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From the
The avian skeletal
Insulin-like growth factor I (IGF-I),
The effects of IGF-I
overexpression have previously been studied in cultured cells and in
transgenic mice, but these earlier studies did not address the effects
of IGF-I expression on skeletal muscle
specifically
(11, 12) . Mathews et al.(11) utilized the metallothionein promoter to drive expression
of an hIGF-I cDNA in transgenic mice resulting in IGF-I overexpression
in a broad range of visceral internal organs and increased
concentrations of IGF-I in serum. These transgenic mice exhibited an
increase in body weight and organomegaly, but only a modest improvement
in muscle mass. Thus, in order to test the effects of IGF-I
overexpression on muscle growth and physiology in vivo, we
reasoned that it would be necessary to target its overexpression
specifically to striated muscle.
The
We describe here the construction of a
myogenic expression vector containing regulatory elements from both the
5`- and 3`-flanking regions of the avian skeletal
Under serum-free
culture conditions, we wanted to determine how IGF-I influenced
myogenic gene activity. Northern blots of total RNA isolated from
myoblasts cultured in growth media with IGF-I demonstrated enhanced
expression of the myogenic basic helix-loop-helix factors such as MyoD
and myogenin in general agreement with Florini and co-workers (40). As
shown in Fig. 3, we observed a correspondence between elevated
IGF-I expression and increased expression of myogenic specified gene
products, such as the intermediate filament protein desmin, and
skeletal
Inclusion of the skeletal
Recently,
genetic truncations of the single murine IGF-I gene
(46, 47) and of the type I IGF receptor
(48) have provided
direct evidence in vivo for the ascribed functions of IGF-I in
skeletal muscle development. Powell-Braxton et al.(46) reported that IGF-I mutant mice showed severe muscular
dystrophy and highly reduced myofibrillar organization in both heart
and skeletal muscle. They observed that the majority of IGF-I mutant
mice die at birth due to respiratory failure likely caused by
incomplete maturation of the diaphragm and intercostal muscles. Liu
et al.(48) reported that mice lacking a functional
allele for the type I IGF receptor exhibited generalized cell
hypoplasia and specifically had a reduced number of myonuclei and
myofibers. In addition to evidence from gene knockout experiments of
the role of IGF-I in the development of skeletal muscle, localized
up-regulation of IGF-I expression has been implicated as a mediator of
stretch-induced myofiber hypertrophy and muscle
regeneration
(8, 9) . Together, these observations lend
support to a model in which IGF-I is a central trophic growth factor
required for the proliferation of myoblasts, the progression of
myogenic differentiation, and subsequent growth and hypertrophy of
myofibers. In addition, they suggest that, depending on the
developmental timing, overexpression of IGF-I in skeletal muscle in
vivo could potentially elicit effects at multiple stages of
skeletal muscle development. Since expression of the skeletal
At present, it is not clear what mechanism(s) underlies the observed
myofiber hypertrophy in SK733 IGF-I 3`-SK transgenic mice, but based on
results from previous studies, it is plausible that overexpression of
IGF-I induced hypertrophy through either one or a combination of
several mechanisms. First, IGF-I is known to elicit numerous effects on
the metabolism of skeletal muscle that are anabolic in nature such as
stimulation of amino acid and glucose uptake and enhancement of net
myofibrillar protein accretion via combined effects on protein
synthesis and degradation (see Refs. 2 and 3 for review). Thus, it
could be that the myofiber hypertrophy observed in the present study
was primarily due to the cumulative anabolic effects of IGF-I. Another
possibility is that overexpression of IGF-I stimulated processes that
are normally involved in muscle regeneration and stretch-induced
hypertrophy. Previous research has established that expression of IGF-I
is increased locally in regenerating muscle
(9) and in muscle
undergoing stretch-induced hypertrophy
(8) , and it is
hypothesized that this increase in IGF-I drives the hypertrophic
response. However, the manifestation of IGF-I's actions on
regeneration and hypertrophy of skeletal muscle may also require the
action of other regulatory factors. For example, Bischoff and
colleagues
(50) have observed in studies of single muscle fibers
with attached satellite cells in vitro that satellite cell
proliferation is refractory to an IGF-I challenge unless the cells are
first exposed to an as yet unidentified factor present in crushed
muscle extract. This suggests exercise or stimuli that naturally result
in myofiber hypertrophy may enhance the phenotype observed with
overexpression of IGF-I in striated muscle. Currently, studies are
ongoing to address these hypotheses.
Data presented are mean ± S.D. for adult male transgenic and
age-matched control mice.
Data presented are
mean ± S.D. for two male SK733 IGF-I 3`-SK and two male
littermate control mice and mean for one female SK733 IGF-I 3`-SK and
one female littermate control.
We thank Dr. Ray Hintz for determining the IGF-I
content of conditioned media samples. We dedicate this study in memory
of Dr. Hal Weintraub.
Volume 270,
Number 20,
Issue of May 19, pp. 12109-12116, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-actin gene was used as a template for
construction of a myogenic expression vector that was utilized to
direct expression of a human IGF-I cDNA in cultured muscle cells and in
striated muscle of transgenic mice. The proximal promoter region,
together with the first intron and 1.8 kilobases of 3`-noncoding
flanking sequence of the avian skeletal -actin gene directed high
level expression of human insulin-like growth factor I (IGF-I) in
stably transfected C
C myoblasts and transgenic
mice. Expression of the actin/IGF-I hybrid gene in
C
C muscle cells increased levels of myogenic
basic helix-loop-helix factor and contractile protein mRNAs and
enhanced myotube formation. Expression of the actin/IGF-I hybrid gene
in mice elevated IGF-I concentrations in skeletal muscle 47-fold
resulting in myofiber hypertrophy. IGF-I concentrations in serum and
body weight were not increased by transgene expression, suggesting that
the effects of transgene expression were localized. These results
indicate that sustained overexpression of IGF-I in skeletal muscle
elicits myofiber hypertrophy and provides the basis for manipulation of
muscle physiology utilizing skeletal
-actin-based vectors.
