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-Actin-IGFBP-4 Fusion Gene Induces Smooth Muscle Hypoplasia
Divisions of Endocrinology and Metabolism (J.W., W.N., Y.E.N., T.L.C., J.A.F.), Pediatric Pathology (D.P.W.), Pediatric Endocrinology (S.D.C.), University of Cincinnati, Cincinnati, Ohio 45267; the Division of Cardiology, Cedars-Sinai Medical Center (B.S.), Los Angeles, California 90048; and the Department of Cell Biology, Neurobiology, and Anatomy, Ohio State University (A.R.S.), Columbus, Ohio 43210
Address all correspondence and requests for reprints to: James A. Fagin, M.D., Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Room 5564, Cincinnati, Ohio 45267-0547. E-mail: faginja{at}uc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-actin 5'-flanking region (SMP2-BP-4) or
-1074 bp, 63 bp of 5'-untranslated region, and 2.5 kb of intron 1 of
smooth muscle -actin (SMP8-BP-4). SMP2-BP-4 mice expressed low
levels of the exogenous IGFBP-4 messenger RNA (mRNA), which was not
specifically targeted to SMC-rich tissue environments, and were
therefore not analyzed further. Six SMP8-BP-4 transgenic lines derived
from separate founders were characterized. Mating of hemizygous
SMP8-BP-4 mice with controls produced about 50% transgenic offspring,
with equal sex distribution. Expression of IGFBP-4 mRNA in
nontransgenic littermates was maximal in liver and kidney. By contrast,
transgenic IGFBP-4 mRNA expression, distinguished because of a smaller
transcript size, was confined to SMC-containing tissues, with the
following hierarchy: bladder > aorta > stomach =
uterus. There was no transgene expression in skeletal muscle, brain, or
cardiac myocytes. The abundance of IGFBP-4 measured by Western ligand
blotting or by immunoblotting, was 8- to 10-fold higher in aorta and
bladder of SMP8-BP-4 mice than in their nontransgenic littermates, with
no change in plasma IGFBP-4 levels. Transgenic mice exhibited a
significant reduction in wet weight of SMC-rich tissues, including
bladder, intestine, aorta, uterus, and stomach, with no change in total
body or carcass weight. In situ hybridization showed
that transgene expression was targeted exclusively to the muscular
layers of the arteries, veins, bladder, ureter, stomach, intestine, and
uterus. Overexpression of IGFBP-4 was associated with SMC hypoplasia, a
reciprocal phenotype to that of transgenic mice overexpressing IGF-I
under control of the same promoter (SMP8-IGF-I). Double transgenic mice
derived from mating SMP8-BP-4 with SMP8-IGF-I animals showed a modest
decrease in wet weight at selected SMC tissues. Although we cannot
exclude that the effects of IGFBP-4 may be IGF independent, these data
suggest that IGFBP-4 is a functional antagonist of IGF-I action on SMC
in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGFBP-4 was originally isolated and cloned from a human osteosarcoma
cell line (4). It exists in biological fluids as a 28-kDa glycosylated
and/or a 24-kDa nonglycosylated form. It consistently inhibits
IGF-mediated cell proliferation of all cell types tested in
vitro. IGFBP-4 does not bind to the cell membrane and is found
associated with connective tissue, although the precise nature of this
interaction is not known. We and others (5, 6, 7) have observed that adult
arterial SMC release IGFBP-4 into serum-free conditioned medium.
IGFBP-4 is subject to proteolysis by a cation-dependent serine
protease that cleaves it only in the presence of IGF-I (7, 8, 9). IGFBP-4
is also expressed in the rat artery wall in vivo, where it
is found primarily in the extracellular compartment. It is important to
note that individual IGFBPs may have opposite activities on IGF action
in vitro according to whether the IGFBP in question is in
solution or bound to the cell membrane or matrix (2). Thus, a more
physiological appraisal of the action of a particular IGFBP requires
that it be expressed in the appropriate tissue setting in
vivo, as it will presumably interact with its putative partners
and be targeted to its natural compartment. In this paper, we report
the characterization of transgenic mice overexpressing IGFBP-4
selectively in smooth muscle through a mouse smooth muscle -actin
(SM--actin) promoter. To our knowledge, together with a companion
paper in which we generated mice overexpressing IGF-I under control of
the same promoter (10), these reports represent the first examples of
overexpression of functional proteins selectively in SMC, capable of
modifying their properties in vivo. Indeed, the SMP8 smooth
muscle -actin promoter directs high levels of expression that are
entirely confined to SMC of all tissue beds. Furthermore, SMP8-BP-4
transgenic mice demonstrate that overexpression of this binding protein
is associated with smooth muscle hypoplasia and support the concept
that IGFBP-4 inhibits IGF action, presumably by preventing IGF access
to its cellular receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-actin to a rat IGFBP-4
(rIGFBP-4) complementary DNA (cDNA) followed by the simian virus 40
(SV40) small T intron and early polyadenylation signal fragment (Fig. 1
). SMP-2 contains -724 bp of the
proximal 5'-flanking region plus 43 bp 5'-untranslated (5'-UT) region
(exon 1). The rIGFBP-4 cDNA contains the entire coding sequence as well
as 184 bp 5'-UT and 318 bp 3'-UT. Plasmid pSMP2 (11) was digested with
BamHI and NcoI to delete a 0.67-kb
chloramphenicol acetyltransferase cDNA fragment; the rat IGFBP-4
cDNA fragment was released by EcoRI from pRBP-4–503 (a gift
from Dr. Shimasaki) and inserted into pSMP2 after filling in both
vector and insert with DNA polymerase I.
