Regulation of synthesis and activity of bovine adrenocortical NADPH-cytochrome P-450 reductase by ACTH

Vol. 128, No. 2, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

April 30, 1985

Pages 650-656

REGULATION

OF SYNTHESIS AND NADPH-CYTOCHROME

ACTIVITY OF BOVINE ADRENOCORTICAL P-450 REDUCTASE BY ACTH

Albert Dee*, Grace Bettie Sue Masters** *

Department **

Received

of Biochemistry, Department

February

Carlson”, Cydnie Smith* and Michael R. Waterman*

University Dallas, TX

of Biochemistry, Milwaukee,

of Texas 75235

Health

Medical College WI 53226

Science

Center

of Wisconsin

18, 1985

Primary, monolayer cultures of bovine adrenocortical cells have been utilized to demonstrate that ACTH regulates the synthesis .and thus the activity of NADPH-cytochrome P-450 reductase. The temporal pattern of induction correlates well with that previously reported for cytochromes and P-450c2,, microsomal steroid hydroxylases serviced by P-450 23@e Results of fi vitro translation studies suggest that ACTH . regulates P-450 reductase synms at the transcriptional level and show that the adrenocortical enzyme is synthesized as the mature form. The action of ACTH to induce P-450 reductase synthesis in the adrenal cortex can be mimicked by dibutyryl CAMP, but not by. phenobarbital which induces P-450 reductase synthesis in liver. 0 1985 Academic Press, Inc.

The both

steroidogenic

mitochondrial

mitochondrial

chain

adrenodoxin are

and

steroid

transport

reduced

is

likely

as liver,

lung

tissues

such

steroid

hydroxylases species.

P-450c2,,

purified

arid

their

or liver

microsomal

identical and

localized

of

activities P-450

by

The

to

in the

is

a

respectively).

reductase

be

reconstituted (6-9).

650

form of

these using

The electron and

hydroxylases

reductase

(3-51,

found cortex

reticulum,

contain

an

steroid

adrenal

distinct

via

reductase

endoplasmic

Both

$1.50

0 1985 by Academic Press. Inc. of reproduction in any form reserved.

P-450

involve

(flavoprotein)

P-450

microsomes

which

can

the

testis

reactions.

NADPH

microsomal

In the

kidney.

and

reductase

NADPH-cytochrome

adrenocortical

each

( P-450, Ta and

Cowright All rights

are

Bovine

CZl-hydroxylase,

0006-291X/85

via

reduced

(1,21.

ovary

hydroxylation

adrenodoxin

protein)

NADPH

which

are

of

cortex,

steroid

hydroxylases

consisting

by

in adrenal

microsomal

(iron-sulfur

flavoprotein

the

pathways

in one

a

other or

two

depending

on

17a-hydroxylase of

cytochrome enzymes

either

have

and P-450 been

adrenocortical

BIOCHEMICAL

Vol. 128, No. 2, 1985 Steroidogenesis (ACTH),

which The

(10). capacity

has both

chronic

and

increased

involves

either

newly

enzyme

to be

(11.13).

In the

present

whether

translation

of RNA

level;

whether

enzyme

cyclic

to

increased

synthesis this

suggesting

the

adrenal

P-450

in a manner

to that

and

other

related

whether

can

regulation

cortex.

RNA

can be

cells,

synthesized

ACTH of

the

from

newly

have

CAMP

been

(such

P-450,7a

ACTH

[and

be

detected at the

to increased

amounts

whether

phenobarbital

is chronically the

which

results regulated

steroidogenic

to

immunoisolating

of

occurs

The

for

leads

thus

as and

CAMP)

NADPH-cytochrome

synthesis

and

pathway.

ACTH

cytochromes

adrenocortical

leads

described

and

analogs of

examined

with

the

thus,

steroidogenic

P-45OC2,,

actions

pathway

of this

isolating

chronic

the

reductase

and

by

and,

reductase;

NADPH-cytochrome similar

or

in

synthesis

cells

protein

synthesis

that

of optimal

immunoisolating

of

steroidogenic

of components

cellular

AMP

increase

on the

P-450,,a

and

we have

P-450 in

acute

by adrenocorticotropin

adrenocortical

and

by

increased

NADPH-cytochrome this

the

study

the

reductase;

of synthesis

vitro,

RESEARCH COMMUNICATIONS

maintenance

hydroxylase

-in

lead

regulates

for

total

steroid

mediated

P-450C2,

is required

radiolabeling

Both

cAh!P)

effects

cytochromes

RNA

dibutyryl

chronic

of both

(11,12).

found

is regulated

bovine

by

the

and

cortex

of cultured

synthesized

translating

acute

regulation

synthesis

detected

adrenal

effect

stimulation

Thus,

also

in the

AND BIOPHYSICAL

P-450 by

--in

vitro

transcriptional and

activity can

indicate --in vivo cytochromes

that by

of

induce the ACTH P-450

enzymes.

