Selective Alterations in Organ Sizes in Mice with a Targeted Disruption of the Insulin-Like Growth Factor Binding Protein-2 Gene
Teresa L. Wood1,
Leslie E. Rogler2,
Maureen E. Czick,
Alwin G.P. Schuller and
John E. Pintar
Department of Neuroscience and Cell Biology Robert Wood Johnson
Medical School University of Medicine and Dentistry New Jersey
Piscataway, New Jersey 08854
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ABSTRACT
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Insulin-like growth factor binding protein 2
(IGFBP-2) is one member of the family of IGF binding proteins
believed to have both endocrine functions elicited by modulating serum
IGF half-life and transport as well as autocrine/paracrine functions
that result from blocking or enhancing the availability of IGFs to bind
cell surface receptors. To clarify the in vivo role of
IGFBP-2, we have used gene targeting to introduce a null IGFBP-2 allele
into the mouse genome. Animals homozygous for the altered allele are
viable and fertile, contain no IGFBP-2 mRNA, and have no detectable
IGFBP-2 in the adult circulation. Heterozygous and homozygous animals
showed no significant differences in prenatal or postnatal body growth.
Analyses of organ weights in adult males, however, revealed that spleen
weight was reduced and liver weight was increased in the absence of
IGFBP-2. In addition, ligand blot analyses of sera from adult IGFBP-2
null males showed that IGFBP-1, IGFBP-3, and IGFBP-4 levels were
increased relative to wild-type mice. These results demonstrate that
up-regulation of multiple IGFBPs accompanies the absence of IGFBP-2
and that IGFBP-2 has a critical role, either directly or indirectly, in
modulating spleen and liver size.
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INTRODUCTION
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Regulation of growth factor and hormone action by soluble
binding proteins has emerged as a common theme for a wide variety of
peptides including insulin-like growth factor (IGF)-I and -II,
transforming growth factor-ß (TGF-ß), activin, epidermal growth
factor (EGF), nerve growth factor (NGF), tumor necrosis factor (TNF),
GH, and CRF (1, 2, 3, 4, 5, 6, 7). Among the best studied of these binding proteins
are the IGF binding proteins (IGFBPs). Six different, high-affinity
IGFBPs have been identified that each derive from a unique member of
the IGFBP gene family (5, 8, 9). Most IGFBPs are found at high levels
in adult circulation where they regulate IGF half-life and availability
(10, 11, 12). In addition, in vitro studies suggest that the
IGFBPs may have autocrine/paracrine actions by localizing IGFs to
specific tissue sites and by modulating IGF binding to cell surface
receptors or to the extracellular matrix (ECM) (5, 9, 13, 14, 15). Although
some members of the IGFBP family are coexpressed in specific cell
types, each of the six members of the IGFBP family has a distinct
expression pattern in both developing and adult tissues (16, 17, 18, 19, 20, 21, 22, 23).
Moreover, the major IGFBPs in adult serum are regulated differentially
(5, 9, 12, 24, 25, 26, 27). These data support the hypothesis that each member
of the IGFBP family has a unique function.
IGFBP-2 is a particularly prominent IGFBP in late fetal and
neonatal serum (28, 29, 30). We have previously reported high IGFBP-2
expression in specific embryonic tissues and cell types (19, 31, 32, 33).
IGFBP-2 mRNA is highly expressed in the epiblast of the egg cylinder
and its derivatives including neuroectoderm, surface ectoderm, and the
sensory placodes. Expression of IGFBP-2 also is found in specific
endodermal derivatives including the developing gut and liver. In
contrast, fetal mesoderm derivatives lack IGFBP-2 expression with the
striking exceptions of the notochord, mesonephric tubules, and a region
of modified mesoderm, termed the anterior splanchnic mesodermal plate
(ASMP), that is the progenitor of the spleen (32). In the adult,
IGFBP-2 is found at significant levels in serum where it is under
complex physiological regulation (5, 9, 12, 14, 24, 25, 26). For example,
liver IGFBP-2 mRNA expression and circulating levels of IGFBP-2 are
increased by IGF-I (34), by hypophysectomy (35), and by conditions that
cause mild insulin deficiency (24). IGFBP-2 is also expressed in the
adult central nervous system (CNS) in the choroid plexus and neural
lobe of the pituitary (19), and IGFBP-2 protein is a component of
cerebral spinal fluid (36).
Despite a large amount of in vitro data, the precise
in vivo function(s) of IGFBP-2 as well as the other IGFBPs
has been difficult to address. The utilization of homologous
recombination in embryonic stem (ES) cells to create null mutations in
mice has resulted in valuable information on the in vivo
function of many molecules including IGF-I, IGF-II, and their receptors
(37, 38, 39, 40, 41, 42, 43, 44, 45). We here report the production and characterization of a mouse
line that carries a null mutation in the IGFBP-2 gene.