()
a
peptide growth factor that is structurally related to proinsulin
(1-4), has a primary role in promoting the differentiation and
growth of skeletal muscle. The effects of IGF-I on myogenic cells
include stimulation of myoblast replication, myogenic differentiation,
and myotube hypertrophy (see Refs. 5 and 6 for review). In
vivo, up-regulation of IGF-I expression in skeletal muscle is
coincident with myotube formation in the developing embryo
(7) ,
stretch-induced myofiber hypertrophy
(8) and muscle regeneration
following injury
(9) , suggesting that IGF-I serves as an
autocrine/paracrine mediator of these processes in skeletal muscle.
Increased biosynthesis and extracellular secretion of IGF-I from
cultured mammalian myoblasts has been shown to be coincident with
myoblast alignment, withdrawal from the cell cycle, and
fusion
(5, 6) . In addition, inclusion of IGF-I in the
media of primary cultures of avian myofibers has been shown to elicit
larger fiber diameters, a near doubling in myosin content and
substantial increases in protein stability and synthesis in comparison
to untreated cultures
(10
-skeletal actin gene is a
member of the actin multigene family which, in vertebrates, is made up
of three distinct classes of actin isoforms termed as
``cytoplasmic,'' ``striated,'' and ``smooth
muscle'' on the basis of their cellular distribution and pattern
of expression in adult tissues
(13, 14, 15) . The
striated actins, -cardiac and -skeletal, are co-expressed in
embryonic heart and skeletal muscle, and examination of the switching
of actin gene expression studied during myogenesis of birds and small
mammals indicated that paired vertebrate sarcomeric cardiac and
skeletal -actin genes are up-regulated sequentially during early
muscle development, whereas only skeletal -actin is maintained at
high levels in adult skeletal muscle but reduced in cardiac
tissue
(13, 15, 16) . At adulthood, skeletal
-actin accounts for approximately 8% of the poly(A) RNA in avian
skeletal muscle
(16) and is expressed in all classes of
myofibers (17). To determine how the avian skeletal -actin gene
was regulated during striated muscle differentiation, our laboratory
(18) and others (19) identified the rough 5` regulatory boundary
at -202 base pairs (bp) which harbors evolutionarily conserved
regulatory elements that accurately initiate skeletal -actin
transcripts and induce transcripts from reporter genes in
differentiated skeletal muscle cells and cardiomyocytes. Mouse
transgenic studies by Petropulous et al.(20) indicated
that the conserved sequences in the proximal 200 bp of the promoter
region were primarily responsible for the avian skeletal -actin
gene's restricted expression pattern in heart and skeletal
muscle. However, these studies also revealed a high degree of ectopic
expression suggesting that additional regulatory elements from the
skeletal -promoter are required for strict striated
muscle-specific expression. Recent research implicates the contiguous
3`-flanking region of the human skeletal -actin gene in directing
correct temporal and spatial expression of skeletal actin-based
transgenes in mice
(21) , and we have recently observed in
transfection assays in vivo that potent activating sequences
are located immediately upstream of the core promoter in the region
spanning -424 to -202 bp upstream of the transcription
start site
(22) .
-actin gene and
its application for overexpression of an hIGF-I cDNA in cultured muscle
cells and transgenic mice. Our study indicates that IGF-I
overexpression in cultured muscle cells caused precocious alignment and
fusion of myoblasts into terminally differentiated myotubes and
elevated the levels of myogenic basic helix-loop-helix factors,
intermediate filament, and contractile protein mRNAs. Transgenic mice
carrying a single copy of this hybrid skeletal -actin/hIGF-I
transgene had hIGF-I mRNA levels that were approximately half those of
the endogenous murine skeletal -actin gene on a per allele basis,
while conferring tissue-specific expression activity. In transgenic
mice we observed that elevated levels of IGF-I in skeletal muscle
caused uniform muscle hypertrophy without increases in overall body
weight or circulating IGF-I concentration.
Construction of
Organization of the avian skeletal -Skeletal Actin/IGF-I Hybrid
Genes-actin
gene
(14, 23) and identification of 5` regulatory
boundaries have been described previously
(18, 24) . In
order to construct hybrid skeletal -actin/IGF-I genes, the 5` core
promoter, upstream activating sequences (-202 to -424),
natural capsite, 5`-UTR (exon 1), first intron, and portions of exon 2
up to the initiation ATG were obtained from 2.3 kb of -SK cloned
into M13mp18
(25) . Site-directed mutagenesis was utilized to
replace the sequences at the initiation ATG of -SK733 and the
hIGF-I cDNA
(26) with an NcoI site based on the
methodology of Kunkel
(27) utilizing single-stranded
uridine-substituted M13 DNA from the BW313
(dut, ung
) host
upon superinfection with helper phage m13K07. Two oligonucleotides were
synthesized such that each contained a central NcoI site
flanked by 12 nucleotides on each side complementary to the non-coding
strand produced by the phagemid. The oligonucleotides were annealed to
the uridine-substituted template and extended with the Klenow fragment
of DNA polymerase I. Double-stranded plasmids were sealed with T4 DNA
ligase, and the synthetic strands carrying the NcoI mutations
were selected upon transformations of a dut
ung
Escherichia coli host,
DH5
. The NcoI-containing mutants were then sequenced to
verify the mutation and the integrity of adjoining sequences. SK733
IGF-I was created by subcloning the full-length hIGF-I
cDNA
(26) , including the native IGF-I 3`-UTR and poly(A)
addition site, into this construction on an
NcoI/HindIII fragment adjacent to the skeletal
-actin NcoI site. A NaeI/HindIII
fragment containing the skeletal -actin 3`-UTR and contiguous
3`-noncoding region was excised from the avian skeletal -actin
genomic clone and then subcloned into
EcoRV/HindIII-digested pBluescript II to form SK 3`.
SK733 IGF-I 3`-SK was then created by subcloning the actin promoter and
hIGF-I cDNA, minus the 3`-UTR and poly(A) addition site from SK733
IGF-I on a BamHI fragment into the BamHI site of SK
3`.
