1 School of Biological Sciences, Stopford Building, University of Manchester,
Oxford Road, Manchester, M13 9PT, UK
2 Endocrine Sciences Research Group, Stopford Building, University of
Manchester, Oxford Road, Manchester, M13 9PT, UK
3 Hannah Research Institute, Ayr, KA6 5HL, UK
* Author for correspondence (e-mail: melissa.westwood{at}man.ac.uk)
Accepted 5 November 2002
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Summary |
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Key words: Insulin-like growth factors, Insulin-like growth factor binding proteins, Apoptosis, Mammary gland
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Introduction |
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The actions of IGFs are modulated by high affinity interactions with a
family of structurally related IGF-binding proteins (IGFBPs), IGFBP-1 to
IGFBP-6 (Jones and Clemmons,
1995). IGFBPs are themselves subject to modification by
proteolysis, post-translational alterations such as phosphorylation and
interactions with cell surface and extracellular matrix components
(Jones et al., 1993
;
Westwood et al., 1997
). IGFBPs
are known to regulate the bioavailability of IGFs in the circulation; however,
their functions at the cellular level are not fully understood. IGFBPs have
been reported to both inhibit and enhance IGF-I action depending on the system
under investigation (Jones and Clemmons,
1995
).
We have demonstrated that IGF-I suppresses apoptosis of primary mammary
epithelial cells in culture (Farrelly et
al., 1999). Mammary gland models also indicate the importance of
IGFs as survival factors in vivo. In studies where transgenic mice overexpress
IGF-I or II specifically in the mammary gland, apoptosis is reduced
(Neuenschwander et al., 1996
;
Hadsell et al., 1996
;
Moorehead et al., 2001
).
Involution, the rapid induction of apoptosis that occurs to remodel the gland
after lactation, was delayed as a result of reduced apoptosis in these
animals. Regulating the availability of IGFs to mammary epithelial cells may
therefore represent a physiological mechanism for initiating apoptosis during
the process of involution.
All six IGFBP mRNAs are present in the mouse mammary gland, although
IGFBP-3 and -5 are expressed most prominently in the stroma and also in ductal
and alveolar epithelium in the pregnant gland
(Wood et al., 2000). An
increase in IGFBP-5 protein expression has been observed in rat milk after 48
hours of involution (Tonner et al.,
1997
). From these studies, it has been proposed that IGFBPs may
regulate apoptosis in the mammary gland by blocking IGF-mediated survival. In
this work, we therefore aimed to determine whether IGFBP-5 can directly
regulate apoptosis by interfering with IGF signalling.
We provide conclusive evidence that IGFBP-5 is expressed during mammary gland involution when apoptosis levels are markedly increased, and demonstrate that IGFBPs are secreted into a cellular compartment where they can modulate IGF actions. We also observed that IGFBPs inhibit IGF-mediated survival signalling to cause apoptosis of mammary epithelial cells. This study therefore demonstrates a functional role for IGFBP-5 in the control of mammary cell survival. Additionally, our results provide a possible mechanism for the initiation of involution in the mammary gland.
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Materials and Methods |
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Protein and DNA extraction from mouse mammary gland tissue
Mammary tissue from ICR mice (Harlan Sera-Labs Ltd, Loughborough, UK) was
collected from 8-week-old virgin animals (V) and mice pregnant for 9 and 15
days after detection of a vaginal plug (P9 and P15). At birth, pup numbers
were normalized to 10 per dam. Mammary tissue was then removed from mice in
full lactation for 9 days (L9), and from mice where the pups had been removed
for up to 5 days following a 9-day lactation period (involution, I1-5). For
immunoblot analysis, tissue was ground to a powder in liquid nitrogen and
homogenized in lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1%
(w/v) Nonidet-P40, 1% (w/v) sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM
Na3VO4, 10 mM NaF, and protease inhibitors (Calbiochem,
Nottingham, UK).
