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Departments of Internal Medicine (L.J.M.) and Physiology (T.M., J.V.S., Z.S., Y.G., L.J.M.), University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3; and Department of Pediatrics, Baylor College of Medicine (A.S., S.K.D., D.R.P.), Houston, Texas 77030
Address all correspondence and requests for reprints to: L. J. Murphy, M.B., Ph.D., Departments of Internal Medicine and Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3. E-mail: ljmurph{at}cc.umanitoba.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to these IGF-dependent effects, an increasing amount of in vitro data suggests that IGFBP-3 exerts effects on cell proliferation and apoptosis that are IGF independent. For example, IGFBP-3 has antiproliferative effects on breast cells that are unresponsive to IGF-I (10) and on mouse fibroblasts that lack IGF-I receptors (11). In addition, proteolytic fragments of IGFBP-3 that have markedly reduced affinity for IGF-I retain antiproliferative effects in vitro (12).
The ability of IGFBP-3 to bind to a variety of other serum proteins, including acid-labile subunit (ALS), plasminogen, fibrinogen, and fibrin, has been reported (13, 14, 15). Cell surface binding sites for IGFBP-3 have also been identified in some cell types (16, 17, 18).
These and other reports suggest that IGFBP-3 and other binding proteins may have biological effects in vivo over and above simply modulation of IGF action (19) and justify studies to ascertain the role of IGFBP-3 in vivo. To this end we have generated Tg mice that overexpress IGFBP-3 at high levels. Here we report phenotypic characterization of these transgenic (Tg) mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tg mice were generated by pronucleus injection of the linearized transgene fragment, devoid of plasmid sequences, into fertilized CD-1 zygotes. The microinjected embryos were transferred into CD-1 foster mice using standard techniques (24). The founders were bred with wild-type CD-1 mice. CD-1 mice, bred in a similar fashion, provided wild-type (Wt), nontransgenic control mice of the same genetic background. All experiments were performed in accordance with protocols approved by the animal care committee of the Faculty of Medicine, University of Manitoba.
Southern blot analysis
The presence of the transgene was detected by Southern blot
analysis of tail DNA. Filters were hybridized with a human (h) IGFBP-3
cDNA fragment of the transgene under stringent conditions. For
determination of transgene copy number, serial dilution of tail DNA
from homozygous Tg mice were analyzed by dot-blot hybridization and
quantified densitometrically. The data were compared to a standard
curve of human IGFBP-3 cDNA generated in a similar fashion.
IGFBP-3 and IGF-I assays
IGFBP-3 was measured using an immunoradiometric assay from
Diagnostics Systems Laboratories, Inc. (Webster, TX).
Total plasma IGF-I was measured using a rat IGF-I RIA with an assay kit
from the same source. To detect the presence of IGFBP-3 in the ternary
complex, serum from Tg mice was chromatographed on a Sephacryl S-300HR
column.
RNA extraction and ribonuclease protection assays (RPAs)
RNA was extracted from a variety of tissues from 50- to
60-day-old mice using the single step method described by Chomczynski
and Sacchi (25). The concentration of RNA was determined
spectrophotometrically, and the integrity of the RNA in all samples was
documented by visualization of the 18S and 28S ribosomal bands after
electrophoresis through an 0.8% formaldehyde/agarosis gel. Two
different RPAs were used to measure IGFBP-3 messenger RNA (mRNA)
abundance. One RPA was species specific and measured only the
transgene-derived human IGFBP-3; the other assay measured both mouse
and human IGFBP-3 mRNA. To generate radiolabeled complementary RNA
(cRNA) for the nonspecies-specific RPA, the plasmid (pBluescript SK)
containing a full-length human IGFBP-3 cDNA was digested with
SphI. A 326-bp fragment of coding sequences, nucleotides
647–973 was cloned into the pGEM-7Z vector. This sequence has over
90% homology with the mouse IGFBP-3 sequence, and both human and mouse
IGFBP-3 mRNAs were detected using this assay. Radiolabeled cRNA (SA,
0.5 x 109 cpm/µg) was generated by
transcription with T7 polymerase using
[
-32P]CTP or
[-32P]UTP. An RPA specific for the
transgene-derived IGFBP-3 mRNA was also developed using a 267-bp
fragment containing sequence corresponding to the 3'-end of hIGFBP-3
cDNA and the bovine GH polyadenylase signal region of the
transgene. Total RNA (20 µg) was denatured at 78 C and hybridized
with 106 cpm radiolabeled cRNA in solution at 55
C for 16 h as previously described (25). After
hybridization, T2 RNase was added to digest unbound unprotected RNA.
