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Hannah Research Institute, Ayr, United Kingdom KA6 5HL; and the Department of Medicine, University of Birmingham (A.L.), Birmingham, United Kingdom B15 2TT
Address all correspondence and requests for reprints to: Dr. David J. Flint, Hannah Research Institute, Ayr, United Kingdom KA6 5HL. E-mail: flintd{at}main.hri.sari.ac.uk
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Abstract |
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Introduction |
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During involution of the mammary gland, induced by removal of the suckling young, we identified the appearance in milk of an IGF-binding protein (IGFBP) (5). We hypothesized that as IGF-I has been described as an important survival factor for mammary epithelial cells (7, 8, 9) and thus can inhibit involution of the mammary gland after removal of the young, the epithelial cells produce an IGFBP to inhibit IGF-mediated cell survival in order for mammary cell death to occur. As such, this IGFBP could play a key role in the programed cell death known to occur during involution of the mammary gland.
To investigate this hypothesis further, we sought to identify the precise IGFBP produced and its site of production and to characterize control of its synthesis and secretion. As cessation of milk production leads to rapid changes in the serum concentrations of PRL, 17ß-estradiol, progesterone, and IGF-I, we concentrated our initial studies on these hormones. We also included corticosterone and GH, because both have been implicated in the control of mammary involution (4, 5, 10).
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Materials and Methods |
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Hormones
Recombinant bovine GH was a gift from Monsanto (St. Louis, MO),
and ovine PRL-19 (oPRL-19) was a gift from the NIDDK. Recombinant human
IGF-I was obtained from Bachem UK (Saffron Walden, Essex, UK).
Antiserum to IGF-I (capable of blocking IGF-I actions in
vivo) was a gift from Dr. S. Hodgkinson (Ruakura, New
Zealand).
Study 1
Lactating rats were treated commencing on days 12–14 of
lactation by removing the sucking young (litter removed). A control
group was allowed to continue nursing their young. Animals were killed
by cervical dislocation 24 or 48 h later, and the abdominal
inguinal mammary glands were dissected out. A portion of tissue was
stored in liquid N2 for preparation of RNA, whereas a
second portion was stored at -80 C for in situ
hybridization studies as described below. Some animals were
anesthetized with 0.3 ml sodium pentobarbitol (60 mg/ml; Sagatal, RMB
Animal Health, Dagenham, UK), after which the dam received 1 U oxytocin
(Intervet UK, Cambridge, UK) to induce milk ejection. Milk was obtained
from the mammary gland by gentle pressure and stored at -20 C until
used for the detection of IGFBPs by Western ligand blotting (11) and
Western immunoblotting as described below.
Study 2: hormonal control of IGFBP synthesis and secretion in
lactating rats
Lactating rats were treated on day 14 of lactation by daily
administration of 17ß-estradiol (100 µg in safflower
oil/injection). A second group of animals had teats on one side sealed
with tissue glue (Vetbond, Vet Drug Co., Falkirk, UK), and pup number
was reduced to six (one per unsealed gland). After treatment for 2
days, litters were removed from these groups for 4 h to allow milk
to accumulate before milking as described above.
Study 3: hormonal control of IGFBP synthesis and secretion after
litter removal
Animals had litters removed on day 14 of lactation (time zero)
and received one of the following treatments for 2 days, all given by
sc injection: recombinant bovine GH (1 mg in 20% PVP/injection) twice
daily, PRL (1 mg in 20% polyvinylpyrrolidone (PVP)/injection) twice
daily, hydrocortisone (1 mg in safflower oil/injection) once daily,
progesterone (5 mg in safflower oil/injection) once daily, or
anti-IGF-I serum (1 ml/injection) once daily or were left untreated.
All of these treatment doses were based on previous studies in which
they have been shown to be effective. The anti-IGF-I serum was
administered to inhibit the potential effects of the increase in serum
IGF-I that occurs after litter removal. This antiserum has previously
been shown to inhibit IGF-I action at the doses used in this study
(12). After treatment for 2 days, a milk sample was obtained from the
upper abdominal mammary glands, as described above, and stored at -20
C. The animals were then killed by cervical dislocation, and the lower
abdominal mammary glands were removed and stored in liquid
N2 for subsequent RNA isolation.