For analysis of DNA integrity, tissue was ground to a powder in liquid nitrogen and homogenized in 100 mM NaCl, 10 mM EDTA, 300 mM Tris, 200 mM sucrose, pH 8.0, 0.65% SDS before incubation for 30 minutes at 60°C. Following proteinase-K (Roche, Lewes, UK) digestion for 1 hour at 55°C, DNA was extracted using phenol chloroform as described previously (Pullan et al., 1996). Equal amounts of DNA were separated on a 1.2% agarose gel, stained with ethidium bromide and photographed.
Substrata and cell culture
Collagen-I-coated dishes were prepared by incubating dishes overnight at
4°C with rat tail collagen at 8 µg/cm2. The plates were
washed extensively with phosphate-buffered saline before use.
Growth-factor-reduced Matrigel (Becton Dickinson, Oxford, UK) was used to coat
dishes at 14 mg/ml.
Primary mammary epithelial cells were prepared from 14.5-18.5-day pregnant
ICR mice as previously described (Pullan
and Streuli, 1996) and plated on different substrata in nutrient
mixture F-12 (Biowhittaker, Wokingham, UK) containing 10% heat-inactivated
fetal calf serum (Biowhittaker), 1 mg/ml fetuin (Sigma), 5 ng/ml EGF (Harlan
Sera-Labs) and 1 µg/ml hydrocortisone (Sigma). After 48 hours, the medium
was removed and fresh F-12 medium was added. After a further 24 hours, cells
were serum-starved overnight in Dulbecco's modified Eagle's medium/nutrient
mixture F-12 medium (DF-12; Biowhittaker).
To investigate the IGFBP expression profile of primary mammary epithelial
cells in vitro, the serum-free medium removed after the 24-hour starvation
process was concentrated (tenfold) and prepared for ligand blot analysis. To
investigate whether IGFBP was secreted basally or apically, cells were grown
on basement membrane (growth-factor-reduced Matrigel) in DF-12 differentiation
medium containing 1 µg/ml hydrocortisone, 5 µg/ml insulin and 3 µg/ml
prolactin for 4 days. After this time, the medium was replaced with serum-free
medium or serum-free medium containing 2.5 mM EGTA to open tight junctions
(Barcellos-Hoff et al., 1989).
After 10 minutes, the medium was removed and was concentrated for ligand and
immunoblot analysis of casein expression.
FSK-7 mouse mammary epithelial cells
(Kittrell et al., 1992) were
cultured in DMEM:F12 medium supplemented with 2% fetal calf serum. For
signalling experiments, FSK-7 or primary mammary epithelial cells were treated
with 1 nM IGF-I±IGFBP as appropriate for 15 minutes after
serum-starvation. Following treatment, cells were harvested in lysis buffer as
above.
Apoptosis assay
Primary cultures of mammary epithelial cells grown on collagen I were
serum-starved overnight and then were treated for 24 hours with 1 nM
IGF-I±IGFBP as appropriate. Following treatment, the medium was removed
and detached cells were pelleted by centrifugation. Attached cells were
trypsinised and combined with detached cells. Cells were cytospun onto
polysine slides (Merck, Poole, UK), and fixed in 3.7% paraformaldehyde for 10
minutes. Apoptosis was quantified by examining the nuclear morphology of cells
stained with 4 µg/ml Hoechst 33258 (Molecular Probes, Leiden, The
Netherlands). Each experiment was repeated at least three times and in each
experimental condition more than 1000 single cells were scored for
apoptosis.
Immunoprecipitations
For immunoprecipitations with mammary gland tissue, tissue lysate was
incubated with 4 µl IGFBP-5 antibody for 16 hours at 4°C and then 50
µl protein A-Sepharose beads (Zymed Laboratories, Cambridge Bioscience,
Cambridge, UK) were added for a further 2 hours. For primary mammary
epithelial cells, cell lysate was incubated with 1 µg IRS-1 antibody for 1
hour at 4°C then 50 µl protein A-Sepharose beads were added for a
further 4 hours.
