1 Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge
CB2 1QP, UK
2 Clinical Research Department, Christie Hospital NHS Trust, Manchester M20 4BX,
UK
* Author for correspondence (e-mail: cjw53{at}mole.bio.cam.ac.uk)
Accepted 9 April 2003
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SUMMARY |
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Key words: LIF, Mammary development, STAT3, Apoptosis, ERK, Mouse
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INTRODUCTION |
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We have identified a number of transcription factors that regulate
apoptosis in epithelial cells of mouse mammary gland during the normal
physiological process of post-lactational regression. Our work, and that of
others, has highlighted the particular importance of the STAT family of
transcription factors in regulating adult mammary gland development. STAT5a,
which is induced by prolactin, promotes differentiation of epithelial cells
and is important for lobuloalveolar development during pregnancy
(Liu et al., 1997;
Teglund et al., 1998
). By
contrast, STAT3 is pro-apoptotic and is a crucial mediator of post-lactational
regression (Chapman et al.,
1999
). Using conditional gene targeting, we have shown that in the
absence of STAT3, involution is delayed for several days, owing to a reduction
in apoptosis, and this is associated with elevated levels of p53 (TRP53 -;
Mouse Genome Informatics) and p21, precocious activation of STAT1, and failure
to induce IGFBP5. However, unlike STAT5, the physiological inducer of STAT3
has not been identified. The aim of this study was to identify this inducer of
STAT3 in mammary gland, and thereby to establish the identity of the
physiological signal that initiates the involution/apoptotic switch -; the
crucial first step in remodelling of the mammary gland.
STAT3 is activated in response to a number of cytokines that share a common
transmembrane gp130 protein receptor subunit
(Heinrich et al., 1998). These
include members of the interleukin (IL) 6 cytokine family, such as IL11,
ciliary neurotrophic factor, oncostatin M, cardiotrophin and leukaemia
inhibitory factor (LIF). STAT3 can also be activated by other factors,
including prolactin, in a cell-type-specific manner.
In previous studies, we have used a cell culture model of mouse mammary
gland (KIM-2) to observe signalling pathways that influence differentiation
and apoptosis of mammary epithelial cells. These cells can be induced to
differentiate, express milk proteins and undergo apoptosis upon withdrawal of
lactogenic hormones (Gordon et al.,
2000). Using these cells, we found that a number of cytokines
activated STAT3, the most potent of these being LIF. LIF signalling is
mediated mainly by the SHP-2/Ras/extracellular signal regulated kinase (ERK)
pathway, the PI-3K/Akt pathway and JAK/STAT pathways, and can be inhibited by
members of SOCS and PIAS family of proteins
(Bousquet et al., 1999
;
Duval et al., 2000
). Among the
many effects of this cytokine, LIF regulates endocrine functions of the
hypothalamo-pituitary-adrenal axis and utero-placental unit (reviewed by
Gadient et al., 1999), and maintains embryonic stem cell pluripotentiality
(Ernst et al., 1999
),
haemopoiesis and neural differentiation
(Kim and Melmed, 1999
;
Schwartz et al., 1999
;
Bousquet et al., 1999
).
Moreover, LIF can induce both differentiation and apoptosis in the M1 myeloid
cell line (Minami et al.,
1996
; Tomida et al.,
1999
).
To establish whether there is a physiological role for LIF in mammary
gland, we have used Lif-;/-; mice
(Stewart et al., 1992).
Unexpectedly, mice with an engineered null mutation for LIF develop normally
and exhibit a rather mild phenotype. Lif-;/-; mice are
smaller compared with control heterozygous and wild-type littermates. This
mild phenotype has been attributed to partial functional redundancy between
IL6 family members occurring as a result of overlapping expression patterns
and the sharing of the common gp130 (Il6st -; Mouse Genome Informatics)
subunit. Female Lif-;/-; mice, however, are unable to
support pregnancy because of defective blastocyst implantation
(Stewart et al., 1992
;
Escary et al., 1993
). We have
overcome this infertility with intra-peritoneal injection of LIF post-coitus.
Thus, for the first time we have been able to study complete mammary
development in LIF-deficient animals.