Cell Culture
The murine myoblast line
CC(28) was utilized for generation of
stable transfectants. Stable transfection of myoblasts was achieved by
co-transfection of skeletal
-actin/IGF-I constructs with the drug
selectable-vector EMSV-hygromycin. The EMSV-hygromycin vector was
created by cloning the hygromycin resistance gene
(29) into the
EMSV expression plasmid
(30) . Transfection and selection with
hygromycin B (150 µg/ml) were performed as described
previously
(31) . Stable transfectant myoblasts were maintained
in Dulbecco's modified Eagle's medium containing 20% fetal
calf serum, 25 µg/ml gentamycin sulfate, and 150 µg/ml
hygromycin B (Boehringer Mannheim, Mannheim, Germany). Myoblasts were
switched to Dulbecco's modified Eagle's medium containing
0.05% (w/v) bovine serum albumin to permit myogenic differentiation and
study of the expression activity of the skeletal -actin/IGF-I
hybrid gene.
Generation of Transgenic Mice
One-cell mouse
embryos resulting from matings of FVB strain mice (Taconic, Germantown,
NY) were microinjected with approximately 2 pl of linearized,
gel-purified DNA (2 ng/ml) representing the gene constructs of interest
as described previously
(32) . After microinjection, embryos were
transferred to pseudopregnant females. Founder mice and subsequent
generations were screened for the presence of transgenes by Southern
blot analysis of genomic DNA
(33) . Transgene copy number was
determined by comparing the hybridization signal for 10 µg of mouse
genomic DNA to that of known quantities of purified DNA constructs
diluted in 10 µg of salmon sperm DNA using dot-blot hybridization.
Hybridization signal was quantitated using a Betascope model 603 blot
analyzer (Betagen, Waltham, MA).
RNA Isolation and Northern Blot Analysis
Total RNA
was isolated from cells and tissues by selective precipitation from
phenol-extracted, guanidine thiocyanate homogenates as described
previously
(34) . Northern blots were prepared by size
fractionation of total RNA samples on 1% agarose, 2.2 M
formaldehyde gels, and subsequent capillary transfer to GeneScreen
nylon membranes (DuPont NEN). After transfer, RNA was cross-linked to
the membrane by exposure to approximately 120,000 µJ UV (254
nM), and the membranes were then baked at 80 °C for 2 h.
All prehybridizations and hybridizations were performed in 50% (v/v)
formamide, 5 SSPE, 5
Denhardt's (0.1% (w/v)
bovine serum albumin, 0.1% (w/v) polyvinylpyrrolidone, 0.1% (w/v)
ficoll), 1% (w/v) SDS, 10% (w/v) dextran sulfate, 200 µg/ml sheared
salmon sperm DNA. Northern blots were hybridized with either
P-labeled DNA or antisense RNA probes as denoted for
specific probes (see figure legends). Hybridizations with DNA probes
were carried out for 16 h at 45 °C with 10
cpm/ml
probe. Membranes were subsequently washed 2 30 min in 2
SSPE, 1% (w/v) SDS at 68 °C and then 1
30 min in 0.5
SSPE, 0.1% (w/v) SDS at 55 °C. Blots were subsequently
exposed to x-ray film (Kodak X-Omat AR) at -70 °C with
intensifying screens.
Immunohistochemistry and IGF-I
Radioimmunoassay
IGF-I concentrations in conditioned media were
determined by radioimmunoassay as described previously
(35) .
Immunostaining of CC myoblasts was performed
using a 1:200 dilution of rabbit anti-hIGF-I antiserum
(35) as
the primary antibody and a goat anti-rabbit IgG immunoperoxidase
staining kit (Vectastain, Burlingame, CA). IGF-I concentrations in acid
extracts of skeletal muscle and acid/ethanol extracts of serum were
determined using a radioimmunoassay kit (Nichols Institute Diagnostics,
San Juan Capistrano, CA). Acid extracts of skeletal muscle were
prepared essentially as described previously
(4) . Briefly,
frozen hind limb muscle was powdered under liquid N
and
then homogenized 1:5 (w/v) in 1 M acetic acid using a Polytron
tissue homogenizer. Homogenates were incubated on ice for 2 h and then
centrifuged at 3,000 g for 15 min at 4 °C. The
supernatant fraction was removed to a new tube, and the pellet was
re-extracted by disbursing into 5
volume of 1 M acetic
acid and incubation on ice for 2 h. The mixture was centrifuged at
3,000
g for 15 min at 4 °C. Both supernatant
fractions were then pooled and lyophilized. The lyophilized extract was
reconstituted in 0.1
volume of 50 mM Tris-Cl (pH 7.8)
and then clarified by centrifugation prior to radioimmunoassay.
Staining of Myofibers for Morphometric
Analysis
The superficial gluteus and gastrocnemius (female only)
muscles were dissected immediately after sacrifice and affixed to a
1-cm diameter cork sheet with OCT (Miles) freezing media with the aid
of a dissecting microscope for proper orientation. Muscle samples were
then frozen by immersion into a container of isopentane cooled in
liquid N for 10 s. Frozen muscle samples were stored in
humidified (i.e. one small chip of wet ice) glass
scintillation vials at -70 °C until sectioning in the
cryostat. Tissues were allowed to warm up to approximately -15
°C in the cryostat prior to sectioning, and sections were taken at
6 µm. Staining for succinate dehydrogenase activity was performed
as described previously
(36) . Following staining for succinate
dehydrogenase activity the sections were fixed in formol saline, washed
in distilled water, and coverslipped using an aqueous mounting medium.
Morphometric analysis of succinate dehydrogenase-stained myofibers was
conducted with the aid of a computer-based image analysis system
running Optimas image analysis software (Bioscan, Edmonds, WA). Four
fields per animal, encompassing at least 290 total myofibers, were
analyzed. Myofibers in the superficial gluteus muscle exhibited three
distinct staining intensities for succinate dehydrogenase and were thus
classified as high, medium, or low for data analysis
(37) .
RESULTS
Skeletal
In order to examine
these parameters, we constructed two IGF-I expression vectors based on
the avian skeletal -Actin 3`-UTR Enhances the Accumulation
of IGF-I mRNA through mRNA Stabilization-actin gene which is normally activated during
withdrawal from the cell cycle and myoblast fusion
(16) .
