Selective inhibition by sodium butyrate of the glucocorticoid induction of glycerol phosphate dehydrogenase in glial cultures

Vol.

93, No.

April

29,

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1980

INHIBITION

SELECTIVE

1297-1304

BY SODIUM BUTYRATE OF THE GLUCOCORTICOID INDUCTION

OF GLYCEROL PHOSPHATE DEHYDROGENASE IN GLIAL CULTURES Daniel

Weingarten

and Jean de Vellis

Laboratory

of Nuclear Medicine and Radiation Mental Retardation Research Center, Brain Research Institute, and Departments of Anatomy and Psychiatry, UCLA School of Medicine, University of California, Los Angeles, Los Angeles, California 90024

Received

March

Biology,

4,198O

SUMMARY: We examined the effect of millimolar concentrations of the short chainfatty acid, n-butyrate, on the induction of two cytoplasmic enzymes in the C6 rat glioma cell line. Hydracortisone addition to the culture medium produced a 2- to 3.5-fold induction of glycerol phosphate dehydrogenase after 24 h, and norepinephrine produced a E-fold increase in lactate dehydrogenase activity in this time period. Five mM sodium butyrate completely abolished the glucocorticoid-mediated response, while the catecholamine-mediated response was unaffected. This selective inhibition was rapid and reversible, and did not affect the level of cytoplasmic steroid binding. Furthermore, high doses of hydrocortisone did not overcome the inhibition. Finally, this phenomenon was also seen in primary cultures of pure oligodendrocytes.

INTRODUCTION In a variety sodium

butyrate

lism,

effect

exert

of NaB shared

diverse

by all

lines,

histone

activity hyperacetylation

histone

acetylation

is

(8,9).

Recent

on NaB-induced

the

nucleosomal

NaB, sodium Abbreviations: HC, hydrocortisone.

proteins

The suggestion

role

shows that

in regulating

structure butyrat6;

types

underlies

changes.

work

cell

metabo-

specific

biochem-

reported (6)

to date

exists

any of the wide

has been made that

GPDH, glycerol

due to yet

that

number

of

an important

transcriptional

hyperacetylated

has been altered

is

most probably

proof

chromatin

of

morphology,

The only

No conclusive

cellular

activity

(l-5).

histone (7).

concentrations

upon cellular

vertebrate

observed for

millimolar

effects

of nucleosomal

of deacetylase

NaB-induced

cell

and gene expression

hyperacetylation

inhibition the

(NaB)

differentiation,

ical the

of vertebrate

chromatin

(10)

by the criterion

of

phosphate

dehydrogenase;

0006-291X/80/081297-08$01.00/0

1297

Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

93, No.

4, 1980

BIOCHEMICAL

DNAse I cleavage, attack

in which

is generally

activity

By this

hyperacetylation These rate

observations

evidence

supports

of their

cytoplasmic

While

the

it

alter

view

nature

that

of and accessibility

via

a cyclic causes

increased

GPDH),

dehydrogenase in the

(HC) and related

rat

'acceptor'

the most

interest with act

rise protein

in the

laboratory

C6 glial

tumor

glucocorticoids

--de novo RNA and protein (16,17),

and a classical

presence

of 5 mM NaB, the

synthesis, steroid increase

mRNAs (12).

is

still

role

(13),

(e.g.,

of their A second

(see

(16).

is

receptor-mediated in GPDH level

1298

of They

processes AMP

references).

enzyme,

inducible

Furthermore,

the continual

class

of cyclic

15 for

cytoplasmic

line

modifying

transcriptional level

by

of events.

glycerol

NAD+Z-oxidoreductase,

cell

(14).

is altered

series

intracellular

with

elucidated.

interactions

a different

of

directing

sites)

to be precisely

in-

a matter

candidate

sites.