Surprisingly, in the light of the functional redundancy between IL6 family members in other tissues, we show that LIF is the sole activator of STAT3 in mammary gland. Furthermore, we observed perturbed development that was associated with a decline in phosphorylated ERK1/2 and changes in progesterone receptor (PR) status. These findings are consistent with a dual role for LIF as the physiological activator of STAT3-mediated apoptosis during involution and ERK1/2-mediated branching morphogenesis early in mammary development.
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MATERIALS AND METHODS |
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Cell culture
The conditionally immortalised murine mammary epithelial cell line, KIM-2,
was maintained as previously described
(Gordon et al., 2000). The
cells were grown to confluence in growth medium, MM (F12/DMEM supplemented
with 10% FCS, 5 µg/ml insulin, 5 ng/ml EGF, and 5 µg/ml linoleic acid)
and maintained at confluence for one day before treatment. For
differentiation, MM was replaced 24 hour after cells had reached confluency by
differentiation medium (DM) (F-12/DMEM supplemented with 10% FCS and the
lactogenic hormones, 5 µg/ml insulin, 1 µg/ml prolactin, 40 ng/ml
dexamethasone). Cells were cultured in DM for 12 days by which time cells
expressed markers of a fully differentiated phenotype.
Detection of apoptosis
Apoptosis studies was performed using annexin V staining assessed either by
flow cytometry or in situ. Undifferentiated KIM-2 cells were maintained at
100% confluence for 1 day and then incubated with either MM or MM + LIF
(Peprotech, UK) with or without U0126 (10 µM, Promega, UK) for 24 hours.
For annexin V flow cytometry, KIM-2 cells were harvested as previously
described (Clarkson et al.,
2000). For annexin V in situ, cells were stained with 1 µg/ml
FITC-annexin V and after 15 minutes incubation in the dark, the plastic slides
were photographed using an inverted epi-fluorescence microscope.
RT-PCR analysis
RNA was extracted from tissue using the TRIzol reagent (Promega, UK) and
cDNA synthesis was performed using the Superscript cDNA synthesis kit (Gibco,
URL, UK). The following forward and reverse primer pairs were used for
specific amplification: Lif,
5'-CTGTGGCTGTCATTGTTGGCGTGGTA-3' and
5'-ATCGGCGGCGGGGTTCGTTA-3'; Lifr,
5'-GGCTCTGGAACCTTGGGCAAAACTG-3' and
5'-GCCTGCACTGCTCCAACCTCCTGTA-3'; gp130,
5'-TCGGAGGAGCGGCCAGAAGAC-3' and
5'-ATCAGCCCCCGTGCCAAGAGC-3'; C/EBP,
5'-ACCCGCGGCCTTCTACGA-3' and
5'-GCGCCCTTTTCTCGGACTGT-3'; and cyclophilin,
5'-GACGCCACTGTCGCTTTTCG-3' and
5'-CTTGCCATCCAGCCATTCAGTC-3'.
The relative expression of LIF was compared between mammary tissues at different time points by real time RT-PCR using an ABI PRISM 7700 sequence detection system (Taqman) according to the manufacturer's instructions. Primers and probes were designed using the Primer Express v5.0 software (Applied Biosystems, Warrington, UK). The probe was labelled with 5'FAM and 3'TAMRA. LIF primers and probe sequences and the final concentrations they were used at were: mouse LIF forward, 5'-CCCCTGTAAATGCCACCTGT (300 nM); mouse LIF reverse, 5'-CTTCTCTGTCCCGTTGCCAT (300 nM); mouse LIF probe, FAM-5'-CATACGCCACCCATGCCACGG -;3'TAMRA (200 nM).
In addition, the endogenous control 18S ribosmal RNA was assayed using primers and probe from Applied Biosystems. Probe and primer optimisation and real time PCR was performed using the manufacturer's recommended conditions. Standard curves were generated by serial dilution of a standard preparation of total RNA isolated from mouse uterus. Data are expressed in arbitrary units relative to the level of the same gene in this standard RNA. cDNA was produced from each mammary gland sample by reverse transcription using 5 µg of total RNA with 200IU Superscript RT (Invitrogen, Paisley, UK) according to the manufacturer's instructions. The expression values obtained were normalised against those from the control ribosomal 18S to account for differing amounts of starting material.
