Rapid Activation
of Insulin-Like
Growth Factor Binding Protein-5
Gene Transcription
during Myoblast
Differentiation
Peter
Rotwein,
Payton
L. James,
and Kou Kou
Departments
of Medicine and Biochemistry
and Molecular Biophysics
Washington
University School of Medicine
St. Louis, Missouri 63110
INTRODUCTION
Insulin-like
growth
factor
binding
proteins
(IGFBPs) comprise
a family of secreted
proteins
that bind insulin-like
growth factors-l
and -II (IGF-I
and -II) with high affinity and potentially
modulate
their biological
effects. We have demonstrated
previously that IGFBP-5, the most conserved
of the
six known IGFBPs, is expressed
in muscle cells in
the developing
embryo and during the terminal differentiation
of several myogenic
cell lines. In this
study we show that an IGF-I analog that binds
minimally
to IGFBPs potently enhances
the differentiation of the stringently
controlled
inducible
C2
myoblast (C2l) cell line and identify IGFBPQ as the
sole IGFBP secreted during C2l differentiation.
We
find that induction
of IGFBP-5 mRNA and protein is
coincident
with the onset of myogenin
gene expression
and occurs secondary
to the rapid activation of IGFBP-5 gene transcription.
By transient
gene transfer experiments
we demonstrate
that a
1994 base pair segment of the IGFBP-5 promoter
is
very active in directing
expression
of the reporter
gene luciferase in C2l myoblasts.
A promoter
fragment containing
only 155 nucleotides
of 5’-flanking
DNA retained
more than 70% of maximal activity
and mediated
at least part of the differentiationdependent
rise in IGFBP-5
gene transcription.
Within this active segment
are several potential
binding
sites for muscle-enriched
transcription
factors. Our results show that induction
of IGFBP-5
expression
is an early event in the myogenic
differentiation
of the C2l cell line and suggest that one
function
of this IGFBP is to modulate
IGF-induced
differentiation.
C2l cells are thus an excellent
in
vitm
model for elucidating
the developmental
factors that control
IGFBP-5 gene transcription
and
action in skeletal muscle. (Molecular
Endocrinology 9: 9X+923,1995)
One of the more perplexing problems in the biology of
the insulin-like growth factors (IGFs) is the role of the
IGF binding proteins (IGFBPs). Depending on the cellular context and the experimental
system, members
of this family of six secreted proteins have been shown
to provide a storage depot for IGF-I and IGF-II (reviewed in Ref. 1) to both enhance and inhibit IGF
action (reviewed in Refs. 2 and 3) and to have biological effects that are independent
of the two growth
factors (4, 5). The difficulties in defining specific functions for each of the IGFBPs have been exacerbated
by the presence of several binding proteins in most
biological
fluids (l), and by the production
and
secretion of multiple IGFBPs by cultured cells (2).
We have demonstrated
recently that the C2 myogenie cell line expresses IGFBP-5 mRNA and protein
during its terminal differentiation
(6). IGFBP-5 appears
to be the only IGFBP produced
by C2 cells and by
another rapidly fusing muscle cell line, F3 azamyoblast
clone b (6). We now report that in C2l myoblasts
[a clonal derivative of C2 cells, which are dependent
on insulin or IGF-I for initiating terminal differentiation
(7)], an IGF-I analog that binds minimally to IGFBPs (8)
potently enhances differentiation.
In conjunction
with
this observation
we find that C2l cells express
IGFBP-5 as their sole IGFBP, indicating
that one
function
of this binding
protein is to modify the
differentiation-promoting
effects of the IGFs. Rapid
activation of IGFBP-5 transcription
accounts for the
massive increase in IGFBP-5 mRNA and protein that
accompany
C2l differentiation.
Gene transfer experiments indicate that the IGFBP-5 promoter is very active after transfection into C2l myoblasts and demonstrate that the proximal promoter mediates at least
part of the stimulation
of gene transcription
during
differentiation.
C2l cells are thus an excellent in vitro
model for identifying
the mechanisms
that control
IGFBP-5 gene expression during development
and for
defining the actions of this binding protein in muscle.
o&xi-9909/95/$3.00/0
Molecular
Copyright
Endocrinology
0 1995 by The Endocrine
Society
913
MOL
914
END0
.1995
Vol 9 No. 7
RESULTS
Induction
of C21 Myoblast
and an IGF-I Analog
Differentiation
by IGF-I
Inducible C2 myoblasts (C21 cell line) were selected by
Pinset et al. (7) as a subclone of C2 cells (7) that
required only insulin (or IGF-I) to differentiate in vitro
and did not differentiate in the presence of fetal calf
serum (7,9). As shown in Fig. 1, C2l cells differentiated
readily when incubated
in serum-free
medium with
graded concentrations
of IGF-I or des- [l-3] IGF-I, an
analog that has markedly
decreased
affinity for
IGFBPs but not for the IGF-I receptor (8, 10). Both
IGF-I and des-[l-3]
IGF-I stimulated terminal differentiation to the same extent, as evidenced by the equivalent maximal induction of creatine kinase enzymatic
activity and myotube formation after treatment for 72
h. Incubation of C2l cells in serum-free medium with
1.6 ~.LM insulin (7, 9) resulted in a similar induction of
myotube fusion and creatine kinase activity (400 U/g
protein at 72 h). By both biochemical
and morphological criteria, des-[l-3]
IGF-I was at least 16 times more
potent than IGF-I (half-maximal
concentration
for inducing creatine kinase activity: IGF-I, -6 nM, des-[l-3]
IGF-I, co.37 nM; maximal myotube formation: IGF-I,
-6-11
nM, des-[l-3]
IGF-I, -0.37 nM). Based on the
marked difference
in potency
between
IGF-I and
des- [l-3] IGF-I, these results indicate that C2l myoblasts secrete IGFBPs that appear to counteract
or
modify the differentiation-promoting
actions of IGFs
on these cells.
