Vol. 166, No. 3, 1990 February 14, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1205-1212

ACIDIC AND BASIC FIBROBLAST

EXPRESSED

BY SKELETAL

GROWTH FACTOR mRNAS ARE!

MIJSCLE

SATELLITE

CELLS

JeanineAlterio*, Yves Courtois*,JacquesRobelin**, DanielBechet**and IsabelleMarte:lly*** 1 *Unit6 deRechercheGerontologique no118INSERM, meWhilhemParis75016,France ** INRA, 63122Ceyrat,France ***LaboratoiredeMyog6neseet RCgt%r&ation Musculaire(MYREM), Universid ParisVal deMame,av G6nCraldeGaulle,Cr6teil94010 , France Received

December

19,

1989

SUMMARY:

We postulated that Fibroblast Growth Factor (FGF) involved in fetal or regenerative morphogenesis of skeletal muscle originated from this tissue. Using a bovine retina cDNA probe encoding acidic :FGF, we showed that growing muscles from bovine fetuses express this mRNA, but that this expression is reduced in neonate muscles. Cultures of proliferating satellite cells isolated from adult rat muscles expressed aFGF mRNA strongly but bFGF mRNA weakly: these mRNAs disappeared in cells differentiated into myotubes. 10-7M 12-0-tetradecanoyl phorbol -13-acetate (TPA) increased aFGF mRNA expression in both proliferating and differentiated satellite cells. Contrastingly, proliferating L6 myogenic cells only expressed aFGF mRNA significantly under TPA treatment. Therefore, the satellite cells did seem to be a possible source for FGF, especially aFGF, which might regulate the myogenic process. QI.990Academic Press,Inc.

The proliferation of mesodermic tissue is known to be regulated by FGF , especially bFGF.

In vitro , a- and bFGF where observed to stimulate cellular in primary cultures of myoblasts and myogenic cell lines (see review 1). Furthermore, FGF is also known to inhibit the myogenic differentiation of embryonic myoblasts and myogenic cell lines (2.3) and of rat satellite cells (4).

proliferation

The addition of FGF to cells at the postmitotic expression

(5)

and their

content

of

the

stage induced reductions of mRNA specific

muscle protein

creatine

phosphokinase (CPK) (6). In adult animals, skeletal muscles regenerate through the activation of undifferentiated

myogenic cells called satellite cells (7), which proliferate

and

differentiate into myotubes in vitro, in the same way as do fetal myoblasts. Satellite cell proliferation is stimulated by FGF (4.8, and our observation). In mdx mouse mutants, whose muscles are continuously regenerating, bFGF was detected *To whom reprint

requests

should

be addressed. 0006-291X/90 1205

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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AND BIOPHYSICAL

in basal membranes of muscle fibers at a normal non dystrophic

RESEARCH COMMUNICATIONS

much higher concentration than in

muscles (9).

These results show the key role of FGF in regulating growth and the morphogenesisof mesodermic-derived tissue such as skeletal muscle. The origin of this growth factor, found principally in the extracellular matrix of growing or regenerating muscles (9). is widely misunderstood. In this study, we postulated that myogenic cells

generate

such growth factors, at least in part. We therefore examined developing muscles and cultured myogenic cells to see if they were capable of transcribing genes encoding FGF. EXPERIMENTAL