METHODS

AND

MATERIALS

Primary monolayer cultures of bovine adrenocortical cells were prepared as previously described (14) and grown to confluency. The confluent monolayers were removed from antibiotic-containing medium and 24 hours later an experiment was begun. Cell cultures were maintained in the presence or absence of ACTH (1 PM). At various times following35initiation of ACTH treatment, total cellular protein was radiolabeled with S-methionine (New England Nuclear) as previously described (14). From other cultures total cellular RNA was isolated and translated in a reticulocyte lysate cell-free system (14). Newly synthesized NADPH-cytochrome P-450 reductase was immunoisolated from total newly synthesized proteins obtained by either procedure. Purified pig liver NADPH-cytochrome P-450 reductase and the IgG fraction raised against this protein were prepared as described previously 651

Vol. 128, No. 2, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

NADPH-cytochrome c reductase were (15). activities determined in post-mitochondrial supernatani fractions prepared from cultured cells (16) using standard assay procedures (171. Protein concentrations were measured according to Bradford (18). Electrotransfer of cellular proteins and immunoblot analysis were performed as previously described (11,19). Responsiveness of cell cultures to ACTH was determined by cortisol measurements using radioimmunoassay (20).

RESULTS The

ability

P-450

reductase

Fig.

1.

In

of the

to recognize lane

immunoisolated enzyme, liver

1 it

by

having

this

weight

enzyme

to compete

in

of ACTH

as does

the

two

microsomal

preliminary

evidence

the

the

protein

that

protein liver

of 100 I.L~ purified

pig

2).

purified

Thus,

and

the

was

concluded

RNA

from

based

ability

on

the

of

purified

that

anti-pig

to study

the

bovine

size

RNA

as

the

occurs

which

isolated

treated

with 2-fold.

mature,

ACTH, Also, purified 652

rate

24 hours

of synthesis

increase

is

in synthesis

P-45O,7,

(111,

To

obtain

and

services. might

adrenocortical system. P-450

reductase,

is maximal

at 24 hours.

the

synthesized

enzyme.

in

(12) it

newly

shown

at

translation

NADPH-cytochrome

is

12 and

in synthesis

from

P-450

approximately

P-450C21

increase

ACTH

the the

optimizes

the

was

to between

Thus,

cells.

in an --in vitro

synthesized

is approximately

response

hydroxylases

as to whether

NADPH-cytochrome

At 24 hours,

reductase

steroid

cells

of

in

of cytochromes

synthesis

newly

cells

in control

synthesis

in transcription,

same

in

pig

be utilized

synthesis

in synthesis

P-450

time

increase

it

could

treatment.

that

NADPH-cytochrome

using

protein

the

increase

2 times

to direct

of

adrenocortical

maximal

approximately

seen

the

Addition (lane

is illustrated

radiolabeled

with

of 78,000.

reductase

NADPH-cytochrome

predominant

immunoisolation,

pattern

initiation

increase

the

immunoisolation

this

liver

enzyme

co-electrophoreses

P-450

bovine

The

following

that

pig

adrenocortical

immunoisolated

with

temporal

reductase 2.

seen

against

enzyme.

The

Fig.

bovine

weight

the

NADPH-cytochrome

adrenocortical

the

this

of

DISCUSSION

prepared

antibody

inhibits

molecular

of

is

a molecular

reductase

liver

antibody

AND

This

agrees

the

same

involve

cells

and

an used

In Fig.

3 it is

determined Again,

the

reductase with

earlier

is

Vol.

128,

0

'1

No. 2, 1985

BIOCHEMICAL

0

2

2

AND

BIOPHYSICAL

RESEARCH

03

1234567

12

COMMUNICATIONS

34

56

7

Immunoisolation of bovine adrenocortical NADPH-cytochrome P-450 w: re uctase using an IgG fraction raised against pig liver NADPH-cytochrome of newly synthesized, radiolabeled P-450 reductase. Lane 1, immunoisolation P-450 reductase from bovine adrenocortical cells treated with ACTH (1 i.rM) for 36 hours. Lane 2, Identical to lane 1 except immunoisolation was carried P-450 reductase which comp&es out in the presence of 100 ug pig liver against the radiolabeled enzyme. The arrow indicates the position of migration of the purified pig liver enzyme on the same gel as determined by staining with Coomassie blue. lmmunoisolation of newly synthesized bovine adrenocortical H cytochrome P-450 reductase from cells maintained for various tines in the presence or absence of ACTH. Lane 1, 0 time; Lanes 2.4 atld 6 from cells maintained for 12,24 and 36 hours, respectively, in the absence of ACTH; Lanes 3.5, and 7 from cells maintained for 12.24 and 36 hours. respectively, in ihe presence of ACTH (1uM). In all samples, immunoisolation The arrow was carried out from the same amount of total radioactivity. indicates the position of migration of purified pig liver P-450 reductase on the same gel.