The protected hybrids were denatured and separated by electrophoresis
through a 5% polyacrylamide/urea gel. Dried gels were exposed to
Kodak XAR film (Eastman Kodak Co., Rochester,
NY) at -70 C for 12–24 h. A riboprobe for mouse cyclophilin
(Ambion, Inc., Austin, TX) was used as the internal
standard in some RPA assays. Century RNA markers from Ambion, Inc., were used to determine the size of the protected
fragment.
Western and ligand blotting
Sera (2 µl) from Tg and Wt mice were analyzed on a 10%
SDS-polyacrylamide gel and transferred to nitrocellulose membrane. For
immunodetection, membranes were incubated with biotinylated goat
anti-hIGFBP-3 antibody (Diagnostics Systems Laboratories, Inc.) at 4 C overnight. After washing, the membrane was
incubated with streptavidin-horseradish peroxidase conjugate
(Life Technologies, Inc., Burlington, Canada). Detection
of immune complexes was achieved using an enhanced chemiluminescence
Western blotting kit (Amersham Pharmacia Biotech, Baie
d’Urfe, Canada).
For ligand blotting, the membrane was incubated with [125I]IGF-I, (500,000 cpm; NEN Life Science Products, Boston, MA) at 4 C overnight. The membrane was subsequently washed four times with Tris-buffered saline (pH 7.6), 0.1% Tween 20, and exposed to Kodak XAR film at -70 C for 24–72 h.
Statistical analysis
Data are expressed as the mean ± SEM.
Student’s t test was used for single comparisons between Tg
and Wt mice. For determining statistical differences between multiple
groups, an ANOVA followed by Dunnett’s t test was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A Southern blot of genomic DNA from Tg mice is shown in Fig. 1
. The DNA was restricted with four
endonucleases. With each of the enzymes used, a different digestion
pattern was apparent with the two DNA samples, indicating a difference
in the transgene insertion sites in the two strains of mice. The
transgene copy numbers were 9 and 21 for PGKBP-3 and CMVBP-3 mice
respectively.
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). Using a human IGFBP-3
immunoradiometric assay, cross-reaction with endogenous mouse IGFBP-3
was apparent. The IGFBP-3 in levels in Wt mice were about 0.3
µg/ml. In serum from the CMVBP-3 and PGKBP-3 Tg mice the levels of
human IGFBP-3 were similar in male and female mice and were
approximately 17-fold higher than those measured in Wt mice. There was
no significant difference in IGFBP-3 levels between CMVBP-3 and PGKBP-3
mice. Chromatographic analysis of sera from Tg mice indicated that the
majority of the IGFBP-3 was present as a 150-kDa complex (Fig. 3).
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). IGF-I levels were significantly
increased in both PGKBP-3 and CMVBP-3 Tg mice compared with Wt mice.
There were no significant differences in the IGF-I levels between the
CMVBP-3 and PGKBP-3 strains of Tg mice. Among the individual Tg mice,
there was a significant correlation between IGFBP-3 levels and IGF-I
levels (r2 = 0.62; n = 21; P
< 0.01).