PROCEDURES

Bovine muscle RNA preparation: Four muscles, longissimus dorsii (LD) , tensor faciae latae (TFL), masseter (M) and rectus abdominis (RA),were taken from bovine fetuses aged 103,152 and 250 days, and from newborn animals. Total guanidinum-LiCl procedure of RNA was isolated by a modified version of the Chigwin et al. (10). Cell cultures: Satellite cells were prepared as previously described (1 l), with the following modifications: rat satellite cells were dissociated from muscle fibers by incubation for 2 hrs at 37°C in 0.15% pronase in Ham’s F12 medium buffered with 1OmM HEPES. After exhaustive washing, satellite cells suspended in Dulbecco Modified Medium containing 10% fetal calf serum and 10% horse serum (Gibco were seeded on gelatinized petri dishes at the concentration of 2~10~ cells per cm1 and incubated in 5% CO2 at 37°C. The L6 cell line was grown as described previously (11). In some experiments, O.luM of the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA, Sigma) was added to cultures for 2 to 24hrs. The drug was prepared in DMSO (Merck) and the final concentration of the solvent represented 0.01% in TPA-treated cultures. The same DMSO concentration was added to control cultures. DNA synthesis was measured on 4 different cultures after a 6 hr incubation with 0.2 uCi /ml of methyl-3H-Thymidine (87Ci/mmol, Amersham). Cultures were dissolved in 0.5M NaOH. Incorporated radioactivity was determined by liquid scintillation counting. Proteins were determined according to Lowry in aliquots of the same samples. Under our experimental conditions, the soluble 3H-Thymidine which was not incorporated into DNA constituted 10% of total radioactivity. The activity of creatine phosphokinase (CPK) was measured with a Merckotest kit (Merck). Cells were lysed in 50mM Tris HCl pH 7.5, and 1mM phenyl methyl sulfonyl fluoride (Sigma), and extracts were sonicated before the assays. Endogenous myokinase was inhibited by the addition of 3mM Ap5A:pl,p5-Di(adenosine-5’)pentaphosphate (Sigma). Each point represent 3 separate measurements. Cellular mRNA isolation: Satellite cells were lysed during proliferation or differentiation into myotubes, in buffer containing 4M guanidinum isothyocyanate, 1M l3-mercaptoethanol and 0.5% Sarkosyl. Total RNA was isolated by ultracentrifugation on a CsCl cushion according to standard procedure (12). Poly (A+) mRNAs were purified by affinity chromatography on Hybound-Map (Amersham) according to supplier’ s instructions. Probes: The Acidic FGF cDNA probe is a 4.0 Kb bovine aFGF cDNA cloned into lambda gtll (13). The cDNA insert, which sequence contained the entire protein coding sequencefor bovine aFGF (14). The basic FGF cDNA probe was a gift from J. Abraham and J. Fiddes (California Biotechnology Inc. Mountain View, CA). This probe was a 1.4 Kb bovine bFGF cDNA cloned into pBR 322 (15). The cDNA insert contained the entire protein coding sequence for bovine bFGF. The (GAPDH) obtained from glyceraldehyde-3-phosphate dehydrogenase cDNA probe 1206

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AND BIOPHYSICAL

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Dr. Blanchard (Depart. Molec. Biol., Montpellier Univ., France) was a 1.3 Kb rat GAPDH cDNA cloned into PUC 18 (16). This probe was used as internal standard and also to estimate the loading and transfer efficiency of RNA samples. Northern Blot analysis: Northern blot of Poly (A+) mRNA purified from satellite cells was prepared as previously described (13) except that RNA was transferred onto Genescreen Plus membrane (DuPont de Nemours, France). The RNA blot was prehybridized at 42’C in a buffer containing 50% formamide. 1% SDS, 1M NaCl, 10% dextran sulfate and lOOug/ml salmon DNA. Blot was then incubated in the same buffer at 42°C with specific probes labeled with 32P by nick translation ( lo6 cpm/ml). The blots were washed twice for 20 min in 2xSSC at 60°C and processed by autoradiography. Dot blot analysis: Total RNAs extracted from bovine skeletal muscles or Poly(A+) mRNA from rat satellite cells were dissolved in a buffer containing 50% deionized formamide and 6% formaldehyde. Genescreen Plus membranes were loaded, either with 4ug mRNA prepared from satellite cell cultures, or with 2 to 0.25 ug of total RNA extracted from bovine muscles. The exact quantity of RNA loaded onto the membranes was measured by reflection-absorption at 260nm using a Shimadzu scanning densitometer. The RNAs were hybridized as for Northern blot analysis . After hybridization with a specific probe, the blots were washed twice for 20 min in 1xSSC at 42’C for bovine muscle RNAs, and in 2xSSC at 60°C for satellite cell mRNAs . The membranes were then subjected to autoradiography. RESULTSAND DISCUSSION When dot

