Vol. 9, 155-163,
February
Cell Growth & Differentiation
1998
Vasopressin and Induce Myogenesis
Insulin-like Growth Factors in Serum-free Medium’
Synergistically
Simona Minotti, Bianca Maria Scicchitano, Clara Nervi, Sigfrido Scarpa, Marco Lucarelli, Mario Molinaro, and Sergio Adamo2
differentiation factors growth factors.
Dipartimento
the effects and the mechanisms of action of AVP and IGFs on myogenic cells have been investigated under conditions allowing growth and differentiation of myogenic cells in a simple serum-free medium. Under these conditions, L6 and L5 myogenic cells slowly proliferate and do not undergo differentiation (less than I % fusion up to 7 days). AVP rapidly (2-3 days) and dose-dependently
Introduction Terminal differentiation of myogenic cells is regulated, at least in vitro, by hormones and growth factors (henceforth collectively referred to as hormones). Fibroblast growth factor and transforming growth factor 13are potent inhibitors of myogenic differentiation (1-3), both of which act on relatively early steps of the differentiation process. Other factors such as adrenocorticotropic hormone, platelet-derived growth factor BB, -y-melanocyte-stimulating hormone, leukemiainhibitory factor, and other cytokines have been reported to primarily affect myoblast proliferation and have shown vanable effects on myogenic differentiation (4-6). IGF-I,3 IGF-Il, and insulin (IGFs) display unique properties, because they stimulate, rather than inhibit, myogenic differentiation in several myogenic cell lines and primary cultures of myoblasts and satellite cells (1 , 7, 8). One exception is represented by the widely used C2 cell line, which does not seem to respond to exogenous lGFs, basically because C2 cells secrete large
induces
amounts
di Istologia
ed Embnologia
Medica
[S. M., B. M. S., C. N.,
M. M., S. A.] and Dipartimento di Biotecnologie Cellulari ed Ematologia e I Istituto di Clinics Chirurgica [S. S., M. L], Universit#{224} “La Sapienza,” 00161
Rome,
Italy
Abstract
Terminal differentiation of myogenic cells has long been known to be positively regulated by insulin-like growth factors (IGFs). Arg8-vasopressin (AVP) has been recently
reported
differentiation.
Creatine
to potently
Induce
In the present
the formation
myogenic
study,
of multinucleated
myotubes.
kinase activity and myosin accumulation
are
up-regulated by AVP. Insulin or IGF-I or IGF-II, at concentrations that cause extensive differentiation in serum-containing medium, induces a modest degree of differentiation in serum-free medium. The
presence
of AVP and of one of the IGFs
in the synthetic medium induces maximal differentiation of L6, L5, and satellite cells. The expression of both myogenin and Myf-5 is dramatically stimulated by AVP. Our results indicate that AVP
induces a significant in the absence
level of myogenic
of other factors.
differentiation
Furthermore,
they
suggest that to express their full myogenic potential, IGFs require the presence of other factors normally present in serum and fully mimicked by AVP. These studies support the conclusion that terminal myogenic differentiation
may depend
on the presence
and
of
Received 3/4/97; revised 10/27/97; accepted 12/8/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-
cate this fact. 1 Supported by Grant 418 from Telethon Italy. The financial support of Consiglio Nazionale delle Richerche, Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologica (Grant 96.00651 .39 to S. A.) and As-
Italiana
edged. 2 To whom
requests
Ricerca
sul Cancro
for reprints
should
(grant
to M. M.) is also
be addressed,
it.
(9). It is remarkable
of
that
the
related
peptides
(1 2, 13).
MyoD,
Myf-5,
and
MRF4)
share
homology
within
a basic helix-loop-helix motif that mediates dimenization and binding to a consensus CANNTG sequence (E-box) present in the promoter of many muscle-specific genes. Each of the MRF genes can activate myogenesis when expressed in nonmyogenic cells (1 5, 16).
acknowl-
at Dipartimento
di
Istologia ed Embriologia Medica, via Scarpa 14, Universit#{224} “La Sapienza,” 00161 Rome, Italy. Phone: 39-6-49766756; Fax: 39-6-4462854; E-mail:
[email protected]
the absence
The mechanism by which IGFs and AVP stimulate myogenesis is likely mediated by the enhancement of the transcniptional activity of MRF genes (1 2, 14). MRF proteins (myogenin,
sociazione
hormones
than
differentiative action of the IGFs is accompanied by a mitogenic action (1). It has been proposed that the effects of IGFs on myogenic cell differentiation and proliferation are mediated by the IGF-I receptor, which exhibits the binding specificity IGF-l > IGF-Il > insulin (1, 10, ii). Recently, AVP and related peptides have been shown to constitute a new family of positive regulators of the terminal differentiation of myogenic cells (L6, mouse satellite cells; Refs. 1 2 and 13). By interacting with V1-type receptors, AVP causes dose-dependent stimulation of the fusion of myoblasts into myotubes and of the expression and accumulation of myogenin and Myf-5 products at both the RNA and protein level. Other markers of myogenic differentiation, such as myosin, nicotinic acetylcholine receptor function, and subunit expression, were also found to be stimulated by AVP
strongly
simultaneous
of these
rather
The abbreviations used are: IGF, insulin-like growth factor; AVP, arg8vasopressin; CK, creatine kinase; FBS, fetal bovine serum; MRF, rnyogenesis-regulatory factors; mAb, monoclonal antibody. 3
155
156
Hormonal
Control of Myogenic
The recent
discovery
genic
differentiation
their
effects
Differentiation
of AVP and related factors
with
makes
those
peptides
it interesting
of the
lGFs
and
as myo-
to compare
to
analyze
their
respective mechanisms of action. Such a study is complicated by the presence of serum in the culture medium, which contains mitogens,
a number which,
differentiated genic
phenotype.
cells
induced
This
medium.
chemically
report
that
can
carefully
controlled
of other
level
factors,
stimulate
the
type in cultured
true
prompted
for
myo-
us to use a
the proliferation
be modulated
including
a significant
absence
have
in which
hormones,
under
to induce tically
reasons
medium
of myoblasts
of specific
is particularly
differentiation is classically the concentration of serum in the
These
defined
differentiation
factors, including expression of the
(1 , 8), whose
in culture
by decreasing
culture
tion
of more or less defined in general, repress the
AVP.