Stimulation
of IGFBP-5
Myoblast Differentiation
Expression
during
C2l
To identify the IGFBPs produced
by C2l myoblasts,
we first analyzed conditioned
culture medium by ligand blot. Figure 2A shows the appearance
of a -29kilodalton (kDa) IGFBP starting within 12 h of initiation
of differentiation
by addition of insulin (Fig. 2A). A
Western blot of the same conditioned
medium using
anti-IGFBP-5
antiserum identified this binding protein
(Fig. 2B), although this method was less sensitive than
ligand blotting. Although an IGFBP of -46 kDa (nearly
the same mobility as IGFBP-3) also was apparent by
ligand blots at the 48 and 72 h time points, no IGFBP-3
could be detected at 72 h by immunoblotting,
even
though it could be seen readily in mouse serum (Fig.
3). Since in some experiments
the -46- kDa IGFBP
was recognized
by anti-IGFBP5
antiserum
(Fig. 28
and data not shown), it is possible that this band
represents a dimer of IGFBP-5 and another secreted
protein. Therefore, IGFBP-5 is the predominant
IGFBP
induced during C2l cell differentiation.
We next examined the time course of IGFBP-5 gene
expression during C2l differentiation.
As shown in Fig.
4, a minimal amount of IGFBP-5 mRNA was produced
by proliferating
C2l cells, and little was expressed
when cells were made quiescent
by incubation
in
Dulbecco’s
modified
Eagle’s medium plus 60 nM
transferrin for 24 h. By contrast, within 24 h of addition
of insulin (1 PM) to the serum-free medium, abundant
IGFBP-5 mRNA was detected, and mRNA levels continued to rise for up to 72 h. As also shown in Fig. 4, the
time course of accumulation
of IGFBP-5 transcripts
paralleled
the increase in myogenin
mRNA levels.
Thus, the expression of both genes appears to be an
early event in C2l cell differentiation.
Activation
of IGFBP-5 Gene
C2l Myoblast Differentiation:
Transcriptional
Control
Expression
Evidence
during
for
The appearance
of IGFBP-5 mRNA and protein during
the terminal differentiation
of C2l cells could reflect
transcriptional
activation of the IGFBP-5 gene and/or
posttranscriptional
events. We examined
potential
transcriptional
mechanisms
by performing ribonuclease protection experiments
with 32P-labeled IGFBP-5
genomic antisense probes and RNA isolated from C2l
cell nuclei at intervals after exposure of cells to insulincontaining serum-free medium. As seen in Fig. 5, low
levels of nuclear IGFBP-5 mRNA could be found in
quiescent cells. By 8 h after exposure of cells to differentiation medium, a 2.5 to 3-fold rise in the abundance of nuclear IGFBP-5 transcripts was evident, and
mRNA levels progressively increased during the ensuing 40 h. In addition, a rise in nascent IGFBPB mRNA
was detected with the exon 3 probe beginning 8-12 h
after onset of differentiation
(Fig. 5B). As identical results were seen with probes from both exons 1 and 3
(compare Fig. 5A and 5B), it seems likely that the rise
in nuclear IGFBP-5 mRNA reflects enhanced
gene
transcription
rather than release of a block to mRNA
elongation. Studies with the exon 1 probe also showed
that in C2l ceils the second of two transcription
start
sites identified in the mouse IGFBP-5 gene (11) was
preferred.
In other ribonuclease
protection
experiments using cytoplasmic
RNA isolated from the same
cells, a similar pattern of IGFBP-5 mRNA induction
was seen, although it was delayed in onset until 12 h
after initiation of differentiation
(data not shown). Thus,
IGFBP-5 gene transcription
appears to be activated as
an early event in C2l myoblast differentiation.
We also examined the effects of differentiation
on
IGFBP-5 mRNA stability through use of the selective
inhibitor of RNA polymerase
II elongation,
5,6-dichloro-1 -p-o-ribofuranosylbenzimadazole
(DRB). Addition of DRB to proliferating or differentiating
cultures
of C2l cells led to a progressive decline in IGFBP-5
mRNA abundance
(Fig. 6) with a calculated t,,* for
both experiments
of 11-12 h. The effectiveness of
DRB was further ascertained by measuring the t,,, of
myogenin
mRNA from differentiating
cells. As also
shown in Fig. 6, the abundance
of myogenin transcripts decreased in cells exposed to DRB (calculated
t,,, of -20 min). Thus, differentiation
does not lead to
an alteration in IGFBP-5 mRNA stability.