blots

of RNA extracted from four

muscles of bovine fetuses of

different ages were analyzed with a aFGF cDNA probe (Fig l), aFGF mRNA was abundant in Longissimus dorsii. detected in all four muscles and was especially The strength of its hybridization signal diminished during fetal development and was almost undetectable in neonate muscles. As the amount of RNA loaded onto membranes was UV-controlled, fetal

development

the

was significant.

The

diminution reason

1234

A

B

of why

the

hybridization

signal

1234

a b c d

it C d

a b C

it C

d

d

-1:

C

D

Dot blot analysis of poly(A+) mRNA extracted from fetal and newborn bovine skeletal muscles. Total RNAs extracted from fetal muscles (A: 103 days; B : 152 days and C: 250 days) ‘or from newborn skeletal muscles (D) were analysed by dot blot, as described in Experimental Procedures; to detect acidic FGF gene transcripts, 2ug (1). lug (2). 0.5ug (3) and 0.25ug (4) total RNA purified from (a) longissimusdorsii, (b) tensor faciae latae, (c) masseter and (d) rectus abdominis were hybridized with aFGF cDNA aa probe . 1207

during

aFGF mRNA was present for a

Vol. 166, No. 3,

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1990

AND BIOPHYSICAL

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3H Thymidine pmol Img prot 400

-

300

--

200

--

100

.-

10-2

CPK x U/mg

prot

+0 0

4

2

6

6 days

of

10

12

14

culture

Fig. 2 ; Determination of proliferation and differentiation stages of rat satellite cells in culture. The rate of DNA synthesis was determinedby

measuring 3H-Thymidine incorporation into cells after 6 hr incubation and creatine phosphokinase(CPK) was estimatedin cell extracts at different times after plating, as describedin Experimental Procedures.

longer time in Longissimus dorsii fetal skeletal musclesdo To facilitate

is not known. This result proved, however, that

express aFGF mRNA, especially during

growth .

the study of FGF mRNA expression during the myogenic

process, we used primary cultures of rat satellite cells, which in vitro mimic fetal myogenesis in vivo. Under our standard culture conditions (fig 2). satellite cells in primary cultures proliferated during the first 6 days after plating. 3H-Thymidine incorporation into DNA was maximal at the 4th day of culture. This agrees with the After the proliferative period, data we obtained earlier by image analysis (17). cellular differentiation

commenced

and

fusion took place,

leading to the

formation of myotubes which contracted spontaneously on the 12th day of culture. Differentiation

of

cultures

was routinely

recorded

by

the

appearance of

muscle-specific gene products such as CPK. As a function of these results, RNAs were extracts on the 4th and 14th days of culture. They will be referred to below as proliferating cell and myotube RNAs respectively. Messenger RNAs were analyzed by Northern blot hybridization, with cDNA of

retina bovine aFGF and brain bovine bFGF as probes (Fig 3). The presence of a single aFGF mRNA species was shown in lane A3, where 1Oug mRNA from proliferating cells was applied to the blot (fig 3-A). The hybridization band for satellite cell extracts correspondedto 4.0 Kb, as the aFGF mRNA found in bovine retina (lane A2). An additional 2.5 Kb mRNA was detected in bovine retina, and its characterization is presently under investigation. On the other hand, no hybridization signal was detectable with mRNA prepared from myotubes (lane A4) in spite of the increased quantity of mRNA

applied (14 vs 10 ug).

1208

Vol.