In this
conditions,
of myogenic
and
by the
addi-
study, AVP
we
is able
differentiation
in the
and that AVP and IGFs synergis-
expression
myogenic
of the differentiated
pheno-
cells.
Results Morphological Differentiation Effects of AVP and Insulin. cells/cm2 to DMEM insulin
in DMEM (0.1
third
or both
M)
for several
slowly
added,
Under
of culture
at 5000 plating
AVP (0.1
and
serum-free
regardless
plated 24 h after
medium).
were
days.
proliferated, day
L6 cells were
FBS and shifted
1 % BSA (serum-free
+
cultured
+ 1 0%
the
in serum-free
were
conditions,
of the treatment, medium,
or
ui)
cells
cells
and by the
the cultures
that
suIts (Fig. 2) indicate that AVP (0.1 LM) stimulated heavy chain accumulation to a higher extent than insulin,
whereas
maximal
expression was both hormones. The time
stimulation
induced
course
of myosin
by simultaneous
of differentiation
myosin 0.1 M
heavy
chain
treatment
with
of L6 cell cultures
treated
with 0.1 .1.M AVP or 0.1 M insulin or both in serum-free medium is shown in Fig. 3A. The data indicate that AVPtreated cells underwent a rapid burst of fusion during the third day of treatment and, by the end of the 4th day of treatment, reached approximately 40% fusion. Conversely, no significant amount of fusion was detected in control cells during
the same
period,
was measurable ous
presence
starting reaching
and
a modest
in insulin-treated of AVP
and
percentage
cultures.
insulin
induced
after the second day of approximately 80% fusion
of fusion
The simultaneextensive
fusion,
hormone treatment and by the end of the fourth
day. No significant change in the percentage of fusion of control or hormone-treated cultures occurred after the fourth day, up to the seventh day (data not shown). Analysis of total nucleVmicroscopic field during the period examined mdicated no striking difference among the various culture conditions, confirming that none of the treatments significantly affected L6 cell proliferation in the absence of serum (Fig. 3C). Comparison of the effects of AVP and insulin in the absence and in the presence of FBS shows that in the latter condition, AVP induced a higher degree of fusion than in serum-free medium (Fig. 3, B versus A); insulin promoted extensive differentiation (at variance with its effect in serum-
had received
free
cleated
degree. Furthermore, it may be noted that in the presence of 5% FBS, hormone-induced L6 cell differentiation was a slower process than in the absence of serum. Serum-con-
AVP alone displayed the presence of multinumyotubes (Fig. 1 B). No fusion was detectable in cultures (Fig. 1A). Insulin-treated cells (Fig. iC) pre-
control sented
a very
modest
percentage
of fusion,
whereas
exten-
sive fusion (approximately 75%) was obtained in the presence of both AVP and insulin (Fig. iD). Insulin-treated cultures consisted mostly of mononucleated myoblasts, with rare, thin myotubes containing less than 6 nucleVfiber. Conversely, in cultures treated with 0.1 .tM AVP, frequent, rather large myotubes containing an average of 12 nuclei/fiber were present. Larger myotubes (average, 23 nucleVfiber) formed in cultures treated with both AVP and insulin. Parallel expeniments
conducted
morphological
with
L5 cells
pattern
percentage
of fusion
confirmed
that
for
both
a significant presence
Evaluation microscopic proliferation Although verify
of
of the
iE)
(Fig.
in serum-free
by the medium.
the
study
of myogenesis, response
cells under the conditions insulin or their combination medium
age of myosin
of this study. on satellite
“Materials
to
of satellite
The effect of AVP cell differentiation
cells and
valid
of interest
cultures
by evaluating
chain-expressing
(see
represent
it was
of primary
was assessed
heavy
treatment
on either L6 or L5 cell
conditions. L6 and L5
or in
the percent-
after
Methods”).
72 h of The
taming
and control
medium
re-
cells
induced
differentiated
L6 cells
to a significant
to proliferate
to a much
higher extent than in serum-free conditions (Fig. 3, D versus C). However, in the presence of 5% FBS, neither AVP nor insulin induced significant effects on cell number at the concentration used, in agreement with our previous data (1 2, 13). The dose dependency of the effect of AVP alone or in combination
with
insulin
in serum-free
medium
is reported
in
Fig. 4A. A significant effect of AVP on cell fusion was evident at a concentration of 0.1 nM, reaching a plateau at the highest
AVP concentrations though
on higher
the
various
used. values
fixed concentration
differentiation
by AVP and potentiated
or of their combination
the hormonal
hormone
of myogenic
insulin
under these culture cell lines such as for
serum-free
Calculation L5 cultures
of the total number of nuclei (unfused + fused) per field (Fig. iF) did not suggest dramatic effects of
either hormone
models
a superimposable
L6 and
level
of both cell lines was induced simultaneous
yielded
of differentiation.
medium),
AVP
A similar
of fusion,
of insulin
(0.1
concentrations
dose was
M)
(Fig.
dependency,
measurable
was present 4A).