(glycerol-3-phosphate:

interaction

respective

nuclear

the

body of

sites,

possibility

phosphorylation

the

DNA binding

to regulate

and -in

'acceptor'

in the

through

the

--in vivo

via

site

accessory

alter

The great

promising

has yet

and appear

in our

proteins'

nuclear

interaction

AMP-regulated

nuclear

of protein-DNA

our

receptors

types.

to the critical

catecholamines,

Specifically, phate

of these

function

increased

to membrane

which

by the

by hormonal

both

of hormones,

specific

the specificity

receptor's the

with

NaB could

to be regulated

cell

an important,

accessory

that

further

the steroid

bind

transcriptional

transcription

of synthesis,

class

DNA is

playing

of this

rate

this

of the

appears

known

in different

of transcription

specificity

The knowledge

the

that

proteins

hormones,

whether

proteins

chromosomal

NaB (10)

us to investigate

receptors

the chemical

The nature

to DNAse I

increased

to increase

of proteins the

rate

controversy,

led

hormones

of a number

creases

with

NaB is thought

of specific

Steroid

vitro,

COMMUNICATIONS

of DNA in chromatin

to be correlated

test

RESEARCH

mechanism.

of synthesis

agents.

BIOPHYSICAL

susceptibility

believed

(11).

AND

phos-

EC 1.1.1.8;

by hydracortisone this

presence

induction of the

mechanism induced

(18).

involves steroid In the

by hydracortisone

Vol.

93, No.

4, 1980

BIOCHEMICAL

was completely

abolished.

(EC 1.1.1.27)

AND

In contrast,

by catecholamines

(16)

BIOPHYSICAL

the

RESEARCH

induction

was not

COMMUNICATIONS

of lactate

affected

by this

dehydrogenase concentration

of NaB. MATERIALS

AND METHODS

Cells and Cell Culture: The C6 glioma cell line was originally established from a chemically induced tumor in rat brain (19). The 2BD subclone was used in these experiments. All cultures were grown at 37°C without antibiotics in a humidified atmosphere of 95% sir/5% C02. Culture medium was Ham's F-10 containing 10% fetal calf serum (Irvine Scientific Co., Irvine, CA). Medium was changed 1 d prior to any experimental treatment. All drugs were first added to the medium, the pH adjusted to 7.4, and the medium then filter-sterilized prior to addition to the cultures. All experiments were carried out on confluent cultures (lo-12 d). Primary cultures of pure oligodendrocytes from fetal rat brains were prepared as described (20) and maintained in basal medium Eagle's (with Hank's salts) in the same atmosphere as C6 cells. Enzyme Assays: GPDH and lactate dehydrogenase determined at 30°C (17.21). One unit of enzvme is which causes the oxidation of 1 nmol of NADHjmin at is expressed as units of enzyme activity/mg of total determinations (22) and enzyme assays were done in as an average + standard deviation.

activities were defined as that amount 30°C. Specific activity Protein cell protein. triplicate and expressed

Cytoplasmic Receptor Assay: A high speed cytosol preparation (105,000 x g) was made from C6 cells treated with or without 5 mM NaB. The amount of specific binding of [3H]dexamethasone, corrected for protein taken from cell homogenates, was determined as previously reported (23), except that the charcoal used to separate free from bound hormone was coated with albumin to prevent adsorption of cytosol proteins on the charcoal. C6 cells were incubated Incorporation of E3H]leucine into Total Protein: for 30 min at 37'C with 2-4 uCi/ml [3H]leucine in leucine-free Ham's F-10 medium supplemented with 10% dialyzed fetal calf serum and 0.26 pg/ml leucine. Cells were processed as previously described (17). Whole Cell Uptake 10 ~J.M[3H]hydrocortisone presence or absence of cold phosphate-buffered then sonicated for 20 scintillation counting

of [3H]hydrocortisone: C6 cells were incubated with in complete medium for 1 h at 37°C either in the 5 a$1 NaB. Cells were then washed 4 times with icesaline, harvested in phosphate-buffered saline, and of this suspension were taken for liquid s. Aliquots and protein determination. RESULTS

Effect of C6 cells

of Sodium

Butyrate

increased

GPDH activity

mM NaB in the GPDH basal with

medium resulted

level

remained

5 mM NaB followed

on GPDH Induction: nearly

in a complete

unaffected. by a rapid

4-fold block

Pretreatment medium wash did

1299

Hydrocortisone over

treatment

of the induction of the not

Five

a 24 h period.

inhibit

cells

while for

the

24 h

subsequent

GPDH

Vol.