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actin to the rIGFBP-4 cDNA followed by the SV40
early polyadenylation signal fragment. SMP8 contains -1074 bp of the
5'-flanking region, 63 bp of 5'-UT, and the 2.5-kb first intron of
SM--actin. A 0.24-kb Sau3A1 fragment of SV40-pA from
pSMP2 was cloned into pRBP-4–503 at the BamHI site,
resulting in plasmid pRBP-4-SV. A 3.6-kb SMP8 fragment, released
from pSMP8 by digestion with BamHI and filled in by Klenow,
was partially digested with HindIII and cloned into
pRBP-4-SV at the HindIII and EcoRv sites, so that
rat IGFBP-4 fused to SV40 early polyadenylation signal is driven by
SMP8.
Generation of transgenic mice
The SMP8-BP-4 fusion gene was released from pSMP8-BP-4 by
XhoI and BamHI restriction before microinjection,
and that fragment was isolated and purified as previously described
(10). The male pronuclei of fertilized eggs from FVB-N mouse strains
were microinjected with 2 pl linearized DNA at the transgenic mouse
facility of the University of Cincinnati. Microinjected eggs were
implanted into the oviduct of pseudopregnant female mice and carried to
term. Positive founders were identified by Southern blotting and bread
to wild-type FVB-N mice for propagation of the line. Heterozygotes and
nontransgenic progeny from F1 and subsequent generations were selected
by Southern blotting of EcoRI-restricted genomic DNA, as
previously described (10). Hybridization was performed with a rat
IGFBP-4 cDNA labeled by random priming (Prime-It II kit, Stratagene, La
Jolla, CA). The transgene was identified as a unique 1.3-kb band.
Transgene copy number was calculated by quantitative Southern blotting,
using known amounts of the SMP8-BP-4 construct added to nontransgenic
mouse genomic DNA as a standard according to the following formula:
8100 bp (size of SMP8-BP-4) x 5 µg/6 x 109
bp/copy = 6.75 x 10-6 µg/copy = 6.75
pg/copy.
To examine the effects of overexpression of IGFBP-4 in SMC-rich tissues, where IGF-I levels were also increased, the SMP8-BP-4 mouse line f23928 was crossed with the recently characterized SMP8-IGF-I transgenic mouse line f23988 (10). The wet weights of SMC tissues of double transgenic offspring were compared with those of single transgenic age- and sex-matched controls, as described below.
RNA isolation and Northern blot analysis
Total RNA was isolated from tissues by a single step, acid
guanidinium thiocyanate-phenol-chloroform extraction method. Northern
blots were performed as previously described (10). Briefly, 5 µg
tissue total RNA was gel-separated, transferred to Nylon membrane, and
then hybridized with random primed rat IGFBP-4 cDNA. For
standardization, blots were rehybridized with either human
glyceraldehyde-3-phosphate dehydrogenase cDNA, for comparisons within
the same tissue type, or 18S ribosomal RNA, for comparisons between
different tissues.
Allometry of SMP8-BP-4 transgenic mice
Transgenic mice and their nontransgenic littermates were killed
by CO2 asphyxiation. After determining the body weight,
blood was collected by cardiac puncture, and serum was stored at -80 C
until use. Organs of interest, except the aorta, were dissected, rinsed
in ice-cold PBS, tissue-blotted, weighed, and immediately frozen in dry
ice. The contents of the stomach and small intestine were flushed out
with PBS before weighing. A section of the arterial vessel, from the
aortic arch to the level of femoral fork, was excised and placed in
PBS. Adhering fat and connective tissue from the adventitia were
scraped off under surgical microscope, and the vessel was rinsed with
PBS to remove residual blood. Organs were immediately placed on dry ice
and stored at -80 C until use.
Tissue extraction and RIA for IGF-I
Tissues were placed in 1 ml ice-cold 1 M acetic acid
and immediately homogenized for 1 min on ice using a Polytron PT3000
(Brinkmann Instruments, Westbury, NY) at full speed. After standing on
ice for 2 h, the tissue extracts were centrifuged in siliconized
microcentrifuge tubes at 18,000 x g for 1 h at 4
C. The supernatants were then concentrated to 2–3 µg/ml protein
through Centricon-3 (Amicon, Beverly, MA) devices at 4 C. The
concentrated extracts were mixed in a 3:1 ratio with a 4 x mobile
phase solution consisting of 0.8 M acetic acid and 0.4
M trimethylamine for 1 h at room temperature
before HPLC separation of binding proteins from IGFs. The mixture was
centrifuged and then filtered through 0.22-mm nitrocellulose filters to
remove protein precipitates. HPLC separations were performed on a
Waters size exclusion column Protein-Pak 125 (Waters Associates,
Milford, MA) with the detector set at 1.0 absorbance. Samples were
eluted isocratically at 0.5 ml/min, and 1-min fractions were collected.