Both
when
ala
along with in the
ab-
sence and in the presence of insulin, AVP stimulated L6-C5 myoblast fusion with an ECso 0.3 nM. A similar experiment was conducted to investigate the insulin concentration dependency of L6 cell fusion in serum-free medium, both in the absence and in the presence of a fixed concentration (0.1 p.ri) of AVP (Fig. 4B). In the presence of AVP, insulin significantly stimulated
L6
cell
fusion
at concentrations
30
ni,
and
maximal stimulation of fusion occurred at insulin concentrations in the 0.1-3 tM range. A modest but reproducible stimulation of L6 cell differentiation occurred at the same insulin concentrations, also in the absence of AVP (Fig. 4B). Microscopic evaluation of total nuclei in the experimental conditions reported for Fig. 4, A and B, did not indicate
Cell Growth
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B
CtrI
.-
‘‘.
.,
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tM
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L51
Fig. 1 . Morphological analysis of L6 and L5 cells cultured in serum-free medium. A-D, photomicrographs of L6 cultures at the third day in serum-free medium (fourth day of culture) in the absence (A) or in the presence of0.1 M AVP (B), 0.1 tM insulin (C), and AVP and insulin (D). Similarly prepared cultures of L6 and L5 cells were microscopically counted, and the results were expressed as the percentage of fusion (E) and the total (unfused + fused) nucleVmicroscopic field (F).
157
158
Hormonal
Control
of Myogenic
Differentiation
50
Ctrl
-.---
Satellite
cells
-U
40 it? 4)
pM
Ins, 0.1 pM Ins+AVP
-v--
I 00
!;
30
U
-A-
AVP,0.1
-
I-
a) > ..
80 60
‘
(I)
0 0. C.)
1::
20
a)
10
0 400 Ctrl
AVP
Ins
AVP Ins
+
Fig. 2. Effect of AVP and insulin on the differentiation of mouse satellite cells in serum-free medium. Primary cultures of satellite cells were shifted to serum-free medium and treated with 0.1 M AVP and/or 0.1 M insulin as indicated. After 72 h, the cells were fixed and subjected to immunocytochemical analysis using the anti-myosin heavy chain mAb MF2O.
!
300
200
‘5
C.) C
I 00 0
0123456012345678 significant
of either
effects
on L6 cell shown),
proliferation except
nuclei
a
modest
by insulin
effect
at 10 and
presence was
of these
4, C and
in Fig.
D, and
of 0.1
induced
.LM
be noted
(Fig.
that the biphasic
to
their
attributed were
prevented
IGF-II
effect with
above
effects
medium,
in the
reaching
from
previous
by
whose
differentiation obtained (data
results
from initial
seeding
2,500 density,
L6 cells
fusing.
more
similar
not shown).
verify
this
were
cells
possibility,
densities already
or higher
in the presence
unable of AVP.
was
L6
up to four described,
than
those
dis-
by IGFs. The under fusion-
of AVP
± IGFs),
occurred
increased
however,
even
to induce
extensive
as
stepwise fusion
of AVP and
of
our findings,
IGFs
(Fig. 5A). After
was
obtained
signal
was
from
biochemical were
of culture
by
IGF-l (3
activity
of CK,
without
enhanced in the presence treated cells, the specific lation),
compared although
of The
of myosin
in L6
investigated by imheavy chain mAb medium,
induced
A weak
insulin
positive
(0.1 MM). Again,
one of the IGFs induced heavy chain (Fig. 5A).
which
hormone
was
extremely
additions,
low
in
was strongly
of 0.1 LM AVP (Fig. 5B). In insulinactivity of CK was also significantly
to that we
cells.
nM) and
of controls
constantly
found
lower than those in AVP-treated for fusion, simultaneous addition
synergistically 5B).
markers analyzed.
in serum-free
0.1 p.M AVP-treated
medium
enhanced
CK
+
induced
specific
also
medium was the anti-myosin
the combination of AVP and any significant accumulation of myosin The
and
1 % BSA) did not display detectable heavy chain, whereas a strong signal
cells
evidence
Level
on the accumulation
3 days
(DMEM of myosin
control
on Myosin
differentiation
cells grown in serum-free munoblot analysis using
values noted
at the highest
IGFs
substantiate
serum-free
medium,
fusion
effect
and
myogenic
terminal
MF2O
be
by L6 cells in se-
fusion-competent
of myoblast
of L6 cells
(com-
could
in serum-containing
cells/cm2;
at IGF-I
medium
Effect ofAVP Activity To further
evident
nuclei
was strongly enhanced not shown) indicate that
density
in the absence
IGF
(1 7). It is to
of serum)
attained even To
maintained
to 20,000 lGFs
data
1 0 nM; data
that
permissive conditions (e.g., a progressive acceleration the initial
4D) at conat higher
of total
presence
cell densities
cultures
(Fig.
of the curves,
densities
such
played
In the
Fig. 3. Time course of the differentiation (percentage of fusion, A and B) and proliferation (nuclei/field, C and D) of L6 cells cultured in serum-free medium (left panels) or in the presence of 5% FBS (right panels). All cultures were seeded on day 0 at the same initial density (5000 ceIIs/cm) in DMEM + 10% FBS and shifted 24 h later to either serum-free medium c4 and C) or 5% FBS medium (B and D). #{149}, control (no hormone addition); ., 0.1 M AVP; A, 0.1 j.tM insulin; V, AVP + insulin. The arrows indicate the time of hormone addition.
of cell fusion
reversed
cultures were set up at various initial times the density used in the experiments thus
as shown
of insulin.
of the IGFs in serum-free
to the lower
rum-free
that
stimulation
stimulation
concentrations effect
investigated
day of culture
not
in total
f.LM.
with
shape
(up to 2.5-fold
The modest
(data
for insulin (Fig. 4B), was likely related of these factors at the highest con-
for IGF-l and IGF-II than to the mitogenic effect or IGF-II
was
4C) and
This
in agreement
centrations
30
significant
of 1-1 0 n.
concentrations,
pared
factors
AVP,
tested (1 0-1 5%)
experiments described, insulin and readily available alternative IGF-I and IGF-Il (1). The dose-
compared
by IGF-I
centrations
or in combination,
increase
IGF-l and IGF-Il. In the was used as an inexpensive to the physiological ligands dependent
alone
at all concentrations
for
induced
hormone,
very high
levels
(over
20-fold
CK specific L6 cells. of AVP
stimu-
activity
As already and insulin
of CK activity
(Fig.