Schematic representations of the two -actin/hIGF-I hybrid
constructions that were utilized for the studies reported herein are
shown in Fig. 1A. These IGF-I expression vectors were
evaluated for developmental expression, IGF-I biosynthesis, and their
influence on myogenesis in stably transfected myogenic
CC cells. Examination of IGF-I mRNA content
was evaluated by hybridizing Northern blots with a
P-labeled human IGF-I cDNA probe to total RNA isolated
from myoblasts that were switched from growth to differentiation media
as displayed in Fig. 1B. An approximate 1 to 2 orders of
magnitude increase in IGF-I mRNA accumulation was detected in fused
C
C myotubes containing SK733 IGF-I 3`-SK in
comparison to the expression of SK733 IGF-I. The avian skeletal
-actin promoter displayed developmentally restricted expression
with the IGF-I 3`-UTR, but replacement with the skeletal -actin
3`-UTR and contiguous 3`-noncoding sequences led to dramatically
increased expression of IGF-I mRNA upon myogenic differentiation.
Figure 1:
The avian skeletal -actin 3`-UTR
and contiguous 3`-noncoding region fostered robust expression of
skeletal -actin/IGF-I hybrid genes in differentiated
CC myoblasts. A, schematic diagram of
the skeletal
-actin/IGF-I hybrid gene constructions show the avian
skeletal -actin promoter -424 to +1 (angled
lines), the natural capsite (+1), and the 5`-UTR, (exon 1,
cross-hatch, 60 bp), first intron (line, 123 bp),
portions of exon 2 up to the initiation ATG (cross-hatch, 15
bp), and human IGF-I cDNA (open box, 504 bp). The SK733 IGF-I
construct contains the IGF-I 3`-UTR (filled box, 240 bp),
while the SK733 IGF-I 3`-SK construct contains the skeletal -actin
3`-UTR (mini boxes, 310 bp) and contiguous 1.5 kb of noncoding
sequences (vertical lines) as shown above, but not drawn to
scale. B, Northern blots of total RNA samples (10 µg)
isolated from CC myoblasts cultured in either
growth media (G) or differentiation media (D) were
hybridized with a
P-labeled hIGF-I cDNA probe (26). RNA
samples isolated from myoblasts cultured in differentiation media were
harvested 48 h after switching from growth to differentiation media.
C, stably transfected C
C myoblasts
carrying the skeletal
-actin/IGF-I hybrid genes described in A were grown to approximately 80% confluence in growth media and
then switched to differentiation media and cultured for 4 days.
Differentiated cultures were immunostained with a rabbit anti-hIGF-I
antiserum (generous gift of Dr. Ray Hintz, Stanford University) and a
goat anti-rabbit IgG immunoperoxidase kit (Vectastain, Burlingame, CA)
and then photographed at equal magnification. Conditioned media from a
population of stable transfected myoblasts were assayed for IGF-I
(Table I).
The steady state levels of different mRNAs reflect the balance
between the rate of synthesis of new mRNA and the rate of mRNA
degradation. We asked if one of the regulatory roles of the skeletal
-actin 3`-UTR is to impart mRNA stability. A transcription
blocker, actinomycin D (8 µg/ml), was added to differentiated
myogenic cultures in order to measure relative mRNA stabilities by
monitoring total mRNA content after inhibiting RNA synthesis. Timed
samples were removed for Northern blot analysis as shown in
Fig. 2
. Transcripts containing the natural IGF-I 3`-UTR were
found to turn over rapidly (half-life of less than 1 h) in contrast to
skeletal -actin transcripts which had greater stability with a
half-life estimated to be more than 6-8 h under conditions with
actinomycin D, a highly toxic drug (see Schwartz (38)). These data
indicate that the avian skeletal -actin 3`-UTR conferred increased
mRNA stability to a heterologous RNA species.
Figure 2:
The avian skeletal -actin 3`-UTR
increased the half-life of hIGF-I mRNA in CC
myoblasts transfected with skeletal
-actin/IGF-I hybrid genes.
Pooled populations of CC myoblasts stably
transfected with the actin/IGF-I constructs described in Fig. 1 were
grown to approximately 80% confluence and then switched to
differentiation media. After 48 h, actinomycin D (8 µg/ml) was
added followed by isolation of total RNA at 0, 4, 6, 8, and 12 h later.
Northern blots of total RNA samples (10 µg) were hybridized with a
P-labeled hIGF-I cDNA probe (26). Hybridization signal was
quantified by densitometry and expressed relative to the hIGF-I mRNA
levels at the time of actinomycin D addition for each construct to
depict rates of hIGF-I mRNA turnover.
Expression of IGF-I in Stably Transfected Myoblasts
Causes Precocious Myoblast Fusion and Enhances Myogenic Gene
Expression
Previous studies indicate that the progression of
myoblasts through the terminal differentiation pathway might be
directed by the autocrine/paracrine action of
IGFs
(39, 40) . We observed in confluent cultures that
myoblasts containing SK733 IGF-I and SK733 IGF-I 3`-SK exhibited a
greater degree of fusion after switching to differentiation media as
compared to control myoblasts (Fig. 1C).
Immunoperoxidase staining of myogenic cultures revealed increased
production of immunologically reactive IGF-I in myotubes of stably
transfected myoblasts, but not in control CC myoblasts stably transfected with an EMSV-hygromycin resistance
gene (Fig. 1C). The most intense staining resided with
the expression of the SK733 IGF-I 3`-SK construct. These data provide
qualitative evidence that increased IGF-I expression coincided with
enhanced myotube formation. After determining that SK733 IGF-I and
SK733 IGF-I 3`-SK were effective in driving IGF-I expression in muscle
cells, we quantified the biosynthesis of this factor by
radioimmunoassay of conditioned tissue culture media ().
Levels of IGF-I in control cultures were no greater than 0.6 ng/ml. In
comparison, cultures stably transfected with the vector SK733 IGF-I
3`-SK had accumulated levels of IGF-I in culture media that were at
least 150 times greater than control myoblasts after 4 days in culture.
Cultures stably transfected with SK733 IGF-I produced IGF-I at
approximately 5% the level of cultures containing the SK733 IGF-I 3`-SK
vector. These results suggest that substitution of the skeletal
-actin 3`-UTR and contiguous noncoding region for the native IGF-I
3`-UTR significantly enhanced IGF-I expression.