Western blot analysis
Total protein was extracted from frozen mammary gland tissue or from KIM-2
cells in RIPA protein extraction buffer (50 mM Tris-HCl, 150 mM NaCl, 1%
NP-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.5), supplemented with protease
and phosphatase inhibitors [10 µg/ml aprotinin, 1 µM pepstatin, 1 µM
leupeptin, 1 mM phenylmethlysulfonyl fluoride (PMSF), 1 mM
Na2VO4]. The samples were homogenised then further
disrupted by passage through a 21-gauge needle (between eight and 10 times).
These were subsequently incubated on ice for 30 minutes and centrifuged at
9500 g for 20 minutes at 4°C. Supernatants were
transferred to a fresh tube and protein concentration was measured with the
BCA colorimetric assay (Pierce). Samples (15-30 µg/lane) were run on
SDS-polyacrylamide gels, blotted onto PVDF membranes and incubated with
blocking solution (5% MarvelTM) in TBS with 0.1% Tween 20 for 1
hour. Membranes were incubated with primary antibody diluted in blocking
solution overnight at 4°C and specifically bound antibody was detected
using horseradish peroxidase-conjugated secondary antibodies in conjunction
with a chemiluminescent substrate (ECL; AP Biotech). Antibodies were obtained
as follows: STAT3, tyrosine phosphorylated STAT3 (pSTAT3), STAT1, tyrosine
phosphorylated STAT1 (pSTAT1) and phosphorylated ERK1/2 (pERK1/2) from New
England Biolabs; ERK1/2 from Transduction Labs; tyrosine phosphorylated
STAT5a/b (pSTAT5) from Upstate Biotechnology; p21 from Pharmingen; STAT5,
Bcl-x (Bcl2l -; Mouse Genome Informatics) and Bax from Santa Cruz; p53 (CM5
antibody) was a gift from Prof. David Lane (University of Dundee, UK); and
ß-casein was a gift from Dr Bert Binas (Berlin).
Immunohistochemistry
Immunohistochemistry for pSTAT3 was carried out with the rabbit polyclonal
antibody (New England Biolabs) and the peroxide-based Envision+TM
system (Dako, Ely, UK). Mammary gland sections (5 µm) were deparaffinized
and subjected to antigen retrieval in a pressure cooker for 1 minute
(high-pressure) and 9 minutes (low pressure) in 10 mM citrate acid buffer (pH
6.0). Endogenous peroxidase activity was inactivated by incubation in 1%
hydrogen peroxide in water for 20 minutes. Sections were rinsed with TBS (25
mM Tris at pH 7.6, 130 mM NaCl) and blocked with 20% normal goat serum in TBS
for 30 minutes, then incubated for 1 hour with primary antibody (diluted 1/100
in 5% normal goat serum in TBS), washed in TBS and incubated with
HRP-conjugated EnvisionTM polymer for 30 minutes. Sections were
then washed with TBS and incubated with diaminobenzidine (0.5 mg/ml in 48 mM
Tris, 0.038 M HCl and 10 mM imidazole at pH 7.6 containing 0.02% hydrogen
peroxide) until they turned brown. After washing with water, samples were
counterstained with Haematoxylin, dehydrated and mounted. Immunohistochemistry
for the progesterone receptor (PR) was carried out using a rabbit polyclonal
anti-PR antibody (Dako, Ely, UK), a biotinylated goat anti-rabbit IgG second
antibody and the ABC detection system (both supplied by Vector Laboratories).
Briefly, 5 µm sections of mammary tissue were dewaxed, rehydrated and
endogenous peroxidase blocked by incubating sections in 0.2% hydrogen peroxide
in methanol. Antigen was retrieved by pressure cooking for 1 minute and 30
seconds at high pressure and the sections rinsed in phosphate-buffered saline
(PBS, pH 7.0). Nonspecific binding was blocked by incubating the sections in
10% goat serum, 0.1% bovine serum albumin and avidin in PBS for 1 hour.
Sections were incubated for 1 hour with the primary antibody (diluted 1:50 in
10% normal goat serum, 0.1% BSA and biotin in PBS), washed twice with PBS and
incubated with biotinlyated goat ant-rabbit IgG antibody diluted 1:200 in
NGS/BSA solution for 35 minutes. After washing in PBS, the ABC reagent was
applied to the sections for 35 minutes, washed and incubated with
diaminobenzidine for 8 minutes. Sections were counterstained in Haematoxylin,
dehydrated and mounted. PR expression was quantitated by counting at least
1000 lobulo-alveolar cells across several randomly selected high power fields
and expressing the number of positively stained nuclei as a percentage of the
total counted.