Cell Growth
80
C 0 (I)
c)
60
$\c’
0
0) CU C
40
a)
20
0.
s>
Bt
& Differentiation
159
I
/*
-
0 0
0.1
1
10
[AVP],
100
0
0.1
1
10 100 1K 10K
(Insulin],
nM
nM
B C
80
C
D 160
0
U)
2
60
0
0) (U C
120 20
5 0.
80 AA.&..A
A
0
0
A
0.3
3
(IGF-l],
A
-
30
0
0.3
nM
3
30
(IGF-Il],
0
nM
40
Fig. 4. Dependency of L6 differentiation (percentage of fusion) on the concentration of AVP, lGFs, and their combinations in serum-free medium. L6 cells were seeded at 5000 cells/cm2 in DMEM + 10% FBS, and at 24 h, the cultures were shifted to serum-free medium, and the hormones were added. The cultures were fixed and stained on the fifth day of culture. A, increasing concentrations of AVP without (0) or with (#{149}) 0.1 ,.M insulin. B-D, increasing concentrations of insulin and IGF-l or IGF-ll without (Lx) or with (A) 0.1 M AVP, respectively.
Expression
of the MRF Genes
Differentiation of L6 cells the MRF gene myogenin. ments
on the
indicate of IGF-I,
Myogenin
steady-state
level
of the
myogenin
the myogenin
transcript
in Fig. 6B. The
is shown
detectable whereas
in control
of the accumulation
of
or 0.1 j.tM AVP-stimulated
myogenin
Fig. 5. Effect of AVP and/or IGFs on the accumulation of myosin and the CK activity in serum-free medium. A, immunoblot of extracts of L6 cells cultured for 3 days in the absence (Ctrl’) or in the presence of 0.1 M AVP, 0.1 LM insulin, 3 nM IGF-l, 3 n.i IGF-ll, or their combinations. MHC Std, a myosin standard run along with the experimental samples. B, CK-specific activity of extracts of L6 cells cultured in serum-free medium in the absence (Ctri’) or in the presence of 0.1 M AVP, 0.1 f.LM insulin, or both.
transcript
a strong effect of AVP and a relatively modest effect insulin, and IGF-II (in order of potency), whereas the level of expression is evident in AVP + insulin-
maximal treated cells (Fig. 6A). The time course cells
and Myf-5
is accompanied by expression of The effects of the various treat-
0
transcript
was
un-
in cells grown for 24 h in DMEM + 10% FBS, a low level of myogenin mRNA was present in
whereas
Myf-5
interesting
to
shown
evident
in AVP-treated
signal
expressed
The presence protein
was
occurred in 5%
investigated
cells
same
time,
displayed
none
in serum-free
localization
me-
of myogenin
by immunofluorescence myogenin-specific mAb of culture in serum-free
a very weak
of the
earlier
(48 h).
and the subcellular
also
ing of L6 cells, using the 1 8; Fig. 7). After 3 days control
much
FBS
IGFs
and diffuse
induced
stainF5D (Ref. medium,
signal.
a strong
(Lane
early
in the
of myogenin
(19).
investigate
in L6 cells
the
under
in L6 cells
hormonal
of the
(12,
regulation
the conditions
in Fig. 6C, no expression
vations tectable
by AVP
AVP
upstream
is not
of this Myf-5
B). At the same with
culture
insulin
(Lane I) strongly induced versely, none of the IGFs
At the
F; and IGF-II,
effect,
of Myf-5
Lane
time,
(Lane
study.
As
product
in proliferative with our previous
medium obser-
AVP
alone (Lane
(Lane
was demedium C), or in
G), or IGF-II
Myf-5 mRNA expression. alone (insulin, Lane 0; IGF-l, of serum.
it is
Myf-5
gene
E), IGF-I
H) significantly
in the absence
20), of
(1 2), whereas a very slight expression level in L6 cells 1 8 h after shifting to serum-free
combination
in the
or with
to be expressed
to act
myogenin
(24 h) than
localized AVP
are known
was observed in L6 cells growing (10% FBS; Lane A), in agreement
dium
with
and
Comparison with the data obtained in the presence of 5% FBS (1 2) confirmed that induction of the expression of
cultures.
correctly
in cells treated (Fig. 7).
MyoD
MyoD
expression
was
positive
somite
Whereas
in the level
mRNA
and
developing
control L6 cells at 1 , 2, and 3 days after shifting the cultures to serum-free medium. At the same times, a strong increase of myogenin
a strong
nucleus was elicited plus one of the lGFs
stimulated
ConLane
the expression
1&’j
Hormonal
Control
of Myogenic
Differentiation
the positive
A
genic
role played
by AVP
cells (12) opened
addition
to
present
IGFs,
may
results
sence
of
positively
indicate
other
in the differentiation
the possibility that
factors,
regulate
AVP,
of myo-
that other
factors,
in
myogenesis.
but
not
significantly
IGFs,
Our
in the
stimulates
ab-
myogenic
differentiation. We found
Myogenin
our plex
media
were
factors.