Northern blot analysis of RNA
Samples of total cellular RNA (20 µg) were separated by
electrophoresis in 1.2% agarose gels containing 2.2 M
formaldehyde (13) and transferred to Biotrans membrane (ICN, Amsterdam,
The Netherlands). These were then hybridized to a labeled antisense RNA
probe for rat IGFBP-5 (a gift from Dr. S. Shimasaki, Department of
Molecular Endocrinology, The Whittier Institute, La Jolla, CA). Blots
were exposed to a phosphor screen, and the image was scanned in a
Molecular Dynamics PhosphorImager 445SI and analyzed with ImageQuant
software (Molecular Dynamics, Sunnyvale, CA. Results were standardized
for the amount of 28S ribosomal RNA in each sample, as previously
described (14).
In situ hybridization studies
Frozen sections of rat mammary gland obtained from control
lactating animals or after litter removal for 48 h were probed for
IGFBP messenger RNA (mRNA). Sections were dehydrated in 100% ethanol
twice for 5 min each time, then fixed in 2% formaldehyde in 100
mM Tris-HCl and 50 mM EDTA, pH 8.0, for 5 min.
The sections were rinsed in distilled water for 5 min, and the tissue
was permeabilized in 10 µg/ml proteinase K, 50 mM EDTA,
and 1200 mM Tris-HCl, pH 8.0, for 30 min at 37 C. After
fixation in 2% formaldehyde solution, sections were rinsed in water,
then in 100 mM triethanolamine, pH 8.0, for 2.5 min and
acetylated for 10 min at room temperature in 100 mM
triethanolamine, pH 8.0, and 0.0025% (vol/vol) acetic anhydride. The
sections were rinsed briefly in 2 x SSC (standard saline citrate)
and dehydrated in a graded ethanol series (50%, 70%, 95%, 100%, and
100%) for 3 min each, dried under vacuum, and stored desiccated at
-20 C.
Antisense and sense RNA probes were prepared by incubating 1 ng template DNA, 200 µCi [35S]UTP, 50 mM GTP/CTP/ATP, 20 U RNA polymerase, 20 U ribonuclease inhibitor, 5 mM dithiothreitol (DTT), 2 mM spermidine, 10 mM NaCl, 6 mM MgCl2, and 40 mM Tris-HCl, pH 7.5, in a final volume of 20 µl at 37 C for 2 h. Deoxyribonuclease I (1 U) was then added at 37 C for 15 min, the reaction was stopped by adding 100 mM EDTA, pH 8.0, and the labeled probe was separated from unincorporated label using a Sephadex G-50 column.
Sections were prehybridized overnight at 55 C in a humidified chamber in 100 µl hybridization buffer consisting of 0.5 mg/ml transfer RNA, 0.5 mg/ml Torula yeast transfer RNA, 10 mM DTT, 50% formamide, 10% dextran sulfate, 0.2 mg/ml Ficoll 400, 0.2 mg/ml PVP, 0.2 mg/ml BSA, 1 mM EDTA, 300 mM NaCl, and 10 mM Tris-HCl, pH 8.0. Sections were then hybridized with 1 x 107 cpm 35S RNA probe in 70 µl hybridization buffer under a coverslip overnight at 55 C in a humidifed chamber.
After hybridization, the coverslips were soaked off in 4 x SSC and treated with 20 µg/ml ribonuclease A in 500 mM NaCl, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.0, for 30 min at 37 C. The sections were then washed as follows: twice in 2 x SSC and 1 mM DTT for 10 min at room temperature, once in 0.5 x SSC for 20 min, and once in 0.1 x SSC and 1 mM DTT at 65 C for 60 min and rinsed once in 0.1 x SSC and 1 mM DTT. Sections were dehydrated for 3 min each in 50% ethanol in 0.1 x SSC-1 mM DTT, 70% ethanol in 0.1 x SSC-1 mM DTT, 95% ethanol, and 100% ethanol. After vacuum drying for 30 min at room temperature, the sections were exposed to ß-Max Hyperfilm (Amersham International, Little Chalfont, UK) for 3 weeks to estimate the extent of hybridization. The sections were subsequently coated in NTB-2 emulsion (IBI, Cambridge, UK) diluted 1:2 with water, exposed at 4 C, developed, counterstained, dehydrated, mounted, and examined under dark- and lightfield microscopy.