The IGF-I-spanning fractions 19–22 were pooled, and 50 mg/ml BSA in a
1/100th volume mobile phase were added as a carrier protein. The pooled
fractions (
2 ml) were concentrated in Centricon-3 at 4 C. Small
aliquots were pipetted into polystyrene tubes to evaporate the solvent
in a Speed-Vac (Savant Instruments, Farmingdale, NY) for 2 h with
no heating.
Recombinant human IGF-I (Austral Biologicals, San Ramon, CA) was iodinated according to the method described by Hill et al. (12). The specific activity of the purified [125I]iodyltyrosyl-IGF-I was estimated at 320 µCi/µg. For the IGF-I RIA, pooled HPLC fractions of each tissue extract were resuspended in 0.03 M sodium phosphate (pH 7.4) containing 0.05% Tween-20, 0.02% NaN3, 0.02% protamine sulfate, and 0.01 M EDTA and assayed using a double antibody precipitation, nonequilibrium assay, as previously described (10). The IGF-I antiserum was provided by Dr. L. E. Underwood (Chapel Hill, NC), and recombinant human IGF-I was used as a standard. Tissue extracts and rodent sera gave displacement curves parallel to the human IGF-I standard.
Western ligand blotting and immunoblotting
To determine tissue and serum contents of IGFBPs, frozen tissues
(bladders and aortas) were homogenized in PBS with 12 mM
EDTA and 1 mM phenylmethylsulfonylfluoride in a Polytron
(model PT3000) at full speed on ice. Extracts were centrifuged at
100,000 x g for 1 h at 4 C, and the supernatant
was further concentrated in a Centricon-3 device (Amicon, Danvers, MA).
Aliquots of the concentrated extracts were taken for protein assay. For
Western ligand blotting, serum samples (8 µl each) were run on a
7.5–20% gradient Laemmli gel, and tissue extracts were run on
10–20% gradient or 10% Laemmli gels under nonreducing conditions.
After blotting, the nitrocellulose membrane was sequentially incubated
with PBS-0.1% Tween-20 and PBS-1% BSA, and then with 24 µCi
[125I]IGF-I in 150 ml PBS containing 1% BSA-0.1%
Tween-20 at 4 C for 16 h. After washing, the blots were dried and
exposed to x-ray film at -70 C. For tissue immunoblotting, the
extracts were run on the same gels as those used for ligand blotting,
but under reduced conditions. The blots were blocked with TBS solution
containing 0.05% Tween-20 and nonfat dry milk and incubated with a
1:5000 diluted rabbit antirat IGFBP-4 antiserum (raised against a
synthetic peptide corresponding to amino acids 80–100 of the mature
rat IGFBP-4 protein) at room temperature for 16 h. Membranes were
washed and then incubated with a 1:2500 diluted goat antirabbit
antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) for 1 h at
room temperature. The blots were detected with SuperSignal reagent
(Pierce, Rockford, IL).
In situ hybridization
In situ hybridization was performed as previously
described (10). Tissues dissected from animals at the indicated ages
were fixed in 4% paraformaldehyde, saturated overnight with 30%
sucrose in PBS, and frozen in OCT (Miles, Elkhart, IN). Cryostat
sections (7 µm) were mounted on silane-coated slides. Sense and
antisense complementary RNA probes for rat IGFBP-4, which hybridizes to
both endogenous mouse and transgenic rat IGFBP-4 messenger RNA (mRNA),
and for a transgene-specific SV40 3'-UT/polyadenylase signal sequence,
which recognizes only the exogenous mRNA, were labeled with
[35S]UTP, using a commercially available kit (Stratagene,
La Jolla, CA). For generation of the antisense IGFBP-4 riboprobe, the
pRBP-4-SH (gift from Dr. Shimasaki) plasmid was linearized with
SmaI and transcribed with T7 RNA polymerase. The product was
493 bases long, 449 of which were complementary to transgene mRNA (Fig. 1
, probe A). The sense IGFBP-4 riboprobe was 491 bases long and was
obtained after linearizing the same vector with HindIII and
transcribing with T3 RNA polymerase. Transgene-specific riboprobes were
obtained from the pSV40-pA plasmid. The antisense probe was transcribed
from HindIII-linearized plasmid with T7 polymerase,
generating a 351-base riboprobe, 150 bases of which were complementary
to the transgenic sequence (Fig. 1, probe B). The 343-base sense
riboprobe was generated from BamHI-linearized pSV40-pA with
T3 polymerase. Hybridization was performed with a total of 5 x
105 to 1 x 106 cpm in a final volume of
30 µl/slide. The sections were hybridized overnight at 42 C, treated
with 50 µg/ml ribonuclease A (Sigma Chemical Co., St. Louis, MO) and
100 U/ml ribonuclease T1 (Boehringer Mannheim Biochemicals,
Indianapolis, IN) for 30 min at 37 C, and washed to a final stringency
of 0.1 x standard citrate saline at 50 C. Slides were dipped in
NTB2 emulsion (Eastman Kodak, Rochester, NY), diluted 1:1 with 0.6
mol/liter ammonium acetate, exposed for 10–14 days, and developed in
D19 developer (Kodak). Sections were counterstained in hematoxylin and
eosin and photographed under dark- and brightfield illumination.