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RESULTS
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Targeted Disruption of the Mouse IGFBP-2 Gene
A
clone containing exons 2, 3, and 4 of the mouse
IGFBP-2 gene was isolated from a C57Bl6/J genomic library (Fig. 1A
). To generate a null mutation of the
BP-2 gene by homologous recombination, we designed the targeting vector
to delete essentially all of exon 3 (115 nucleotides, representing 38
amino acids) as well as 0.6 kb of the 3'-flanking intron including the
exon/intron boundary (Fig. 1B
). Addition of the neomycin resistance
gene introduced two novel, diagnostic EcoRI restriction
sites (Fig. 1B
). The targeting vector was linearized at a unique
nonplasmid BamHI site and used to electroporate ES cells.
Seven prospective ES clones were identified initially by PCR screening
(Fig. 1D
) and subsequently were confirmed to have targeted BP-2 alleles
by Southern blot analysis. Six of the seven confirmed recombinant
clones contained a normal chromosome complement while one clone was
triploid. The six clones with normal karyotypes were tested for their
ability to contribute to chimeric mice by microinjection into C57Bl6/J
blastocysts and subsequent transfer of injected blastocysts into
pseudopregnant females. Two ES lines successfully contributed to
chimeric males that transmitted the mutant allele through the germ
line. Progeny heterozygous for the mutated gene, identified by Southern
analysis of tail DNAs, were mated, and offspring from these
heterozygous matings were genotyped as neonates. All genotypes were
represented in the progeny from these matings (Fig. 1E
) in proportions
not significantly different from Mendelian expectations (Table 1
). We have not detected any differences
in viability, fecundity, or longevity in mutant homozygote animals when
compared with wild-type littermates over more than 10 generations.

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Figure 1. Targeted Deletion of the IGFBP-2 Gene
Panel A, Restriction map of the wild-type mouse IGFBP-2 genomic
fragment containing exons 2, 3, and 4. Restriction sites are: B,
BamHI; D, DraI; E, EcoRI;
E47, Eco47II-I; H, HindIII; P, PstI;.T,
Tth 111I. Panel B, Replacement vector for deletion of the IGFBP-2 gene.
The neomycin resistance (NeoR) gene was used to replace a DNA fragment
that contained the majority of BP-2 exon 3 (115 nucleotides,
representing 38 amino acids) as well as 0.6 kb of the 3'-flanking
intron including the exon/intron boundary. The insertion of the NeoR
gene resulted in the addition of two novel, diagnostic
EcoRI restriction sites. The targeting vector was
linearized at a unique nonplasmid BamHI site and used to
electroporate ES cells. Panel C, Predicted structure of mutated IGFBP-2
locus upon homologous recombination. The dashed line
represents the 2.3-kb PCR fragment amplified using an upstream primer
to BP-2 gene sequence outside the targeting vector and a downstream
primer in the neoR gene. Panel D, Ethidium bromide-stained gel showing
PCR analysis of ES clones after electroporation and selection in G418
and gancyclovir. Lanes 17 show the results of PCR amplification of
seven different pools of ES lines. Each pool represents DNAs from five
ES clones. Lanes 2 and 7 show the 2.3-kb amplified product expected
after homologous recombination. Subsequent PCR analysis of individual
ES clone DNAs from each positive pool identified two positive ES cell
clones that were confirmed as homologous recombinants at the BP-2 locus
by Southern hybridization. Panel E, Southern blot of
EcoRI-digested tail DNAs showing that three genotypes
arise from heterozygote crosses. Blot was hybridized with a
random-primed 32P-labeled cDNA probe containing exons 2, 3,
and 4 of the IGFBP-2 gene. +/+, Single 7-kb EcoRI fragment;
EcoRI fragment denotes normal allele; +/-, 7-kb normal
allele and two fragments (4.8 kb, 2.8 kb) that correspond to the mutant
allele; -/-, only the 4.8-kb and 2.8- kb fragments are detected from
the mutant alleles.
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BP-2 mRNA and Protein Are Absent in the Homozygous BP-2 Mutant
Animals
Tissues from adult animals of all three genotypes were analyzed
for IGFBP-2 mRNA and protein by Northern and Western blot analyses
(Fig. 2A
, B). Mice heterozygous for the
mutated allele contained reduced levels of BP-2 mRNA in brain and liver
and of IGFBP-2 protein in serum. The reduction in IGFBP-2 protein and
mRNA occurred whether the mutant gene was maternal or paternal in
origin suggesting that, unlike IGF-II (37) and the
IGF-II/mannose-6-phosphate receptor (46), the IGFBP-2 gene is not
imprinted. Homozygous mutants contained no detectable BP-2 mRNA or
immunoreactive protein of either native size or altered size, which
conceivably could have been produced by alternate splicing around the
neomycin gene (47).

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Figure 2. IGFBP-2 RNA and Protein Levels in Heterozygote and
Homozygote Mutants
A, Northern blot of 20 µg total RNA isolated from brain or liver of
adult wild-type (+/+), heterozygous (+/-), or homozygous (-/-)
IGFBP-2 mutant mice. Blot was hybridized with a random-primed
32P-labeled probe to rat IGFBP-2 and exposed to X-OMAT AR
film. The probe detected a single band at approximately 1.9 kb. Level
of RNA in each lane was verified by reprobing the blot with a
random-primed labeled cDNA to mouse actin which hybridized to a single
RNA species at approximately 2.1 kb. B, Western blot of adult serum
proteins separated by PAGE and incubated with a rabbit antibody to rat
IGFBP-2 (1:1000) followed by an alkaline phosphatase-conjugated
antirabbit secondary antibody as described in Materials and
Methods. The immunopositive band is approximately 30 kDa. Shown
are representative serum samples from two wild-type(+/+), one
heterozygote (+/-), and four homozygote (-/-) mutant mice.