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, upper panel). When the
autoradiographs were analyzed by densitometry, the abundance of intact
IGFBP-3 in sera from Tg mice was increased 4.9 ± 0.2- and
7.7 ± 0.6-fold in CMVBP-3 and PGKBP-3 mice, respectively,
compared with Wt mice. The additional bands detected by ligand blotting
probably represent IGFBP-2 and IGFBP-4. There was no significant
difference in the abundance of these lower mol wt binding proteins
between Tg and Wt mice. Western blotting with anti-hIGFBP-3 antisera
confirmed the presence of transgene-derive IGFBP-3 in the sera from Tg
mice.
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). Because of the sequence similarity
between human and mouse IGFBP-3 cDNAs, this assay detected
endogenous mouse IGFBP-3 mRNA in addition to the hIGFBP-3 mRNA
derived from the transgene. In the Wt mice endogenous IGFBP-3
expression was highest in the kidney, lung, spleen, and heart, with
lower levels of expression in the liver and brain. In each tissue,
IGFBP-3 mRNA abundance was increased in both PGKBP-3 and CMVBP-3 Tg
mice.
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). When the results were
quantified by densitometry, similar levels of IGFBP-3 mRNA were
detected in adipose tissue from CMVBP-3 and PGKBP-3 mice (154.0 ±
9.4 vs. 179.9 ± 12.3 OD area arbitrary units;
P < 0.01). In contrast, hIGFBP-3 mRNA was
significantly more abundant in testicular tissue from CMVBP-3 Tg mice
than in that from PGKBP-3 Tg mice (196.4 ± 4.0 vs.
157.5 ± 9.8 OD area arbitrary units; P <
0.01).
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). As there was a tendency
for litter size to increase in female mice with each subsequent litter,
a careful comparison was made between size of the first litter in Tg
and Wt mice. When 2-month-old virgin PGKBP-3 and CMVBP-3 Tg mice were
mated with Wt male mice, a small, but significant, reduction in litter
size was apparent compared with litters from virgin female Wt mice
(Fig. 8B). Litter size was smaller in PGKBP-3 Tg mice compared with
CMVBP-3 Tg mice. In contrast when male CMVBP-3 and PGKBP-3 Tg mice were
mated with virgin Wt mice there was no significant reduction in litter
size.
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).
PGKBP-3 Tg mice tended to be lighter than CMVBP-3 mice, but the
difference between the two Tg strains was not statistically
significant. A more detailed study was undertaken in litters derived
from crossing Tg mice with Wt mice. In this experimental design, Tg and
Wt mice from the same litter could be directly compared. Both male and
female Tg newborn mice of each strain were significantly lighter than
Wt controls (Fig. 9B). Furthermore, a similar reduction in birth weight
was apparent when Tg pups were born to Wt female mice, indicating that
the effect on fetal growth was a result of fetal, rather than maternal,
transgene expression. For example, both male and female hemizygous Tg
pups derived from the cross of CMVBP-3 male mice with Wt mice were
significantly lighter than male and female Wt pups, respectively.
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. Both male and female mice from each
of the Tg mouse strains were significantly lighter than their Wt
controls during the first 30 days of life. Weight gain in PGKBP-3 mice
of both sexes continued at a rate parallel to that seen in the Wt mice
over the next 2 months, and these Tg mice remained significantly
lighter than their Wt sex-matched controls throughout the study. In
contrast, CMVBP-3 mice of both sexes gained more weight in the period
from 40–60 days than their Wt controls. As a result, male CMVBP-3 mice
did not differ significantly in body weight from the Wt mice at 3
months of age. This phenomenon of delayed weight gain was more marked
in female CMVBP-3 mice, which were significantly heavier than their Wt
controls at 3 months (31.9 ± 1.2 vs. 30.5 ±
0.6 g; P < 0.005).
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). At 6 and 12 weeks of
age in both male and female PGKBP-3 Tg mice all three parameters were
significantly reduced compared with those in Wt mice. A similar, but
less marked, reduction in linear growth was apparent in CMVBP-3 mice.
However, the differences in these parameters between Wt mice and
CMVBP-3 mice did not achieve statistical significance, with the
exception of tail length in 12-week-old male CMVBP-3 mice.