Western immunoblotting for IGFBP-5
Western blots for IGFBP-5 detection were blocked in 3% Nonidet
P-40 in Tris-buffered saline (TBS) for 30 min, 1% BSA in TBS for
2 h, and 0.1% Tween-20 in TBS for 10 min, all at 4 C. Blots were
then incubated overnight at 4 C in a sealed plastic bag with a 1/1000
dilution of antiserum to IGFBP-5 (a gift from Dr. D. R.
Clemmons) in TBS with 1% BSA and 0.1% Tween-20. The blots were
washed for 10 min using four changes of TBS-0.1% Tween and incubated
at room temperature for 45 min with affinity-purified
peroxidase-conjugated antiguinea pig IgG antiserum diluted in TBS, 1%
BSA, and 0.1% Tween-20. The blot was washed as described above and
incubated for 1 min with Enhanced Chemiluminescence reagent (Amersham
International, Little Chalfont, UK), then exposed to Reflection film
(DuPont, Stevenage, UK) at room temperature.
Preparation of casein micelles and whey fractions from milk
Milk samples were diluted 10-fold in 10 mM phosphate
buffer, pH 7.4, and centrifuged at 100,000 x g for 20
min, and the pellet (casein micelles) and supernatant (whey fraction)
were separated by aspiration. Samples were then subjected to Western
ligand blotting as described above to determine the presence or absence
of IGFBP in each fraction.
Statistical analysis
Statistical analyses were undertaken using one-way ANOVA. Where
significant differences were found, Tukey’s multiple comparisons test
was used to determine significant differences between individual
groups. Comparisons of sealed and unsealed glands from the same animals
were made using Student’s paired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that the major IGFBP present in
involuting mammary gland could be detected using an antiserum to
IGFBP-5. To determine whether IGFBP-5 was being synthesized in the
mammary gland, as opposed to being transported from blood into milk,
Northern analysis was performed. Figure 2
clearly demonstrated the dramatic increase in expression of IGFBP-5
mRNA 48 h after involution of the mammary gland commenced. There
was also a modest increase in expression of IGFBP-4, although there was
no evidence of its presence in milk. The site of synthesis of IGFBP-5
was further localized to the mammary secretory epithelial cells by
in situ hybridization (Fig. 3). An increase in synthesis of IGFBP-2
and -4 from epithelial cells was also evident during involution (Table 1). There was, however, no evidence of
IGFBP-2 or -4 in milk. Preliminary in situ hybridization
studies and Northern blotting failed to detect mRNAs for IGFBP-1, -3,
or -6 in either lactating or involuting mammary gland (data not
shown).
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. Levels
of IGFBP-5 were extremely low in milk derived from lactating
mammary gland and were unaffected by administration of 17ß-estradiol
to the lactating dam (to mimic the increase that occurs when estrus
resumes after removal of the suckling litter). 17ß-Estradiol also
failed to influence IGFBP-5 mRNA steady state levels. By contrast, in
lactating animals that had half the mammary glands sealed to allow milk
accumulation to occur in the presence of an unchanged hormonal
environment, there was a 5-fold increase in IGFBP-5 concentrations in
the sealed, but not the unsealed, glands. IGFBP-5 mRNA also increased
in sealed mammary glands compared with that in their unsealed
counterparts (Fig. 4).
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).
Concurrent PRL injections markedly reduced this increase, such that
values were only 5-fold higher than those in lactating animals. By
contrast, treatment with an antiserum to IGF-I (to neutralize the
increase in serum IGF-I that occurs after removal of the litter) or
with GH, progesterone, or corticosterone failed to prevent the increase
in IGFBP-5 that occurred after litter removal.
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). Consistent with their lack of
effect on IGFBP-5 concentrations in milk, treatment with GH,
progesterone, corticosterone, or an antiserum to IGF-I did not
significantly influence steady state levels of IGFBP-5 mRNA. Finally, centrifugation of milk samples to produce a casein micelle fraction and a supernatant or whey fraction demonstrated that the majority of IGFBP-5 was present in the casein micelle fraction (results not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inability of GH to regulate IGFBP-5 was also unexpected, as we have shown that GH can inhibit apoptosis in the rat mammary gland (5). We have also demonstrated that GH and PRL play interactive roles in maintaining mammary gland function and cell survival, although we have previously been unable to offer any mechanistic explanation for this. We would now propose that GH plays its major role by stimulating an increase in serum IGF-I concentrations and perhaps locally produced IGF-I from the mammary stroma, but that this is less potent in maintaining mammary epithelial cell survival unless PRL is present to suppress IGFBP-5 synthesis. In support of this hypothesis, Kleinberg and co-workers (21) have shown that IGF-I is produced by the mammary stroma and that GH stimulates steady state levels of IGF-I mRNA produced by the mammary stroma.