Tissue histomorphometry
Morphometry was performed using NIH Image V1.61, an
image-processing and analysis program for the Macintosh. The arterial
section images of SMP8-BP-4 transgenic mice (line 23942) and their
age-matched nontransgenic controls were color-captured into the
computer from Trichrome-stained sections through the microscope. After
adjusting the image contrast, the area of interest was auto-outlined,
and the regions outside and inside the area were cleared. The lumen
area of the aorta and the thickness of the aortic media were calculated
from a hypothetical perfect circle. The formulas are as follows:
Do = P/
, Ai = P2/4 - A, Di =
2(Ai/)0.5, T = (Do - Di)/2, where Do is the
outer diameter, P is the perimeter, Ai is the lumen area, A is the
measured area, Di is the inner diameter, and T is the thickness of the
muscular area.
Determination of myocyte number and tissue DNA and RNA contents
RNA and DNA were extracted from bladder, intestine, stomach, and
brain tissues of SMP8-BP-4 transgenic mice and their nontransgenic
littermates as previously described (10). The concentrations of the
extracted nucleic acids were measured by UV absorption at 260 nm. To
verify RNA/DNA data, the myocyte number per surface area was directly
counted on hematoxylin- and eosin-stained slides using a grid as a
reference point. For each tissue examined, a minimum of five fields
(x40) of five sections were counted.
Statistics
Statistical analysis was performed using either Student’s
t test or, when more than two groups were compared, one-way
ANOVA. Differences between any two groups selected by the ANOVA were
then analyzed using the Walker-Duncan adaptive procedure.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-actin 5'-flanking region (SMP2), containing a single E box,
upstream of rat IGFBP-4 cDNA (Fig. 1). This choice was made based on
data reported by Foster et al. (11), indicating that the
most proximal E box is sufficient to evoke maximal transcriptional
activity in BC3H1 myoblasts in vitro. However, SMP2-BP-4
mice had only a very modest increase in IGFBP-4 mRNA levels (at best
2-fold) that was not specifically targeted to SMC-rich tissues (data
not shown). We, therefore, generated new fusion genes containing a more
extended 5'-flanking region of mouse SM--actin (Fig. 1). Within the
-1074 bp region of SM--actin, there is an evolutionary conserved
motif that represses expression in nonmyogenic fibroblast cells as well
as six E box motifs mediating high level expression in postconfluent
BC3H1 myoblasts (11, 13, 14). In addition, the new constructs included
the first intron of SM--actin, which has been proposed to contain
additional motifs important for SMC-specific transcription (14).
Generation of SMP8-BP-4 mice and examination of transgene
expression
Six SMP8-IGFBP-4 founder animals were obtained. The percentage of
transgenic offspring in the F1 generation indicated that three of the
six founders were mosaics. In subsequent generations, matings of
hemizygous transgenic mice with controls produced about 50% transgenic
offspring with equal sex distribution. Six transgenic lines were
further propagated for more complete analysis.
The tissue distribution of endogenously produced and transgenic IGFBP-4
mRNA is shown in Fig. 2 (left
panel). Endogenous IGFBP-4 mRNA was abundant in the liver, kidney,
and uterus of nontransgenic mice and was expressed at relatively low
levels in other SM-rich tissues. The IGFBP-4 transgene, identified
because of its lower size, was detected at very high levels in bladder
and aorta and to a lesser degree in stomach and uterus. Note that
although expression of native IGFBP-4 mRNA was readily observed in
aorta, stomach, and bladder, the abundance of the transgenic IGFBP-4
mRNA was severalfold higher than that of the endogenous transcript in
these tissues. Exogenous IGFBP-4 mRNA was observed almost exclusively
in smooth muscle-rich tissues of transgenic mice in all founder lines
tested, although the levels of expression varied between the lines
(Fig. 2, right panel). Western ligand blotting demonstrated
a marked increase in IGFBP-4 protein in bladder and aorta of transgenic
mice from several lines (Fig. 3).
Glycosylated as well as deglycosylated forms of IGFBP-4 were present in
greater abundance, as confirmed by N-glycanase digestion of
bladder and aortic extracts of SMP8-BP-4 mice (Fig. 3D). The
overexpression of IGFBP-4 in bladder and aorta was also confirmed by
Western blotting (Fig. 3E). These data document selective
overexpression of IGFBP-4 in SM-rich tissues. Increased biosynthesis of
the binding protein in SM-rich tissues did not result in a concomitant
rise in serum IGFBP-4 levels, as determined by Western ligand blotting
(Fig. 3C).