BIOCHEMICAL

166, No. 3, 1990 Under

3-A),

bFGF

labeled

same

conditions

cDNA

probe

was applied

band

corresponded retina

was (lane

the bFGF

of

reflected

from

(lane

the amount

B3). of

of mRNA

firstly,

mRNA

when

they

proliferate,

rat brain

The aFGF cultures (20). RNA

mRNA

of cardiac

As internal

onto

with

membrane

but

of satellite

rmt

did

control,

GAPDH 3

they

not

(18),

intensity

of the hybridization

from

satellite

cells

was

lowfer

signal than

have

A

obtained

with

they mutase

that the probe

).

of F9 teratocarcinoma

The

cultures

do express

differentiated cells with

into

as that of

(19)

and of 10 ug of

4 ug of bovine

6

1234

123

f

GAPDH Ejz. 3 : Nothern blot analysis of poly(A+) mRNA purified from in proliferating rat satellite cells culture, and from ceils into myotubes. mRNAs derived from differentiated Polyadenylated proliferating cells (A3 and B2, 10 ug) and from myotubes (A4 and B3, 14ug). and poly(A+) mRNA extracted from bovine retina (A2 and Bl, 5 ug) used as control, were analyzed by Northern blot, as described in Experimental Procedures. RNAs were sucessively hybridized with aPGF (A) and bFGF (B) cDNA probes. To determine the relative amounts of RNA loaded per lane, blots were rehybridized with a GAPDH plasmid probe. The sizes of the 32P-labeled Hind III restriction fragments of Lambda DNA (Al) are indicated. 1209

It

hybridize

cDNA

cells is about the same size (4.OKb)

myocytes

B2).

we showed

in primary

once

(lane

the 7.0 Kb of bovine

phosphoglycerate

the

(Fig

A faintly

set of experiments

of

(Fig

cells

above

3-B).

extracts

cultures

form

that satellite

(fig

from

in another

muscular obtained

loaded

prove

myotubes.

However,

the

signal

results

cell

differentiated

described

blot

was different

in preparation).

hybridization

as those

proliferating

Extracts mRNA

RESEARCH COMMUNICATIONS

to the same Northern

with

These

primary poly(A+)

the

hybridization

Kb which

probe the

et al, manuscript

intensity

aFGF

with

of

of 6.0

Bl).

cDNA

hybridize

(Castella

detectable

to an mRNA

mRNA

with did

the

AND BIOPHYSICAL

retina

Vol.

BIOCHEMICAL

166, No. 3, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

mRNA. According to Goodrich, there is a 90% homology between bovine and rat aFGF cDNA (21). Thus, the fact that less of the bovine cDNA probe was hybridized with rat myogenic mRNA than with bovine retina mRNA might only mean that less aFGF messenger was present in satellite cells. Secondly, our results show that the expressionof bFGF mRNA was only weak in proliferating satellite cells. A 6.0 Kb mRNA species was found in extracts of these cells as in rat brain (22). Whereas a single 7.OKb bFGF mRNA species was found in bovine retina , two distinct mRNA speciesof 7.0 and 3.7 Kb encoded bFGF in bovine brain (15) and capillary cells (23), and several mRNA species were detected in human cells (24,25). In addition, in cells which had differentiated into myotubes, aFGF and bFGF mRNAs disappeared, suggesting that FGF mRNA was down-regulated during the myogenic process. Contaminating non myogenic cells, presumably fibroblasts. which are unavoidable in satellite cell primary cultures, accounted for

about 10% of the total number of cells which we previously

determined under clonal

culture conditions

(17).

Here, however,

despite the presence of a few fibroblasts which might have produced FGF (24,25.26), it is highly improbable that these contaminating cells, which would no doubt be present in both growing and differentiated cultures, participated significantly in aFGF mRNA expression, because it was confined to proliferating cultures and was not detectable in differentiated cultures. The absence of FGF mRNA in myotube cultures raises the possibility of a causal relationship between proliferation ability and the presence of FGF mRNA, since addition of exogenous FGF is known to keep cells in the proliferation cycle and delay differentiation