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RESULTS |
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Lif-;/-; mammary glands are
characterised by delayed involution, diminished apoptosis and a lack of
pSTAT3
In order to address whether LIF is required for the activation of STAT3
that normally occurs within 12 hours of involution in vivo, pregnancy was
established in Lif-;/-; mice by injecting LIF around the
time of implantation. We then examined mammary glands from
Lif-;/-; mice after forced involution. Pups born to
Lif-;/-; mothers fed and grew normally indicating that
lactation is not substantially perturbed in the absence of LIF. However, the
absence of LIF had dramatic effects on involution
(Fig. 2A). Two days after
forced involution, the alveoli of Lif+/-; glands had
started to collapse, with reappearance of adipocytes. Morphologically
apoptotic epithelial cells accumulated mainly in the alveoli, with some shed
into the open lumen. Similar to Stat3-null mice
(Chapman et al., 1999), the
alveoli of Lif-;/-; mice at the same stage remained intact
and extended with milk. In contrast to Stat3-;/-; mammary
glands, however, a considerable number of cells were shed into the lumen,
although there was no evidence of tissue remodelling. This may be a
consequence of reduced collagen deposition around ductal and alveolar
structures in the absence of Lif (data not shown). Western blots for
cleaved caspase 3, a late event in the apoptotic cascade, confirmed that
apoptosis was indeed reduced in the absence of LIF
(Fig. 2C).
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The above result was confirmed by immunohistochemistry with a specific anti-phosphotyrosine STAT3 antibody (Fig. 2B). pSTAT3 immunostaining was evident in the nuclei of epithelial cells lining the alveoli in 2 day involuting glands of Lif+/-; animals. By contrast, pSTAT3 staining was absent in the vast majority of cells in the LIF-deficient glands, although occasional positive cells were seen.
We also examined the expression of a known downstream target of STAT3 in
mammary gland, C/EBP (Hutt et al.,
2000
) (data not shown). C/EBP
was upregulated at day 2
involution in control (Fig. 2D,
lane 5), but was not induced in Lif-;/-; (lane 6) or
conditional Stat3-;/-; (lane 4) mammary tissue, thus
providing further evidence for the abrogation of STAT3 activity in mammary
glands lacking a LIF signal.
These data combined indicate that LIF is the principal physiological
activator of STAT3 during involution. This is further supported by the recent
observation of Zhao and colleagues who showed that IL6, a cytokine that also
signals through gp130, does not activate STAT3 during mammary involution
because in IL-6 deficient mammary glands, STAT3 is phosphorylated normally
(Zhao et al., 2002).
Regulation of STAT1, STAT5 and ß-casein gene expression in
Lif-;/-; involuting glands
LIF deficiency is not an exact phenocopy of STAT3 deficiency. We therefore
investigated the molecular changes associated with the absence of LIF during
involution. The activities of STAT3 and STAT5 have a reciprocal pattern of
regulation during mammary gland development. We investigated the levels of
STAT5a in Lif-;/-; compared with
Lif+/-; glands by western blot analysis. Consistent with
Stat3-null mammary tissue, no differences were observed in the levels
of STAT5 protein between Lif-;/-; and control animals,
whereas levels of pSTAT5 were higher in the Lif-;/-;
compared with Lif+/-; tissue
(Fig. 3A). This is reflected in
the higher levels of ß-casein seen in the Lif-;/-;
glands and provides further evidence of a delay in involution in the absence
of LIF.
|
Molecular analysis of apoptosis-related proteins in involuting
mammary glands in the absence of LIF
Bcl2 family members have been shown to be regulated at the onset of
involution (Heermeier et al.,
1996; Li et al.,
1997
) and Bcl-xL is regulated by STAT3 in some tissues.
However, it is not clear if this anti-apoptotic member of the Bcl2 family of
proteins is directly regulated by STAT3 in the involuting mammary gland
because only subtle changes were detected in levels of Bcl-xL in
Stat3-null mammary tissue (Chapman et al.,
1999
). No significant differences were observed in levels of
Bcl-xL or Bax in Lif-;/-; compared with
Lif+/-; mice, with the exception of tissue from one animal that had
elevated levels of Bax (Fig.