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Developmental Expression of Other IGFBPs
In several previous gene disruption experiments, ablation of one
member of a gene family has resulted in compensation by one or more
other members of the gene family. Functional redundancy has been
demonstrated for several gene families including the muscle
transcription factors Myo D, Myf-5 and myogenin (48, 49, 50, 51), the retinoic
acid receptors (52, 53, 54), the src family of tyrosine kinases (55), and
the MAD family of transcription factors (56). In some cases,
compensation by another family member has been accompanied by an
increase in expression of the corresponding gene (48, 57). Of potential
significance for interpretation of the BP-2 mutant, previous studies
have shown substantial overlap between the patterns of BP-2 and BP-5
expression during prenatal development (19, 23). To determine whether
any changes in BP-5 levels accompanied the BP-2 mutation during this
period, levels of BP-5 mRNA in heads and trunks of embryonic day 13.5
and 17 BP-2 heterozygote and homozygote littermates were determined by
solution hybridization (Fig. 3
). Each RNA
sample was hybridized concurrently with both
32P-labeled BP-5 and actin antisense riboprobes
to ensure accurate normalization (see Materials and
Methods). No significant differences were observed in levels of
IGFBP-5 RNA in homozygote vs. heterozygote embryos at either
embryonic age. However, since RNA was isolated from large fetal
regions, it is possible that regional or cellular changes in BP-5 RNA
levels could have been missed in these experiments. Since previous
studies also demonstrated specific expression patterns for other IGFBP
genes during embryonic development (16, 17, 23), we extended the
analysis of BP-2 minus embryos to determine whether tissue-specific
expression patterns of BP-5 as well as other BPs were altered in the
absence of BP-2. In situ hybridization was used to analyze
expression patterns of the other IGFBPs and of IGF-II in BP-2
homozygous mutant embryos during midgestation. To date, we have
observed no ectopic expression or gross change in the spatial pattern
or level of mRNAs for any IGFBPs or for IGF-II in e15 embryos (data not
shown).

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Figure 3. Analysis of IGFBP-5 RNA Levels in BP-2 Homozygote
and Heterozygote Embryos
The relative levels of IGFBP-5 mRNA in heads (H) or trunks (T) of
heterozygous and homozygous littermates are shown at embryonic days
13.5 and 17. Total RNA was isolated from heads and trunks of homozygote
and heterozygote littermates. Each embryo was genotyped by Southern
analysis of placental DNA. Individual RNA samples (5 µg from each
head or trunk) were hybridized with both 32P-labeled mouse
BP-5 and mouse ß-actin riboprobes. Resulting protected bands were
excised from the dried gels and counts per min were determined from
scintillation counting. BP-5 measurements were corrected based on the
actin mRNA levels to normalize for variation in RNA amounts between
samples. Graph shows mean ± SE for each group.
Three to five animals were analyzed per group. Students
t test was performed to determine significance between
heterozygote and homozygote embryos for each region at each age. No
significant differences were observed in levels of BP-5 RNA in
homozygote vs. heterozygote embryos at these ages.
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Body Growth in IGFBP-2 Null Mutants
Gene targeting of IGF-I, IGF-II and the type I IGF receptor have
all resulted in prenatal growth deficits (37, 38, 39, 40, 43). IGF-I deficiency
results in additional defects in postnatal growth (38, 39, 40, 43). To
determine whether the absence of IGFBP-2 resulted in altered prenatal
growth, mice from multiple heterozygote crosses were weighed at birth
(Fig. 4
). There was no difference in
birth weights among the three genotypes, suggesting that prenatal
growth was not affected by the mutation. Mice of all genotypes were
then weighed daily until postnatal day 14 (p14) and then every 23
days through p35. Body weights of both male (Fig. 4A
) and female (Fig. 4B
) wild-type and homozygous mutant mice, as well as heterozygous mice
(data not shown), were indistinguishable during this period,
demonstrating that the extent of postnatal growth was also unaffected
by the BP-2 mutation.

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Figure 4. Postnatal Growth Curves of Wild-Type and BP-2
Homozygous Mutant Mice
Male (A) and female (B) body weights of mice from heterozygous matings.
Mice derived from heterozygote matings were weighed daily beginning on
the day of birth (p0) through day 14 and then every second or third day
until day 35.
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Organ Weights in IGFBP-2 Homozygous Null Mutants
Analysis of transgenic mice that overexpress IGF-I or IGF-II
has shown that these peptides induce growth of specific organs
(58, 59, 60, 61, 62). To determine whether absence of IGFBP-2 caused selective
changes in organ growth, numerous organs from mutant and wild-type
adult males were weighed (Table 2
).
