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Lehrstuhl für Molekulare Tierzucht und Haustiergenetik (E.W.) and Medizinische Klinik II, Klinikum Großhadern (M.M.W.), Ludwig-Maximilians-Universität, D-81377 München, Germany; Abteilung Innere Medizin II, Sektion Nephrologie, Medizinische Klinik und Poliklinik, Universität Ulm (P.M.J.), D-89081 Ulm, Germany; Forschungszentrum für Milch und Lebensmittel Weihenstephan, Technische Universität München (H.S., A.D.), D-85350 Freising-Weihenstephan, Germany; Research Center for Developmental Medicine and Biology, School of Medicine, University of Auckland (B.H.B.), Auckland, New Zealand; Abteilung Biotechnologie in der Tierproduktion, Interuniversitäres Forschungsinstitut für Agrarbiotechnologie (U.B., G.B.), A-3430 Tulln, Austria; and University of Veterinary Science (L.F.), H-1400 Budapest, Hungary
Address all correspondence and requests for reprints to: Dr. Eckhard Wolf, Lehrstuhl für Molekulare Tierzucht und Haustiergenetik, Ludwig-Maximilians-Universität, Feodor-Lynen-Str. 25, D-81377 München, Germany. E-mail: ewolf{at}lmb.uni-muenchen.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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S1-casein (S1-cas) gene. Western
blot analysis of milk from S1-cas-hIGF-I transgenic
rabbits demonstrated production of high amounts of mature hIGF-I
peptide (7.6 kDa). Quantitative analysis by RIA revealed hIGF-I levels
between 50 and 300 µg/ml milk. Recombinant hIGF-I purified from the
milk of S1-cas-hIGF-I transgenic rabbits bound to IGF-I
receptors on human IM-9 lymphoblasts and stimulated DNA synthesis by
growth-arrested MG-63 human osteosarcoma cells as efficiently as hIGF-I
produced in Escherichia coli. Ligand blot analysis of
milk serum revealed the presence of 45-kDa, 30-kDa, and 23-kDa
IGF-binding proteins (IGFBPs). The 30-kDa IGFBP was shown to be IGFBP-2
by immunoprecipitation using an antiserum raised against human IGFBP-2.
Secretion of IGFBP-2 was markedly stimulated by hIGF-I overproduction
in S1-cas-hIGF-I transgenic rabbits. The latter
displayed slightly increased milk yield, but no significant changes in
total protein content or overall milk protein composition, and reared
their offspring without any problems or clinical signs of impaired
welfare, even after multiple lactations. Our results indicate that high
amounts of biologically active hIGF-I can be produced in the mammary
glands of S1-cas-hIGF-I transgenic rabbits. Local
production of hIGF-I in mammary tissue is associated with increased
secretion of IGFBP-2, which may prevent major biological effects by
high levels of hIGF-I on the mammary gland. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although recombinant human IGF-I (hIGF-I) can be produced in
Escherichia coli (E. coli), the mammary glands of
transgenic livestock could be an alternative source to produce large
amounts of this peptide. We therefore generated transgenic rabbits
carrying hybrid gene constructs in which a synthetic DNA coding for
hIGF-I was placed under the transcriptional control of regulatory
elements isolated from the bovine S1-casein
(S1-cas) gene. High amounts of correctly processed
hIGF-I could be purified from the milk of these rabbits to a nearly
homogenous form (16). In the present study, we investigated receptor
binding and biological activity of hIGF-I synthesized by the mammary
glands of S1-cas-hIGF-I transgenic rabbits (MGTR) as
compared with E. coli-derived hIGF-I. In addition, we
studied effects of hIGF-I overproduction in the mammary gland on milk
yield, overall milk protein composition and IGF-binding protein (IGFBP)
levels in milk.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S1-cas-hIGF-I transgenic rabbitsS1-cas gene,
were produced by pronuclear DNA-microinjection as previously described
(16). Animals investigated in this study were founder animals described
in our initial report (16), as well as their F1-offspring.
