First published online 23 December 2002
doi: 10.1242/jcs.00263
Insulin-like growth factor binding protein 5 and apoptosis in mammary epithelial cells
Emma Marshman1,2,
Kirsty A. Green1,
David J. Flint3,
Anne White1,2,
Charles H. Streuli1 and
Melissa Westwood1,2,*
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
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Fig. 1. An increase in IGFBP-5 expression occurs concomitantly with DNA
fragmentation during mammary gland involution. (A) Whole tissue lysates from
different stages of development were analysed by 15% SDS-PAGE and ligand
blotting with I125-IGF-I. Tissue was taken from virgin animals (V);
mice pregnant for 9 and 15 days after detecting a vaginal plug (P9 and P15);
mice in full lactation for 9 days (L9); and mice where the pups had been
removed for up to 5 days following a 9-day lactation period (I1-5). Samples of
recombinant IGFBP-1, -3 and -5 were included as reference markers and the
E-cadherin levels of the samples were analysed by immunoblotting (IB) to
confirm equal epithelial cell content. (B) Lysates from duplicate samples were
immunoprecipitated (IP) with an antibody against IGFBP-5 prior to
I125-IGF-I ligand blotting. (C) DNA extracted from mammary gland
tissue from different stages of development was separated on a 1.2% agarose
gel and was stained with ethidium bromide. Results are representative of three
sets of tissue.
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Fig. 2. IGFBPs are secreted from the basal surface of primary mammary epithelial
cells grown on basement membrane. Cells were cultured on reconstituted
basement membrane for 6 days in differentiation medium. Medium was replaced
with serum-free medium (CON) or serum-free medium containing 2.5 mM EGTA for
10 minutes. Medium was then removed and concentrated before ligand blot
analysis to detect IGFBPs and immunoblotting to detect secreted ß-casein.
IGFBPs could not be detected in the basement membrane gel in the absence of
cells (BM alone). Casein immunoblots and ligand blots are representative of
three separate experiments.
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Fig. 3. IGFBP-5 and IGFBP-3 reverse IGF-I mediated suppression of apoptosis in
primary mammary epithelial cells. Following serum-starvation, cells were
either treated with (A) 1-10 nM IGFBP-5 or (B) 1-10 nM IGFBP-3 alone or in
combination with 1 nM IGF-I. FCS and SFM indicate cells maintained in
serum-containing and serum-free growth medium, respectively. The number of
apoptotic cells was determined using Hoechst 33258 staining and counting of
cells with typically apoptotic nuclear morphology. Data are means±s.d.
from three independent experiments.
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Fig. 4. IGFBP-3 and IGFBP-5 inhibit IGF-I-mediated IRS-1 phosphorylation in mammary
epithelial cells. (A) FSK-7 mammary epithelial cells were serum-starved
overnight and treated with either 1 nM IGF-I or a combination of IGF-I and 5
nM IGFBP-5 or IGFBP-3. Control cells (CON) were maintained in serum-free
medium without treatment. Immunoprecipitates were separated by 6% SDS-PAGE and
analyzed by immunoblotting with a phosphotyrosine antibody or IRS-1 to
indicate equal total protein levels. (B) Primary mammary epithelial cells were
treated with 1 nM IGF-I±10 nM IGFBP-5 as above. Results are
representative of three experiments.
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Fig. 5. IGFBP-5 and IGFBP-3 inhibit IGF-I and IGF-II-mediated PKB phosphorylation
in primary mammary epithelial cells. Cells were serum-starved overnight before
treatment with (A) 1-10 nM IGFBP-5±1 nM IGF-I; (B) 1-10 nM
IGFBP-3±1 nM IGF-I; and (C) 5 nM IGF-II±10 nM IGFBP-5 or -3.
Control cells (CON) were maintained in serum-free medium without treatment.
Cell lysates were separated by 10% SDS-PAGE and analyzed by immunoblotting
with antibodies specific for phosphorylated PKB (Ser473) or total PKB to
indicate equal protein levels. Results are representative of three
experiments.
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Fig. 6. IGFBP-5 and IGFBP-3 inhibit IGF-I and IGF-II-mediated FKHRL1
phosphorylation in primary mammary epithelial cells. Cells were serum-starved
overnight before treatment with (A) 1 nM IGF-I±10 nM IGFBP-5; and (B) 5
nM IGF-II±10 nM IGFBP-5 or -3. Control cells (CON) were maintained in
serum-free medium without treatment. Cell lysates were separated by 8%
SDS-PAGE and analyzed by immunoblotting with an antibody that recognises both
the phosphorylated (*) and non-phosphorylated form of FKHRL1.
Immunoblotting for total PKB was used to indicate equal protein levels.
Results are representative of three experiments.
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Fig. 7. A model for IGFBP regulation of apoptosis. IGFBP-5 is secreted from the
basal surface of mammary epithelial cells where it is suitably placed to
interact with IGFs. This interaction prevents IGF-mediated activation of the
type I IGF receptor (IGF-IR) and inhibits (-) phosphorylation of insulin
receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3K) and protein
kinase B (PKB). In the inactive state, PKB is unable to phosphorylate
downstream effectors such as forkhead transcription factors (e.g. FKHRL1)
leading to FKHRL1 activation (+), induction of apoptosis and subsequently,
involution.
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© The Company of Biologists Ltd 2003