Specific organs analyzed included liver, kidney, heart, lung, and
spleen. Since previous studies have shown that weight of some, but not
all, organs correlate with changes in body weight (59, 60, 61), organ
weights from the two groups were analyzed both directly or as a
function of body weight. Using either analysis, spleen weights of
IGFBP-2 homozygous mutants were significantly less than that of
wild-type animals (P
0.001; Table 2
). Weights of
kidneys and hearts were significantly reduced only when analyzed as a
function of body weight (P < 0.001; Table 2
). In
contrast, liver weights in the BP-2 mutant animals were significantly
higher than those of the wild-type animals both directly
(P < 0.001) and as a function of body weight
(P
0.005; Table 2
).
Since the phenotype of several knock-out lines of mice have differed
depending on the strain background (38, 63, 64), we have continued
backcrossing the BP-2 mutation into the C57BL/6J mouse strain. Analysis
of BP-2 minus and wild-type mice after seven generations of
backcrossing revealed that BP-2 minus spleens were approximately 80%
normal size (n = 4 +/+; 5 -/-), similar to the reduction seen
previously in the outbred line of mice.
Analysis of Spleen Deficit in BP-2 Null Mutant Mice
The spleen arises developmentally from the ASMP, a region of
the splanchnic mesoderm (65). Early studies of the mouse mutant
Dominant Hemimelia demonstrated that a defect in the ASMP
resulted in loss of the spleen (65) and, recently, it was determined
that expression of the homeobox gene, Hox11, in the ASMP is
essential for spleen formation (66, 67). Our previous studies showed
expression of BP-2 in the ASMP during the embryonic period critical for
spleen formation (32). To determine whether loss of BP-2 resulted in a
deficit in early spleen growth, we weighed spleens from normal and BP-2
minus neonatal animals at postnatal day 12. The weight of BP-2 mutant
spleens (31.7 ± 1.7 mg; n = 7) was not significantly
different from those of normal mice (30.8 ± 0.8 mg; n = 6)
at this age.
To begin to address whether the reduced weight of the adult BP-2 null
spleens resulted in functional deficits, spleens from normal and BP-2
mutant mice were analyzed for histological alterations and for
alterations in lymphocyte numbers. No gross histological abnormalities
were observed in fixed sections from BP-2 mutant and wild-type spleens
that were analyzed by light microscopy after hematoxylin and eosin
staining. To assess whether the reduction in spleen size was correlated
with a reduction in lymphocyte number in the BP-2 null mutant mice,
spleens from four normal and four BP-2 homozygous mutant male mice were
isolated, weighed, and homogenized to obtain lymphocytes (68). The
total number of lymphocytes per spleen was reduced in the BP-2
homozygous mutants (7.5 x 107 ± 0.4) as
compared with wild-type mice (1 x 108 ±
0.8; P = 0.05) in proportion to the observed reduction
in spleen weights.
Serum Levels of Multiple IGFBPs Are Increased in IGFBP-2 Minus
Mice
Circulating levels of IGFBPs in normal and BP-2 mutant animals
were assessed using ligand blots. Serum was collected from age-matched
(from 48 months) adult male normal (n = 11) and BP-2 homozygous
mutant (n = 20) mice. Prominent serum bands that bound both
[125I]-IGF-II and
[125I]-IGF-I corresponded to predicted sizes
for the mouse BP-3 doublet and mouse BP-4 (Fig. 5A
). When the intensities of the
radiolabeled bands were compared with Coomassie stained gels of
identical samples, it was concluded that there was a 2.5-fold elevation
of BP-3 and BP-4 levels in BP-2 mutant serum. Western immunoblots of
the same serum samples confirmed the identity of the doublet as BP-3
and further demonstrated elevation of circulating BP-3 (Fig. 5B
).

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Figure 5. Serum Levels of Other IGFBPs in Adult IGFBP-2
Homozygous Mutant Mice
A, Ligand blot showing adult serum IGFBPs after PAGE and
incubation with 125I-IGF-II. Samples are identical to those
seen in Fig. 2B . Prominent serum bands that bind to IGF-II correspond
to predicted sizes for the IGFBP-3 doublet at 3943 kDa
(arrowhead) and to IGFBP-4 at 24 kDa
(arrow). Based on comparisons with Coomassie-stained
gels of identical samples, levels of both BP-3 and BP-4 are elevated in
the homozygous BP-2 mutant mice. B. Western immunoblot to detect
IGFBP-3 protein in serum samples shown in panel A. A second set of the
serum samples shown in panel A were separated by PAGE and used for
Western immunoblotting with a rabbit polyclonal antibody to rat IGFBP-3
(1:500) as described in Materials and Methods. The
antibody detected a doublet at 3943 kDa that was more intense in the
BP-2 homozygote mutant serum compared with wild-type serum.