Western blot analysis
Milk from S1-cas-hIGF-I transgenic rabbits and
controls was defatted by centrifugation (15,000 x g; 4
C; 10 min). To obtain milk serum, skim milk was ultracentrifuged
(100,000 x g; 4 C; 10 min). Milk serum was diluted 1:5
with Tris-buffered saline (TBS; pH 7.3) and subsequently mixed with an
equal volume of 2 x sample buffer [5% glycerol; 10%
2-mercaptoethanol; 4% SDS; 10 mM Tris-HCl, pH 6.8],
boiled (5 min), and loaded on a 5% stacking/15% separating
SDS-polyacrylamide gel. Electrophoresis was performed in a
Mini-Protean® II Dual Slab Cell (Bio-Rad, Munich, Germany)
for 5 min at 100 V and then for 45 min at 180 V. Proteins were
transferred to Hybond-C nitrocellulose (Amersham, Braunschweig,
Germany) using a horizontal semidry electroblotting system (Sartorius,
Göttingen, Germany). Immunodetection of hIGF-I was performed
according to Amersham’s ECL Western blotting protocols using a
monoclonal antibody specific for hIGF-I (MCA 520; batch 177A; Serotec,
Oxford, UK; dilution 1:125) and a horseradish peroxidase-labeled rabbit
antimouse IgG-antibody (A 9044; Sigma, Deisenhofen, Germany; dilution
1:500).
RIA for IGF-I
IGF-I concentrations in rabbit milk were measured by a
double-antibody RIA in which excess hIGF-II is added to block the
interference of IGFBPs. The antiserum used (no. 878/4) has a low
cross-reactivity with hIGF-II (<0.05%). The assay was validated for
milk according to the recommendations of Blum & Breier (17) and Bang
et al. (18). Briefly, milk samples were defatted by
centrifugation (15 min; 5,000 x g; 4 C). The skim milk
was then diluted and incubated in assay buffer containing the antiserum
at a final dilution of 1:105,000 and 25 ng/tube of hIGF-II (Eli Lilly,
Indianapolis, IN) for 30 min at room temperature. All other assay steps
were identical to the method described previously (19). The minimal
detectable concentration was 0.04 ng/ml. The recovery of hIGF-I added
to milk samples from control rabbits was 81 ± 11% (n = 12).
The intraassay and interassay coefficients of variation were 4.8% and
8.1%, respectively.
[125I]IGF-I-binding assays
Specific binding of [125I]IGF-I to IGF-I receptors
already has been demonstrated in human IM-9 lymphoblasts (20). A linear
relationship between IGF-I binding and IM-9 cell concentration was
obtained over the range of 1 x 106 to 1 x
107 cells/ml. IM-9 cells were grown in continuous
suspension culture in RPMI 1640 medium (Biochrom, Berlin, Germany)
supplemented with 25 mM HEPES, 10% FCS (Seromed, Munich,
Germany), 200 mM glutamine, 100 U/ml penicillin G and 10
µg/ml streptomycin. Medium was changed every 3 days.
In the present experiments, we incubated 5 x 106 IM-9
cells in binding buffer with 0.078 ng/ml [125I]hIGF-I
(Saxon, Hannover, Germany; specific activity: 256 µCi/mg) and
increasing concentrations of unlabeled hIGF-I from the mammary glands
of S1-cas-hIGF-I transgenic rabbits (purified as
described in Ref.16) or produced by E. coli (a generous
gift by Dr. Th. Müller, Ciba Geigy AG, Basel, Switzerland). The
binding buffer was 50 mM HEPES, 120 mM NaCl, 5
mM KCl, 1.2 mM MgSO4, 10
mM CaCl2, 10 mM dextrose, 15
mM CH3COONa, 0.1% BSA, pH 7.8. All chemicals
were from Merck (Darmstadt, Germany); BSA was from Behring (Marburg,
Germany). Binding equilibrium was achieved after 1 h at 15 C. The
specificity of [125I]IGF-I binding was further
demonstrated by the inhibitory action of the monoclonal antibody
IR-3 (Oncogene Science, Uniodale, NY), directed against the
-subunit of hIGF-I-receptors (21) (data not shown). Cell-bound and
free intact activity were counted in an automatic 
-counter
(Berthold, Munich, Germany) with 70% efficiency. The concentration of
unlabeled peptide yielding a 50% inhibition of
[125I]IGF-I binding (IC50) was determined
from competition-inhibition curves. Based on the Cheng-Prusoff
relationship (22), IC50 is inversely related to receptor
affinity.
Evaluation of biological activity
Purified hIGF-I from MGTR was compared with E.
coli-derived hIGF-I for its ability to stimulate DNA synthesis by
growth-arrested, IGF-I-responsive MG-63 human osteosarcoma cells (23).