!i?i!s:

lmmunoisolation of newly synthesized adrenocortical P-450 reductase directed by RNA isolated from bovine adrenocoitical cells maintained in the presence or absence of ACTH for various times. Lane 1, 0 time; Lanes 2 and 3 from cells maintained for 24 and 48 hours, respectively, in the absence of ACTH; Lanes 4-7 from cells maintained for 12,24,36 and 48 hours. resoectivelv, in the oresence of ACTH In all samples, immunoisolatidn was carried out fro& the same amount (1O.J). of total radioactivity. The arrow indicates the position of miaration of purified pig liver P-450 reductase on the same gel.

reports synthesized

indicating as

that its

mature

rat

liver

form

(21).

tJADPH-cytochrome

653

P-450

reductase

is

Vol. 128, No. 2, 1985

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE

1

NADPH-Cytochrome c t?eductase Activity in Post Mitochondrial Supernatant Fractions from Cultured Bovine Adrenocortical Cells Time

-ACTI

+ACTH

(nmollmlnlmg 12 h 24 h 36 h

48 h 60 h

-

I-resh gland

In

both

synthesis

of

2-fold

types

approximate appears the

activity

of

in

2-fold

activity

we

and

in

binding

reductase after

36

hours.

of both

FAD

seen

However,

initiation

of ACTH

FMN,

the

Table

well

activity.

this

increase

in

correlates

Perhaps and

a modest

As

parameters

24 hours

at

only

reductase.

synthesis

about

observe

P-450

increase

maximizes

synthesis

5.8 5.2

experiments,

the

to maximize

4.0 5.6 7.0

= 15.2 rrniol/mln/mg

NADPH-cytochrcme

increase

proteln)

2.4 3.4 3.1 4.2 3.3

in 1 the

with

the

an

synthesis

treatment

difference cofactors

while

is

related

required

for

to this

enzyme. It

is

interesting

approximately activity induction

On

of

the

has

cultured arld

seems

cyclic

that

reductase

the is limited

The

results

reductase

provide

further

limited

to

2 and

phenobarbital

NADPH-cytochrome this

in

lung

xenobiotic

as

determined

increase

As

effect

of

no

shown

P-450 by

the

P-450 has

(23).

phenobarbital

on

the

Figure

reductase

of this

4,

levels

analysis, enzyme.

on

to

reductase

effect in

immunoblot

level

leads

in

while Thus,

cytochrome

it

P-450

to liver. presented

in

this

is synthesized indication liver.

NADPH-cytochrome (Figs.

liver,

NADPH-cytochrome

analogs

inductive

rat

hand,

on

cells

in of

activity

no effect

AMP

that

other

adrenocortical

ACTH

note induction

reductase

phenobarbital

P-450

2-fold

a

(22).

to

that

that

form

phenobarbital

reductase this

indicate

as the mature

Furthermore, P-450

3) and

study

effect

it is

NADPH-cytochrome

in adrenocortical induction

is

regulated

is mediated 654

that

of

in adrenal by

this

that

shown

cyclic

tissue

and

enzyme

synthesis cortex

AMP

(Fig.

by

is of

ACTH 4).

In

Vol.

128,

BIOCHEMICAL

No. 2, 1985

AND

12

lmmunoblot analysis ovme adrenocortical cells Lane 1, no for 24 hours. Lane 3, dibutyryl (1 NJ; phenobarbital treatment (2 migration of purified pig liver

respect,

hydroxylases their

and

stimulating

P-450

reductase

thought

to

this

in

factors

levels

and

is

Presumably,

enzyme.

(phenobarbital)

CAMP-mediated regulate

( 10,24)

.

factors),

found

of

for

Likewise,

in

have

interesting

gene

has

one the

this

in

tissue

to

hand, other

maintenance

found

in is

the

and hand,

to

be

physiological and

thus

induce

reductase

which

examine

cells, to

is

microsomal

thought

capacity

of

granulosa

been

enzyme

the

steroid

P-450

any

the

be

the

on

ACTH

analogs

addition,

this

adrenocortical

NADPH-cytochrome

will

, on

expression

require

CAMP

In it

other

which

enzyme

found. Thus,

like

(19,25).

ubiquitous

tissues.

xenobiotics or

an

P-450 all

enzymes

hormone synthesis

be

cytochrome same

related

COMMUNICATIONS

34

responds

endogenous

optimal

follicle

enzyme

the

RESEARCH

of NADPH-cytochrome P-4SO reductase levels maintained in the presence of various effecters treatment (control); Lane 2, ACTH treatment cyclic AMP treatment (1 mM); Lane 4, mM). The arrow indicates the position of P-450 reductase.

Pi?:

this

BIOPHYSICAL

to gene

be encoding

regulated factors tissue

by (cAMP specific

gene.

ACKNOWLEDGM,ENTS

(BSM)

This work and by

was Grant

supported l-624

by from

The

USPHS grants Robert A. 655

AM Welch

28350 (MRW) Foundation.

the

and

HL30050

Vol. 128, No. 2, 1985

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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