Induction
of IGFBP-5
Gene
Transcription
in Differentiating
0
.Ol
C21 Myoblasts
.l
Concentration
0.37 nM Des [l-3]
IGF-I
915
1
10
loa
(nM)
0.36 nM IGF-I
Fig. 1. Induction
of Differentiation
in C2l Myoblasts
by IGF-I and des-[l-3]
IGF-I
The top pane/ shows results of a dose-response
curve for creatine kinase enzymatic
activity in extracts
of C2l cells incubated
for 72 h in serum-free
medium
with 60 nM transferrin
and graded concentrations
of either IGF-I or des-[l-3]
IGF-I. The bottom
pane/ shows phase-contrast
photomicrographs
of C2l myoblasts
incubated
for 72 h in serum-free
medium with 60 nM transferrin
and the indicated
concentrations
of IGF-I or des- [l-3] IGF-I. Photographs
were taken (at 10 x objective)
using a Nikon WX-IIA
controller
and a 35 mm camera.
MOL
916
END0
Vol 9 No. 7
. 1995
A. Ligand
lmmunoblot
Blot
S
kDa
92-
%
DM
UD
4
t
I2
24
J&&
48
72
UD
Ligand blot
72
S
IGFBP-3
*
’
B. Immunoblot
c2
72
kDa
DM
UD
4
I2
24
48
72
72
-46kDa-
+
-3OkDa-
c
=b
-21
2l-
UD
hr
hr
92694630-i
7
9
kDa -
IGFBP-5
’
Fig. 3. C2l Myoblasts
Do Not Secrete
IGFBP-3
Mouse
serum
(1 ~1) and 50 ~1 of the same C2l cellconditioned
media used in Fig. 2 were electrophoresed
through
a 12.5% SDS-polyactylamide
gel under nondenaturing conditions
and transferred
to nitrocellulose.
The left
panel shows a luminograph
after the filter was probed
with
anti-rat
IGFBP-3
antisera
followed
by horseradish
peroxidase-conjugated
secondary
antibody
and ECL. IGFBP-3
is
detected
in serum (S), but not in C2l cell-conditioned
media (UD or 72 h). The right panel shows a ligand blot of the
same samples
used in the leff panel. The -46-kDa
band
(small arrow,
see also Fig. 2) seen in the CPI-conditioned
media obtained
72 h after insulin treatment
migrates
more
slowly
than
IGFBP-3
in mouse
serum.
Molecular
size
markers
are indicated
to the side of each panel.
21-
Fig. 2. Secretion
of IGFBP-5
by Differentiating
C2l Myoblasts
A, Autoradiograph
of a ligand blot of conditioned
media
(50 PI/lane) from C2l cells after being made quiescent
for 24
h (lane UD), and at 4, 12, 24, 48, and 72 h after the onset of
differentiation.
The abundance
of the -29
kDa band, detected using 1251-labelad
IGF-II, increased
progressively
with
differentiation,
starting
at the 12-h time point. Exposure
to
x-ray film was for 48 h at -80 C. 8, Western
blot of the same
conditioned
media used in panel A with anti-IGFBP-5
antiserum. A single band of -29 kDa is detected
only in media
from differentiating
C2l cells. For both panels A and B, the
positive
control
in the leti lane was derived
from C2 cells
exposed
to serum-containing
differentiation
medium for 72 h.
DM, Unconditioned
serum-free
medium.
Molecular
size
markers
are indicated
to the left of each panel.
Analysis of Promoter Function
The results described
above indicated that the rise
in IGFBP-5 mRNA levels during C21 differentiation
was secondary to activation of IGFBP-5 gene transcription. Accordingly,
we next sought to identify the
promoter
regions mediating
this effect. Transient
transfection
experiments
were performed using chimerit IGFBP-5-luciferase
plasmids. We have shown
in previous studies using Hep G2 cells [which produce IGFBP-5 mRNA constitutively
(12)] that transfection of IGFBP5-1004
Luc, a fusion plasmid containing 1004 base pairs (bp) of 5’-flanking
DNA and
the initial 120 nucleotides
of exon 1, stimulated
the
highest level of luciferase activity. The same plasmid
was active when transfected
into proliferating
C2l
myoblasts;
luciferase values were >l OO-fold higher
than those obtained with a promoterless
lucifease
plasmid and were -17%
as high as was seen with
an IGF-II promoter-luciferase
fusion gene (Fig. 7).
Luciferase
activity directed
by IGFBP5-1004
Luc
was stimulated
by 3.3 If: 0.6 fold (mean + SD) when
transfected
cells were incubated
in differentiation
medium for 46 h before harvesting.
As shown previously with C2 cells (13), promoter activity directed
by the chicken acetylcholine
a-receptor
gene rose
markedly (15.3 t 5.5 fold) during C2l cell differentiation,
while IGF-II promoter
activity remained
constant (Fig. 7).
To determine whether the IGFBP-5 promoter accurately directed transcription
after transient transfection
into C2l cells, the mRNA cap site of the fusion gene
IGFBP5-1004
Luc was analyzed by ribonuclease
protection assay, using RNA extracted from transfected
cells that had been incubated in growth or differentiation medium. As shown in Fig. 8, an appropriately
sized protected
band was seen in transfected
cells
that had been exposed to differentiation
medium. Surprisingly, the length of this fragment corresponded
with mRNAs initiating at site 1, rather than site 2, the
preferred transcription
stat-t site in C2l cells (see Fig.