used
of other
L6 and
factors,
of hormones
More
in
or less
by several
cell proliferation
in Ref. 8). Using
absence
of the effects
on several
of myogenic
viewed
groups
comfor the
and differentiation
L5 cells,
myogenic
we found
(re-
that
differentiation
in the
was
af-
fected by the particular preparation of BSA used. Indeed, whereas one of the commercial preparations of BSA that we tested
behaved
was toxic
B
12 and
AVP
CtrI
used
in this
in the presence
BSA preparations, under control
18%
preparation
cific
in
and
study,
of two
one
additional
the percentage of fusion was conditions, 45 and 55% in the
presence of 0.1 LM AVP, and 30 and 34% in the presence of 0.1 j.tM insulin, respectively. We selected a commercial BSA
123
0123
as the one we have
for our cells,
commercial
Myogen
reproducibility
depends
serum-free
analysis
rRNA
that
system
that
of serum serum-free
-
allowed
hormones
were
minimal
present.
differentiation
unless
Furthermore,
accurate
spe-
removal
when cells were shifted from 10% FBS medium to medium seemed to be critical, particularly for the
reproducibility
of the effect
as 0.5%
FBS to serum-free
increment
of the percentage
insulin serum-free
of IGFs.
In fact,
medium of fusion
of as little
in a significant
in the presence
(an approximately 4-fold medium, insulin-treated
MM
addition
resulted
of 0.1
increase compared cultures; data
to not
shown).
C
Under
these
tiation
ABC
DEFGHI
Myf-5
,-,
absolute
terms
strongly
stimulated
1%
fusion
FBS controls;
lated
inhibit,
lGFs were considered
and hormones in that myogenic differentiation”
“unique
they stimulate, (1). Our recent
among
growth
rather than discovery of
controls
over
to any major
These
results
differentiation
markers
but
nor insulin
extent;
confirmed
such
as
20%
and
are
stimu-
(b) they in the
syner-
presence
by measuring
myosin
heavy
in
of insulin
cell differentiation
differentiation
were
was
(less than
on the effect
IGF-I/IGF-II
myogenic
60%)
and approximately
results
neither
(a)
activated
other
chain
CK
or
activity.
The
effects
of AVP
and
IGFs
on myogenic
parameters
expression together,
of Myf-5 and myogenin was investigated. these results suggest that the mechanism
of AVP
involves
regulatory
are similar
the
genes.
act upstream declines
induction
It is worth
of myogenin,
when
that
to those
terminal
entiation
of the
recalling and that
of myogenin
observed
expression
that Myf-5
differwhen
the
Taken of action of these
is known
its expression
to
in L6 cells
(21 , 22). These
increases
data and the observation that the onset of differentiation in the presence of AVP and insulin is faster than in the presence of AVP alone
factors
differen-
of FBS in
to the control
on L6, L5, and satellite
differentiation
of AVP.
versus
compared
Fig. 3). Our
in that:
gistically
Fig. 6. Northern blot analysis of the expression of myogenin and Myf-5 in L6 cells. A, L6 cell cultures, seeded as usual in DMEM + 10% FBS, were shifted at 24 h to serum-free medium and cultured for an additional 72 h without (Ctr!) or with 0.1 M AVP, 0.1 tM insulin, AVP + insulin, 4 ni IGF-l, and 4 n IGF-ll. Hybridization of the blot with an rRNA probe was used for the purpose of normalization. B, time course of the effect of 0.1 M AVP on the expression of myogenin. Control cells at day 0 (24 h of culture in DMEM + 10% FBS) and at 1 , 2, and 3 days of culture in serum-free medium are shown; 0.1 MM AVP-treated cells at 1 , 2, and 3 days of hormone treatment in serum-free medium are shown. Ethidium bromide staining was used to verify equal loading of the samples. C, L6 cells were cultured for 24 h in DMEM + 10% FBS (Lane A) and then shifted to serum-free medium, treated with hormones as indicated, and incubated for an additional 18 h (Lanes B-I). Lane B, control; Lane C, 0.1 M AVP; Lane D, 0.1 tM insulin; Lane E, AVP + insulin; Lane F, 3 n.i IGF-l; Lane G, AVP + IGF-l; Lane H, 3 n.i IGF-ll; Lane I, AVP + IGF-ll.
AVP-dependent
fusion
when
in serum-free
or IGF-I/IGF-II
rRNA
conditions,
(40%
5%
unexpected
Discussion Until recently,
culture
of L6 cells was lower than in the presence
(Fig. 3A) may
help explain
effect
of the combined
treatment
pared
to that
alone)
Some those similar
with
AVP
discrepancy
of Ewton to
ours,
seems
with
reported
and
on the expression
to exist
et a!. (23). Using they
the relatively AVP
between
a serum-free that
both
modest
IGF-I
our data and medium
IGF-I
(corn-
of Myf-5.
and
very IGF-Il
Cell Growth
161
Insulin
IGF-lI
IGF-I
Ctrl
& Differentiation
,
I
1.
,
‘ ‘,
AVP
AVP
AVP+IGF-l
,
a 9
#{149}!#_
S
+
AVP
IGF-ll
I
I,
S
Insulin
+
,
.%
C
a
#{149}; “
*.
,
%
‘
‘.
a
.4’
\
.,‘
‘
1 -
Fig. 7. lmmunofiuorescence (Ctr() or with the following
stimulated
myogenic
formation have
analysis hormones:
and
differentiation
OK activity.
shown
that
differentiation
IGF-I
and/or medium.
obtained
a significant
recently,
induces
first
proliferation
of L6 cells
In addition, level
of both myotube Engert et a!. (24)
in terms
More
hypertrophy
rum-free
in L6 cells. L6 cells, seeded and cultured as described, IGF-ll; and 0.1 M insulin or their combinations.
of the expression of myogenin 0.1 M AVP; 4 n IGF-l; 4 n
it must
and
cultured
be noted
of fusion
in control
then
that
may
in se-
(or
a
that
they
when
possibility the
cells
could are
be the incomplete
shifted
from
the
removal
that
as little
effect of insulin plating densities longed different bilities those The
from
those
reported
explain
in this
origin
the mechanism
and
been extensively
studied
muscle
Conversely, on myogenic
before
shifting
in hormone
effects
study.