The proposal that IGFBP-5 is important in regulating apoptosis has also been made after the use of a lateral cross-screening strategy that identified IGFBP-5 as a gene product that was up-regulated dramatically in both involuting mammary gland and prostate (22). Indeed, IGFBP-5 expression is also increased in the involuting thyroid (23) and in ovarian follicles undergoing atresia (24).
Although our studies reveal an excellent correlation between IGFBP-5 expression and apoptosis in the mammary gland, our immediate goal is to explore a causal relationship between the two. If our hypothesis is correct, we have to propose that IGFBP-5 produces an inhibitory, rather than augmentory, effect on IGF action. A number of studies have provided support for such an inhibitory effect of IGFBP-5 on the actions of IGF-I (25, 26, 27, 28).
In our study we have also demonstrated an interaction of IGFBP-5 with the micelle fraction of milk. IGFBP-5 is protected from degradation by interaction with the extracellular matrix (29), although this also leads to a reduction in the affinity of IGFBP-5 for IGF-I and is thus proposed to be part of a transport system for IGF-I delivery to cells, augmenting, rather than inhibiting, the actions of IGF-I. We are, however, intrigued by the possibility that IGFBP-5 may be interacting with the casein micelles present in milk. IGFBP-5 binds with high affinity to hydroxyapatite (a crystalline form of calcium phosphate) in bone (30), and casein micelles also possess calcium phosphate nanoclusters (31). In addition, IGFBP-5 has been shown to be phosphorylated (32), and it contains a region that is a consensus sequence for the true mammary casein kinases.
Proteolysis of IGFBP-5 was evident in milk, although the majority of IGFBP-5 was intact. This is surprising in the involuting mammary gland, where a number of proteolytic enzymes are active. However, IGFBP-5 has recently been shown to interact with plasminogen-activating inhibitor-I, and this may serve to protect it from proteolysis in an analogous fashion to the effect of extracellular matrix (33). Such an interaction of IGFBP-5 and plasminogen-activator inhibitor-I in the mammary gland is worthy of investigation. We also cannot rule out possible interactions of IGFBP-5 with the mammary cell surface, as has been proposed for endothelial cells (34) or the possibility that this involves interaction with an IGFBP-5 "receptor" (35).
Finally, the demonstration that PRL is capable of repressing expression of IGFBP-5 mRNA in the mammary gland provides an interesting comparative model for the study of PRL signaling in the mammary gland, as considerable information exists with respect to the ability of PRL to stimulate gene expression (see Ref. 36 for review), but little is known about how it acts to repress mRNA expression. The promoter region of IGFBP-5 contains consensus binding sites for activating protein-1 (AP-1) and AP-2 (37, 38), and AP-1 increases dramatically during involution (39). Whether repression of AP-1 or AP-2 by PRL provides a mechanistic explanation for this phenomenon remains to be seen. The IGFBP-5 promoter also contains a CACCC box capable of binding the progesterone receptor (40). However, the inability of progesterone to influence IGFBP-5 mRNA expression in the mammary gland in our study is readily explained by the loss of progesterone receptor expression, and thereby progesterone responsiveness, that occurs in the mammary gland during lactation (41).
In conclusion, we have demonstrated for the first time a dramatic increase in the concentration of IGFBP-5 mRNA in the secretory epithelial cells of the involuting mammary gland and its secretion into milk. PRL, which is a major inhibitor of apoptosis, also inhibited IGFBP-5 synthesis. In addition, teat sealing, which leads to the accumulation of milk in the absence of changes in circulating hormones, led to an increase in IGFBP-5 synthesis and secretion. This suggests that locally produced, as yet unidentified, factors also play a role in regulating IGFBP-5 expression. We are currently investigating the potential role of IGFBP-5 in the regulation of apoptosis using in vitro mammary culture systems and a transgenic animal approach involving expression of IGFBP-5 on a mammary-specific promoter.
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Acknowledgments |
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Footnotes |
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Received January 27, 1997.
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