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, A–C, and E). By contrast, expression
of the SMP8-BP-4 transgene mRNA, detected with the same probe, was
targeted to medial aortic SMC and the SMC layer of the small intestine
(Fig. 4, D and F). When examined with a transgene-specific riboprobe
complementary to vector-derived sequences from the SV40 3'-UT region of
the construct, expression was entirely confined to smooth muscle,
further confirming the cell specificity of transcription directed by
this promoter. Figure 5 shows examples of
transgene expression in the SMC layers of the uterus, stomach, ureter,
vena cava, aorta, and urinary bladder.
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). The
surface area and thickness of the aortic medium were also significantly
lower in SMP8-BP-4 transgenic mice compared with those in age- and
sex-matched nontransgenic controls (Table 1). The SMP8-BP-4 transgenic line 23942
had low expression of transgenic IGFBP-4 in the intestine (Fig. 2, left panel), and correspondingly, there was no change in
length or wet weight of small bowel. Line 23928 did have significant
transgene expression in the bowel as well as marginally decreased
intestinal weight (not shown). The lower SMC mass in the SMP8-BP-4 mice
is due to a decrease in cell number and not to hypotrophy, as
demonstrated by RNA/DNA ratios (Table 2).
However, the admixture of other cell types in the tissues we examined
may skew these ratios toward normality (this is less likely to be a
factor in the artery wall, as these were stripped of adventitia).
Additional evidence was, therefore, obtained by histomorphometry.
Myocyte counts of x40 magnified sections of bladder and aorta of
transgenic and nontransgenic mice were not significantly different in
the IGFBP-4 transgenics (five fields of five sections from the
respective tissues of four separate transgenic and nontransgenic mice
were examined). As the total mass of SMC tissue was decreased in
SMP8-BP-4 mice (Table 1), this indicates that there are fewer cells
overall.
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-actin promoter
(SMP8-IGF-I mice). These animals exhibited remarkable SMC hyperplasia,
with a distinct pattern of tissue remodeling (10). This manifested in
marked thickening of the SMC layers of the bladder and artery wall,
whereas the uterine horns and small bowel grew primarily in length. To
determine whether overexpression of IGFBP-4 can antagonize these
effects, heterozygous SMP8-IGF-I and SMP8-IGFBP-4 mice were crossed,
and the double transgenics were analyzed. These animals had
overexpression of both IGF-I and IGFBP-4 in smooth muscle, to a similar
degree as that found in their respective single transgenic parents (not
shown). However, the wet weight of SMC-rich tissues in the double
transgenics was only modestly decreased and was significant only in
aorta of males and in uterus and intestine of females (Table 3: as determined by ANOVA). Moreover, as
the estrous cycle of the females was not synchronized, we cannot assume
that changes in uterine weight were directly due to overexpression of
IGFBP-4.
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Thus, the higher tissue IGFBP-4 levels decrease SMC mass without
interfering with IGF-I gene expression or tissue IGF-I abundance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the function of IGFBP-4 in SMC in vivo, we used
the SM--actin promoter to target its expression. As SM--actin
gene expression is primarily restricted to SMC of adult rodents and
rabbits (19, 20, 21), its promoter seemed well suited to the task. We first
focused on the SMP2 and SMP8--actin promoter constructs to direct
expression to SMC of the different tissue beds. Although the SMP2
fragment contained regulatory regions capable of directing high levels
of expression to SMC in vitro (11), it was almost without
effect in vivo. By contrast, the SMP8 promoter directed
robust levels of transgene expression specifically to SMC, as reported
here and in a companion paper (10). This indicates that regulatory
regions between -724 and -1074 bp of SM--actin are important for
SMC-specific expression in vivo. Alternatively, critical
sequences may lie within the first intron of the SM--actin gene that
were not present in SMP2 but were included in the SMP8 construct. The
phenotypic changes in the SMP8-IGF-I (10) and SMP8-IGFBP-4 mice were
confined to SMC. During development, SM-actin is transiently
expressed in skeletal and cardiac muscle and only becomes restricted to
SMC during late fetal maturation (19, 23, 24). The lack of effect of
overexpression of IGFBP-4 on cardiac or skeletal muscle mass indicates
either that the SMP8 promoter does not entirely recapitulate the
pattern of expression of the endogenous gene product during fetal life
or that the binding protein is without measurable effect when
overexpressed during that discrete period of time (embryonic day 11.5
to birth). Notably, the type I IGF receptor is expressed in skeletal
muscle and heart at the appropriate period of rat embryogenesis
(25).