5A). The reason for this discrepancy
is not known. In
addition, when quantitated, the abundance of the protected RNA fragment from differentiating
cells was at
least 6 times greater than the chimeric mRNA protected in proliferating C2l cells. Thus, it is likely that the
net increase in luciferase enzymatic activity presented
in Fig. 7 underestimates
the relative rise in IGFBP-5
fusion gene expression during C2l differentiation.
We next examined a series of chimeric IGFBP-5luciferase plasmids to define the regions responsible
for the differentiation-dependent
stimulation
of promoter function. As summarized
in Fig. 9, plasmids
containing from 156 -4100 bp of IGFBP-5 5’-flanking
DNA all directed a similar 2.2- to 2.8-fold increase in
luciferase activity in differentiating
C2l cells compared
Induction
of IGFBP-5
c2
72
Gene
Transcription
in Differentiating
C21 Myoblasts
c21
’ P UD 4
12 24 48
917
DISCUSSION
72 ’
-28s
-18s
Fig. 4. Induction
of IGFBP-5
mRNA Expression
during C2l
Myoblast
Differentiation
The top panel shows an autoradiograph
of a Northern
blot using total cellular
RNA (5 pg/lane)
isolated
from C2l
cells when proliferating
(lane P), after being made quiescent for 24 h (lane UD), and at 4, 12, 24, 48, and 72 h after
the onset of differentiation.
The RNA was isolated
from the
same cells described
in the legend to Fig. 2. The blot was
hybridized
concurrently
with 32P-labeled
probes for mouse
IGFBP-5
and rat myogenin
cDNAs.
RNA from C2 myoblasts exposed
to differentiation
medium
for 72 h (lane C2
72) represents
a positive
control
(6). Autoradiographic
exposure
was for 24 h at -80 C with intensifying
screens.
The middle
panel shows
the ethidium
bromidestained
RNA gel before transfer.
Molecular
size markers
are indicated to the left of the top pane/. The bottom
panel shows
quantification
of the results by densitometry
and demonstrates
that the time course
of accumulation
of both
mRNAs
is similar.
with proliferating
cells, although
promoter strength
varied by a factor of 2.5 among the five plasmids
tested. A recombinant
plasmid with 75 bp of Y-flanking DNA (IGFBPS75
Luc) was ~20% as active as
IGFBPS1004
Luc in proliferating
myoblasts
and
showed a small and not statistically significant rise in
activity in differentiating
cells. Recombinant
plasmid
IGFBPS31
Luc, with 31 bp of 5’-flanking
DNA, was
minimally active, giving essentially background values.
In this paper we show that IGFBP-5 gene transcription
is rapidly induced during the terminal differentiation
of
the C2l myoblast cell line and demonstrate
through
use of an IGF-I analog that one function of IGFBP-5 in
C2l cells is to modulate the differentiation-promoting
actions of the IGFs. We find that IGFBP-5 gene expression is activated within 8 h of exposure of C2l cells
to differentiation
medium, as inferred by analysis of
nuclear IGFBP-5 transcripts (Fig. 5). Coupled with the
relatively long t,,, of IGFBP-5 mRNA in C2l cells
(11-12 h, Fig. 6), the progressive enhancement
of transcription is sufficient to stimulate a massive rise in
both mRNA expression (Fig. 4) and protein secretion
into the conditioned culture medium (Fig. 2). While the
mechanism of transcriptional
activation has not been
ascertained, the proximal promoter of the gene mediates at least part of this effect, as fusion plasmids
containing
only 156 nucleotides of 5’-flanking DNA still
showed a differentiation-dependent
rise in reporter
gene expression (Fig. 9).
Previous studies from our laboratory have demonstrated that IGFBP-5 is produced by muscle cells in
vitro and in vivo (6, 11) and is expressed during muscle
development
in the embryo (14). We had initially identified IGFBP-5 as a novel IGFBP that was secreted
during C2 myoblast differentiation
(15), purified the
protein from C2 cell-conditioned
media (6), and cloned
it from C2 and azamyoblast cDNA libraries (6). Based
on results described
here, it is likely that transcriptional mechanisms are responsible for the induction of
IGFBP-5 expression both in developing muscle and in
myogenic cell lines.
The IGFBP-5 gene forms part of a conserved linkage
group that includes IGFBP-2, fibronectin,
and villin
(16). In the mouse these genes map to the proximal
pat-i of chromosome
1, and in the human to 2q33-q36
(16, 17). In the mouse genome igfbpli and igfbp2 are
separated by only 5 kilobases (16), yet their patterns of
expression are generally not concordant.
In the rodent
embryo IGFBP-5 mRNA is found in myotomal cells
derived from the somite, coincident
with their initial
developmental
appearance,
and subsequently
is detected in muscle cells throughout the embryo, as well
as in other tissues (14). By contrast, IGFBP-2 is not
localized to embryonic
skeletal muscle (14). Thus,
locus-regulating
&-acting
sequences do not appear
to control the coordinate expression of these two related and physically linked genes. The mechanisms
directing high-level expression of IGFBP-5 in muscle
may therefore be promoter-specific.