Immunoblot and ligand blot analysis
Immunoprecipitates or tissue/whole cell lysates were subjected to SDS-PAGE
and transferred to 0.2 µm nitrocellulose membrane (BioRad, Hemel Hempstead,
UK). Membranes were blocked in 3% nonfat milk (Marvel) for 1 hour and
incubated with antibodies to IGFBP-3 (1:1000), IRS-1 (1 µg/ml), phospho-PKB
(1:1000), PKB (1:1000), FKHRL1 (1:750), E-cadherin (1:3000) or casein (1:4000)
overnight at 4°C. Membranes were then incubated with secondary antibody
(Amersham, Buckinghamshire, UK; 1:3000) for 1.5 hours at room temperature.
Proteins were visualized using Supersignal West Femto (Perbio, Tattenhall, UK)
or ECL (Amersham). In each of the studies presented, the results are typical
of at least three independent experiments.
For ligand blot analysis, proteins were subjected to SDS-PAGE under non-reducing conditions. Following transfer to nitrocellulose membrane, the membrane was incubated in 0.5% (w/v) sodium azide and 1% (v/v) Nonidet-40 in phosphate-buffered saline (PBS) for 30 minutes at 4°C. The membrane was then blocked in 1% bovine serum albumin in PBS/0.5% Tween for 1 hour at room temperature before incubation with 125I-IGF-I (150,000 cpm/ml) for 3 hours. The ligand blot was then washed and exposed to film for 72 hours at -70°C.
Statistical analysis
Analysis was performed using the statistical software package Instat2
(GraphPad Prism, San Diego, USA). Comparisons of apoptosis values gained by
each method were analysed using Student's t-test. A P-value
less than 0.05 was considered to indicate statistical significance.
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Results |
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To confirm that IGFBP-5 expression is increased coincidently with apoptosis, DNA integrity was also assessed in samples from the same mammary glands used for protein analysis (Fig. 1C). DNA ladders were not observed in samples taken from virgin, pregnant and lactating mice; however, DNA internucleosomal fragmentation was marked at involution day 1, increasing at involution day 2. Thus, the profile of DNA fragmentation mirrors IGFBP-5 expression during mammary involution suggesting a link between IGFBP-5 secretion and apoptosis induction.
Mammary epithelial cells secrete IGFBPs in culture from the basal
surface
In vivo, IGFBP-5 must have ready access to IGFs if it is to inhibit
IGF-mediated survival. We therefore examined whether IGFBPs are secreted
basally or into the lumen of cultured mammary pseudo-alveoli. Mouse mammary
epithelial cells (MECs) grown on basement membrane in the presence of
prolactin form partially differentiated alveolar-like structures with their
basal cell surfaces orientated outwards towards the medium and their apical
surfaces towards a sequestered lumen
(Barcellos-Hoff et al., 1989).
This model attempts to recapitulate mammary epithelial morphogenesis.
Secretion of proteins from the basal surface can be detected in the medium
surrounding these cells, and apical secretion into the lumen can be determined
when medium is analyzed after the tight-junctions are opened using EGTA
(Barcellos-Hoff et al.,
1989
).
Medium from differentiated MECs grown on basement membrane was replaced with EGTA-containing medium or medium alone before the medium was removed, and concentrated. Ligand blotting revealed the predominance of an IGF-I binding species that co-migrated with recombinant IGFBP-5 (Fig. 2). A faint band corresponding to the molecular weight of glycosylated IGFBP-3 was also detected. After EGTA-treatment, when the lumenal contents were released into the medium, levels of IGFBP-5 were not elevated indicating that MECs are able to secrete IGFBPs basally. To act as a positive control for tight junction opening, the distribution of casein was also measured and a protein of the same molecular weight as ß-casein was found to be present only in EGTA-treated-primary cultures.
|
These results provide evidence that not only is IGFBP-5 expressed in mouse mammary gland and that its level of expression is increased at involution, but also that IGFBP-5 is synthesized by mammary epithelial cells and secreted basally into a tissue compartment where it could physiologically regulate IGF-I-mediated survival in vivo.