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In separate experiments, sets of adult male normal and homozygote
mutant animals (three to five animals, age 58 months) were assayed by
ligand blotting using a gel system modified from that shown in Fig. 5
(see Materials and Methods). Serum samples from three
representative animals (two homozygote; one normal) revealed increased
levels of the 3845 kDa doublet (confirmed as BP-3) and a 24-kDa band
identified by size as BP-4 (Fig. 6A
). In
addition, an enlarged image of the region that corresponds to 3032
kDa shows BP-2 in the two wild-type samples and absent in the
homozygous sample (confirmed by immunoblotting, data not shown). In
addition, levels of two additional bands (arrows) of lower
molecular mass than BP-2 appeared increased in the homozygous serum.
The lower of these bands was identified as IGFBP-1 by immunoblotting
(data not shown), while the additional radiolabeled band was increased
in the region of the gel where a glycosylated form of BP-4 has been
reported (69, 70). To determine whether the increases in serum levels
of BP-3 and BP-4 were due to induction of the respective mRNAs for
these proteins, mRNA expression levels of BP-3 and BP-4 were analyzed
by Northern blotting. After adjustment for hybridization to a control
RNA, no significant differences were observed in liver RNA expression
levels for either BP-3 or BP-4 in the homozygote BP-2 mutant animals as
compared with wild-type animals (data not shown).

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Figure 6. Multiple BPs In BP-2 Region Are Up-Regulated In
Adult Mutant Serum
Two different sets of adult male wild-type and homozygote mutant
animals (three to five animals of each genotype) were assayed by ligand
blotting using an expanded electrophoresis procedure to optimize
separation in the 25- to 40-kDa protein range (see Materials and
Methods). Three representative samples (two homozygote; one
wild-type) are shown after ligand blotting with 125I-IGF-I.
The upper doublet at 3943 kDa was confirmed by Western immunoblotting
to be BP-3 and the lower band (at 24 kDa) corresponds to the known
molecular mass of BP-4. The increased gel resolution reveals several
bands in the region from 2933 kDa. A band corresponding to BP-2 is
seen in the +/+ sample only but is absent from the homozygote mutant
sera. In addition, two additional bands of lower molecular mass than
BP-2 are detectable in the homozygote mutant serum samples. These bands
correspond to BP-1 and phosphorylated BP-4.
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DISCUSSION
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We report the targeting of a null allele to the mouse IGFBP-2
locus and the production of mouse lines in which the capability to
produce IGFBP-2 has been eliminated. These strains represent the first
in which one member of the IGFBP family has been genetically
inactivated. Homozygote mutant animals are viable and fertile and can
be distinguished from wild-type mice histopathologically by a 25%
reduction in spleen size and a 15% increase in liver size. Although
IGFBP-2 is expressed in the progenitor of the spleen during
midgestation development (32), comparison of spleen weights in BP-2
null mutants at neonatal ages suggests that the reduction in spleen
size in the BP-2 null mutant adult animals is not due to a
developmental growth defect. Since BP-2 is expressed in the adult
spleen (71), the reduction in spleen size in the BP-2 (-/-) animals
may be the result of local actions of BP-2 on pubertal spleen growth or
on adult spleen function. Previous studies have shown that spleen size
is acutely responsive to changes in circulating levels of IGF-I in
transgenic mice that overexpress IGF-I (59, 61), mice selected on the
basis of high or low circulating levels of IGF-I (72), and mice or rats
given exogenous IGF-I (73, 74, 75, 76). In all cases, high circulating levels
of IGF-I resulted in increased spleen weight. These data suggest that
the reduction in spleen weight in the BP-2 null mutant mice may be the
result of decreased availability of IGF-I in these mice. Initial
analyses of IGF-I serum levels in the BP-2 null mutants showed no
significant differences in total IGF-I levels from the wild-type mice
(A. J. DErcole, personal communication). Moreover, we observed
no substantial differences in body growth in the BP-2 minus animals,
supporting the conclusion that the absence of BP-2 has not resulted in
significant alterations in circulating IGF levels. This suggests that
the absence of BP-2 may result either in decreased transport of
circulating IGF-I to the spleen or in decreased autocrine/paracrine
actions of IGF-I in the spleen itself.
If BP-2 is required in spleen function, we postulate that the absence
of BP-2 will cause specific deficits in spleen immune responses.
Previous reports have demonstrated an increase in the ratio of CD4+ to
CD8+ T cells in both rats and monkeys after infusion of exogenous IGF-I
(73, 77). We have determined that total lymphocyte numbers are reduced
in the spleens of the BP-2 mutant mice as compared with control mice,
and that the reduction is proportional to the observed decrease in
spleen weight. Preliminary studies analyzing the proportion of
lymphocyte subsets suggest that the CD4 to CD8 ratio may be increased
in the BP-2 minus spleens. Additional preliminary analyses of the
immune response to herpes virus infection suggest that the BP-2 minus
spleens are able to respond to viral challenge as assessed by increased
spleen weight and lymphocyte number; however, levels of cytokine
production by the mutant spleen lymphocytes may be altered. Future
experiments are designed to correlate deficits in spleen function with
pathogenesis of viral infection in the BP-2 mutant mice. However, since
ratios of lymphocyte populations and response to viral challenge differ
significantly in different mouse strains, we have postponed further
analyses of spleen function in the BP-2 null mutants until backcrossing
the mutation 10 generations into the C57Bl6/J strain has been
achieved.