MG-63 cells (CRL 1427; batch F-11035; American Type Culture Collection,
Rockville, MD) were grown to confluence in MEM with Earle’s BSS
(Seromed), nonessential amino acids (NEAA; Seromed), and 10%
heat-inactivated FCS (Biochrom). Confluent cultures were trypsinized
and cells were seeded onto 24-well plates (Nunc, Wiesbaden-Biebrich,
Germany) in MEM with NEAA and 0.4% FCS (5 x 104
cells/well). After 24 h, the medium was removed, the cells were
washed once with Ca2+- and Mg2+-free PBS (37
C), and then cultured in serum-free MEM for 48 h. The quiescent
cells were then washed once with PBS and 400 ml of medium MCDB104
(GIBCO, Eggenstein, Germany) containing 0.1% BSA (A-7906; Sigma), 0.1
mM dexamethasone (Sigma), 1 mg/ml transferrin (GIBCO) and
various concentrations of hIGF-I. After 22 h, the cells were
pulsed with [3H]-thymidine (TRK 120; Amersham; 0.4
µCi/ml) in MCDB104 with 0.1% BSA for 1 h. The cells were then
washed twice with ice-cold PBS and twice with ice-cold 10% trichloro
acetic acid (TCA). The completely dried TCA-precipitated material was
counted with a ß-scintillation counter 12 h later (2
min/sample).
Ligand blot analysis of IGFBPs in milk
Milk serum was obtained as described before, fractionated by
12% SDS-PAGE under nonreducing conditions and blotted onto
nitrocellulose membranes (Millipore, Ann Arbor, MI). The blots were
blocked in 1% fish gelatin (Amersham) and incubated with
[125I]hIGF-II (106 cpm/blot). Binding
proteins were visualized by autoradiography (X-OMAT 228 film; Eastman
Kodak Company, Rochester, NY) for 96 h (24). The signals on the
film were quantified by densitometry (Image Master 1D Software, Version
1.20; Pharmacia Biotech, Uppsala, Sweden). IGFBPs were further
characterized by deglycosilation and by immunoprecipitation using a
specific rabbit antiserum to human IGFBP-2 (kindly provided by Dr. M.
Elmlinger, Universitätskinderklinik Tübingen,
Tübingen, Germany). For deglycosilation of IGFBPs, 10 µl milk
serum were incubated with 2 µl Endoglycosidase F/N-Glycosidase F
(Boehringer Mannheim, Mannheim, Germany) for 3 h at 37 C before
ligand blot analysis. For immunoprecipitation, 2 µl
anti-IGFBP-2-serum were added to 10 mg washed Protein A (Pharmacia) and
preincubated for 1 h at room temperature. Subsequently, 100 µl
milk-serum containing 0.5 µl of a mixture of protease inhibitors
(Complete, Boehringer Mannheim) were added and incubated at room
temperature for 3 h. Then the samples were centrifuged and the
pellets were washed twice with PBS, resuspended in nonreducing sample
buffer, and boiled for 5 min. After centrifugation, the supernatant was
subjected to ligand blot analysis as described above.
Determination of milk yield, total milk protein, and overall milk
protein composition
Milk was obtained from lactating rabbits using a mechanical
milking device specifically developed for quantitative milk extraction
(25). Milk yield was determined on at least 20 days per lactation, and
mean daily milk yield was calculated. Does harboring the
S1-cas-hIGF-I transgene (n = 21) were compared with
nontransgenic controls (n = 5) and with
S1-cas-chymosin transgenic does (n = 6) (26). Total
milk protein was determined using the BCA Protein Assay Reagent
(Pierce, Rockford, IL). Overall milk protein composition was analyzed
by SDS-PAGE under reducing conditions as described in Ref. 27. The gels
were stained with Coomassie Brillant Blue G-250.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S1-cas-hIGF-I transgenic rabbits belonging to different
lines detected high amounts of hIGF-I of correct size (Fig. 1
). The concentrations of hIGF-I determined by RIA in
milk samples from three transgenic rabbits at different stages of
lactation ranged from 50–300 µg/ml (Fig. 2), whereas
IGF-I levels measured in milk from control rabbits were below 0.8
µg/ml (data not shown). Although hIGF-I levels determined in milk
from an S1-cas-hIGF-I transgenic founder doe (no. 4001)
were relatively constant throughout lactation, two F1 does (no. 5255,
no. 5326) from a different line (4117) displayed a 2- to 4-fold
increase in hIGF-I secretion from day 4 to day 21 of lactation.
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, maximal binding of [125I]IGF-I averaged 10 ± 0.7%
with nonspecific binding of less than 1%. Competition with
[125I]IGF-I by hIGF-I from MGTR (IC50 = 10
ng/ml) and from E. coli (IC50 = 12 ng/ml) was
not different. Recombinant insulin was 10-fold less potent in
competition than hIGF-I (data not shown).