-actin.
Figure 3:
Overexpression of IGF-I in muscle
cell-stimulated myogenic gene activity. Control CC myoblasts and myoblasts stably transfected with the actin/IGF-I
hybrid gene SK733-IGF-I 3`-SK were grown to approximately 80%
confluence and then switched to differentiation media (day 0).
Replicate plates of control C
C myoblasts were
switched to differentiation media (day 0) with and without 40 ng/ml
hIGF-I. Total RNA was isolated at -1, 0, 1, 2, 3, and 4 days
after switching to differentiation media. Northern blots of total RNA
samples (10 mg) were probed for human IGF-I (26), murine MyoD (51),
murine myogenin (MyoG, 52), murine desmin (53), and murine skeletal
-actin (54) mRNAs using P-labeled cDNA probes.
DMEM, Dulbecco's modified Eagle's medium;
BSA, bovine serum albumin.
Actin/IGF-I Transgene Expression in Mice Results in a
Localized Increase in IGF-I and Myofiber Hypertrophy
A line of
mice harboring a single copy of the skeletal -actin/IGF-I
transgene (SK733 IGF-I 3`-SK), which was shown to be strongly expressed
in differentiated cultures of myogenic CC cells (see Fig. 1and Fig. 3), was utilized for study
of the effects of overexpression of IGF-I in skeletal muscle. The level
and tissue specificity of transgene expression in this line of mice was
similar to that of the endogenous skeletal
-actin gene being
restricted to striated muscle and more abundant in skeletal muscle than
in heart ( Fig. 4and Fig. 5). An abundant 1.1-kb hIGF-I
mRNA was detected on Northern blots of total RNA from skeletal muscle
of transgenic mice that was not present in control mice
(Fig. 5A). Expression of the transgene in this single
copy line of mice was approximately 50% that of the endogenous skeletal
-actin gene in hind limb muscle of adult mice
(Fig. 5C). High level transgene expression in the
present study resulted in concentrations of IGF-I in acid extracts of
skeletal muscle from transgenic mice being at least 47-fold greater
than in wild type mice (). Interestingly, IGF-I
concentrations in serum of transgenic mice were not elevated relative
to age-matched wild type mice () leading us to conclude
that the hIGF-I resulting from transgene expression did not enter the
circulation in appreciable quantities. This conclusion is further
supported by the lack of an effect of transgene expression on body
weight () or endogenous IGF-I expression in liver
(Fig. 5A).
Figure 4:
The SK733 IGF-I 3`-SK hybrid gene is
expressed specifically in striated muscle of transgenic mice. A
Northern blot of total RNA (20 µg) isolated from individual tissue
pools of 2 female and 2 male (except as noted below) SK733 IGF-I 3`-SK
transgenic mice was hybridized to a P-labeled DNA probe
derived from the 3`-UTR of the avian skeletal
-actin gene (16).
Abbreviations for individual tissues are: T, testis (n = 2 male mice); S, seminal vesicles (n = 2 male mice); U, uterus (n = 2
female mice); SP, spleen; S, stomach; L,
liver; K, kidney; P, pancreas; SM, skeletal
muscle; LU, lung; H, heart; and B, brain.
Hybridization and washing conditions are described under
``Materials and Methods.''
Figure 5:
Expression of the single-copy SK733 IGF-I
3`-SK hybrid gene in transgenic mice is approximately one-half of the
level of the endogenous murine skeletal -actin gene. A,
total RNA (20 µg) from pooled hind limb muscle and liver of
transgenic (tg) and nontransgenic littermate control
(ntg) mice was hybridized to a P-labeled hIGF-I
cDNA probe (26). A and B show blots of RNA from ntg
(lane 1) and tg (lane 2) skeletal muscle and from ntg
(lane 3) and tg (lane 4) liver tissues. The
hybridization signal in liver depicts expression of the endogenous
mIGF-I gene. B, the blot depicted in A was stripped
and subsequently hybridized with a
P-labeled murine
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe to
demonstrate approximately equal RNA loading. C, total RNA
pooled (n = 2) from skeletal muscle of age-matched
control mice (ntg) and adult transgenic (tg) from
female (lanes 1 and 2) and male (lanes 3 and
4) mice was hybridized simultaneously to murine skeletal
-actin (54) and hIGF-I (26) cDNA probes labeled to approximately
the same specific activity by polymerase chain reaction. Analysis of
the hybridization signal by direct counting on a Betascope (Betagen,
Waltham, MA) indicated that expression of the SK733IGF-I 3`-SK hybrid
gene in skeletal muscle was approximately 50% that of the endogenous
murine skeletal -actin gene on a per allele basis. Hybridization
and washing conditions are described under ``Materials and
Methods.''
Morphometric analysis was performed on
cross-sections of the superficial gluteus muscle stained for the
mitochondrial enzyme succinate dehydrogenase (I,
Fig. 6
). Myofibers were grouped based on their succinate
dehydrogenase staining intensity as either high, medium, or low
intensity as has been described previously
(37) . Results
indicate that overexpression of IGF-I in skeletal muscle resulted in
hypertrophy of myofibers regardless of succinate dehydrogenase staining
intensity. This was evidenced by increases in the cross-sectional area
of myofibers that ranged from 115% for high intensity succinate
dehydrogenase fibers in a female transgenic to 17% for low intensity
succinate dehydrogenase fibers in male transgenic mice as compared to
wild type littermates. In addition to an increased cross-sectional area
of myofibers, morphometric analysis revealed a trend toward a higher
proportion of high intensity succinate dehydrogenase myofibers
(e.g. 31.4% versus 10.9% in female mice) and a lower
proportion of low intensity succinate dehydrogenase myofibers (e.g. 52.1% versus 71.7% in female mice) in transgenic
versus littermate wild type mice. A previous study
(37) has established that high intensity succinate dehydrogenase
staining of myofibers correlates with fibers classified as either 2A or
2X based on myosin ATPase staining, whereas medium intensity succinate
dehydrogenase staining correlates with type 1 fibers and low intensity
succinate dehydrogenase staining corresponds to type 2B fibers. These
data indicate that IGF-I overexpression induced a shift toward more
oxidative fiber types (likely types 2A and 2X), in addition to
increasing the relative cross-sectional area of all classes of
myofibers.