In contrast to the reduction in spleen size in the BP-2 null mutant
mice, the livers of these mice show a significant increase in wet
weight. The induction in liver weight in the BP-2 null mutants may be
due to local autocrine/paracrine actions of BP-2 in liver growth since
BP-2 is expressed at high levels in liver from the onset of its
development. The mechanisms by which the loss of BP-2 results in
opposite effects on spleen and liver size are unclear. Since attainment
of normal liver size is particularly dependent on GH (58, 60), one
possibility would be that slightly elevated GH levels in the IGFBP-2
null mice, insufficient to alter body size, could produce selective
overgrowth of the liver. In fact, GH levels in both urine and serum are
not significantly different between wild-type and IGFBP-2 mutant
genotypes, with a tendency toward lower levels in the mutants (T.
L. Wood, A. G. P. Schuller, and J. E. Pintar,
unpublished observations). It thus seems more likely that the
differential phenotypes in spleen and liver reflect differential
functions of BP-2 in the growth and/or function of each organ. Data
from in vitro studies have supported a role for the IGFBPs
both in blocking and enhancing actions of IGFs. The ability of the
IGFBP to inhibit or enhance IGF action has been proposed to depend upon
whether the binding protein is free in solution or is cell surface or
ECM associated, respectively (5, 9, 13, 14, 15). IGFBP-2 has the potential
to bind cell surface integrins through its carboxy-terminal RGD
sequence (5, 9, 14). In addition, a recent study has shown that BP-2,
once bound to IGF-I or IGF-II, can bind to heparin and ECM (78).
Whether IGFBP-2 predominantly enhances or inhibits IGF actions in
vivo is unknown; however, the data presented here suggest that its
role may be distinct in different tissues.
Targeted mutations in IGF-I (38, 39, 40, 43), IGF-II (37), and the type I
IGF receptor (38, 39) all result in fetal growth deficits. In contrast,
deletion of the IGF-II/M6P receptor (41, 42) results in fetal
overgrowth due to excess IGF-II. Since the IGFBPs are believed to
modulate the half-life and cellular actions of the IGFs, and since
IGFBP-2 is prominently expressed during embryonic development, we
predicted that the absence of circulating BP-2 might result in
alterations in fetal growth. This prediction was not supported by the
observed phenotype of the BP-2 minus mice that had neonatal weights
indistinguishable from wild-type littermates. In addition, the
expression of BP-2 in numerous sites in the embryo predicts a role for
this IGFBP in development of many tissues where no gross phenotype was
observed in the BP-2 null mutant mice. Several implications of this
observation can be mentioned. First, we and others have shown that all
other IGFBPs are expressed throughout the midgestational period in the
embryo proper and thus may be able to compensate for the lack of
IGFBP-2 (16, 17, 18, 19, 20, 23). In addition, other IGFBPs are also potentially
available to the developing fetus from the decidualizing regions of the
uterus (21). For example, IGFBP-3 is found lining the endothelial cells
of the decidua, while IGFBP-1 lines the uterine lumen as early as
embryonic day 8 in the rat. Thus, compensation for IGFBP-2 absence
would not necessarily require an increase in levels of other IGFBPs,
although in the adult BP-2 knockout mice, we did observe induction of
circulating levels of several other members of the IGFBP family
including IGFBPs-1, -3, and -4.
If the induction of the other IGFBPs in the BP-2 deletion mutant
represents functional compensation, then multiple knockouts will be
required to demonstrate additional roles for the IGFBPs. The necessity
of multiple knockouts to delineate the functions of gene products that
arise from multiple gene families has been repeatedly demonstrated. For
example, while mice containing an ablation of only one isoform of the
retinoic acid receptor (RAR-
-2) are normal, ablation of all
-RAR
isoforms results in lethality (52, 53). In addition, genetic disruption
of individual members of the src family of tyrosine kinases (Src, Fyn,
and Yes) each produces a mild phenotype but combinatorial deletions of
any two of these genes results in a more severe phenotype (55).
Finally, genetic ablation of MyoD, which itself can induce muscle
differentiation in fibroblast lines after transfection, has no dramatic
effect on skeletal muscle differentiation; neither does ablation of a
second transcription factor, Myf-5 (79). However, a double mutant of
MyoD and Myf-5 produces a complete absence of skeletal muscle (49).
These examples of functional compensation between related gene products
provide a potential framework for understanding the restricted
alterations in the IGFBP-2 mice that have thus far been observed.