IGF-I-mediated survival is inhibited by IGFBP-5 and -3
To determine if IGFBPs act as physiological regulators of IGF function, we
investigated whether IGF-mediated survival of primary MECs could be modulated
by exogenous IGFBPs. Since IGFBP-5 was the most prominent IGFBP expressed in
the mammary gland in vivo, we examined its effect on survival and apoptosis.
Although expression of IGFBP-3 could not be detected in vivo, it was secreted
by primary cultures so the effects of IGFBP-3 on IGF-mediated survival were
also investigated. Apoptosis, determined by changes in nuclear morphology, was
quantified in primary MECs exposed to IGF-I in the presence or absence of
IGFBPs for 24 hours (Fig. 3). Serum withdrawal resulted in a fourfold increase in the number of cells
undergoing apoptosis; but this was reversed by inclusion of 1 nM IGF-I
(28.5±4% vs 7.9±1.6%; P=0.001). This confirms the
importance of IGF-I as a survival factor in MECs
(Fig. 3A,B)
(Farrelly et al., 1999).
|
Recent studies have indicated that IGFBPs may act independently of IGFs to
induce apoptosis in other cell systems
(Nickerson et al., 1997;
Perks et al., 2000
); however,
when MECs were incubated with 10 nM IGFBP-5 or -3 alone apoptosis was not
significantly increased compared to serum-starved cells. A modest survival
effect was seen although this was not statistically significant. Apoptosis was
not maximal in these cells since apoptosis in the absence of serum can be
further induced by other pharmacological agents (data not shown).
When IGFBP-5 or -3 was co-incubated with IGF-I, apoptosis was significantly
higher than in cells incubated with IGF-I alone (21.3±5% vs
7.9±1.6%; P0.01) and the percentage of apoptotic cells was
not significantly different from cells grown in serum-free medium
(Fig. 3A,B). The concentrations
of IGFBPs and IGFs used are physiologically relevant given that there is
greater than tenfold molar excess of IGFBP-5 compared with IGF-I in the
involuting mammary gland (Tonner et al.,
2000
). Inclusion of 5 nM IGF-II suppressed apoptosis and this was
completely inhibited by 10 nM IGFBP-5 or -3 (data not shown).
These results demonstrate that although IGFBP-5 and IGFBP-3 do not induce apoptosis themselves, they can abrogate IGF-I and IGF-II-mediated survival.
IGF-initiated signalling is inhibited by IGFBP-5 and -3
To understand the mechanism responsible for IGFBP effects on IGF-initiated
survival, we examined changes in phosphorylation of key signalling molecules.
We concentrated on the insulin-receptor substrate/phosphatidylinositol
3-kinase/protein kinase B pathway because 1 nM IGF-I induced phosphorylation
of this pathway but did not induce mitogen-activated protein kinase
phosphorylation at this concentration (data not shown). In FSK-7 mouse MECs,
IGF-I (1 nM, 15 minutes) stimulated a dramatic increase in insulin-receptor
substrate 1 (IRS-1) phosphorylation (Fig.
4A, lane 2) which was inhibited in cells co-incubated with 5 nM
IGFBP-5 or 5 nM IGFBP-3 (Fig.
4A, lanes 5,6). Similarly, IGFBP-5 inhibited IGF-I-mediated IRS-1
phosphorylation in primary cultures of MECs
(Fig. 4B). Neither IGFBP-5 nor
-3 had independent effects on IRS-1 phosphorylation
(Fig. 4A, lanes 3,4, and data
not shown).
|
To determine if these signalling changes were reflected by alterations in
downstream effectors, phosphorylation of the serine/threonine kinase protein
kinase B (PKB) at Ser473 was investigated
(Fig. 5). PKB phosphorylation
was stimulated when MECs were incubated with 1 nM IGF-I for 15 minutes
(Fig. 5A,B, lane 3) to a
similar level seen with cells grown in the presence of serum (data not shown).
IGF-initiated PKB phosphorylation was however inhibited in the presence of
5-10 nM IGFBP-5 and 1-10 nM IGFBP-3 (Fig.