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). A
comparison of the receptor-ligand affinities with the dose-response
relationships for [3H]-thymidine incorporation suggested
that hIGF-I from MGTR and from E. coli stimulate growth via
high-affinity binding to IGF-I receptors.
Effects on IGF-binding proteins
Ligand blot analysis of milk serum from
S1-cas-hIGF-I transgenic rabbits and controls revealed
the presence of different IGFBPs with apparent molecular mass of 45
kDa, 30 kDa, and 23 kDa. The concentration of the 30-kDa IGFBP was
markedly (3- to 5-fold) increased in milk samples from transgenic
rabbits, whereas the levels of the 45-kDa and 23-kDa IGFBPs were not
significantly different between the two groups (Fig. 4).
After deglycosilation, the molecular mass of the 45-kDa IGFBP was
reduced to 39 kDa (Fig. 5A), as can be expected for
IGFBP-3. In contrast, the molecular size of the 30-kDa IGFBP remained
unchanged (Fig. 5A), making the presence of N-glycosilated IGFBP-4
unlikely. After immunoprecipitation of milk serum with an antiserum
specific for human IGFBP-2, a single band (30 kDa) was detected in the
subsequent ligand blot analysis (Fig. 5B), indicating that the IGFBP
upregulated in S1-cas-hIGF-I transgenic rabbits
represents IGFBP-2.
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S1-cas-hIGF-I
transgenic does was slightly, but not significantly, greater than that
of S1-cas-chymosin transgenic rabbits and controls (Fig. 6). When data from the latter two groups were pooled,
the increase in milk yield of the hIGF-I overexpressing does reached
the borderline of statistical significance (167 ± 9
vs. 138 ± 13 g/day; P = 0.08). Total
protein content of milk from controls tended to decrease from early to
midlactation and to increase again by the end of lactation. In milk
from S1-cas-hIGF-I transgenic rabbits, total protein
concentration was rather constant throughout lactation (Fig. 7A). The two groups were not significantly different in
any stage of lactation investigated. SDS-PAGE of skim milk revealed the
typical milk protein pattern described recently by Baranyi et
al. (27) with no obvious differences between
S1-cas-hIGF-I transgenic rabbits and controls, except
for the recombinant hIGF-I, which was clearly visible in
Coomassie-stained gels (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S1-cas-hIGF-I transgenic rabbits as a model
system. Our previous study demonstrated that expression of this
transgene is restricted to the mammary gland and that large amounts of
correctly processed hIGF-I are secreted into milk and can be purified
to a nearly homogenous form (16). The present study includes
quantitative analysis of hIGF-I production throughout lactation and
evaluation of its biological activity in terms of receptor binding and
stimulation of DNA synthesis by growth-arrested MG-63 human
osteosarcoma cells. Our results demonstrate that hIGF-I secretion is
high throughout lactation, with an increase from early to midlactation.
The levels of hIGF-I in milk from transgenic rabbits (50–300 µg/ml)
measured by specific RIA were lower in comparison with our previous
measurements by Western blot analysis (up to 1 mg/ml; Ref.16) of the
same samples. This is not surprising, given the semiquantitative nature
of the Western blot methodology. However, our RIA has been shown to be
free from interference by IGFBPs (17) and was rigorously validated for
the rabbit milk from this study in accordance with the recommendations
from the 3rd International Symposium on IGFs (18). The level of hIGF-I
production in the mammary glands of transgenic rabbits was in the same
order of magnitude as observed with other transgenes controlled by
S1-cas regulatory sequences (26; for review, see Ref.28).
Binding of purified hIGF-I from MGTR to IM-9 lymphoblasts was not
different from that of E. coli-derived hIGF-I. In adherent
human fibroblast monolayers, the addition of unlabeled IGF-I in
concentrations between 0.5 and 10 ng/ml resulted in a paradoxical
increase in [125I]IGF-I binding that was caused by IGFBPs
on the cell surface and their release into the medium during the
binding assay (29, 30). In these experiments, unlabeled IGF-I between
25 and 300 ng/ml was required to displace [125I]IGF-I
binding; however, neither IR-3 nor insulin inhibited IGF-I binding.
Although IGFBPs were detected in IM-9-conditioned medium (data not
shown), we did not observe any paradoxical increase in
[125I]IGF-I binding. [125I]IGF-I was
displaced from IM-9 cells in a dose-dependent manner by hIGF-I, by the
specific type I receptor monoclonal antibody IR-3, and with a
10-fold lower potency also by insulin. Therefore, we suppose that our
IM-9 cell assay is suitable to determine IGF-I binding to type I IGF
receptors without significant interference by membrane-associated
IGFBPs. In addition to receptor binding, the biological activity of
hIGF-I from MTGR was shown by its mitogenic effect on MG-63 cells.