As evidenced by results of gene transfer studies into
C2l cells, a 1004 bp IGFBP-5 promoter segment is
most active, although the proximal 156-bp fragment of
the promoter directs -70% of maximal transcriptional
activity
in myoblasts and is responsible for at least part
of the differentiation-dependent
rise in gene expression (Fig. 9). Within this latter region are potential binding sites for two muscle-enriched
transcription
factors
MOL
918
END0
Vol 9 No. 7
. 1995
A Exon
I
’0
Proliferafing
-DRB
fDRB
/ 4 12 24”l 4 12 24’
‘0
Differentiating
-DRB
+ORB
I 4 12 24
4 12 24’
-probe
-tx
-
site I
tx site 2
Fig. 6. The Stability of IGFBP-5
mRNA Remains
Constant
during C21 Myoblast
Differentiation
The top panels show autoradiographs
of Northern
blots
using total cellular RNA (15 pg/lane for proliferating
cells, 5
pg/lane for differentiating
cells) isolated at intervals
(0, 1, 4,
12, and 24 h) after addition
of DRB or vehicle to the culture
medium, as described
in Materials
andMethods.
Proliferating
cells were at -50%
of confluent
density at the start of treatment, and differentiating
myoblasts
had been preincubated
for 24 h in serum-free
medium plus insulin before addition
of
DRB. Autoradiographic
exposures
were for 16 h (proliferating
cells) or 4 h (differentiating
cells) at -80 C with intensifying
screens.
Positions
of IGFBP-5
and myogenin
mRNAs
and
molecular
size markers
are indicated.
The bottom
panels
show the ethidium
bromidestained RNA gels before transfer. The t,,* of IGFBP-5
mRNA in each experiment
was calculated to be 11-12 h, while the t,,, of myogenin
was -20
min.
(Fig. 10). Adjacent sites for myocyte nuclear factor
(MNF), a newly described
member
of the HNF-3/fork
-probe
-we-mRNA
--mRNA
head family
that is induced
in differentiating
muscle
cells
(18),
are found
between
-146
and
-131
(..CCCCACCCCCACCCC..).
This region
also encom-
passes AP2 sites (19), and in preliminary experiments
we have shown that nuclear proteins can bind to this
segment of the promoter (our unpublished
observations). An E box (20, 21) which can potentially bind
dimers
of muscle-specific
basic helix-loop-helix
Fig. 5. Rapid induction
of Nuclear IGFBP-5
RNA in Differentiating
C2l Myoblasts
Autoradiographs
of ribonuclease
protection
experiments
using probes derived from mouse IGFBP-5
exons 1 (A) and 3
(B) (11), and nuclear RNA (5 pg/lane)
extracted
from quiescent (lane Q) or differentiating
(1, 4, 8, 12, 24, and 48 h) C2l
myoblasts.
Results
with both probes
demonstrate
that
IGFBP-5
mRNA accumulates
rapidly after exposure
of cells
to differentiation
medium.
In addition,
panel A shows that the
second of two adjacent
transcription
start sites is used predominantly
in C2l cells, while in mouse kidney both sites are
used equivalently
(11); panel B demonstrates
that both nascent and processed
nuclear
IGFBP-5
mRNA are induced
during C2l cell differentiation.
Autoradiographic
exposures
were for 16 h at -80 C with intensifying
screens.
Maps of the
probes and protected
bands are illustrated
below each autoradiograph.
Induction
of IGFBP-5
Gene
Transcription
in Differentiating
C21 Myoblasts
12000
Undifferentiated
10000
I
n
0
I-x
Differentiated
8000
Ti
0
ST+
t
6050
c%
4000
2
-t
204a0
0
IGF-II
“romot~r
3
Acetvlcholine
iGFRP5-IOCM
Fig. 7. Activation
of IGFBP-5
Promoter
Function
in Differentiating
C21 Myoblasts
Recombinant
plasmids
were transfected
into C21 myoblasts, differentiation
was initiated,
and luciferase
and 6-galactosidase
activities
were determined
in whole cell extracts,
as described
in Materials
and Methods.
The mean 2 SD of
four to six experiments
is presented.
Luciferase
activity directed by IGFBP!5-1004
increased
by 3.3 t 0.6 fold in differentiating
compared
with proliferating
cells, and activity
directed
by the chicken
acetylcholine
(Y promoter
rose by
15.3 + 5.5 fold. Expression
of the mouse IGF-II promoterluciferase
plasmid was constant,
as described
previously
for
C2 myoblasts
(13).
proteins such as myogenin, myoD, myf-5, and MRF-4
(22, 23) as well as other ubiquitously
expressed nuclear proteins
(20), is located
at -56 to -51
(CAACTG), within the DNase I footprint detected with
Hep G2 nuclear extracts
(12). Although
a nearconsensus
MEF2 site overlaps the TATA region at
-32 to -23 ~CTAllTAAAAG;
the consensus
is
CTA(T/A),TA(G/A)
(24)], it is unlikely that members of
this family-of transcription
factors will be able to bind
to this sequence, since the underlined T, missing in the
motif found in the IGFBP-5 promoter,
is absolutely
required for MEF2 binding (24). The role of these muscle-enriched
transcription
factors in the induction of
IGFBP-5 gene expression during myoblast differentiation remains to be established.
Our results do not exclude the possibility that insulin
(or IGF-I), rather than a differentiation-specific
factor,
directly stimulates IGFBP-5 transcription.