3A). The reason for this discrepancy is not clear.
P53 is upregulated in Stat3-;/-; glands and similarly
is upregulated in the Lif-;/-; mammary tissue compared
with controls (Fig. 3A). P53 is
a major regulator of apoptosis although its role in mammary involution is not
clear because strain-dependant differences have been observed in
p53-;/-; mammary glands. p21 has been shown to be a target
of STAT3 at the transcriptional and translational level in some cell types. It
has been proposed that p21 is a survival signal in many types of cell and that
active caspase 3 cleaves p21 when apoptosis is induced
(Kwon et al., 2002). We showed
in conditional Stat3-;/-; glands that p21 is upregulated
(Chapman et al., 1999
).
However, no changes were observed in the expression of p21 in
Lif-;/-; animals (Fig.
3A). This suggests that the expected upregulation of p21 in the
absence of STAT3 is balanced by cleavage of p21 caused by a downregulation of
survival signals in the absence of LIF receptor activation.
Interestingly, there was a significant decrease in the amount of pERK in the absence of LIF (Fig. 3A). Thus, LIF regulates pERK1/2 mediated survival signals in addition to a pSTAT3-dependent apoptotic stimulus. This regulation of pERK1/2 by LIF during involution prompted us to examine levels of pERK throughout a mammary developmental cycle (Fig. 3B). Phospho-ERK1/2 levels were highest in virgin and mid-pregnant glands. Notably, pERK levels were diminished during involution when pSTAT3 levels were highest, suggesting that at this stage LIF signals primarily through STAT3.
LIF deficiency results in aberrant ductal and alveolar
morphogenesis
As pERK levels were highest early in the mammary developmental cycle, when
LIF was also highly expressed, we determined whether the absence of LIF had
consequences for lobuloalveolar development. Precocious alveolar development
occurred in Lif-;/-; glands during early pregnancy
(Fig. 4A). This coincided with
a significant reduction in pERK1/2 activity compared with
Lif+/-; controls and an increase in pSTAT5 levels,
confirming the precocious alveolar development and suggesting that LIF-induced
pERK1/2 may contribute to normal development of the gland at this time. There
was no significant difference in the levels of pSTAT3, which was barely
detectable at day 8 pregnancy (Fig.
4B). Because LIF is expressed at significant levels in the mammary
glands of virgin animals, it was interesting to determine if the absence of
LIF had consequences for postnatal mammary development. In
Lif-;/-; mammary glands as early as 4 weeks of age, ductal
elongation was markedly reduced and the ducts were thicker
(Fig. 4C). In addition to a
reduction in growth rate in the absence of LIF, the morphology of the terminal
end buds was abnormal, these being more rounded and disorientated.
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Changes in PR status during pregnancy in LIF deficient mammary
glands
The progesterone receptor (PR) is downregulated during pregnancy and it has
been suggested that PR status reflects proliferation status
(Clarke et al., 1997). Because
we observed an increase in the number of mitotic cells in mid-pregnancy (data
not shown), we determined the number of PR-positive cells in sections from
Lif-;/-; and Lif-;/+ glands. Using
immunohistochemistry, we found that at day 8 of pregnancy, the percentage of
cells expressing PR in glands from the LIF deficient mice was almost half that
seen in glands from the heterozygotes (9.7±0.8 versus 16.6±1.6;
P=0.018 by Student's t-test). By comparison, there was no
significant difference in virgins (52.9±1.8 mean±s.e.m. versus
65.1±10.0 mean±s.e.m.). This suggests a growth restricting role
for LIF during pregnancy. Other workers have previously shown that there are
no ovarian defects in the Lif-;/-; mice and that
progesterone levels during early pregnancy are normal
(Chen et al., 2000
;
Song et al., 2000
).