The IGFBP-4 overexpressed in SMC-rich tissues remained entirely paracrine, as plasma levels of IGFBP-4 were not increased in the transgenic animals. Both glycosylated and unglycosylated forms were overproduced. SMP8-BP-4 mice exhibited decreased weight of smooth muscle-containing tissues: i.e. aorta, bladder, stomach, uterus, and intestine. There was no alteration of expression of other IGFBPs in these tissues (based on Western ligand blotting) or of IGF-I itself, indicating that the observed changes were due to the individual action of increased IGFBP-4 and not to compensatory changes in expression or abundance of other members of the IGF system. The effects on smooth muscle were selective, as total body weight and those of other organs were unaffected. Overexpression of IGF-I by SMC in vivo is associated with increased cell mass, with characteristics dependent on the tissue microenvironment. IGF-I regulates SMC number through its mitogenic effects as well as by serving as an inhibitor of apoptosis (1). It is likely that the effects of IGFBP-4 were due to its IGF-binding activity and consequent prevention of ligand activation of the SMC type I IGF receptor. However, preliminary data on IGFBP-4 null mice indicate that, contrary to expectation, disruption of IGFBP-4 function leads to lower (10–15%) postnatal weight, an effect that becomes apparent by postnatal day 7 (26). The precise mechanisms of this paradoxical finding are still under investigation. Furthermore, although the effect of IGFBP-4 deletion on total body weight has been reported, information on its impact on expression of other binding proteins and on growth of individual organs and tissues is still pending.
When SMP8-IGF-I and SMP8-IGFBP-4 were crossed, the double transgenic animals overexpressed both gene products in smooth muscle, i.e. IGF-I and IGFBP-4, to a similar degree as in their single transgenic ancestors. However, there was only a modest inhibition of SMC mass that was only significant in selected tissues. Thus, IGFBP-4 cannot completely overcome the effects of very high levels of tissue IGF-I. This may be due to the stoichiometry of these two compounds in the tissues of this particular cross. Alternatively, we cannot exclude that IGFBP-4 may have effects that are independent of its interaction with IGF-I, although to our knowledge, there is no evidence of such an effect in vitro. It is more likely that the modest phenotype may be due to activation of the IGFBP-4 protease, which is known to be IGF-I dependent (7, 8, 9, 12, 27), leading to increased proteolysis of IGFBP-4 and partial negation of its presumed inhibitory properties. We have been unable to obtain information on whether the protease is more active in the double transgenics, because of nonspecific cross-reactivity of an abundant 16-kDa mouse protein with the IGFBP-4 antibody that prevents visualization of the cleavage products by Western blotting. Nevertheless, the role of this IGF-I-dependent protease remains an important issue, as tissue IGF-I levels increase remarkably in certain pathophysiological situations, such as vascular injury and bladder hypertrophy. It remains to be seen whether overexpression of a cleavage-resistant form of IGFBP-4 results in a more powerful antagonism of tissue IGF-I action.
Received September 11, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-actin gene contains
evolutionarily conserved sequence motifs within a functional promoter.
J Biol Chem 265:16667–16675-Smooth muscle actin is transiently expressed in embryonic rat
cardiac and skeletal muscles. Differentiation 39:161–166[CrossRef][Medline]
-vascular but not the 
-enteric isoform in differentiating
striated myocytes. J Cell Biol 109:2929–2937This article has been cited by other articles:
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 A. Zhang, X. Liu, J. G. Cogan, M. D. Fuerst, J. A. Polikandriotis, R. J. Kelm Jr., and A. R. Strauch YB-1 Coordinates Vascular Smooth Muscle {alpha}-Actin Gene Activation by Transforming Growth Factor {beta}1 and Thrombin during Differentiation of Human Pulmonary Myofibroblasts Mol. Biol. Cell, October 1, 2005; 16(10): 4931 - 4940. [Abstract] [Full Text] [PDF]
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 J M Fleming, B J Leibowitz, D E Kerr, and W S Cohick IGF-I differentially regulates IGF-binding protein expression in primary mammary fibroblasts and epithelial cells J. Endocrinol., July 1, 2005; 186(1): 165 - 178. [Abstract] [Full Text] [PDF]
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S. V. Subramanian, J. A. Polikandriotis, R. J. Kelm Jr., J. J. David, C. G. Orosz, and A. R. Strauch Induction of Vascular Smooth Muscle {alpha}-Actin Gene Transcription in Transforming Growth Factor {beta}1-Activated Myofibroblasts Mediated by Dynamic Interplay between the Pur Repressor Proteins and Sp1/Smad Coactivators Mol. Biol. Cell, October 1, 2004; 15(10): 4532 - 4543. [Abstract] [Full Text] [PDF]
