Vasopressin and Insulin-like Growth Factors Synergistically Induce

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have been investigated under conditions allowing growth and differentiation of myogenic cells in a simple serum-free medium. Under these conditions, L6.
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

A .‘

B

CtrI

.-

‘‘.

.,

%

.

.‘

.

_

,‘‘

‘_

..‘

.

.

a ,

.%

..

‘1:_:: : “

#{149}

‘: :



.

.,

‘.

.

#{149}.l

,

;

-#{149}

:

S

-

‘:

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C ‘

.

,‘%

.

.

;b_

-

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.



.

.

0

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.

,

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,t. #{149}.“..

.

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.

:

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AVP+Ins

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#{149} #{149}‘

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a

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%

0.1 pM ..-.

__,_

#{149}

.

,. .

#{149}::1 0#{149}

‘S -

..

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%#{149}

.-..

Insulin

I..

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-

‘l

#{149}:

:

.‘.

‘? #{149}

S

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:.#{149} .

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‘‘:

:%

c .#{176},

#{149}..-:.- ,.

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.‘

#{149},

... --.#{149}

,-

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‘:‘.#{149}

:-;::3’:::::’:;

..#{176}

-

.

,

#{149};

:

..

#{149} *

#{149}.

‘C

-#{176}%

%

tM

-

‘ -,..

:

.

-

...

.

-

-

AVPO.1

& Differentiation

-

-

..

..,

,

,..

.

#{149} a

.

..

I 00 200 80

.

1’ a

160

::

:1

20

0)

40

0

0 c’

cp

CO

CO

0

\ #{149}

I

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-