All these
between
(reviewed
of the
AVP.
On the other
levels dined
as gestational
have
not
disputed.
hand,
an unsuspected
AVP skeletal
were muscle;
age increased
of
induces
measured its
role
were well known (29), and high in extracts
concentration
(30). Furthermore,
of de-
Hanley
of the during
cells
of the
that
peptide a finding
origin
of AVP
skeletal
myogenic
they
factors;
myogenic
muscle
are the
to
presence
phenotype.
our study, differentiation
differentiate
to of
and
proliferate
myogenic
cells
exposed
sufficient
such
factors
represses
the ex-
By establishing
real
on the contrary, supports the of at least some myogenic
cell culture models (such as those used in this study, which do not secrete large amounts of differentiation factors) depends rather
on the presence than the absence
of specific of proliferative
by using
we
have
induce
consistently
carefully
maximal
of myogenic elucidate
ergism
found
of differentiation factors
pathways.
that
factors
myogenic
have
Research
differentiation signals.
controlled
culture
such as fusion, and myogenin IGFs whose
differentiation.
distinct
signals
receptors
is in progress
and
condi-
accumulaexpression,
AVP
represent
synergistic
actions
These
families
two
and distinct in our
laboratory
sigto
the level and the molecular mechanism of the synbetween AVP and lGFs in regulating the expression of
the myogenic
an indeed no primary
developmental
growth
control conditions, view that terminal
naling
muscle are evident in the clinical conby either reduced or increased levels of
of immunoreactive embryonic
lGFs
relevance of the effect of AVP (1 2, 26, 27) is not univocally
for a hormone whose effects in the adult has been recently reported for endothelin-i human
and
in Ref. 25), and their role is basically
established. On one hand, several reports indicate modest effect of AVP on adult muscle (28), and disorders of skeletal ditions characterized
amounts
families
of action
question
peptide)
unless
In conclusion,
possi-
our results
of a vasopressin-like nervous system,
tions and measuring parameters tion of myosin, CK activity, Myf-5,
groups.
development
the physiological cell differentiation
the
observed that initial in this study or pro-
results
the differences
by the other
increases
medium
medium
presented
in skeletal
significantly
in serum-containing
to serum-free
could
FBS
on fusion. We have also above those examined
culturing
the cultures
as 0.5%
on the
thought
spontaneously
pression
me-
dium (20% FBS medium; Ref. 24) to the serum-free medium. This possibility is particularly relevant in light of our observation
light
vasopressin-like
It is commonly
of serum
serum-containing
cast
the presence sympathetic
development.
conditions
(serum-free medium). As already pointed out, one possible factor that could explain these discrepancies is the origin of the BSA used by these groups (see previous discussion). Another
eta!. (31) reported in the mammalian
were treated for 3 days without
Materials
phenotype.
and
Methods
Synthetic AVP and related peptides, bovine insulin, CK assay kit, TnReagent, and other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). IGF-l and IGF-ll were purchased from Chemicon (Temecula, CA) and from Intergen (Purchase, NY). Quick-Hyb Northern blot hybridization solution was obtained from Stratagene (Heidelberg, Germany). Fatty acid-free BSA (Boehringer Mannheim) was selected by comparing BSA from different commercial sources, on the basis that no signs of toxicity appeared even after long-term culture of L6 cells, and no
162
Hormonal
Control of Myogenic
Differentiation
fusion could be detected in the control conditions. The anti-myosin heavy chain mAb MF2O (32) was a kind gift of Dr. D. Fishman (Cornell University, New York, NY). The anti-myogenin Ab F5D (18) was kindly provided by Dr.
W. E. Wright (University
Cell Cultures.
blots with a ribosomal Collection,
Rockville,
18S RNA (rRNA) probe (American MD).
Type Culture
of Texas, Dallas, TX).
Subcloning
and
characterization
of L6 rat myogenic
Acknowledgments
cells (33) was reported previously (26). Cells of the subclone CS (L6-C5), a clone that had shown significant differentiation ability when cultured under appropriate conditions (26), were used throughout this study. The cells were routinely seeded at the density of 5000 cells/cm2 (unless otherwise indicated) in DMEM supplemented with 100 units/mI penicillin, 100 g/ml streptomycin, and 10% heat-inactivated FBS. Twenty-four h after plating, cuitures were extensively washed with DMEM and shifted to serum-free medium consisting of DMEM supplemented with BSA, with or
We thank G. Cossu and M. Fiszman for critical reading of the manuscript and F. Naro and A. Di Noi for helpful advice and discussion. Mouse
without
2. Clegg, C. H., Linkhart, T. A., Olwin, B. B., and Hauschka, S. D. Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor. J. Cell Biol., 105: 949-956, 1987.
other
additions.
Media
were
changed
every
3 days,
as appropri-
ate. Preliminary experiments performed with a medium composed of DMEM supplemented with BSA allowed us to asses the minimal concentration (1 %) of BSA compatible with survival and good morphology of the cells. Furthermore, it was found that BSA preparations obtained from different commercial sources were widely different from one another when
satellite
cells were
kindly
provided
by M. Coletta
and L Capece.
References 1 . Florini, J. R., Ewton, D. 1, and Magri, K. A. Hormones, growth factors, and myogenic differentiation. Annu. Rev. Physiol., 53: 201-216, 1991.