Figure 6:
Expression of the SK733 IGF-I 3`-SK hybrid
gene in transgenic mice-induced muscle hypertrophy. Hind limbs from
adult (8 months of age) littermate transgenic and control mice (female)
were skinned and photographed with a ruler marker in millimeters at
equal magnification prior to dissection. Cross-sections were taken from
the approximate midpoint of the superficial gluteus and gastrocnemius
muscles, stained for succinate dehydrogenase (36), and then
photographed at the same magnification (see ruler in
photographs). Data for morphometric analysis of fibers in the
superficial gluteus muscle are presented in Table
III.
DISCUSSION
Targeting IGF-I Expression to Striated Muscle
In
most vertebrates, skeletal -actin is the predominant striated
actin isoform expressed in adult skeletal muscle, whereas it is
expressed at a much lower level in cardiac muscle in which the
-cardiac isoform predominates
(5) . Results from previous
studies utilizing transient transfection assays have suggested that the
regulatory elements responsible for cell type restricted and
developmental expression reside within the proximal 202 bp of the avian
skeletal -actin gene promoter
(18, 19) , and, in
general, these observations were corroborated in earlier transgenic
mouse studies
(20, 41, 42) . However, Petropoulos
et al.(20) also reported that transgenes driven by
either the avian skeletal -actin proximal promoter (-197 to
+27 bp) or more extensive 5`-flanking sequence (-2200 to
+27 bp) exhibited ectopic expression in several transgenic lines
in addition to increased expression in adult heart relative to skeletal
muscle. We observed in the present study that inclusion of the skeletal
-actin 3`-UTR, along with approximately 1.5 kb of contiguous
3`-flanking sequence in a chimeric actin/IGF-I transgene dramatically
increased its expression in cultured myotubes and resulted in
expression being restricted to striated muscle in transgenic mice.
Moreover, this transgene's level of expression in skeletal muscle
was similar to that of the endogenous murine skeletal -actin gene.
Together, these data suggest that the elements required to mimic the
level and tissue specificity of expression for the endogenous murine
skeletal -actin gene are contained within the SK733 IGF-I 3`-SK
construct.
-actin 3`-UTR and
contiguous 3`-flanking sequence in a chimeric actin/IGF-I construct
dramatically increased steady state mRNA and protein levels in cultured
myotubes, and our data suggest that a primary mechanism whereby the
skeletal -actin 3`-UTR increased expression of the actin/IGF-I
hybrid gene in vitro was through increasing mRNA half-life.
This was not surprising since we recently demonstrated the selective
turnover of the cytoplasmic 
-actin versus stabilization
of striated -actin mRNAs in primary myogenic cultures, and
sequence differences between these evolutionarily conserved actin mRNA
isoforms in their 3`-UTRs have been implicated in determining the
intrinsic half-life and cellular content of the respective
mRNAs
(43) . However, the degree to which the skeletal
-actin 3`-UTR influences the level of transgene expression in
vivo is less clear. Brennan and Hardeman
(21) reported that
substituting a CAT reporter with 3` SV40 sequences for the human
skeletal -actin coding and 3` contiguous flanking sequences
decreased expression by less that 50%. In addition to putative effects
of the 3`-UTR on mRNA half-life, recent research suggests that the
3`-flanking regions of the human striated actin genes contain elements
that further restrict expression of transgenes driven by either the
human skeletal or cardiac -actin promoter to striated muscle as
well as influencing their developmental pattern of
expression
(21, 44) . We have also observed that the
aberrant expression of transgenes driven by the avian skeletal
-actin proximal promoter (i.e. -202 to -11
bp) can be eliminated by substituting the skeletal -actin 3`-UTR
and approximately 1.5 kb of contiguous noncoding sequence for the
SV40-derived 3` sequences (data not shown). Thus, in addition to the 5`
promoter and upstream region, the 3`-noncoding regions of the striated
actin genes contain regulatory regions necessary for appropriate
developmental and tissue-restricted expression of skeletal
-actin-based transgenes, and we conclude that avian skeletal
-actin-based expression vectors comprised of the skeletal
-actin proximal promoter, 3`-UTR and contiguous 3`-flanking
sequence can be utilized to target high level expression of
heterologous transgenes specifically to striated muscle.
Overexpression of IGF-I Enhances Muscle
Growth
Numerous studies in vitro have established that
IGF-I elicits pleiotropic effects on myogenic cells including
stimulation of myoblast replication and myogenic differentiation (see
Refs. 5 and 6 for review). We observed that myoblasts transfected with
SK733 IGF-I 3`-SK expressed higher levels of muscle-specific mRNAs than
did myoblasts treated with 40 ng/ml IGF-I even though the concentration
of IGF-I in media conditioned by these cells for 48 h was lower. This
observation suggests that the IGF-I derived via a sustained release
autocrine/paracrine mechanism was more effective as a myogenic stimulus
than exogenous IGF-I administered as a bolus. One possible mechanism
underlying this observation is that a myoblast-derived insulin-like
growth factor-binding protein(s) (IGFBP) might potentiate the effects
of autocrine/paracrine-derived IGF-I. In this regard, we did observe
that expression of IGFBP-5, an IGFBP that is specifically induced upon
differentiation of CC myoblasts
(45) ,
was coordinately up-regulated with overexpression of IGF-I in vitro (data not shown). Although beyond the scope of this report, these
observations suggest that the model of overexpression of IGF-I in
myoblasts may provide novel insights into the potential role of IGFBPs
in regulating myoblast proliferation and differentiation.
-actin gene is normally not observed until relatively late in
muscle development (i.e. after myoblast
fusion
(15, 16) ), overexpression of IGF-I in vivo via a skeletal -actin-derived vector is not likely to elicit
dramatic effects on myoblast proliferation or differentiation. We
observed in the present study that transgenic overexpression of IGF-I
elicited pronounced hypertrophy of all classes of myofibers. In
addition, results also indicated that overexpression of IGF-I induced a
shift in myofiber type toward more oxidative fiber types. This latter
observation is consistent with findings from a previous study which
indicated that growth hormone treatment of hypophysectomized rats,
presumably acting through increased expression of IGF-I, elicited an
increase in the relative proportion of type I myofibers
(49) .