IGF-I has
been shown to induce elastin gene transcription
through an element in the proximal promoter that superficially resembles the putative MNF/AP2 site noted
above (43). In FRTL-5 thyroid cells, incubation
with
optimal doses of insulin (50 rig/ml) for 24 h caused a
rise in IGFBP-5 mRNA abundance,
although the induction of gene expression was modest, being several-fold less than the 6.4x increase seen with IGF-I (25).
The mechanisms
responsible
for the rise in IGFBP-5
mRNA levels in FRTL-5 cells have not been established. By contrast, in human fibroblasts,
treatment
with IGF-I or IGF-II enhanced media concentrations
of
IGFBP-5 but had little effect on transcript abundance
919
(26). In addition, as we have shown previously (6),
in differentiating
C2 myoblasts
concentrations
of
IGFBP-5
mRNA increased
markedly
before any
change occurred in the secretion of IGF-II, and there
was little insulin or IGF-I in the medium (15, 27). Thus,
although not conclusive, it appears that in muscle cells
(and in fibroblasts) neither insulin nor IGF-I or -II directly modulate IGFBP-5 gene expression.
What are the potential roles of IGFBP-5 in muscle
and why is this protein rapidly synthesized
and secreted in differentiating
myoblasts in vitro? One possibility is to block or modify the stimulatory actions of
IGF-I or IGF-II on terminal differentiation
(28, 29) by
either sequestering
the growth factors from their receptors or by functioning
as a storage depot. This
inhibitory role, which is similar to that postulated for
several other IGFBPs that are synthesized in L6 myoblasts (30, 31), is suggested by results obtained with
des-[l-31
IGF-I. This IGF-I analog binds minimally to
IGFBP-5 (8) and is more potent than IGF-I in stimulating morphological
and biochemical
differentiation
(Fig. 1). Additionally,
IGFBP-5 could modulate other
aspects of differentiation,
such as myoblast fusion,
potentially by binding to the extracellular
matrix (32),
since cell-matrix
interactions
are critical for normal
differentiation
(33-35).
In summary, the experiments
described in this paper show that activation of IGFBP-5 gene transcription
is an early event in the differentiation
of the C2l cell line
and confirm that induction of IGFBP-5 expression is
common to several rapidly differentiating
muscle cell
lines. Since the extent of production of IGF-II has been
shown to control the rate of myoblast differentiation in
vitro (29) and correlates with myotube formation in vivo
(36), analysis of the regulation of IGFBP-5 gene transcription and action in muscle cells should provide
insight into the roles of the IGF system in muscle
development,
growth, and regeneration.
MATERIALS
AND METHODS
Materials
Enzymes,
including
restriction
endonucleases,
ligases,
and polymerases,
were
from
Life Technologies-GIBCO
(Gaithersburg,
MD), Perkin-Elmer/Cetus
(Norwalk,
CT), New
England Biolabs (Beverly,
MA), and United States Biochemical (Cleveland,
OH). Deoxyribonucleotide,
ribonucleotide,
and dideoxyribonucleotide
triphosphates
were purchased
from LKB-Pharmacia
(Piscataway,
NJ) and United
States
Biochemical.
Radionuclides
were from Amersham
(Arlington
Heights,
IL) and DuPont-New
England Nuclear (Boston,
MA).
Nitrocellulose
membranes
were obtained
from Schleicher
&
Schuell (Keene, NH). Plasmid Bluescript
was from Stratagene
(La Jolla, CA), and plasmid pGL2 basic from Promega-Biotec
(Madison,
WI). Materials
for DNA purification
were purchased
from Bio 101 (La Jolla, CA) and Qiagen
(Chatsworth,
CA).
Reagents
for cell culture (fetal bovine serum, newborn
calf
serum, and media) were from Life Technologies-GIBCO.
Oligonucleotides
were prepared
at the Washington
University
Protein
and Nucleic
Acids
Chemistry
Laboratory.
Other
chemicals
were reagent
grade and were purchased
from
commercial
suppliers.
MOL
920
END0
. 1995
Vol 9 No. 7
Cell Culture
-220
transgene-
-200
-180
-160
- 140
-120
-100
IGFBP-5-
-80
‘uc#*rasB CDNA
‘ZOZ
--
Pmbe
pmlec~e9lbam
-
Fig.
4M”l
1ca “l, Lx PNB2. rnlGFBP~5
205 nt. LaS1181, w.nrge”e
8. Identification
of a Single Transcription
Start Site in
C2l Myoblasts
Transfected
with IGFBP5-1004
Luc
RNA was harvested
from C2l cells at 72 h after transfaction, after 48 h of incubation
in either growth
(U) or differentiation (0) medium.
Results are shown of a solution-hybridization ribonuclease
protection
experiment
using 10 pg RNA/
lane and the probe depicted
in the lower part of the figure.
RNA from nontransfected
C2l cells was included
as a positive
control and tRNA as a negative
control. The protected
band
in transfected
cells (indicated
by the arrow marked
“transgene”) is 205 bp long and represents
transcripts
initiating
at
the more 5’+.tarl
site (site l), while the 100 bp protected
fragment
labeled IGFBP-5
corresponds
to transcripts
initiating at start site 2 (see Fig. 3A). Autoradiographic
exposure
was for 36 h at -80 C with a DuPont Lightening
Plus intensifying screen. A DNA sequencing
ladder was used to calibrate the results, as indicated
by the lanes marked
G and A.