93, No.

BIOCHEMICAL

4, 1980

AND

EIOPHYSICAL

RESEARCH

COMMUNICATIONS

1.5

N

'0 x .5 $ 1.0 h E"

0

2 g 0.5 (3 In f

1= lst24hrs2nd24hrs-

0

2

’

DAY 2 DAY 0 DAY I NaB NaB HC HC HC +NaB +NaB 1. Effect of 5 mM Na8 on the hydrocortisone (1.38 PM) induction of C6 cells. In two experimental conditions (5th and 6th vertical cells are pretreated for 24 h with NaB. The solid dash (-) simply changing the medium. The first vertical bar represents basal GPDH while the 2nd vertical bar shows 24 h hydrocortisone-induced GPDH The 3rd vertical bar shows the effect of NaB on basal GPDH levels. vertical bar shows GPDH levels 24 h after the concurrent administraNa8 and hydrocortisone.

HC

Figure GPDH in bars), represents levels levels. The 4th tion of

NaB

Basal GPDH levels at Day 0 (start of experiment) and Day 2 (48 h later in confluent C6 cells exposed to medium alone are shown (0). Hydro=F cortisone (1.38 PM)-induced GPDH levels on Day 1 (24 h induction) and Day 2 (48 h induction) are shown (0). The GPDH levels of cells first exposed to hydrocortisone for 24 h, then washed with medium, and finally kept in medium for 24 additional h are shown (A). The GPDH levels of cells first exposed to

hydrocortisone for 24 h, then washed with medium, and finally medium containing 5 mtl NaB are shown (A).

induction effect

by hydrocortisone of NaB appears

the enzyme were This

indicates

analysis

that

receptors,

the

50 $1;

this

would

them to leak

To rule result

concentration

had no effect

24 h challenge

the

induced

levels

of

NaB (Fig.

extracellular

a concentration

media. of NaB as low as (Fig.

3),

of NaB on hydrocortisone

a partial

saturation

blockade

1300

2).

made GPDH

an effect

of hydrocortisone upon the

Thus,

newly

of enzyme induction

out

in only

the

in

1).

with

inactivate

into

that

inhibition

(Fig.

Furthermore,

preferentially

reveals

a significant

metabolism

24 h period

by a subsequent

cause

0.1 mM was ineffective.

following reversible.

NaB does not

nor

Dose-response 0.5 mM caused

to be fully

unaffected that

enzyme molecules

in the

maintained

while

of glucocorticoid

in the medium was raised of GPDH induction

(data

not

to shown).

Vol.

93,

No.

4, 1980

BIOCHEMICAL

HCNaB-

AND

+ -

EIOPHYSICAL

+ 5mM

+ ImM

RESEARCH

+ 0.5mM

COMMUNICATIONS

+ O.lmM

Figure 3. Effect of NaB concentration on the hydrocortisone induction of GPDH in C6 cells. Cultures were treated with 1.38 PM HC for 24 h. NaB was added to the cultures simultaneously with HC.

The butyrate

inhibition

of the

5 mM NaB and hydrocortisone an initial

challenge

with

was not different

added

replaced

bined

the

with

in which

of complete

notion

of the

to occur

GPDH level

the

initial

rapidity

assayed

for

24 h after 24 h later

24 h (Fig.

of induced

of the

When

hydrocortisone

media

inhibition

rapidly.

to a culture

the

hydrocortisone-free

knowledge

the 5 mF1 NaB, this

appears

simultaneously

hydrocortisone,

from a culture

was simply with

were

induction

challenge 2).