Table:
IGF-I
concentrations in media conditioned by stable transfectant muscle cells
Table:
Body weight and IGF-I concentrations in serum
and skeletal muscle of SK733 IGF-I 3`-SK and age-matched control mice
Table:
Morphometric analysis of succinate
dehydrogenase-stained myofibers in the superficial gluteus muscle of
SK733 IGF-I 3`-SK and littermate control mice
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 J. Huard, Y. Li, and F. H. Fu Muscle Injuries and Repair: Current Trends in Research J. Bone Joint Surg. Am., May 1, 2002; 84(5): 822 - 832. [Full Text] [PDF]
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E. R. Barton, L. Morris, A. Musaro, N. Rosenthal, and H. L. Sweeney Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice J. Cell Biol., April 1, 2002; 157(1): 137 - 148. [Abstract] [Full Text] [PDF]
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 N. WINN, A. PAUL, A. MUSARO, and N. ROSENTHAL Insulin-like Growth Factor Isoforms in Skeletal Muscle Aging, Regeneration, and Disease Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 507 - 518. [Abstract] [PDF]
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B. L. Awede, J.-P. Thissen, and J. Lebacq Role of IGF-I and IGFBPs in the changes of mass and phenotype induced in rat soleus muscle by clenbuterol Am J Physiol Endocrinol Metab, January 1, 2002; 282(1): E31 - E37. [Abstract] [Full Text] [PDF]
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A. J. Forhead, J. Li, R. S. Gilmour, M. J. Dauncey, and A. L. Fowden Thyroid hormones and the mRNA of the GH receptor and IGFs in skeletal muscle of fetal sheep Am J Physiol Endocrinol Metab, January 1, 2002; 282(1): E80 - E86. [Abstract] [Full Text] [PDF]
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J. Tureckova, E. M. Wilson, J. L. Cappalonga, and P. Rotwein Insulin-like Growth Factor-mediated Muscle Differentiation. COLLABORATION BETWEEN PHOSPHATIDYLINOSITOL 3-KINASE-Akt-SIGNALING PATHWAYS AND MYOGENIN J. Biol. Chem., October 12, 2001; 276(42): 39264 - 39270. [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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O. Halevy, A. Krispin, Y. Leshem, J. P. McMurtry, and S. Yahav Early-age heat exposure affects skeletal muscle satellite cell proliferation and differentiation in chicks Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2001; 281(1): R302 - R309. [Abstract] [Full Text] [PDF]
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 K. Fukushima, N. Badlani, A. Usas, F. Riano, F. H. Fu, and J. Huard The Use of an Antifibrosis Agent to Improve Muscle Recovery after Laceration Am. J. Sports Med., July 1, 2001; 29(4): 394 - 402. [Abstract] [Full Text] [PDF]
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 Y.-J. Kim, S. Noguchi, Y. K. Hayashi, T. Tsukahara, T. Shimizu, and K. Arahata The product of an oculopharyngeal muscular dystrophy gene, poly(A)-binding protein 2, interacts with SKIP and stimulates muscle-specific gene expression Hum. Mol. Genet., May 1, 2001; 10(11): 1129 - 1139. [Abstract] [Full Text] [PDF]
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M. Brink, S. R. Price, J. Chrast, J. L. Bailey, A. Anwar, W. E. Mitch, and P. Delafontaine Angiotensin II Induces Skeletal Muscle Wasting through Enhanced Protein Degradation and Down-Regulates Autocrine Insulin-Like Growth Factor I Endocrinology, April 1, 2001; 142(4): 1489 - 1496. [Abstract] [Full Text]
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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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M. A. Lawlor and P. Rotwein Coordinate Control of Muscle Cell Survival by Distinct Insulin-like Growth Factor Activated Signaling Pathways J. Cell Biol., December 4, 2000; 151(6): 1131 - 1140. [Abstract] [Full Text] [PDF]
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M. A. Lawlor and P. Rotwein Insulin-Like Growth Factor-Mediated Muscle Cell Survival: Central Roles for Akt and Cyclin-Dependent Kinase Inhibitor p21 Mol. Cell. Biol., December 1, 2000; 20(23): 8983 - 8995. [Abstract] [Full Text]
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E. E. Dupont-Versteegden, R. J. L. Murphy, J. D. Houle, C. M. Gurley, and C. A. Peterson Mechanisms leading to restoration of muscle size with exercise and transplantation after spinal cord injury Am J Physiol Cell Physiol, December 1, 2000; 279(6): C1677 - C1684. [Abstract] [Full Text] [PDF]
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L. A. SCHILDMEYER, R. BRAUN, G. TAFFET, M. DEBIASI, A. E. BURNS, A. BRADLEY, and R. J. SCHWARTZ Impaired vascular contractility and blood pressure homeostasis in the smooth muscle {alpha}-actin null mouse FASEB J, November 1, 2000; 14(14): 2213 - 2220. [Abstract] [Full Text]
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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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 P. M. CORVA and J. F. MEDRANO Diet effects on weight gain and body composition in high growth (hg/hg) mice Physiol Genomics, June 29, 2000; 3(1): 17 - 23. [Abstract] [Full Text] [PDF]
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M. A. Lawlor, X. Feng, D. R. Everding, K. Sieger, C. E. H. Stewart, and P. Rotwein Dual Control of Muscle Cell Survival by Distinct Growth Factor-Regulated Signaling Pathways Mol. Cell. Biol., May 1, 2000; 20(9): 3256 - 3265. [Abstract] [Full Text]
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M. Fournier and M. I. Lewis Influences of IGF-I gene disruption on the cellular profile of the diaphragm Am J Physiol Endocrinol Metab, April 1, 2000; 278(4): E707 - E715. [Abstract] [Full Text] [PDF]
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M. J. Fedele, J. M. Hernandez, C. H. Lang, T. C. Vary, S. R. Kimball, L. S. Jefferson, and P. A. Farrell Severe diabetes prohibits elevations in muscle protein synthesis after acute resistance exercise in rats J Appl Physiol, January 1, 2000; 88(1): 102 - 108. [Abstract] [Full Text] [PDF]