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 G. Yang, A. Haczku, H. Chen, V. Martin, H. Galczenski, Y. Tomer, C. R. Van Beisen, J. F. Evans, R. A. Panettieri, and C. D. Funk Transgenic smooth muscle expression of the human CysLT1 receptor induces enhanced responsiveness of murine airways to leukotriene D4 Am J Physiol Lung Cell Mol Physiol, May 1, 2004; 286(5): L992 - L1001. [Abstract] [Full Text] [PDF]
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 E. Isotani, G. Zhi, K. S. Lau, J. Huang, Y. Mizuno, A. Persechini, R. Geguchadze, K. E. Kamm, and J. T. Stull Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse PNAS, April 20, 2004; 101(16): 6279 - 6284. [Abstract] [Full Text] [PDF]
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 P. Delafontaine, Y.-H. Song, and Y. Li Expression, Regulation, and Function of IGF-1, IGF-1R, and IGF-1 Binding Proteins in Blood Vessels Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 435 - 444. [Abstract] [Full Text]
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S. Zaina, L. Pettersson, A. B. Thomsen, C.-M. Chai, Z. Qi, J. Thyberg, and J. Nilsson Shortened Life Span, Bradycardia, and Hypotension in Mice with Targeted Expression of an Igf2 Transgene in Smooth Muscle Cells Endocrinology, June 1, 2003; 144(6): 2695 - 2703. [Abstract] [Full Text] [PDF]
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 S. M. Firth and R. C. Baxter Cellular Actions of the Insulin-Like Growth Factor Binding Proteins Endocr. Rev., December 1, 2002; 23(6): 824 - 854. [Abstract] [Full Text] [PDF]
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J. V. Silha and L. J. Murphy Minireview: Insights from Insulin-Like Growth Factor Binding Protein Transgenic Mice Endocrinology, October 1, 2002; 143(10): 3711 - 3714. [Abstract] [Full Text] [PDF]
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 M. Zhang, E. P. Smith, H. Kuroda, W. Banach, S. D. Chernausek, and J. A. Fagin Targeted Expression of a Protease-resistant IGFBP-4 Mutant in Smooth Muscle of Transgenic Mice Results in IGFBP-4 Stabilization and Smooth Muscle Hypotrophy J. Biol. Chem., June 7, 2002; 277(24): 21285 - 21290. [Abstract] [Full Text] [PDF]
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 W. A. Price, B. M. Moats-Staats, and A. D. Stiles Pro- and Anti-inflammatory Cytokines Regulate Insulin-like Growth Factor Binding Protein Production by Fetal Rat Lung Fibroblasts Am. J. Respir. Cell Mol. Biol., March 1, 2002; 26(3): 283 - 289. [Abstract] [Full Text] [PDF]
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S. Zaina, L. Pettersson, B. Ahren, L. Branen, A. B. Hassan, M. Lindholm, R. Mattsson, J. Thyberg, and J. Nilsson Insulin-like Growth Factor II Plays a Central Role in Atherosclerosis in a Mouse Model J. Biol. Chem., February 1, 2002; 277(6): 4505 - 4511. [Abstract] [Full Text] [PDF]
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C. A. Shoubridge, C.-B. Steeb, and L. C. Read IGFBP mRNA expression in small intestine of rat during postnatal development Am J Physiol Gastrointest Liver Physiol, December 1, 2001; 281(6): G1378 - G1384. [Abstract] [Full Text] [PDF]
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 W. R. P. Agema, J. W. Jukema, S. N. Pimstone, and J. J. P. Kastelein Genetic aspects of restenosis after percutaneous coronary interventions;towards more tailored therapy Eur. Heart J., November 2, 2001; 22(22): 2058 - 2074. [PDF]
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E. P. Smith, A. Kamyar, W. Niu, J. Wang, B. Cercek, S. D. Chernausek, and J. A. Fagin IGF-Binding Protein-4 Expression and IGF-Binding Protein-4 Protease Activity Are Regulated Coordinately in Smooth Muscle During Postnatal Development and After Vascular Injury Endocrinology, October 1, 2001; 142(10): 4420 - 4427. [Abstract] [Full Text] [PDF]
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M. Holzenberger, G. Hamard, R. Zaoui, P. Leneuve, B. Ducos, C. Beccavin, L. Perin, and Y. Le Bouc Experimental IGF-I Receptor Deficiency Generates a Sexually Dimorphic Pattern of Organ-Specific Growth Deficits in Mice, Affecting Fat Tissue in Particular Endocrinology, October 1, 2001; 142(10): 4469 - 4478. [Abstract] [Full Text] [PDF]
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A. Bayes-Genis, R. S. Schwartz, D. A. Lewis, M. T. Overgaard, M. Christiansen, C. Oxvig, K. Ashai, D. R. Holmes Jr, and C. A. Conover Insulin-Like Growth Factor Binding Protein-4 Protease Produced by Smooth Muscle Cells Increases in the Coronary Artery After Angioplasty Arterioscler Thromb Vasc Biol, March 1, 2001; 21(3): 335 - 341. [Abstract] [Full Text] [PDF]
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 M. L. Gimbel, D. Chelius, T. K. Hunt, and E. M. Spencer A Novel Approach to Reducing Postoperative Intraperitoneal Adhesions Through the Inhibition of Insulinlike Growth Factor I Activity Arch Surg, March 1, 2001; 136(3): 311 - 317. [Abstract] [Full Text] [PDF]