Com-

GPDH levels

inhibition

is

with

further

strengthened. Toxicity absolutely

no effect This

shown). highest

of Sodium

upon

suggests

that

effect

is

the

absence

also

overall

suggests

not the

result

Receptor the cytosol

that

1, 6, or 24 h with

synthesis

is

while

rapid

the

of the

(data

unaffected

at the

reversibility

toxicity.

of

In addition,

induction response

not

(Table is

1)

selective

and

of [3H]dexamethasone

to

toxicity.

specific

binding

was completely

5 mM NaB in the medium

1301

for

steroid

1, 6 or 24 h had

proteins

dehydrogenase

NaB-induced

in C6 cells

into

an argument

lactate

The --in vitro

Studies: receptor

with

inhibition

of nonspecific

incorporation

experiments,

of the the

to 5 mM NaB for

protein

not consistent

of inhibition

further

Exposure

[3H]leucine

NaB dose used in these

the

either

Butyrate:

unaffected (data

not

by treatment shown).

at

Vol.

93, No.

BIOCHEMICAL

4, 1980

Table

1.

AND

Differential effect lactate dehydrogenase in C6 cells

of

Controlb Lactate Dehydrogenasea

aExpressed b

Cultures

'Cultures d

5 mM NaBe

2106

1848

80 f 11 cell

+ 105

46 t 2

+ 50

34 * 2

protein.

receive

only

a medium

change.

receive

3 PM norepinephrine

and

1.38

PM hydrocortisone

for

24 h.

3 pM norepinephrine and 1 mM sodium butyrate.

1.38

PM hydrocortisone

for

24 h

of

eCultures in the

receive presence

3 FM norepinephrine and of 5 mM sodium butyrate.

1.38

pM hydrocortisone

for

24 h

values

Whole uptake

are

Cell

of

expressed

Uptake

the

Effect

in

of

Sodium

the

including

pure

cultures

central

of

of

units.

inhibited

the

not

for

The

simultaneous

5-fold

induction

effect

in

Primary

GPDH

glucocorticoid

the

whole

Cultures

is

cell

of (20).

nervous

localof

the

presence

the

is

(21,25), treatment

specific

in

GPDH

tissue

Hydrocortisone

h increased

of

exclusively

induction

cultures

of

on

shown).

system,

primary

24

f S.D.

no

Induction

nervous The

has

oligodendrocytes

cultures

200

GPDH

(24,25).

oligodendroglial to

(data on

systems

3 cultures

NaB

steroid Butyrate

various

of

[3H]steroid:

oligodendrocytes in

totally

of

In

expressed

40

as an average

radiolabeled

Oligodendrocytes:

from

+ 100

1 mM NaBd

receive presence

'All

of

1880

of

Cultures in the

f

ized

whole

COMMUNICATIONS

sodium butyrate on the induction and glycerol phosphate dehydrogenase

38 f 3

as units/mg

RESEARCH

InducedC

990 t 20

Glycerol Phosphate Dehydrogenasea

BIOPHYSICAL

activity

medium

of

of

5 mM NaB

GPDH.

DISSCUSSION The

hydrocortisone

cultures ity

of

ent

with

cellular

of this

induction

oligodendrocytes

is

inhibition the

in

rapid other

GPDH

C6 glioma blocked

to

NaB

removing

reversibility cell

in

specifically

subsequent

observed

changes

of

types

of (3).

1302

The

most fact

cells by

from of

NaB. the

the that

and

primary

The medium

other this

GPDH

reversibilis

recorded inhibition

consist-

Vol.

93, No.

occurs

4, 1980

BIOCHEMICAL

in normal

of malignant This induced

cells

BIOPHYSICAL

indicates

that

it

RESEARCH

is

COMMUNICATIONS

not merely

a peculiarity

transformation. phenomenon

cannot

nonspecific

destruction leakage

as well

AND

simply

toxicity,

be accounted

blockade

(inactivation)

for

of steroid

of either

basal

of the enzyme extracellularly,

by arguments diffusion

or induced

or inadequate

for

into

levels

NaB-

cells,

of the

cytoplasmic

enzyme,

steroid

binding. We have yet histone the

hyperacetylation

nucleus

(lo),

this

steroid bility

are

issue.