In spite of the high levels of bioactive hIGF-I in the mammary glands
of transgenic rabbits, these animals did not show clinical symptoms of
altered function or pathology of the mammary gland (e.g. no
difficulties in rearing their offspring), even after multiple (up to
five) lactations. There was only little increase in milk yield of
S1-cas-hIGF-I transgenic rabbits as compared with
controls or transgenic rabbits expressing
S1-cas-chymosin fusion genes.
The lack of major effects by high levels of locally synthesized hIGF-I
on the mammary glands of S1-cas-hIGF-I transgenic
rabbits contrasts findings of a recent study on transgenic mice in
which expression of hIGF-I or des(1, 2, 3) hIGF-I was directed to the
mammary gland using regulatory sequences from the rat whey acidic
protein (rWAP) gene (31). Overproduction of des(1, 2, 3) hIGF-I, but not
of hIGF-I, was reported to cause abnormal mammary gland development
with progressive changes, including ductile hypertrophy and
disorganization of secretory lobules. Mice suffering from this
pathology had difficulties in rearing their pups after three to four
lactations.
There are several possible explanations for the different effects
observed in S1-cas-hIGF-I transgenic rabbits and
rWAP-des(1, 2, 3) hIGF-I transgenic mice. First, our transgenic rabbits
produce hIGF-I and not the aminoterminally shortened form des(1, 2, 3)
hIGF-I that has reduced affinity for IGFBPs(32) and therefore
higher biological activity (33). The markedly increased activity of
IGFBP-2 in milk from S1-cas-hIGF-I transgenic rabbits
could result from a feedback response of the mammary epithelial cells
to locally produced hIGF-I, preventing major effects of increased IGF-I
levels on the mammary glands of these animals. Stimulation of
IGFBP-2 and a small complex of IGFBP-3 has been reported
previously in human plasma by IGF-I administration (34). We speculate
that this increase in IGFBP-2 may, at least in part, inhibit the local
effects of hIGF-I on the mammary gland. Inhibition of growth of human
breast cancer cells has been demonstrated for IGFBP-1 and IGFBP-3 (35, 36). On the other hand, overexpression of des(1, 2, 3) hIGF-I in
transgenic mice caused altered involution and pathology of the mammary
gland only on a specific genetic background (ICR x B6D2F1), suggesting
that cooperation of the transgene with a specific allele in this
background is necessary to result in a specific pathological phenotype
(31). Hadsell et al. (31) did not report pathology of the
mammary glands from transgenic lines (n = 3) harboring the
rWAP-hIGF-I transgene; however, immunoreactive IGF-I levels in milk
samples from these animals (0.2–0.8 µg/ml) were two orders of
magnitude lower than hIGF-I concentrations in milk from our transgenic
rabbits. Increased activity (6-fold above control) of a 32-kDa IGFBP
was reported only for one of the rWAP-hIGF-I transgenic mouse lines,
whereas levels of this IGFBP were increased, on average, by 20-fold in
a line carrying the rWAP-des(1, 2, 3) hIGF-I transgene. Taken together
with the data from our study, these results suggest that des(1, 2, 3)
hIGF-I may be more potent in stimulating an IGFBP in the range of 30
kDa than authentic hIGF-I. IGFBPs have been detected in milk from
several other species, including human (19), pig (37), cow (38), and
rat (39); however, to our knowledge, the present study is the first to
describe IGFBPs in milk from rabbits.
In summary, our data show that high amounts of biologically active
hIGF-I can be produced in the mammary glands of
S1-cas-hIGF-I transgenic rabbits without compromising
the animals welfare. High local production of hIGF-I markedly
stimulates the secretion of IGFBP-2, which may (at least in part)
inhibit the action of hIGF-I on the mammary gland. Transgenic rabbits
with mammary gland-specific overproduction of hIGF-I are an important
model for studying effects of this peptide on the mammary gland,
thereby complementing recently published observations of rWAP-des(1, 2, 3)
hIGF-I or -hIGF-I transgenic mice, with all advantages of having a
second species model available.
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Acknowledgments |
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Footnotes |
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Received May 16, 1996.
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References |
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IR-1 and IR-3 with insulin
and somatomedin-C receptors. Endocrinology 118:223–226[Abstract]
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