Use of a specific MEK1 inhibitor, U0126, potentiates apoptosis in
undifferentiated KIM-2 cells after LIF treatment without affecting STAT3
activation
As LIF treatment of KIM-2 cells resulted in STAT3 activation
(Fig. 1), we used this culture
model to determine whether LIF could activate ERK also in KIM-2 cells and
whether there is crosstalk between these signaling pathways in the control of
apoptosis, specifically with regard to whether there is any significance in
the downregulation of phospho-ERK during involution. We speculate that pERK1/2
could provide a survival signal that must be downregulated in order for STAT3
to mediate induction of apoptosis. We treated differentiated KIM-2 cells with
LIF and measured levels of activated ERK1/2 and STAT3 by western blotting
(Fig. 5A). Treatment with LIF
resulted in a rapid activation of STAT3 (within 30 minutes) and a delayed
activation of ERK. Maximal ERK activity was attained in the absence of pSTAT3,
providing circumstantial evidence for crosstalk between these molecules. This
was supported by in vivo evidence from Stat3-;/-;
involuting mammary glands (Fig.
5B). In contrast to the decreased levels of pERK observed in
Lif-;/-; glands (Fig.
3A), pERK activity was higher in the absence of STAT3
(Fig. 5B), suggesting that
STAT3 may regulate the levels of pERK.
|
Moreover, there was a modest induction of apoptosis in confluent cultures of KIM-2 cells with LIF, as measured by annexin V labeling (Fig. 1B; Fig. 5C). Treatment with U0126 resulted also in a modest increase in apoptosis (Fig. 5C). However, treatment of KIM-2 cells with LIF and U0126 simultaneously, resulted in a dramatic potentiation of apoptosis, reflected by the significant increase in annexin V-positive cells. These data suggest that LIF activates signaling pathways in mammary epithelial cells which may culminate in either survival or death, depending on their relative balance and that STAT3 cannot induce apoptosis in the presence of a strong survival signal from pERK.
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DISCUSSION |
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It has been suggested that the primary stimulus for epithelial apoptosis in
the regressing mammary gland originates from locally derived factors, as
involution can occur in sealed glands in the presence of normal circulating
levels of hormones (Li et al.,
1997). A number of potential mediators of this stimulus have been
proposed, including mechanical stress of the epithelium arising from milk
stasis, changing concentrations of apoptosis regulators in the milk, and
immune infiltrates. We have previously reported that STAT3 has a crucial role
in initiating apoptosis at the onset of mammary regression, the first
description of a transcriptional regulator of epithelial apoptosis in mammary
gland. In this study, we now establish that the cytokine LIF is the
physiological activator of STAT3 and plays a principal role in the apoptotic
process. The complete absence of pSTAT3 in Lif-;/-; glands
indicates that, somewhat surprisingly, other cytokines including members of
the IL6 family do not compensate for the lack of LIF signaling. The similarity
between Stat3-;/-; and Lif-;/-;
phenotypes in involution and the failure to induce C/EBP
expression, a
known transcriptional target of STAT3, in LIF-deficient glands further
supports this conclusion.
Recently, it has been shown that LIF expression was induced by haemodynamic
overload in the adult mammalian heart
(Wang et al., 2001). Therefore
it is possible that mechanical stretch could induce the expression of LIF,
which in an autocrine manner, could then activate STAT3 and promote apoptosis.
Alternatively, LIF expression could be induced by the accumulation of a
secreted factor in the lumen during milk stasis. It has been proposed that
TGF-ß3 may activate STAT3 during involution
(Nguyen and Pollard, 2000
).
Overexpression of TGFß3 in transgenic mammary glands caused precocious
apoptosis and elevated levels of pSTAT3 in alveolar epithelium. However, it is
unclear whether TGF-ß3 directly activates STAT3 or whether it has a
permissive role, through inhibition of a negative regulator of STAT3. It will
be of interest to determine where TGF-ß3 lies on the LIF/STAT3 axis.
Although it is not known how LIF expression is regulated during
development, evidence from uterine tissue or breast cancer cell lines suggest
that LIF expression could be regulated by oestrogen or progesterone
(Bhatt et al., 1991;
Cullinan et al., 1996
;
Bamberger et al., 1998
). In
addition, the similarities between LIF and estrogen receptor
knockout
glands (Bocchinfuso et al.,
2000
) would suggest that either estrogen is controlling LIF
expression during early development or that both those factors act
simultaneously to induce mammary growth.