The mouse C21 myoblast
cell line (7) was plated at 1 x lo4
cells/ml
onto gelatin-coated
plastic tissue culture
dishes
and incubated
at 37 C in a 95% air, 5% COP incubator
in
Dulbecco’s
modified
Eagle’s
medium
supplemented
with
10% heat-inactivated
fetal calf serum and 10% newborn
calf serum
(growth
medium).
Proliferating
cells were isolated at 70-80%
confluent
density.
Cells were made quiescent
by incubation
for 20-24
h in Dulbecco’s
modified
Eagle’s medium
with 60 nM transferrin.
Differentiation
was
initiated
by incubation
in Dulbecco’s
modified
Eagle’s medium with-60
nM transferrin
and 1 FM insulin (7) or with 60
nM transferrin
plus graded
concentrations
of IGF-I
or
des-[l-3]
IGF-I (see Fig. 1).
Analysis
of Secreted
IGFBPs
Serum-free
differentiation
medium
was collected
at intervals
after conditioning
by cultured
cells and was clarified
by low
speed centrifugation
at 4 C. Aliqouts
(50 ~1) were electrophoresed under denaturing,
nonreducing
conditions
by sodium
dodecvl
sulfate-oolvacrvlamide
ael elactroohoresis
(12.5%
resolving
gel), and the proteins
were transferred
to 9.2 PM
nitrocellulose
membranes
by electroblotting
using a Bio-Rad
(Hercules,
CA) semidry
transblot
apparatus.
Western
ligand
blots were performed
as described
(6). Filters were washed
after overnight
incubation
with 4 x lo6 cpm of 1251-labeled
IGF-II at 25 C and were exposed
to x-ray film for 48 h at -80
C. For immunoblots,
filters were incubated
for 2.5 h at 25 C
with a 1:lOOO dilution
of guinea
pig anti-human
IGFBP-5
antiserum
(26) or a 1:250 dilution
of rabbit anti-rat
IGFBP-3
antiserum
(37). After incubation
with either rabbit anti-guinea
pig secondary
antibody
coupled
to horseradish
peroxidase
(1:4000
dilution)
for IGFBP-5,
or goat anti-rabbit
immunoglobulin
G coupled
to horseradish
peroxidase
(1:1500
dilution)
for IGFBP-3,
binding
proteins
were detected
by
enhanced
chemiluminescence
(ECL kit, Amersham).
Analysis
of Creatine Kinase Activity
After 72 h in differentiation
medium,
cells were washed with
PBS and lysed by incubation
in 400 ~1 of 50 mM Tris-2-[Nmorpholino]ethanesulfonic
acic, pH 7.8, 1% Triton X-100 for
10 min at 25 C. Lysates were stored at -80 C and assayed
for creatine
kinase activity
at 25 C according
to the manufacturer’s
instruction
(procedure
no. 47-UV, Sigma Diagnostics, St. Louis, MO). Enzymatic
activity
was normalized
to
total protein content
as assessed
by the BCA protein assay
(Pierce Chemical,
Rockford,
IL). Data represent
the average
of duplicate
experiments.
RNA Isolation and Analysis
Total cellular RNA was extracted
from C2l cells after solubilization in buffer containing
4 M guanidinium
thiocyanate
(38,
39) and was quantitated
by spectrophotometry
at 260 nM. In
all samples
intact ribosomal
RNA bands were visualized
after
electrophoresis.
Nuclear RNA was isolated as described
previously (13, 40). Northern
blots and ribonuclease
protection
assays were performed
following
previously
described
protocols (13). Signals were quantitated
with a Betascope
603
(Betagen,
Thousand
Oaks, CA) or by densitometty
(Molecular
Dynamics
personal
densitometer).
Measurement of IGFBP-5 mRNA Stability
The RNA polymerase
II inhibitor,
DRB (Sigma Chemical
Co,
St. Louis, MO), was added as a 200-fold
concentrated
stock
in acidified
ethanol to proliferating
cells or to cells after 24 h
Inductron
of IGFBP-5
Gene
Transcription
ECOIU
in Drfferentiating
BarnHI
EcoRl
oooO)
(-1406)
C21 Myoblasts
921
SnJIsad
J-
(-4100)
ww
(-1-W C-79
QQ
Q
40*8%
86&31%
2.2
n=6
61f10%
138+40%
2.3
n=3
Luc
76?9%
21SfSS%
2.8
n=4
LUC
lBQ%
269fSl%
2.7
n40
IGFBPS-156Luc
729~13%
160f38%
2.2
n-5
ICFBPS-WLUC
19Lt2%
30e%
1.s
n-4
-m
lf0.596
Who.696
1.0
n=4
-loin rir--j
c 0.5
< 0.5
n=3
c 0.5
< 0.5
I?=10
plasmip
lGFBF5-41oOLUC
IGFBPs3oooLue
-1406
1GFBP5.1406Lue
-1004
ICFBPS-1004Lnc
IGFBP5.31LW
cl20
.. . . ..\...........\..................
IGFBPS.lMl4Luere"
/--Tq
prOmO&rlaa-LUC
W
Fig. 9. Identifying
the Promoter
Regions
Mediating
the Differentiation-Dependent
Rise in IGFBP-5
Transcription
in C21 Cells
Different fragments
of Y-flanking
DNA were cloned 5’ to a luciferase
reporter
gene. Luciferase
and 8-galactosidase
enzymatic
activities
were measured
after transfection,
as described
in Materials
and Methods.