3. Olson, N. E., Sternberg,
E., Hu, J. S., Spizz, G., and Wilcox, C. Reg-
was morphologically evaluated, both in the absence of hormones (see “Results”). Rat L5 myogenic cells were routinely cultured in F14 medium supplemented with 10% FBS (34). For serum-free medium experiments, 15 cells were cultured as described above for L6 cells, except that gelatin-coated dishes were used. Primary cultures of satellite cells were prepared from young adult CD1 mice as reported previously (12). After 48 h of cuiture, the proliferation medium (DMEM supplemented with 20% horse serum and 5% chick embryo extract) was substituted with the serum-free medium described above, and hormones were added as appropriate. Measurement of Myoblast Fusion and Growth. May GrunwaldGiemsa-stained cultures were evaluated for cell fusion. Cells were considered fused only if cytoplasmic continuity and at least three nuclei were
ulation of myogenic differentiation by type J. Cell Biol., 103: 1799-1806, 1986.
present in each myotube. Greater than 10 randomly selected fields and greater than 300 total nuclei were counted for each sample. The ratio between the number of nuclei in myotubes versus the total number of
7. de Ia Haba, G., Cooper, G. W., and Elting, V. Hormonal requirements for myogenesis in vitro: insulin and somatotropin. Proc. NatI. Acad. Scm. USA, 56: 1719-1723, 1966.
nuclei
8. Flormni, J. R. Hormonal 577-598, 1987.
differentiation
of L6-C5 cells and in the presence
per microscopic
field was expressed
as the percentage
of fusion.
Each experimental point represented in the graphs is the mean ± SD of the counts obtained from four to nine independent samples obtained in at least two separate experiments. CK Assay. Cells were homogenized in 30 ma HEPES and 1 mi EDTA (pH 7.2), and the 20,000 x g supernatant was used to measure CK activity according to Szasz et a!. (35). Protein content of cell extracts was meas-
ured as described Immunochemistry.
previously
(36).
Myogenin
expression
was
analyzed
in cuitured
cells after fixation in 4% paraformaldehyde in PBS and permeabilization in 0.5% Triton X-100 in PBS. The monolayers were washed in 1% BSA in PBS and incubated overnight at room temperature with the undiluted
(3 transforming
growth
factor.
4. De Angelis, L, Cusella De Angelis, M. G., Monaco, L, Raschell#{224}, G., and Cossu, G. Pro-opiomelanocortin gene is expressed in post-implantation mouse embryos and enhances growth potential of myogenic cells. Dev. Dyn., 198: 265-272, 1993. 5. Yablonka-Reuveni, Z., Balestreri, T. M., and Bowen-Pope, D. F. Regulatmon of proliferation and differentiation of myoblasts derived from aduft mouse skeletal muscle by specific isoforms of PDGF. J. Cell Biol., 111: 1623-1629, 1990. 6. Austin, L, and Burgess, A. W. Stimulation cufture by leukemia inhibitory 101: 193-197, 1991.
factor
and other
of myoblast cytokines.
control of muscle growth.
proliferation J. Neurol.
In Scm.,
Muscle & Nerve, 10:
9. Tollefsen, S. E., Sadow, J. L, and Rotwein, P. Coordinate expression of insulin like growth factor II and its receptor during muscle dlfferentlation. Proc. NatI. Aced. Sci. USA, 86: 1543-1547, 1989. 10. Ewton, D. Z., Falen, S., and Flonni, J. R. The type II IGF receptor has low affinity for IGF-l analogs: pleiotypic actions of lGFs on myoblasts are apparently mediated by the type I receptor. Endocrinology, 120:115-124, 1987. 11 . Cohick, W. S., and Clemmons, D. R. The insulin-like Rev. Physiol., 55: 131-153, 1993.
growth factors.
Annu.
supernatant of F5D hybridoma cells (18). After extensive washing with 1% BSA in PBS, the cells were incubated for 1 h at room temperature with fluorescein-conjugated goat antimouse immunoglobulmn (Cappel Labora-
12. Nervi, C., Benedeth, L, Minasi, A., Molinaro, M., and Adamo, S. Argmnmne-vasopressmn induces differentiation of skeletal myogenic cells and up-regulates myogenin and myf-5. Cell Growth Differ., 6: 81-89,
tories; diluted 1:50; Ref. 18).
1995.
Sarcomeric myosin expression was determined by Western blot analysis using the MF2O mAb, and detection was performed by the enhanced chemiluminescence method (Amersham) according to the manufacturer’s instructions. The same mAb was used for the immunocytochemical staining of myosin heavy chain in primary satellite cell cultures. The reaction was developed as reported previously (1 2), using a sheep antimouse immunoglobulin, horseradish peroxidase-linked antibody obtained from Amersham (Milan, Italy). RNA Preparation and Northern Blot Analysis. Total RNA was iso-
lated from the cells by the Tri-Reagent procedure as indicated by the manufacturer. Equal amounts of total RNA (30 g) were separated by electrophoresis in 0.66 M formaldehyde-i .2% agarose slab gel, transferred to Nytran membranes (Schleicher & Shuell, Hayward, CA) by capillary blotting. and cross-linked to the blots by UV irradiation (37). Other procedures were as described in Ref. 12, except that Quick-Hyb was used as prehybridization and hybridization buffer. The rat myogenin probe (1 500 bp), kindly provided by Dr. W. E. Wright, and the human Myf-5 probe (1250 bp; Ref. 20) were the EcoRl restriction fragments of the respective
by ethidium
cDNA
clones. Equal loading of the samples was verified bromide staining of the gels (37) or by hybridization
13. Nervi, C., Benedetti,
L, Donchenko,
V., Naro, F., Di Noi, A., Molinaro,
M., and Adamo, S. Regulation of skeletal muscle differentiation arginine-vasopressin. In: S. Waxman (ed.), Differentiation Therapy, 193-197, Rome: Ares Serono Symposia Publications, 1995.
by
pp.
14. Flormni, J. R., Ewton, D. Z., and Rcof, S. L Insulin-like growth factor-i stimulates terminal myogenic differentiation by induction of myogenin gene expression. Mel. Endocrinol., 5: 718-724, 1991.
