Endocrinol Japon

ACTH

1992, 39 (4), 377-383

Increases

Expression

of

c-fos,

in the Dexamethasone-treated

c-jun

and ƒÀ-actin

Genes

Rat Adrenals

MOTOTSUGUOHNO*,**, HISAOSEO*, TSUNEOIMAI*,**, YOSHIHARUMURATA*, NORIHIROMIYAMOTO*, YASUYUKI SATOH**, HIROOMIFUNAHASHI**, HIROSHITAKAGI** ANDNosuo MATSUI* *DepartmentofEndocrinologyand Metabolism , Research InstituteofEnvironmentalMedicine,NagoyaUniversity,Nagoya 464-01, and **TheDepartmentof SurgeryII, Nagoya University School of Medicine, Nagoya 466, Japan

Abstract. Our recent finding that ACTH increases c-fos mRNA in the adrenal gland of hypophysectomized rats indicates that the gene product FOS may play an important role(s) in mediating the action of ACTH.

However,

hormones

other

hypophysectomy than

ACTH

dexamethasone-treated

employed

and

in

may modify

the

that effect

study

causes

of ACTH.

the

disappearance

Thus,

in the

of

present

trophic

investigation

,

rats were used. Since FOS functions only when it dimerizes with JUN (the

product of c-jun gene), the changes in the levels of c-fosand c-jun mRNAs were studied together with that of ƒÀ-actin

mRNA

which

is also

affected

by

ACTH.

Northern

blot

analysis

was

employed

to determine

the

mRNA levels. It was demonstrated that ACTH increases the mRNAs coding c-fos and c-jun in the adrenal glands of dexamethasone-treated, ACTH-suppressed rats. The c-fos mRNA was not detectable before the

ACTH

maximum

being

After observed

ACTH

administration,

at 30min

after

ACTH.

the mRNA At 180min

levels post

were

transiently

ACTH,

the level

increased returned

the unstimulated

level. The mRNA coding c-jun was detectable before ACTH administration

increased

after

post

rapidly

ACTH

was

approximately c-jun

and ƒÀ-actin

Key

ACTH cortical

administration. level

words:

c-fos,

ACTH

still

with maximal

higher

than

the

stimulation unstimulated

at 30min. level.

However, The

the mRNA

changes

, to

and it also

level at 180min

in ƒÀ-actin

mRNA

were

the same as those of c-jun mRNA. These results suggest that increased expression of c-fos, genes

by

ACTH

c-jun, ƒÀ-Actin,

stimulates steroidogenesis cells and their proliferation.

Rat

may

adrenal,

of the When

play

an

important

role

in

ACTH.

adrenorats are

mediating

its

(Endocrinol

steroidogenic promotes

action

Japon

enzymes DNA

39:

the

adrenals.

377-383,

1992)

(cytochrome

synthesis

and the

tion to the animals results in increased secretion of corticosterone and hypertrophy of the adrenocor-

increases c-fos mRNA in the adrenal

tical cells. stimulates

product of the c-fos gene [FOS] may play an

also for

Received: March 6, 1992 Accepted: May 15, 1992 Correspondence to: Dr. Hisao SEO, Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya

464-01, Japan.

ACTH-mediated

not been elucidated. we showed that ACTH

hypophysectomized

important

these

P-450) However,

mechanism

It has been shown that ACTH the transcription of mRNAs coding

in

[1-5].

hypophysectomized or ACTH secretion from their pituitary is suppressed by dexamethasone, the adrenals undergo atrophy. ACTH administra-

effects has Recently,

involved

on

rats

and

role(s) in mediating

suggested

ACTH

transiently

gland of that

action

the

[6].

Since FOS has been shown to dimerize with JUN (a product of the c-jun gene) and transactivates gene expression

by binding

to the

specific

DNA

sequ-

ence [7-10], changes in the expression of the c-jun gene were studied in the Hypophysectomy employed

present in the

investigation. previous study

OHNO

378

impairs the secretion of not only ACTH but other trophic hormones and could modify

et al.

Rats were killed was collected for

also the

effect of ACTH on adrenocortical cells. Thus, in this study, dexamethasone-treated rats were used instead of hypophysectomized rats.

sterone. cleaned

by decapitation and trunk blood later determination of cortico-

The adrenal of adherent

mediately

in

liquid

glands were fat, weighed,

nitrogen

and

excised frozen

st0red

at

and im-80℃

for RNA isolation.