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). The relative liver weight was
consistently larger in CMVBP-3 Tg mice than Wt mice, whereas it was
similar in female PGKBP-3 and Wt mice, or slightly reduced in the case
of 6-week-old male PGKBP-3 mice. Relative kidney weight was
significantly reduced in CMVBP-3 mice, but was only marginally reduced
in PGKBP-3 mice. In both PGKBP-3 and CMVBP-3 Tg mice there was a
significant reduction in absolute brain size. The reduction in relative
brain weight was more marked in CMVBP-3 mice than in PGKBP-3 mice.
There was a modest reduction in the relative weight of the heart in
6-week-old male and female CMVBP-3 mice compared with Wt mice. The
absolute and relative weights of the spleen tended to be greater in
both CMVBP-3 and PGKBP-3 mice than in Wt mice (Fig. 11). The most
striking differences between the CMVBP-3 and PGKBP-3 mice were in terms
of epididymal fat pad and testicular weight. Relative weight of the
testes was markedly reduced in CMVBP-3 mice compared with Wt mice,
whereas it was not reduced in PGKBP-3 mice. CMVBP-3 mice had
significantly more epididymal fat than Wt mice, whereas PGKBP-3 mice
and Wt mice did not differ in this regard.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The circulating levels of IGFBP-3 in the Tg were comparable to those seen in human serum and 17-fold higher than those measured in the Wt mouse circulation. Although there is a high degree of sequence homology between mouse and human IGFBP-3 proteins, a mouse IGFBP-3 standard was not used in the human IGFBP-3 immunoradiometric assay. The absolute level of endogenous IGFBP-3 in mouse circulation is unknown. However, when sera from Tg and Wt mice were analyzed by ligand blots there was an approximately 5- to 7-fold increased abundance of intact IGFBP-3. IGFBP-3 degradation products were also markedly increased in Tg mice. Thus, in these Tg mice very high levels of expression of the transgene were achieved with each of the promoters used.
The increase in circulating IGFBP-3 in Tg mice was accompanied by an increase in plasma IGF-I levels; however on a molar basis the increase in IGF-I levels was less than the increase in IGFBP-3. This would suggest that there would be excess unsaturated IGFBP-3 present in the circulation of the Tg mice and probably reduced free IGF-I levels. The majority of the human IGFBP-3 detected in the plasma from Tg mice was present as a 150-kDa complex. This would be consistent with the recent observation that IGFBP-3/ALS binary complexes can form under appropriate physiological conditions (27). It also indicates that human IGFBP-3, which is structurally similar to mouse IGFBP-3, is able to bind to mouse ALS.
Despite the very marked increase in circulating IGFBP-3 levels in Tg mice, the effect on growth was modest. The reduction in birth weight in the IGFBP-3 Tg mice, about 7–10% depending upon the gender and the strain, was less than the 8–16% seen in the IGFBP-1 Tg mice previously generated in this laboratory (28). As the PGK promoter was used to generate IGFBP-1 Tg mice, a similar tissue pattern and timing of transgene expression would be expected for PGKBP-3 mice. However, the circulating levels of IGFBP-1 in PGKBP-1 Tg mice were much lower (10–60 ng/ml) (28) than the levels of IGFBP-3 observed in PGKBP-3 mice. Postnatal growth retardation was less marked in the PGKBP-3 mice compared with the PGKBP-1 Tg mice. For example, at 40 days the reductions in body weight in male and female IGFBP-1 Tg mice compared with that in Wt mice were 5.2 and 8.4 g (28) compared with 4.7 and 2.4 g, respectively, for PGKBP-3 mice.
Interestingly, postnatal growth retardation was less marked in CMVBP-3 Tg mice than in PGKBP-3 mice despite similar circulating IGFBP-3 levels and tissue IGFBP-3 mRNA abundance. CMVBP-3 mice demonstrated a propensity to adiposity that was clearly demonstrable in the second month of life. Body weight growth curves for male CMVBP-3 and PGKBP-3 Tg mice were not statistically different until after 40 days of age. In female Tg mice the difference between the two strains was apparent slightly earlier.