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G. Zhao, R. L. Sutliff, C. S. Weber, J. Wang, J. Lorenz, R. J. Paul, and J. A. Fagin Smooth Muscle-Targeted Overexpression of Insulin-Like Growth Factor I Results in Enhanced Vascular Contractility Endocrinology, February 1, 2001; 142(2): 623 - 632. [Abstract] [Full Text] [PDF]
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L. Fernandez-Celemin and J.-P. Thissen Interleukin-6 Stimulates Hepatic Insulin-Like Growth Factor Binding Protein-4 Messenger Ribonucleic Acid and Protein Endocrinology, January 1, 2001; 142(1): 241 - 248. [Abstract] [Full Text] [PDF]
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J. B. Pucilowska, K. K. McNaughton, N. K. Mohapatra, E. C. Hoyt, E. M. Zimmermann, R. B. Sartor, and P. K. Lund IGF-I and procollagen alpha 1(I) are coexpressed in a subset of mesenchymal cells in active Crohn's disease Am J Physiol Gastrointest Liver Physiol, December 1, 2000; 279(6): G1307 - G1322. [Abstract] [Full Text] [PDF]
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M. Holzenberger, P. Leneuve, G. Hamard, B. Ducos, L. Perin, M. Binoux, and Y. Le Bouc A Targeted Partial Invalidation of the Insulin-Like Growth Factor I Receptor Gene in Mice Causes a Postnatal Growth Deficit Endocrinology, July 1, 2000; 141(7): 2557 - 2566. [Abstract] [Full Text] [PDF]
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 M. R. SCHNEIDER, H. LAHM, M. WU, A. HOEFLICH, and E. WOLF Transgenic mouse models for studying the functions of insulin-like growth factor-binding proteins FASEB J, April 1, 2000; 14(5): 629 - 640. [Abstract] [Full Text]
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 A. Bayes-Genis, C. A. Conover, and R. S. Schwartz The Insulin-Like Growth Factor Axis : A Review of Atherosclerosis and Restenosis Circ. Res., February 4, 2000; 86(2): 125 - 130. [Abstract] [Full Text] [PDF]
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F. M. Faraci and C. D. Sigmund Vascular Biology in Genetically Altered Mice : Smaller Vessels, Bigger Insight Circ. Res., December 3, 1999; 85(12): 1214 - 1225. [Full Text] [PDF]
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 J. Van Wyk Insulin-Like Growth Factors and Skeletal Growth: Possibilities for Therapeutic Interventions J. Clin. Endocrinol. Metab., December 1, 1999; 84(12): 4349 - 4354. [Full Text]
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N. Miyakoshi, C. Richman, X. Qin, D. J. Baylink, and S. Mohan Effects of Recombinant Insulin-Like Growth Factor-Binding Protein-4 on Bone Formation Parameters in Mice Endocrinology, December 1, 1999; 140(12): 5719 - 5728. [Abstract] [Full Text]
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T. C. Nichols, T. d. Laney, B. Zheng, D. A. Bellinger, G. A. Nickols, W. Engleman, and D. R. Clemmons Reduction in Atherosclerotic Lesion Size in Pigs by {alpha}V{beta}3 Inhibitors Is Associated With Inhibition of Insulin-Like Growth Factor-I-Mediated Signaling Circ. Res., November 26, 1999; 85(11): 1040 - 1045. [Abstract] [Full Text] [PDF]
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D. R. Clemmons, G. Horvitz, W. Engleman, T. Nichols, A. Moralez, and G. A. Nickols Synthetic {alpha}V{beta}3 Antagonists Inhibit Insulin-Like Growth Factor-I-Stimulated Smooth Muscle Cell Migration and Replication Endocrinology, October 1, 1999; 140(10): 4616 - 4621. [Abstract] [Full Text]
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 K. Wallner, C. Li, M. C. Fishbein, P. K. Shah, and B. G. Sharifi Arterialization of human vein grafts is associated with tenascin-C expression J. Am. Coll. Cardiol., September 1, 1999; 34(3): 871 - 875. [Abstract] [Full Text] [PDF]
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 T. Gustafsson, P. Andersson, Y. Chen, J. O. Magnusson, and H. J. Arnqvist Interaction of angiotensin II and the insulin-like growth factor system in vascular smooth muscle cells Am J Physiol Heart Circ Physiol, August 1, 1999; 277(2): H499 - H507. [Abstract] [Full Text] [PDF]
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S. Maeda, R. L. Sutliff, J. Qian, J. N. Lorenz, J. Wang, H. Tang, T. Nakayama, C. Weber, D. Witte, A. R. Strauch, et al. Targeted Overexpression of Parathyroid Hormone-Related Protein (PTHrP) to Vascular Smooth Muscle in Transgenic Mice Lowers Blood Pressure and Alters Vascular Contractility Endocrinology, April 1, 1999; 140(4): 1815 - 1825. [Abstract] [Full Text]
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C. Rees, D. R. Clemmons, G. D. Horvitz, J. B. Clarke, and W. H. Busby A Protease-Resistant Form of Insulin-Like Growth Factor (IGF) Binding Protein 4 Inhibits IGF-1 Actions Endocrinology, October 1, 1998; 139(10): 4182 - 4188. [Abstract] [Full Text] [PDF]
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Z.-H. Yang, Y. Kitami, Y. Takata, T. Okura, and K. Hiwada Targeted Overexpression of CCAAT/Enhancer-Binding Protein-{delta} Evokes Enhanced Gene Transcription of Platelet-Derived Growth Factor {alpha}-Receptor in Vascular Smooth Muscle Cells Circ. Res., September 14, 2001; 89(6): 503 - 508. [Abstract] [Full Text] [PDF]
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