Materials Animal

and

Preparation of RNA

Methods

Total

treatment

tion Male Wistar were

rats,

purchased

Shizuoka

weighing form

the

Experimental

Cooperative

approximately closed Animal

Union

of

was isolated acid

in a single

guanidium

method

step

the

Shizuoka,

greater

than

extrac-

thiocyanate

[11]. The amount

phenol

of RNA was

estimated by optical density at 260nm. of absorption at 260 versus 280nm

Agricultural

(Hamamatsu,

an

chloroform

180g,

colony

RNA

by

The ratio was always

1.75.

Japan). The rats (four per cage) were maintained under

controlled

temperature

(26•Ž)

(12h of light and 12h of darkness).

and

Probes

lighting

Rat pellets and

Human

water were given ad libitum. Each group consisted of four

rats.

To

all rats

[13]

and

c-fos cDNA [12], human c-jun cDNA chicken ƒÀ-actin

cDNA

[14]

were

used

as

except the control group, dexamethasone (Decadron, Banyu, Tokyo, Japan; 8mg/2 ml) was

probes. The respective cDNA inserts were purified by electrophoresis after digestion with appropriate

intraperitoneally

restriction

administered

at

a

dose

of

4

enzymes.

mg/kg body weight (BW) for three days (Fig. 1). As shown in Fig. 1, ACTH (Cortorosyn, Daiichi,

with

[ƒ¿-32P]cICTP

land

Nuclear,

Tokyo, Japan;

using

toneally killed

25IU/2 ml)

at a dose at

5.

15.

was given intraperi-

of 50IU/kg 30.

60

and

BW.

The

rats

180

min

after

were

a

Purified (SA,

Boston,

random

(Boehringer

cDNAs

were

3,000Ci/mmol; MA)

to

primed

Mannheim,

labelled.

New

about

DNA

Eng-

108cpm/ƒÊg,

labelling

Germany)

kit

[15].

the

Analysis of mRNA

injection of ACTH.

Northern analysis

blot hybridizations

of

mRNAs.

were

Twenty ƒÊg

of

employed RNA

for

extracted

from the adrenal glands of rats was subjected to electrophoresis after

through

denaturation

1% (w/v) agarose

for

60min

at

50•Ž

in

gels 1M

glyoxal, 50% (v/v) dimethylsulphoxide and 10mM phosphate buffer (pH 7.0). After electrophoresis, the RNA

was transferred

from

the gel to a Magna

Graph Membrane (New England Nuclear, Boston, MA, U.S.A.) according to the manufacture's instructions [16]. The membrane was prehybridized in

a solution

mM

containing

NaCl,

phosphate;

pH

Denhardt's Fig. 1.

Experimental protocol. To all rats except the control group, dexamethasone was administered intraperitoneally at a dose of 4mg/kg BW for 3 days. Twenty-four hours after the

final dexamethasone, ACTH (50IU/kg BW) was injected intraperitoneally. Then, the rats were

killed

at

5,

15,

30,

60

and

180

min.

1mM 7.4),

5•~SSPE

EDTA

(1•~SSPE=180

and

5×Denhardt's

solution=0.02%

polyvinylpyrrolidone

10mM

and

Sodium

solution

(1×

ficoll, 0.02% (w/v) 0.02%

(w/v)

bovine

serum albumin (fraction V, crystallized and globulin free; Sigma, St Louis, MO, U.S.A.)), 0.1% (w/v) sodium dodecylsulphate (SDS), herring sperm DNA

nheim,

(0.1ƒÊg/ml,

Germany)

Boehringer-Mannheim,

and

50%

Man-

(v/v)

formamide

ACTH INDUCTION

(Merck,

Darmstadt,

performed ing 5 x

SSC

sodium

citrate;

0.1%

SDS,

mide

and

cpm/ml The

Table

was

pH

7.0),

2 •~

sperm

Denhardt's

DNA,

32P-labelled

membranes

50•Ž

in

finally

(v/v)

forma-

probes

at

5

X-AR

were

4

for

pre-existing

(106

SDS

tometry

with

an

Medica

Corticosterone

assay

Serum

corticosterone

radioimmunoassay

Japan)

on

2

were

in

the

0 .1

exposed

•~

(ƒÀ-actin)

to Values

membranes other

h.

in

0.1 •~

by

densi-

*,

the

means•}SD.**,

P