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K. L. Haugk, H.-M. P. Wilson, K. Swisshelm, and L. S. Quinn Insulin-Like Growth Factor (IGF)-Binding Protein-Related Protein-1: An Autocrine/Paracrine Factor That Inhibits Skeletal Myoblast Differentiation but Permits Proliferation in Response to IGF Endocrinology, January 1, 2000; 141(1): 100 - 110. [Abstract] [Full Text] [PDF]
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 C. Rommel, B. A. Clarke, S. Zimmermann, L. Nuñez, R. Rossman, K. Reid, K. Moelling, G. D. Yancopoulos, and D. J. Glass Differentiation Stage-Specific Inhibition of the Raf-MEK-ERK Pathway by Akt Science, November 26, 1999; 286(5445): 1738 - 1741. [Abstract] [Full Text]
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M. C. DELAUGHTER, G. E. TAFFET, M. L. FIOROTTO, M. L. ENTMAN, and R. J. SCHWARTZ Local insulin-like growth factor I expression induces physiologic, then pathologic, cardiac hypertrophy in transgenic mice FASEB J, November 1, 1999; 13(14): 1923 - 1929. [Abstract] [Full Text]
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 A. Shiotani, B. W. O'Malley Jr, M. E. Coleman, and P. W. Flint Human Insulinlike Growth Factor 1 Gene Transfer Into Paralyzed Rat Larynx: Single vs Multiple Injection Arch Otolaryngol Head Neck Surg, May 1, 1999; 125(5): 555 - 560. [Abstract] [Full Text] [PDF]
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Z.-M. Wang, M. Laura Messi, M. Renganathan, and O. Delbono Insulin-like growth factor-1 enhances rat skeletal muscle charge movement and L-type Ca2+ channel gene expression J. Physiol., April 15, 1999; 516(2): 331 - 341. [Abstract] [Full Text] [PDF]
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G. McKoy, W. Ashley, J. Mander, S. Y. Yang, N. Williams, B. Russell, and G. Goldspink Expression of insulin growth factor-1 splice variants and structural genes in rabbit skeletal muscle induced by stretch and stimulation J. Physiol., April 15, 1999; 516(2): 583 - 592. [Abstract] [Full Text] [PDF]
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A. Musaro and N. Rosenthal Maturation of the Myogenic Program Is Induced by Postmitotic Expression of Insulin-Like Growth Factor I Mol. Cell. Biol., April 1, 1999; 19(4): 3115 - 3124. [Abstract] [Full Text] [PDF]
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P. W. Flint, A. Shiotani, and B. W. O'Malley Jr IGF-1 Gene Transfer Into Denervated Rat Laryngeal Muscle Arch Otolaryngol Head Neck Surg, March 1, 1999; 125(3): 274 - 279. [Abstract] [Full Text] [PDF]
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A. Zetser, E. Gredinger, and E. Bengal p38 Mitogen-activated Protein Kinase Pathway Promotes Skeletal Muscle Differentiation. PARTICIPATION OF THE MEF2C TRANSCRIPTION FACTOR J. Biol. Chem., February 19, 1999; 274(8): 5193 - 5200. [Abstract] [Full Text] [PDF]
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E. R. Barton-Davis, D. I. Shoturma, A. Musaro, N. Rosenthal, and H. L. Sweeney Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function PNAS, December 22, 1998; 95(26): 15603 - 15607. [Abstract] [Full Text] [PDF]
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M. Renganathan, M. L. Messi, and O. Delbono Overexpression of IGF-1 Exclusively in Skeletal Muscle Prevents Age-related Decline in the Number of Dihydropyridine Receptors J. Biol. Chem., October 30, 1998; 273(44): 28845 - 28851. [Abstract] [Full Text] [PDF]
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J.-L. Liu, A. Grinberg, H. Westphal, B. Sauer, D. Accili, M. Karas, and D. LeRoith Insulin-Like Growth Factor-I Affects Perinatal Lethality and Postnatal Development in a Gene Dosage-Dependent Manner: Manipulation Using the Cre/loxP System in Transgenic Mice Mol. Endocrinol., September 1, 1998; 12(9): 1452 - 1462. [Abstract] [Full Text]
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D. S. Criswell, F. W. Booth, F. DeMayo, R. J. Schwartz, S. E. Gordon, and M. L. Fiorotto Overexpression of IGF-I in skeletal muscle of transgenic mice does not prevent unloading-induced atrophy Am J Physiol Endocrinol Metab, September 1, 1998; 275(3): E373 - E379. [Abstract] [Full Text] [PDF]
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G. R. Adams and S. A. McCue Localized infusion of IGF-I results in skeletal muscle hypertrophy in rats J Appl Physiol, May 1, 1998; 84(5): 1716 - 1722. [Abstract] [Full Text] [PDF]
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E. Gredinger, A. N. Gerber, Y. Tamir, S. J. Tapscott, and E. Bengal Mitogen-activated Protein Kinase Pathway Is Involved in the Differentiation of Muscle Cells J. Biol. Chem., April 24, 1998; 273(17): 10436 - 10444. [Abstract] [Full Text] [PDF]
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P. Kaliman, J. Canicio, P. R. Shepherd, C. A. Beeton, X. Testar, M. Palacín, and A. Zorzano Insulin-like Growth Factors Require Phosphatidylinositol 3-Kinase to Signal Myogenesis: Dominant Negative p85 Expression Blocks Differentiation of L6E9 Muscle Cells Mol. Endocrinol., January 1, 1998; 12(1): 66 - 77. [Abstract] [Full Text]
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 Y. Tsurumi, S. Takeshita, D. Chen, M. Kearney, S. T. Rossow, J. Passeri, J. R. Horowitz, J. F. Symes, and J. M. Isner Direct Intramuscular Gene Transfer of Naked DNA Encoding Vascular Endothelial Growth Factor Augments Collateral Development and Tissue Perfusion Circulation, December 15, 1996; 94(12): 3281 - 3290. [Abstract] [Full Text]
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G. R. Adams and F. Haddad The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy J Appl Physiol, December 1, 1996; 81(6): 2509 - 2516. [Abstract] [Full Text] [PDF]
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