The fact

doses

nuclear

Finally,

selective.

protein

role.

under

that

the resistance the effect

Previously,

acetylation

way in our

for

the

that

of a nuclear play

the

a role

in the of the

to directly

deal

massive total

unavaila-

induction

of LDH to

of gene expression

(26);

in

receptor-hormone

LDH induction

protein

steps

despite

steroid

regulation

indeed

associations

of the

of the catecholamine

of NaB on the

does not

exists

the possibility

is

whether

binding

laboratory

blockade

sites

we reported

phosphorylation

the

to the cytoplasmic

suggests

notably

DNA-protein

to investigate

complete

acceptor

most

If the defect

of nuclear

distal

currently

(50 uM) already

NaB shows that

increased

process

Studies

mechanism,

a critical

of alteration

induction

of critical

complex.

underlying

NaB can be used as a new tool

glucocorticoid steroid.

the

plays

as a result

then

with

to determine

this

is study

is

associated suggests

with that

in the phenomenon.

ACKNOWLEDGEMENTS We thank Dr. J. Sorrentino recipient of a Bank of America were supported by DOE Contract

for his helpful advice. D. W. is a Gianini Foundation Fellowship. These studies DE-AM03-76-SF00012 and USPHS Grant HD-05615. REFERENCES

:: ;: i:

Leder, Wright, Prasad, Fishman, Biochem. Reeves, Riggs, Nature

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7. 8. 1:: i:: ii: 15. :;: 18. 19. E: 22. 23. 24. 25. 26.

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AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Sealy, L., and Chalkey, R. (1978) Cell 14, 115-121. Ruiz-Carillo, A., Wangh, L.J., and Allfrey, V.G. (1975) Science 190, 117-128. Allfrey, V.G. (1971) Histones and Nucleohistones, Plenum, New York. Simpson, R.T. (1978) Cell 13, 691-699. Weintraub, H., and Groudine, M. (1976) Science 193, 848-856. Buller, R.E., and O'Malley, B.!d. (1976) Biochem. Pharmacol. 25, l-12. Yamamoto, K.R., and Alberts, B.M. (1976) Ann. Rev. Biochem. 45, 721-746. Baxter, J.D. (1976) Pharm. Ther. B. 2, pp. 605-659, Pergamon Press, Oxford. Kumar, S., McGinnis, J.F., and de Vellis, J. (1980) J. Biol. Chem., in press. de Vellis, J., and Inglish, 0. (1973) Progress Brain Res. 40, 321-330. McGinnis, J.F., and de Vellis, J. (1978) J. Biol. Chem. 253, 8483-8492. de Vellis, J., McEwen, B.S., Cole, R., and Inglish, 0. (1974) J. Ster. Biochem. 5, 392-393. Benda, P., Lightbody, L., Sato, G., Levine, L., and Sweet, W. (1968) Science 161, 370. McCarthy, K.D., and de Vellis, J. (1980) J. Cell Biol., in press. Breen, G.A.M., and de Vellis, J. (1975) Exp. Cell Res. 91, 159-169. Lowry, O., Rosebrough, N., Farr, A., and Randall, R. (1951) J. Biol. Chem. 193, 265-275. Baxter, J.D., and Tomkins, G.M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 932-937. Leveille, P., McGinnis, J.F., Maxwell, D.S., and de Vellis, J. (1980) Brain Res., in press. de Vellis, J., McGinnis, J.F., Breen, G.A.M., Leveille, P., Bennett, K., and McCarthy, K. (1977) Cell, Tissue and Organ Cultures in Neurobiology, pp. 485-511, Academic Press, New York. Salem, R., and de Vellis, J. (1976) Fed. Proc. 35, 296.

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