We suspect that mice lacking IGFBP-2 have adapted physiologically to
its absence likely as a result of the up-regulation of other members of
the IGFBP gene family. It is possible, however, that specific
physiological challenge may reveal additional phenotypic alterations
even in the absence of multiple gene targeting events.
 |
MATERIALS AND METHODS
|
---|
Targeting Vector and Generation of Mice with a Null Mutation in
the IGFBP-2 Gene
A mouse genomic clone containing exons 2, 3, and 4 of the
IGFBP-2 gene was isolated from a
library (FIX II vector,
Promega Corp., Madison, WI) that contained 15- to 20-kb
inserts of mouse 129/ReJ genomic DNA. Restriction mapping and partial
sequencing (Sequenase-version 2.0) confirmed the identity of the
genomic clone and provided information for constructing the BP-2
targeting vector. To construct the targeting vector, two fragments from
the mouse genomic clone were subcloned into a pBS (sk)II cassette
(obtained from Dr. Steven Potter, University of Cincinnati, Cincinnati,
OH). The targeting vector contained a positive selection marker, the
neomycin resistance (neoR) gene, and a negative selection marker, the
herpes simplex virus thymidine kinase (HSV-TK) gene, each driven by the
HSV-TK promoter. Initially, a 1.4-kb Dra/Tth 111I fragment was
subcloned at the 3'-end of the neoR gene and, subsequently, a 4.7-kb
Eco47II-I/EcoRI fragment was subcloned between the neoR gene
and the TK gene. The DNA for electroporation was purified by cesium
chloride density centrifugation, linearized at the BamHI
site, phenol-chloroform extracted, and ethanol precipitated before
resuspension in sterile TE.
Linearized DNA was electroporated into ES cells grown to 5060%
confluence on mitomycin C-treated STO fibroblast feeder cells. ES
clones that survived double selection in G418 (220 µg/ml) and
gancyclovir (2 µM) were expanded and analyzed for
homologous recombination at the BP-2 locus by PCR and Southern
analyses. Correctly targeted ES cell clones were used for injections
into blastocysts isolated from C57BL/6J mice. Injected blastocysts were
transferred into the uterine horns of pseudopregnant mothers. Three
resulting chimeric male mice demonstrated germ line transmittance of
the mutant BP-2 allele and were used to generate progeny for analysis.
A line of mice from one of these chimeras was expanded by backcrossing
to both C57BL/6J and 129/ReJ females to generate a mixed strain
(129/C57) background line and an inbred (129) background line carrying
the BP-2 mutation. Genotyping of all mice was by Southern blotting of
tail DNA. All mice were maintained in accordance with the NIH Guide for
the Care and Use of Laboratory Animals.
PCR Amplification and Southern Analysis
DNA was isolated from ES cells by lysis at 37 C for 1520 min
in 0.5 ml 0.05 M Tris-HCl (pH 7.5), 0.05 M
EDTA, 0.1 M NaCl, 5 mM dithiothreitol (DTT),
1% SDS, and 0.5 mM spermidine containing 400 µg/ml
proteinase K followed by incubation overnight at 55 C. The DNA was
extracted with phenol/chloroform and then ethanol precipitated. Tail
DNAs were isolated by incubation overnight at 50 C in 50 mM
Tris (pH 8.0), 100 mM EDTA, and 0.5% SDS containing 30
µg/ml proteinase K followed by extraction with phenol/chloroform and
ethanol precipitated.
Aliquots of DNAs from 510 individual double-selected ES cell clones
were pooled for PCR analysis. Pooled DNA was used for PCR amplification
using an upstream primer designed to a region of intron A of the BP-2
gene that was 5' to sequences included in the targeting vector
(5'-TGCACACAGTTGGCTCTATG-3') and a downstream primer designed to
sequences in the neomycin resistance gene (5'-ACAGACAATCGGCTGCTCTG-3').
Amplification was for 35 cycles under standard conditions using 0.1
µM primer concentrations at an annealing temperature of
55 C. Using these primers, a homologous recombination event resulted in
amplification of a 2.3-kb fragment. Additional reactions were run on
all DNA samples using the same upstream primer and a downstream primer
to sequences in exon 3 of the BP-2 gene (5'-CAGGGAGTAGAGATGTTCCA-3')
that were removed in the construction of the targeted null allele.
Amplification with these primers detected a 1.5-kb band from the normal
endogenous BP-2 allele.
Genotypes of putative positive ES clones and subsequent progeny from
chimera matings were confirmed by Southern analysis. Purified ES and
tail DNAs were digested with EcoRI, phenol chloroform
extracted, ethanol precipitated, and separated by agarose gel
electrophoresis. DNAs were then denatured, blotted onto Genescreen
nylon membranes (NEN Life Science Products, Boston, MA)
and used for hybridization with a random-primed cDNA probe digested
from the parent plasmid with HincII so that it included only
exons 2, 3, and 4 of rat IGFBP-2 (80). Hybridizations were performed
overnight at 42 C in 50% (vol/vol) deionized formamide, 2xSSC (1xSSC
contains 0.15 M NaCl and 0.015
M sodium citrate), 1% SDS, 10% dextran sulfate,
250 µg/ml denatured herring sperm DNA, and 13 x
106 cpm/ml of a
[
-32P]CTP-labeled cDNA probe to rat IGFBP-2
(80). After hybridization, blots were washed twice at 65 C in 2xSSC,
1% SDS followed by two washes in 0.2xSSC, 1% SDS and exposed to
Kodak XAR-5 film (Eastman Kodak Co.,
Rochester, NY) at -80 C with an intensifying screen.