5A,B, lanes 4-6). PKB was not phosphorylated under control
conditions or when cells were exposed to 10 nM IGFBP-5 or -3 alone
(Fig. 5A,B, lanes 1,2).
|
We also determined if IGFBP-5 and -3 could modulate downstream signals mediated by IGF-II (Fig. 5C). PKB phosphorylation was observed with 5 nM IGF-II alone (Fig. 5C, lane 2) and was inhibited by a twofold molar excess of IGFBP-3 and -5 (10 nM) (Fig. 5C, lanes 3,4). Together, the results indicate that IGFBPs can potently modulate intracellular signals mediated by both IGF-I and IGF-II.
A number of substrates for PKB have been proposed; however, the Forkhead
transcription factor FKHRL1, has been particularly implicated in the
modulation of apoptosis (Brunet et al.,
1999; Zheng et al.,
2000
). Phosphorylation by PKB results in FKHRL1 inhibition and
retention in the cytoplasm. In contrast, dephosphorylation leads to FKHRL1
activation, translocation to the nucleus and subsequent transcription of genes
encoding death-activating proteins (Brunet
et al., 1999
). We therefore examined whether IGFs could mediate
the phosphorylation of FKHRL1 in mammary epithelial cells and if this is
inhibited by IGFBPs. In contrast to untreated cells and cells exposed to 10 nM
IGFBP-5, a shift in FKHRL1 mobility, indicative of phosphorylation, was
observed when cells were exposed to 1 nM IGF-I
(Fig. 6A, lanes 1-3). A similar
shift in FKHRL1 mobility has been attributed to phosphorylation in
IGF-I-treated fibroblasts (Brunet et al.,
1999
). When MECs were incubated with IGF-I in the presence of
IGFBP-5, this mobility shift was not observed
(Fig. 6A, lane 4). Similarly,
IGF-II induced a shift in FKHRL1 mobility that was inhibited by 10 nM IGFBP-5
or IGFBP-3 (Fig. 6B).
|
Together, these results demonstrate that, in common with IRS-1 and PKB, FKHRL1 phosphorylation is modulated by IGFBP-5 and -3 and suggest that FKHRL1 may be an important mediator of the actions of IGFs in the mammary gland.
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Discussion |
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The implications of this finding are significant given that IGFs are known
survival factors for several cell types. IGF-I is able to protect cells from
apoptosis under a wide variety of circumstances, including growth factor
withdrawal in haematopoietic and neuronal cells; overexpression of
myc in fibroblasts; chemotherapy, and UV-B irradiation
(Muta and Krantz, 1993;
Harrington et al., 1994
;
Sell et al., 1995
;
Kulik et al., 1997
;
Datta et al., 1997
). IGF-II can
also act as a survival factor; for example, IGF-II suppresses hepatocyte
apoptosis triggered by deregulated N-myc expression and can block cell death
during oncogenesis in vivo (Christofori et
al., 1994
; Ueda and Ganem,
1996
).
IGFBP-5 expression is increased during involution when apoptosis is
maximal
Previous studies have shown that IGFBP-5 increases in rat milk after 48
hours of involution (Tonner et al.,
1997). We now reveal its pattern of tissue expression through a
more extensive developmental analysis in a murine model. Levels of IGFBP are
not greatly altered during the transition from ductal epithelium in the
non-pregnant mammary gland to alveolar epithelium in pregnancy and lactation.
However, IGFBP-5 is dramatically upregulated at day 1 of involution, and was
maximal at days 2 and 3, decreasing at day 5. This profile is consistent with
the pattern of DNA fragmentation, providing correlative evidence for the
involvement of IGFBP-5 in mammary apoptosis. This observation has also been
supported by recent in vivo data (Tonner
et al., 2002
).
IGFBPs are appropriately located to inhibit the actions of IGFs
The previous observation that IGFBP-5 is present in large quantities in
milk (Tonner et al., 1997)
could suggest that the IGFBP-5 expressed in involution may not be able to
access the cellular compartment basal to epithelial cells where the actions of
IGFs take place. We show experimentally that IGFBP-5 and -3 are secreted
basally from the alveolar-like structures formed by MECs in culture. This
result leads us to suggest that IGFBP-5 synthesized and secreted by mammary
epithelial cells, binds to IGF-I within the mammary stroma and prevents an
interaction between IGF-I and the type I IGF receptor (IGF-IR) on the basal
cell surface.
