Loss of glucocorticoid regulation of plasminogen activator activity in anucleate rat hepatoma cells

Vol.

96, No.

October

4, 1980

8lOCHEMlCAL

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Pages

31, 1980

1540-1546

LOSS OF GLUCOCORTICOID REGULATION OF PLASMINOGEN ACTIVATOR ACTIVITY IN ANUCLEATE RAT HEPATOMA CELLS P.A.

Barouski

and T.D.

Department of Human Genetics University of Michigan Medical Received

August

Gelehrter

and Internal Medicine School, Ann Arbor, MI

48109

20,198O

Summary: Glucocorticoids decrease the plasminogen activator activity hepatoma cells through production of an inhibitor. We have examined dexamethasone regulation of plasminogen activator in anucleate rat cells to investigate the role of the nucleus in the steroid regulation this membrane-associated phenomenon. Dexamethasone did not affect the intraor extra-cellular plasminogen activator activity of the and did not induce production of an inhibitor of plasminogen cells, vator. Therefore, glucocorticoid regulation of plasminogen activator ity requires the presence of an intact nucleus. Glucocorticoids tissue

alter (HTC) 1 cells,

culture

Dexamethasone,

a synthetic

extra-cellular

activity

which

catalyzes

active

ogen

the

fibrinolysin,

reported

several

that

an established

line

glucocorticoid, of plasminogen

properties

of rat

decreases activator

both

of the

serum protein,

plasmin.

Seifert

and Gelehrter

appears

the

activ-

cells

intra-

a serine

(1).

and

protease

plasminogen,

to the

(4) have previously

of dexamethasone-induced

(PA) activity

acti-

in hepatoma

hepatoma

(2,3),

conversion

the mechanism

activator

membrane-associated

of rat the hepatoma of either anucleate

inhibition

to be the production

of plasmin-

of an inhibitor

of

PA. Steroid

hormones

scriptional

are

activation

thought

of specific

hormone-receptor

complexes

the complexes

to the chromatin

receptor ing

cytoplasmic

mechanisms 1.

complexes

for

to induce

might

into

also

or membrane steroid

genes

(5).

However,

mediate

cellular

hormone

The abbreviations used are: plasminogen activator.

OOOS-291X/80/201540-0~$01.~~/0 Copyrighr 0 1980 by Academic Press, Inc. AN rights of reproduction in any form reserved.

after

the nucleus

functions. action

cellular

changes

translocation

and site-specific steroid

To investigate

HTC, hepatoma

1540

binding

the

tissue

affect-

alternative

glucocorticoid culture;

of

or hormone-

by directly these

tran-

of cytosol

hormones

changes

, we examined

through

PA,

Vol.

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regulation cause

BIOCHEMICAL

4, 1980

of PA activity

a decrease

induce

glucocorticoid of an intact

in anucleate

in either

the production regulation

AND

the

BIOPHYSICAL

HTC cells.

intra-

RESEARCH

Dexamethasone

or extra-cellular

of a PA inhibitor

in the anucleate

of PA activity

COMMUNICATIONS

in HTC cells

did

PA activity, cells. requires

not or

Therefore, the presence

nucleus.

METHODS Cell culture: HTC cells were grown in spinner culture without antibiotics in Eagle's minimal essential medium for suspension culture (Gibco autoclavable powder) supplemented with 50 mM Tricine, 0.5 g/l NaHC03, 2 mM glutamine, 5% calf serum, and 5% fetal bovine serum (4). Enucleation: HTC cells were enucleated in suspension by centrifugation through a discontinuous Ficoll density gradient in the presence of 10 j.lg/ml cytochalasin B according to a modification of the procedure described by Wigler and Weinstein (6,7). Control cells were centrifuged through a Ficoll gradient in the absence of cytochalasin B, or incubated without centrifugation in 15% Ficoll containing cytochalasin B. Identical results were observed with either treatment of control cells. Efficiency of enucleation was routinely >92% as determined in aceto-orcein stained preparations or by measuring the relative rates of r3H] thymidine incorporation in cytoplasts and whole cells (7). Glucocorticoid treatment: Cells and cytoplasts were plated at densities of 2 x 10b cells and 6 x 10 b cytoplasts per 35 mm tissue culture dish in serum-containing medium plus 0.02% CaC12. After four hours incubation at 37'C, cells and cytoplasts were washed and incubated in serum-free medium containing 0.1% bovine serum albumin, 50 ug/ml neomycin, and 0.01% ethanol with or without 0.1 uM dexamethasone. Plasminogen Activator Assay: Conditioned medium was collected from the cells and cytoplasts after 0, 2, 4, 6, and 16 hours incubation, and detached cells removed by centrifugation. Cell extracts were prepared as previously described (4); protein concentration was determined by the method of Lowry et al. (8). -PA activity was measured in a fibrin plate assay (4) using 2 ug human plasminogen in 16 mm diameter wells of [ lz51] fibrin-coated plates (9); solubilized radioactivity was measured after 6 or 6.5 hours incubation at 37OC. Total radioactivity was the amount solubilized by 50 ug trypsin. The background activity, usually 3 to 6% of the total radioactivity, was the amount solubilized by an assay mixture of plasminogen with either 0.2% triton or unconditioned medium. The PA activity of the sample was corrected for background activity, and expressed as a percentage of the total radioactivity. The cell extracts and the conditioned medium samples have no plasminogenindependent fibrinolytic activity. Dexamethasone added directly to the assay has no effect on fibrinolytic activity. Inhibitor Assay: Samples of cell extract and conditioned medium were also examined for the ability to inhibit urokinase, a plasminogen activator purified from human urine (10). Cell extracts or conditioned medium were incubated in a fibrin-coated well with 1.0 or 1.5 x 10q3 Ploug units of urokinase (10) for thirty minutes at ambient temperature; the PA assay was PA activity of the sample then initiated by the addition of plasminogen. incubated without urokinase was also measured.