15. Olson, N. E. The MyoD family: a paradigm 4: 1454-1461, 1990.
for development?
Genes
Dev.,
16. Wright, W. E., Binder, M., and Funk, W. Cyclic amplification
and target
selection (casting) for the myogenin Biol., 11: 4104-4111, 1991.
Mol.
17. Florini, concentration
18. CuselIa L, Vivarelli,
of the
Buckingham,
binding
site.
Cell.
D. Z., Falen, S., and Van Wyk, J. J. Biphasic of the stimulation of myoblast differentiation Am. J. Physiol., 250: C771-C778, 1986.
J. R., Ewton, dependency
by somatomedins.
either
consensus
De Angelis, E., Farmer,
M. G., Lyons, G., Sonnino, C., De Angelis, K., Wright, W. E., Molinaro, M., Bouche, M.,
M., and Cossu,
G. MyoD,
myogenmn-mndependent
differen-
Cell Growth & Differentiation
tiation
of primordial
myoblasts
in mouse
somites.
J. Cell Biol.,
1 16: 1243-
1255, 1992.
phate
19. Buckingham, 149, 1992.
M. Making
muscle
in mammals.
Trends
20. Braun, T., Bober, E., Buschhausen-Denker, K., and Arnold, H. H. Differential expression genes in muscle cells: possible autoactivation EMBO J., 8: 3617-3625, 1989.
21 . Buckingham, myogenic 22.
regulatory
Mangiacapra,
Paradoxical myogenic 2038-2044,
M. Skeletal muscle factors. Biochem. F. J.,
Roof,
S. L,
8: 144-
G., Kotz, S., Grzeschik,
and
the role
24: 506-509,
D. Z.,
and
Florini,
J. R. of
6:
1992.
Ewton, D. 1, Roof, S. L, Magri, K. A., McWade, F. J., and Florini, J. R. IGF-ll is more active than IGF-l in stimulating L6A1 myogenesis: greater mitogenic actions of IGF-I delay differentiation. J. Cell. Physiol., 16: 277-
cedes differentiation 431-440, 1996.
E. B., and Rosenthal,
in IGF-l-stimulated
N. Proliferation
myogenesis.
J. Cell
Biol.,
Y., Azuma,
S., Toyoda,
Y., Ishikawa,
T., Kumada,
K., K.,
M., and Yazachi, in mice defi-
30. Smith, A., Stephen, A. J., Arkley, M. M., and Mcintosh, N. Immunoreactive argmnmnevasopressmn in human fetal and neonatal skeletal muscle. Early
Hum.
Dev.,
28: 215-222,
1992.
31 . Hanley, M. R., Benton, H. P., Lightman, S. L, Todd, K., Bone, E. A., Fretten, P., Palmers, S., Kirk, C. J., and Michell, R. H. A vasopressin-like peptide in the mammalian sympathetic nervous system. Nature (Lond.), 309: 258-261, 1984. 32. Bader, D., Masaki, T., and Fishman, D. A. Immunochemical analysis of myosmn heavy chain during avian myogenesis in vivo and in vitro. J. Cell 95: 763-770,
1982.
D. Retention
of differentiation
135:
cuitivation
of myogenic
cells. Proc. NatI. Acad. Sci. USA, 61: 477-483,
potentialities
during
prolonged
1968.
S. Transduction
of the
cells. Am. J. Physiol.,
27. Naro, F., Donchenko, V., Minotti, S., Zolla, L, Molinaro, M., and Adarno, S. Role of phospholipase C and D signalling pathways In vasopressmn-dependent myogenic differentiation. J. Cell. Physiol., 171: 34-42, 1997.
mus-
33. Yaffe,
1996.
26. Teti, A., Naro, F., Molinaro, M., and Adamo, arginine-vasopressin signal in skeletal myogenic 265: C1i3-Ci21, 1993.
1,6-biphos-
in cultured
pre-
25. Florini, J. R., Ewton, D. 1, and Coolican, S. A. Growth hormone and the insulin-like growth factor system in myogenesis. Endocr. Rev., 17: 481-517,
of glucose
and hormonal stimulation J., 204: 765-769, 1982.
29. Kurihara, Y., Kurihara, H., Suzuki, H., Kodama, T., Maernura, Nagai, R., Oda, H., Kuwaki, T., Cao, W. H., Kamada, N., Jishage,
Biol.,
1994.
24. Engert, J. C., Berglund,
D. The control
state
Y. Elevated blood pressure and craniofacial abnormalities cient of endothelin-1. Nature (Lond.), 368: 703-710, 1994.
of the 1996.
decrease in myf-5 messenger RNA levels during induction differentiation by mnsulmn-likegrowth factors. Mol. Endocrinol.,
M. J. 0., and Pette,
by developmental
cle tissue. Biochem.
Ouchi,
of myogenic determination by the Myf gene products.
development Soc. Trans., Ewton,
Genet.,
23.
284,
28. Wakelam,
163
34. Scarpa,
S., Uhlendorf,
B. W., and Cantoni,
G. The differentiation
of
L5/A10 myoblast cell line (a subclone of L5 line) is controlled by changes of cuiture conditions. Cell Differ., 17: 105-1 14, 1985. 35. Szasz, C., Gruber, W., and Bemt, E. Creatine kinase in serum: determination ofoptimum reaction conditions. Clin. Chem., 22: 350-356, 1978. 36. Bensadoun, A., and Weinstein, D. The assay of proteins in the presence of interfering materials. Anal. Biochem., 70: 241-250, 1976. 37. Sambrcok,
J., Fritsch, E. F., and Maniatis,
Laboratory Manual. ratory, 1989.
Cold
Spring
Harbor,
T. Molecular
NY: Cold
Spring
Cloning: A
Harbor
Labo-