Results have been normalized
to values
(2 SD) obtained
in proliferating
myoblasts
with IGFBP5-1004
Luc, which has been set arbitrarily
to 100%. The fold increase
in luciferase
activity upon differentiation
is indicated,
as are the number
of experiments
performed
with each plasmid.
Myocyte NuclearFactor
-156
GTGTGAGTTTGCGCTGCAAAGCTCCTTGGCATCC
AGGCCTCTCTT-
E-box
-96
TTGCATGGGTTGGGTGTTGGGGAGCTCAAATTGCAGCT
WGCTGGCAGCCAGGGG
-46
CCGTCTATTTAAAAGCGCCTGCTCGACCAGAGCCCGCAGTCTCTTTGG~CTTCT~
+25
GAGCTAGGAAAGAGCTGCAAAGCTGT
J
1
Fig. 10.
Nucleotide
Sequence
of the Proximal
Part of the Mouse IGFBP-5
Promoter
Potential
sites for binding of muscle-enriched
transcription
factors are indicated.
TATA and CAAT
The two transcription
start sites are marked
by arrows.
of preincubation
in differentiation
medium (final concentration
of DRB, 75 PM), and total cellular RNA was isolated
at l- to
24-h intervals.
The ethanol diluent was added to control cell
cultures, and RNA was isolated over an identical time course.
After Northern
blotting
with IGFBP-5
or myogenin
cDNA
probes,
mRNA abundance
was quantitated
by densitometry
and plotted.
The t,,Z was defined
as the time at which the
signal intensity
reached
50% of the value measured
before
DRB was added to the cells (time 0 in Fig. 6).
Analysis
of Promoter
Function
by Gene Transfer
IGFBP-5-luciferase
fusion genes were generated
by subcloning appropriate
restriction
fragments
and/or
polymerase
chain reaction-derived
DNA fragments
into plasmid
pGL2
basic. The orientation
and nucleotide
sequence
of each recombinant
was validated
before use. Control
plasmids
included pMSV P-gal (41), mouse IGF-II promoter
3-luciferase
(13). and the chicken acetylcholine
cY-promoter
(13). All plasmids for gene transfer
were purified
on Qiagen
columns.
Twenty-four
hours before transfection,
C2l cells were plated
at a density
of 1.25 x 1 O5 cells/60
mm diameter
gelatincoated tissue culture plate, and then were cotransfected
(41)
motifs
are in bold lettering.
with 5 pg of a luciferase
test plasmid and 0.4 kg of pMSV
P-gal DNA. After incubation
for 16-l 8 h, the cells were rinsed
twice with 4 ml of Earle’s balanced salt solution, and either fresh
growth medium or differentiation
medium was added. Cell lvsates were collected
48 h later by incubation for 5 min in 500 bl
oli 7.8. 1 mM dithiothreitol.
1% Triton
of 50 mM Tris-MES.
X-100, followed
by centrifugation
at 1200 x g for 5 min at 4 C
to remove
debris, and were stored at -80 C until analysis.
Luciferase
assays were performed
with 50 ~1 cell extract as
described
(42). The reaction mixture was placed in a Monolight
2010 luminometer
(Analytical
Luminescence
Labs, San Diego,
CA) and the light reaction was initiated by injection of 100 ~1 of
1 mM o-luciferin.
Light emission
was measured
by integration
over the first 10 set of the reaction.
/+Galactosidase
activity
was measured
(41) using 100 ).LI extract. Reactions were terminated by addition of 300 ~1 of 1 M sodium carbonate,
and the
color was assessed at 410 nM. All experiments
were performed
in duplicate
on three to 10 separate
occasions.
Acknowledgments
We thank Dr. John P. Merlie of Washington
University
of Medicine
(St. Louis, MO) for the C2l cells and
School
for the
MOL
922
END0
1995
chicken
acetylcholine
receptor
promoter,
and Dr. Eric N.
Olson of M.D. Anderson
Cancer
Center
(Houston,
TX), for
the gift of the myogenin
cDNA.
We are grateful
for the
assistance
of Dr. Claire E. H. Stewart
with the ligand blots,
and we thank Dr. David Clemmons
and colleagues
at the
University
of North Carolina
(Chapel
Hill, NC) for the antiIGFBPB
antiserum.
We also thank Dr. Shunichi
Shimasaki
of the Scripps
Research
Institute
(San Diego, CA) for the
anti-IGFBP-3
antiserum
and
appreciate
the
helpful
comments
of the reviewers.
Vol 9 No. 7
14.
15.
16.
Received
August
15, 1994. Revision
received
March
6,
1995. Accepted
March 29, 1995.
Address
requests
for reprints
to: Peter Rotwein,
Department of Biochemistry
and Molecular
Biophysics,
Washington
University
School of Medicine,
Box 8231, 660 South Euclid
Avenue,
St. Louis, Missouri
63110.
These studies were supported
in part by NIH Grant 5ROlDK-42748
(to P.R.). Oligonucleotides
were synthesized
at the
Washington
University
Protein and Nucleic Acids Chemistry
Laboratory
under support
of NIH Grant DK-20579.
17.
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