,“\ic~ro.~ic~ce Le//er.v, I26 ( I99 I ) IX 20 (’ 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940,91/$03.50

18

ADONfS030439409100209L NSL 07729

Stimulation of biosynthesis of nerve growth factor by acidic fibroblast growth factor in cultured mouse astrocytes Takashi ‘Research

One’, Hiroko

Lahorarories,

(Received Key word.v

Acidic fibroblast

16 October

growth

Bovine acidic and basic fibroblast a culture

medium

factor; growth

of mouse astrocytes.

later and was further eightfold

Saito’, Toshimitsu

Yoshitomi Pharmawuticul

sustained

over the control

These results indicate

Basic fibroblast

growth

factors

and bFGF)

The NGF

(aFGF

concentration

for 24 h when aFGF stimulates

was contained.

The content

did not proliferate

the biosynthesis

T. Ono, Research

Industries.

358, Japan.

Ltd..

Laboratories.

7 25 Koyata

3-chome.

Pharma-

Iruma-shi.

Saitama

1991: Accepted factor; mRNA: sttmulated to increase

of NGF

mRNA

astrocytes

without

4 February Astrocyte;

(Jupun)

1991) Proliferation

the release of nerve growth compared

factor

to that of the control

in the astrocytes

until after 72 h when treated

of NGF in cultured

Yoshitomi

started

and Kanji Miyamoto’

und -‘A.,aku P.s~,chrulric~Institute. Fukushimu

dose-dependently

in the medium

]71. Certain growth factors affect the proliferation and expression of proteins of cultured astrocytes [ 17, 18, 2 I]. The fibroblast growth factors (FGFs) studied here have neurotrophic effects on central neurons in vitro and in vivo [I. 19, 23, 241. Since the effects of FGFs on NGF have not been known well, however, this study was undertaken to ascertain their influence on the biosynthesis of NGF. Purified astrocytes were obtained from cerebral hemispheres of fetal ICR mice on day 17 of gestation by the method of McCarthy and de Vellis [ 161. Tertiary cultures of astrocytes were maintained in Dulbecco’s modified Eagle medium (DMEM) (43&2100, Gibco) containing 10% fetal bovine serum (Filtron) on collagen-coated cul-

Correspondence:

iJupunj

factor: Nerve growth

Astrocytes have been reported to affect neuronal functions, differentiation, and maintenance [IO, 13, 141. These cells secrete diffusible trophic factors against neurons in culture [2, 201. Nerve growth factor (NGF) is a neurotrophic factor related to the differentiation and maintenance of peripheral sensory and sympathetic neurons [ 121 and central cholinergic neurons [8]. In the culture system, NGF is released from mouse astrocytes [5]. The release of this factor is influenced by the cell growth phase [6] and by the catecholamine concentration in the culture medium

ceutical

Takeki Okumoto’

Ltd., Suirumu

1990; Revised version received 31 January

level after 4 h. The astrocytes

that aFGF

Kishimoto’,

Industrirs.

treated

with aFGF

with FGFs under the conditions

promoting

(NGF)

cultures

m 4 h

peaked

at

employed.

cell proliferation.

ture vessels. After 7 days, the culture reached confluence and more than 90% of the cells contained glial fibrillary acidic protein. The cells were cultured for 7 days more in DMEM containing 0.5% bovine serum albumin (BSA) (RIA grade. Sigma) [7] to minimize the influence of FGFs and other unknown factors in serum. Finally, the cells were cultured in the medium with either bovine acidic FGF (aFGF) or basic FGF (bFGF) (R&D Systems). The culture medium was changed to a fresh one twice a week. The concentration of NGF in the culture medium was assayed by two-site enzyme immunoassay [I I] with the use of a monoclonal antibody against mouse /?-NGF, the same antibody conjugated with /&galactosidase, and chlorophenol red D-D-galactopyranoside (Boehringer Mannheim) as a substrate. Total RNA was prepared by the method of Maniatis et al. [ 151. Poly(A)+ RNA was separated with Messenger Activated Paper (Takara Shuzo), fractionated on 1% agarose-formaldehyde gel, and then blotted onto a Hybond N filter (Amersham). Mouse NGF cDNA was cloned from the cDNA library of male mouse submaxillary glands. The PsfIcleaved fragment of the cDNA of NGF was recloned into plasmid pGEM3. An NGF cRNA probe was prepared with the use of SP6 RNA polymerase and [LY3ZP]CTP. Northern blot hybridization was done with 50% formamide at 53 C. The concentration of NGF in the culture medium ot confluent astrocytes was assayed after 24 h of incubation

0 t 0.01’

0.1 I

10 L

1 1

O’Y

Fig.