(see review l), and since FGF receptors are also known to disappear during myogenesis (27). Several studies have shown that protein kinase C activators such as the phorbol ester TPA alter the expression of various growth factors (28) including bFGF (29.30). We therefore TPA

on aFGF mRNA

concentration

of TPA

investigated the effect of

10s7M expression in growing and differentiated satellite cells. This was chosen because it partially inhibits the proliferation of in preparation). Dot blot analyses of poly(A+) RNA from

satellite cells (Martelly satellite cells treated with TPA or from untreated cells

were performed with

the aFGF bovine cDNA as a probe (fig 4). After 24h of TPA treatment, the level of hybridization in extracts of growing cells rose (fig 4-Al and A2). TPA also induced the expression of aFGF mRNA in differentiated cultures (fig 4-B). The hybridization signal of poly(A+) RNA prepared from the myogenic cell line L6 (fig 4-C) was very weak in proliferating untreated satellite cells (fig 4-Cl) and increased in TPA-treated cells with the duration of treatment. Therefore aFGF mRNA appeared to be positively regulated by TPA. These results also proved that a persistent state of proliferation was not required for aFGF mRNA expression, as TPA treatment increased FGF mRNA expression, not only in proliferating cultures (despite a 40% reduction of proliferation), but also in non-proliferating myotube cultures.

Consequently, the proliferative

capacity

1210

of

myogenic cells

did not

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166, No. 3, 1990

AND BIOPHYSICAL

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Fig. 4 : Dot blot analysis of RNAs extracted from poly(A+) proliferating cells or cells differentiated into myotubes in culture either treated with 1O“M TPA or not treated. Dot blots were analysed for the presence of aFGF mRNA gene trancripts in 4ug poly(A+) mRNA from each of the following: proliferating satellite cells, either not treated (Al), or treated with TPA for 24h (A2); satellite cells differentiated into myotubes, either not treated (Bl), or treated with TPA for 24h(B2); proliferating L6 cells, untreated (Cl), or treated with TPA for either 2h (C2) or 6h (C3). Poly (A+) RNA purified from bovine retina (D) was used as internal control.

necessarily

correlate

underlined

by

significantly

this L6

the

when

the level

with fact

that

stimulated

of aFGF mRNA

proliferating by TPA.

L6 However,

myogenic cell line displayed

cells

expression.

only

expressed

This FGF

was mRNA

compared to other myogenic cells,

several differences including level of

protein kinase C (21), certain L6 sublines did not respond to exogenous FGF stimulation, and differ in their “spare”

receptors for insulin growth factors I and

II (31) and for FGF (Olwin B.B. and HauschkaS.D. unpublishedresults cited in 3, 32 and our personal observations). Here, L6 cells did not express FGF mRNA at the proliferating stage, unlike the satellite cells. In conclusion, we showed that aFGF mRNA was more

strongly expressed

than bFGF mRNA by proliferating satellite cells before their differentiation myotubes. In the satellite cells,

into

the presence of aFGF mRNA coincided with that of

aFGF protein. About 10 times more aFGF was found in 4 day proliferating satellite cell cultures, than in satellite cells which had only just dissociated from muscle or which

had

differentiated

into

myotubes

(Groux-Muscatelli

personal

communication). It was rather surprising to find that myogenic cells synthetized more aFGF mRNA than bFGF mRNA. Acidic FGF with is less ubiquitous than bFGF, was generally found in the nervous system including photoreceptors (33), and more and recently in fibroblasts (26) , cardiac myocytes (34) kidney cells (35) smooth

muscle

aFGF

in muscle

aFGF,

as well

cells

(26).

In addition,

extracellular as bFGF,

However,

matrix. might

bFGF

has more

often

been

detected

than

the present findings suggest that

play a role in myogenesis, possibily through

autocrine action. ACKNOWLEDGMENTS: We wish

to thank

This study was partially C. Rey for

culturing

satellite 1211

supported cells,

Drs

by INSERM J. Abraham

(N0876015). and J.M.

Vol.

166, No. 3, 1990

Blanchard Dreyfus

for for

BIOCHEMICAL

supplying revising

respectively

the english

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the bFGF

editing

and

GAPDH

cDNA

probes,

and

M.

.

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