The growth retardation in the IGFBP-3 Tg mice was apparent in the face of increased plasma IGF-I levels in both strains of Tg mice. Total circulating IGF-I levels were increased 2- to 3-fold in the Tg mice. No attempt was made to assess free IGF-I levels, because it is unclear how precise or accurate such assays would be in the face of very markedly increased IGFBP-3 levels. However, our data, indicating growth retardation in the presence of elevated circulating IGF-I levels, complement the recent reports of normal growth in the presence of markedly reduced total plasma IGF-I levels in mice in which conditional knockout of hepatic IGF-I expression has been achieved (29, 30).
As different promoters were used, the phenotype of the two strains of transgenic mice differed. However, many features were common to both strains. These included reduced birth weight, reduced early postnatal growth, and decreased litter size. The transgene was expressed at high levels in the kidney, heart, and brain of CMVBP-3 mice. These organs were relatively smaller in CMVBP-3 than Wt mice and were also smaller in CMVBP-3 mice than in PGKBP-3 mice, in which transgene expression was similar in all tissues. However, the major differences between the two strains of IGFBP-3 Tg mice was the adiposity that developed in the CMVBP-3 mice after weaning. This was manifested as a marked increase in the size of the epididymal fat pad in CMVBP-3 mice. Although other fat masses were not quantified, gross macroscopic examination of the dissected CMVBP-3 mice suggested that there was a generalized increase in adiposity. This would also be consistent with the increased weight gain observed in CMVBP-3 mice in the second and third months of life.
The mechanism underlying this phenotypic difference in adiposity between the two strains of IGFBP-3 Tg mice is not immediately apparent. The transgene was expressed in mature adipose tissue from both CMVBP-3 and PGKBP-3 mice at similar levels. However, it is possible that different levels of expression could occur in adipoblasts and preadipocytes. IGF-I is known to be important in the differentiation of preadipocytes to mature fat cells (30, 31, 32, 33). We previously reported that PGKBP-1 Tg mice have impaired adipogenesis in response to caloric excess (34). In the PGKBP-1 there is both a reduced number of stem cells (adipoblasts) and impaired differentiation of preadipocytes (34). Although further detailed investigation is required, PGKBP-3 Tg mice, unlike PGKBP-1 mice, do not appear to have smaller fat deposits than Wt mice.
In Tg mice expressing IGFBP-3 driven by the mMT-1 promoter previously generated in this laboratory, no growth retardation was observed (35). However, unlike the PGKBP-3 and CMVBP-3 mice reported here, the level of transgene expression in the mMT-1/IGFBP-3 Tg mice was very low. It is of interest, however, that these mice, like the IGFBP-3 Tg mice reported here had splenomegaly. They also had hepatomegaly (35), which was a consistent feature of the CMVBP-3 Tg mice.
Although some differences in the phenotypes of CMVBP-3 and PGKBP-3 mice were observed, the data reported here clearly demonstrate that overexpression of IGFBP-3 is associated with impaired intrauterine growth and early postnatal growth. Organs such as brain and kidney, where IGF-I appears to have an important role in growth (36, 37) were disproportionately growth retarded by IGFBP-3 overexpression. These modest effects on growth are most likely explicable on the basis of inhibition of IGF action, although it is impossible to exclude IGF-independent growth inhibitory effects of IGFBP-3.
The generation of IGFBP-3 Tg mice reported here should provide a useful model for investigating the in vivo effects of IGFBP-3 in certain disease processes, such as breast and prostate cancer, as well as osteoporosis, where epidemiological studies have suggested that IGFBP-3 may have a role in these conditions in humans.
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Footnotes |
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2 Recipient of Canadian Diabetes Association Postdoctoral Fellowship.
Present address: Animal Health Discovery Research, Pharmacia,
Kalamazoo, Michigan.
3 Recipient of a Medical Research Council Senior Scientist award and
an endowed Research Professorship in Metabolic Diseases.
Received October 25, 2000.
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HindIII DNA size markers are indicated.
