore, the STAT3 transcription factor is
required for significant activation of the C/EBP gene in mammary
epithelial cells from lactation through at least the first 3 days of
involution, by contrast to being dispensable for C/EBP expression in
the liver (Alonzi et al.,
2001).
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between STAT3 and multiple
pro-apoptotic genetic events during the initial phase of apoptosis initiation,
which further affect gene expression during the subsequent phase of tissue
remodeling. Interestingly, expression of C/EBP also correlates with
degeneration of surface mucous cells of the larval stomach during
metamorphosis of Xenopus laevis
(Ikuzawa et al., 2005). Thus,
a role in apoptosis during developmental tissue regression may be an
evolutionarily `old' function of C/EBP.
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.
|
null mammary glands, such as in the regulation of p53 expression
(Chapman et al., 1999;
Humphreys et al., 2002;
Kritikou et al., 2003;
Zhao et al., 2004). These may
be explained in part by the genetic background. We chose to study the
C/EBP null mutation on the C57BL/6 strain background because these mice
do not exhibit increased ductal branching in the absence of C/EBP (data
not shown) as do outbred C57BL/6x129S1 mice
(Gigliotti et al., 2003). In
fact, on the mixed strain background C/EBP deficiency did not result in
a morphological delay of involution, and SGP2 and IGFBP5 expression were
impaired on only Inv d1 and 2, respectively
(Gigliotti et al., 2003). We
had demonstrated that the mRNA expression levels of several genes, including
C/EBP and SGP2, are not modulated during involution in 129S1 mice.
However, the dynamic gene expression patterns we observed in C57BL/6 during
involution were similar to those described for FVB/N mice
(Thangaraju et al., 2004). By
contrast to the 129S1 strain, the C57BL/6 and FVB/N strains are widely used in
the study of mammary gland development, rendering them more suitable for the
analysis of gene deletions on involution.
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.
|
gene is present.
C/EBP can activate IGFBP5 expression through direct promoter regulation
(Ji et al., 2003). Thus, the
dependence of IGFBP5 expression on C/EBP in vivo and the ability of
C/EBP to induce IGFBP5 in HC11 cells are consistent with IGFBP5
representing a direct target gene. In vitro and in vivo IGFBP5 can cause
apoptosis of MEC, involving IGF1 dependent and independent mechanisms
(Flint et al., 2003;
Marshman et al., 2003;
Schneider et al., 2002). Thus,
the target gene IGFBP5 is part of the molecular mechanism by which
C/EBP promotes 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 |
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|
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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