IGFBPs block IGF-mediated survival signalling leading to increased
apoptosis
IGFBP-5 and -3 inhibit IGF-mediated survival in our model. Studies using
cultured preovulatory follicles, osteosarcoma cells and neuronal cells
indicate that IGFBP-3 can inhibit IGF-mediated survival
(Chun et al., 1994;
Niikura et al., 2001
;
Schmid et al., 2001
). To our
knowledge, this is the first report demonstrating that IGFBP-5 can also
regulate IGF-mediated survival.
Recently, a number of studies indicate that IGFBPs may act not only via
sequestration of IGF-I but independently of IGF-I to regulate apoptosis.
IGFBP-3 induces apoptosis in MCF-7 breast cancer cells
(Nickerson et al., 1997) and
accentuates ceramide-induced apoptosis in Hs578T breast cancer cells
(Perks et al., 1999
;
Perks et al., 2000
). However,
the situation appears to be different in normal mammary epithelial cells since
treatment with either IGFBP-3 or -5 alone does not trigger apoptosis beyond
that induced by serum-withdrawal. These differences may alternatively reflect
the time course and IGFBP concentration used.
Our results reveal a possible mechanism for the effects of IGFBPs on
mammary epithelial apoptosis (Fig.
7). IRS-1 phosphorylation is inhibited when IGFBP-5 and -3 and
IGFs are co-incubated. This is in contrast to studies in MCF-7 human breast
carcinoma cells where IGFBP-3, but not IGFBP-5, inhibited IRS-1
phosphorylation (Ricort and Binoux,
2001). We also observed that IGFBPs inhibit both IGF-I- and
IGF-II-mediated signalling via PKB (Fig.
7). PKB has a proven role in promoting survival
(Datta et al., 1997
) and has
been implicated in the regulation of involution. Expression of a
constitutively active form of PKB in the mammary gland of transgenic mice
delays onset of apoptosis after weaning
(Hutchinson et al., 2001
;
Schwertfeger et al., 2001
).
Additionally, sustained phosphorylation of PKB correlates with the reduced
apoptosis and delayed involution observed in transgenic mice overexpressing
IGF-II (Moorehead et al.,
2001
). Thus, modulation of PKB phosphorylation via IGFBP-5
inhibition of IGF-I or -II signalling may be an important mechanism in
involution.
|
IGF-I has been shown to phosphorylate FKHRL1 via PKB in other cell types
such as haematopoietic cells (Brunet et
al., 1999), but this has not been studied in the mammary gland.
Other growth factors, such as epidermal growth factor
(Jackson et al., 2000
) and
vascular endothelial growth factor (Price
et al., 2001
) have been shown to activate FKHRL1 in human breast
cancer cells, but this is the first observation that IGF-I and -II can
phosphorylate FKHRL1 in non-transformed mammary epithelial cells. We also
demonstrate that FKHRL1 phosphorylation is blocked by IGFBP-5 and -3
(Fig. 7). Studies concerning
the effect of FKHRL1 on apoptosis have mainly used ectopically expressed
FKHRL1. The Fas ligand gene was shown to be a target for FKHRL1 transcription
in fibroblasts, cerebellar granule neurons and Jurkat T lymphocytes
(Brunet et al., 1999
); however,
in murine Ba/F3 haematopoietic cells, FKHRL1 induces apoptosis through a
death-receptor-independent pathway that involves transcriptional upregulation
of the pro-apoptotic Bcl-2 family member, Bim
(Dijkers et al., 2000
;
Dijkers et al., 2002
). Although
we cannot rule out other PKB substrates as additional effectors, activation of
FKHRL1 provides a potential mechanism to explain the increase in apoptosis
observed when IGF signalling is inhibited by IGFBPs.
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Conclusion |
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Acknowledgments |
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