1. Effect of FGFs were cultured

medium containing of NGF

on NGF

either aFGF

in the culture

release

from

in 96-well tissue culture medium

(0)

or bFGF

was assayed

(a).

cultured

astrocytes.

plates with 100 ~1 of The concentration

24 h later.

a

1

I

.

24

16

Incubation time (hours)

FGF concentration (nglml) Astrocytes

I

I

I

0

Figures

Fig. 2. Time course cultured

of NGF

in 80-cm’ culture

or without

(0)

release from astrocytes.

flasks containing

IO ng/ml aFGF.

Astrocytes

IO ml of medium

were

with (0)

Figures are mean k S.E.M. (n=4).

are

mean + S.E.M. (n=3).

with either aFGF or bFGF. The FGFs at concentrations from 0.1 to 10 ng/ml raised the level of NGF in a dosedependent way (Fig. 1). Compared to the control cultures, the NGF levels were 210% and 160% with IO ng/ml aFGF and bFGF, respectively. These data were reproducible. Since FGFs have been reported to cause astrocytes in primary cultures to proliferate [21], we studied the relationship between NGF release and astrocyte proliferation by FGFs. With either aFGF or bFGF at 10 ng/ml, the numbers of astrocytes did not change significantly for 72 h in tertiary cultures of astrocytes (Table I). Our findings indicate that FGFs enhance the NGF release from astrocytes, but do not promote cell proliferation under the conditions employed. The levels of both NGF and NGF mRNA were assayed during culturing of astrocytes with IO ng/ml TABLE

aFGF. NGF release started to increase compared to that of the control around.4 h after the addition of aFGF (Fig. 2). The level of NGF mRNA remarkably increased around 2 h and reached a peak after 4 h of incubation (eightfold over the control level when densitometrically quantified), decreasing thereafter (Fig. 3). These findings show that aFGF stimulates the de novo synthesis of NGF in cultured astrocytes. The direct neurotrophic effects of FGFs on neurons are already known [I, 19, 23, 241. Recently, in rat astro-

Cl23456 * P

d

4

I

EFFECT

OF FGFs ON PROLIFERATION

Confluent

astrocytes

sue culture

of tertiary

plates with DMEM

were counted wells (3.9kO.2

culture

were maintained

containing

72 h after the addition

NGF+

OF ASTROCYTES in 24-well tis-

0.5% BSA. Numbers

of aFGF

or bFGF

a

of cells

to the culture

x IO4 cells/cmz). The values are mean&S.E.M.

(n=4).

Substance

Cell count ( x IV/cm’)

aFGF

(IO ng/ml)

4.3kO.2

aFGF.

bFGF None

(IO ng/ml)

4.1 fO.l 4.2+O.I

0, 2. 4, 6. 8 and 24 h (lanes l-6. respectively)

Fig. 3. Time course changes Poly(A)+

in NGF mRNA

of astrocytes

RNA (0.5 fig) from the cells treated

treated

with

with aFGF

for

and 0.01 ilg of poly(A)+

RNA from mouse submaxillary glands (lane C) were blotted onto nylon filter and hybridized with a “P-labeled NGF cRNA probe.

a

20

cyte cultures, bFGF has been reported to stimulate NGF synthesis [22]. This finding was confirmed with the present study using mouse astrocytes. Moreover, we found that FGFs, known to be mitogenic for astrocytes, induced NGF release without concomitant proliferation of astrocytes under the present conditions, and that aFGF stimulated NGF synthesis by astrocytes as well as bFGF. Although aFGF is reported to be 3&100 times less potent than bFGF in promoting the proliferation of mesoderm-derived cells [3] and the differentiation of pheochromocytoma cells [4], our data indicate that aFGF is not less potent than bFGF in stimulating NGF release by astrocytes (Fig. 1). The mechanisms of the effect remain to be investigated. The minimum effective concentration of FGFs for enhancing the release of NGF, 0.1 ng/ml (Fig. I), was lower than the content of FGFs in the brain. (The yields of aFGF and bFGF from bovine brain were 620-7 IO ng/ g and 37-45 rig/g,, respectively [8].) Spranger et al. [22] reported that intraventricular injection of bFGF increased hippocampal NGF mRNA. It is possible that aFGF and bFGF support neuronal survival and function in the central nervous system indirectly by stimulating the biosynthesis of NGF in astrocytes.

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