Expression of LIF is stage specific, being highest at the beginning (virgin
and early pregnancy) and end (involution) of the developmental cycle. We
demonstrate that ERK1/2 is reciprocally activated with respect to STAT3 in the
mammary developmental cycle, pERK levels being highest in virgin and early
pregnancy when pSTAT3 is absent, but markedly reduced at the onset of
involution when STAT3 is induced. We have begun to address the molecular
mechanism of this developmentally regulated suppression of LIF-dependent STAT3
signalling in pregnancy and ERK1/2 signalling in involution. The lack of
pSTAT3 in the presence of high levels of LIF in early developmental stages
suggests that a specific negative regulator of STAT3 is biologically active at
this time. The SOCS proteins are classic negative-feedback regulators of
phosphorylated STAT proteins, of which SOCS3 is the principal target for LIF
signalling (Auernhammer et al.,
1998). Furthermore, SOCS3 is a direct transcriptional target of
STAT3, but not STAT5 (Auernhammer et al.,
1999
) (data not shown). Indeed, of the four SOCS genes studied,
SOCS3 exhibits an expression pattern in early development that is consistent
with a role in suppressing pSTAT3 (data not shown). However, SOCS3 is also
expressed at significant levels during the onset of major remodelling in the
involuting gland, when pSTAT3 levels are still high. This suggests that SOCS3
is not the principal regulator of STAT3 at this stage of involution and that
other mechanisms (possibly other SOCS proteins or protein tyrosine
phosphatases) may regulate STAT3 at this time.
Our observations of pERK1/2 levels in Lif-;/-; and Stat3-;/-; mammary glands and in KIM-2 mammary epithelial cells treated with LIF, suggests that STAT3 promotes the downregulation of pERK1/2 in involution. We propose that the loss of ERK activity during involution is biologically significant and is necessary for STAT3 to mediate its maximal apoptotic function. Thus LIF, through STAT3, may contribute directly to the suppression of its alternate downstream signalling pathway in mammary gland. Identification of a LIF-induced molecular switch raises the question of its biological significance in mammary gland. It is interesting to speculate whether the reciprocal inhibition of STAT3 and activation of ERK1/2 early in mammary development prevents inappropriate induction of cell death as seen in our epithelial cell model.
Throughout early development we observed enlarged ducts similar to
C/EBPß-deficient mammary glands
(Seagroves et al., 1998;
Seagroves et al., 2000
),
suggesting that LIF may mediate these effects via C/EBPß. Moreover, LIF
is known to regulate C/EBPß via ERK
(Aubert et al., 1999
), which is
markedly reduced in LIF knockout mammary glands. Activated ERK1/2 transduces
several signals including growth arrest, differentiation and survival. We
observed no differences in the levels of apoptosis between LIF knockout and
control animals in virgin or pregnancy mammary glands (data not shown). We
also report that proliferation (and associated PR expression) is disrupted in
early development in LIF-deficient mice. Although it is possible that this
difference in PR expression reflects a change in cell fate arising from the
increased number of alveolar cells arising during pubertal development, it is
clear that LIF plays a role in growth arrest and/or differentiation, rather
than apoptosis, early in development.
In conclusion, we have shown that LIF is the physiological activator of STAT3 in mammary gland and we suggest that one function of STAT3 in involution is to contribute to the downregulation of the ERK1/2 survival pathway, thus potentiating the subsequent pro-apoptotic effects of STAT3. We demonstrate a developmentally regulated bifurcation of LIF signaling via ERK and JAK/STAT pathways such that LIF signals mainly through ERK during pregnancy but through STAT3 in involution, to control proliferation/differentiation and cell death, respectively. We speculate that this differential regulation is mediated by factors downstream of LIFR/gp130 that are expressed or regulated at different times in mammary development. SOCS3 expression early in mammary development correlates with suppression of pSTAT3 in virgin and early pregnancy (data not shown). In involution, however, LIF-dependent activation of STAT3 contributes directly to the suppression of pERK, resulting in a shift in the balance of apoptotic and survival signals. Precisely what the downstream targets of the ERK1/2 signal are, remains to be determined. However, our data clearly imply that activation of distinct pathways are required to mediate the effects of LIF at different stages of normal mammary development. The challenge now is to identify both the downstream targets of ERK1/2 in mid-pregnant mammary epithelial cells, and to further elucidate the molecular targets of STAT3 that precipitate involution.
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ACKNOWLEDGMENTS |
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