RNA Analysis
Total cellular RNA was extracted and purified as described by
Chomczynski et al. (81). For Northern analysis, RNA samples
were separated by electrophoresis through 1% agarose gels containing
2.2 M formaldehyde and transferred to Genescreen
nylon membrane. Hybridizations were performed at 42 C in 50% (vol/vol)
deionized formamide, 5x Denhardts (1x Denhardts contains 0.02%
Ficoll, 0.02% polyvinylpyrrolidine, and 0.02% BSA), 5xSSPE (1xSSPE
contains 0.15 NACL, 0.01 [SCAP]M sodium
phosphate and 0.001 M EDTA), 1% SDS, 100 µg/ml
denatured herring sperm DNA, and 13 x 106
cpm/ml of a [
-32P]CTP-labeled cDNA probe to
rat IGFBP-2 (80) or to rat ß-actin (provided by J. Roberts). The
cDNA to rat ß-actin encoded exons 12 and was derived from a
full-length rat cDNA sequence (82). The cDNA inserts were labeled by
random priming and had a specific activity of 109
cpm/ml (Roche Molecular Biochemicals, Indianapolis, IN).
Membranes were washed twice for 15 min in 2xSSPE, 1% SDS at 65 C
followed by twice for10 min in 0.2xSSPE, 1% SDS at 50 C and exposed
to Kodak XAR-5 film using an intensifying screen at -80
C.
Total head and trunk RNAs isolated from e13.5 and e17 BP-2 heterozygous
or homozygous embryos were used for ribonuclease protection assays as
previously described (83). Each RNA sample (5 µg) was hybridized
simultaneously with 32P-labeled RNA antisense
probes to mouse BP-5 and mouse ß-actin. Probes were transcribed from
linearized plasmids containing mouse BP-5 (19) or actin cDNAs (provided
by A. Efstratiadis). Total counts per min in each protected band was
determined by scintillation counting of excised bands. Subsequent
values for BP-5 were corrected based on values for the actin band in
each sample to normalize for variation between samples.
Ligand and Immunoblotting
One to 3 µl serum from each animal was run on 12.5%
polyacrylamide gels using either the Mini-Protean II or the Protean
IIxi system (Bio-Rad Laboratories, Inc., Richmond, CA).
For full size gels, samples were electrophoresed through 1617 cm
resolving gels that were run at 85 V for 1214 h. For minigels,
samples were run through 56 cm gels at 85 V for approximately 2
h.
For all ligand blots, proteins were transferred to a polyvinylidene
fluoride (PVDF) nylon membrane (Millipore Corp.,
Bedford, MA) which provided a signal intensity equivalent to that
observed after transfer to nitrocellulose. Membranes were preblocked
with 1% BSA in Tris-buffered saline (TBS) containing 3% NP-40
and 0.1% Tween-20 before overnight incubation with 400,000 cpm of
125I-IGF-I or 125I-IGF-II
at 4 C. After sequential washes in TBS containing 0.1% Tween-20 and in
TBS alone, blots were exposed to Kodak XAR-5 film for
varying durations ranging from 1 day to 3 weeks.
For immunoblots, serum samples were electrophoresed as for ligand
blotting and electroblotted onto either nitrocellulose or PVDF
membranes. The membrane was preblocked with blotto (5% dry milk in
TBS) and incubated overnight at 4 C in antiserum against either rat
IGFBP-2 (1:1000; antisera provided by M. Rechler)(80) or rat IGFBP-3
(1:500; provided by N. Ling) (84). After washes in TBS and TBS plus
0.5% Tween-20, blots were incubated in secondary antibody
(AP-conjugated Anti-Rabbit IgG; Promega Corp.) for 1
h and washed again in TBS and TBS plus 0.5% Tween-20. Blots were then
visualized by treatment with BCIP(X-phos) and nitroblue tetrazolium
(NBT) in 0.1 M Tris (pH 9.5), 0.1 M NaCl, and
50 mM MgCl2.
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to thank Dr. E. Robertson for providing
the CCE embryonic stem cells, Drs. M. Rechler, N. Ling, and D. Clemmons
for providing IGFBP antibodies, Drs. M. Rechler, P. Rotwein, J.
Roberts, and A. Efstratiadis for providing cDNA clones, and Dr. S.
Potter for providing the gene targeting vector. The authors would also
like to thank Dr. R. Bonneau for assistance with spleen analyses and
Paul Hsu, Kristin Concepcion, Carolyn Wahl, and Jennifer Thompson for
excellent technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: John E. Pintar, Department of Neurobiology and Cell Biology (J.E.P.), Robert Wood Johnson Medical School, University of Medicine and Dentistry New Jersey, 675 Hoes Lane, Piscataway, New Jersey 08854.
This work was supported in part by NIH Grant NS-21970 to J.E.P. and NIH
Grant DK-48103 to T.L.W.
1 Present address: Department of Neuroscience and Anatomy,
Pennsylvania State University, College of Medicine, Hershey,
Pennsylvania 17033. 
2 Present address: Liver Research Center, Department of
Medicine, Albert Einstein College of Medicine, Bronx, New York
10461. 
Received for publication October 7, 1999.
Revision received May 18, 2000.
Accepted for publication May 30, 2000.
 |
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