1541

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cells

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4, 1980

The amount incubated

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

of inhibition in cell extracts or conditioned medium with dexamethasone was calculated by this formula:

d 3

amount

of inhibition

from

x

loo%

This equation corrects for any endogenous PA activity of the sample and measures the inhibitory effect specific to dexamethasone treatment. The comparison of inhibition in control and dexamethasone samples eliminates the potential complications introduced by the presence in either the cell extract or the conditioned medium of inhibitors which are not induced by dexamethasone (11). Materials: Fibrinogen (77% clottable) was purchased from CalbiochemBehring and purified as described by Strickland and Beers (9); fibrinogen, (98.3% clottable) also obtained from Calbiochem-Behring, was used without loo-150 uCi/ml) further purification. [1251] fibrinogen (specific activit was purchased from Abbott; r3H] thymidine (1 mCi/ml) and [ 74 Cl amino acid mixture (100 uCi/ml) were New England Nuclear products. Plasminogen was purified from outdated human plasma by lysine-Sepharose chromatography (12). Dexamethasone was a gift from Dr. Walter Gall of Merck and Company. Ficoll ('lype 400) and cytochalasin B were purchased from Sigma; urokinase B grade, was obtained from Calbiochem-Behring. All other compounds were of reagent grade. RESULTS AND DISCUSSION Anucleate

rat

HTC cytoplasts trypan

hepatoma

still

blue,

adhere

even after

(13)

tained

at levels

comparable

Similar

to L-cell

cytoplasts

density

and active

decreases

of PA activity. both

intra-

PA activity

activity

after after

activity

(lower

relative

to that

or higher

and 2 98% exclude

in serum-free

than,

anucleate

areas

those

acid

medium.

PA

(7) are main-

in intact

HTC cells

remain

density

to areas

of high

are not

hours

cells. capable

of

of lower

was decreased

of incubation

with

of incubation.

cells.

treated

(Figure to less

dexamethasone, Similarly,

with

In the

1542

to glucocorticoid

dexamethasone

PA activity

panel)

of cells

of control

responsive

HTC cells,

and extra-cellular

two hours

panel)

incubation

In intact

(upper

six

dishes,

functions.

plates.

HTC cytoplasts

cellular

detectable

from

membrane-related

of a-aminoisobutyric

(14),

culture

Nevertheless, regulation

to,

several culture

hours

transport

redistributing on tissue

retain

to tissue

sixteen

activity

locomotion,

cells

1).

than

Intra-

50% of control

and was not

extracellular

dexamethasone anucleate

dramatically

PA

was reduced

HTC cells,

however,

Vol.

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INTRACELLULAR

16

8 c 5 F 2 Q

4 o

2

4

6

8

IO

12

14

16

3 50 -

EXTRICELLULAR

d

4

IO OF INCUBATION

TIME

12 14 bows)

16

Figure 1: Time course of dexamethasone effects on intraand extra-cellular HTC cells (circles) and cytoplasts (triangles) were incubated PA activity. for O-16 hours in the presence (closed symbols) or absence (open symbols) 0.1 UM dexamethasone. Triton X-100 extracts (4 Ug protein) of cells or cytoplasts (upper panel) and medium (100 ~1) conditioned by cells or cytoplasts (lower panel) were assayed for PA activity on [lz51J-fibrin plates as described in Methods. Each point represents the average of duplicate assays from a single culture. Total radioactivity solubilized by trypsin was 130,000 cpm/well.

dexamethasone

had even

activity, plasts

was

pressed twice

as

effect

after

two

per

no

to

mg of

much

on

16 hours three

protein

of

times

protein;

and

bation, itor

in

Similarly,

the

In

anucleate dexamethasone

regulate

amino The

regulation

complete of

acid

did transport

failure PA activity

of

from

contrast,

cells,

was

PA

PA activity of

of

intact

cells

the

cells

generally

whole

even

after

not

induce

was

cells,

in

16

after

due

did

hours

tyrosine HTC

anucleate not

urokinase

dexamethasone

(7) of

extra-cellular

cyto-

when

ex-

contained

(7).

medium

respectively.

that

nucleated

inhibitor

conditioned

or The

than

the

cell

intra-

treatment.

but, per

the

greater

A dexamethasone-induced extracts

either

of

1543

2 and not

in 4 hours

incu-

this

inhib-

induce

incubation

cell

(Figure

aminotransferase

2). (Vi),

cytoplasts.

cells to

detected

to

a lack

respond of

to

glucocorticoid

dexamethasone receptors.

or

Vol.

96, No.

4, 1980

BIOCHEMICAL

120

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BIOPHYSICAL

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INTRACELLULAR

f

01

d

E 120

COMMUNICATIONS

r

16 EXTRACELLULAR

:,i,L 2

4

6

6

IO

OF INCUBATION

TIME

12 14

16

(hourd

Figure 2: Time course of dexamethasone induction of urokinase inhibitory activity. HTC cells (circles) and cytoplasts (triangles) were incubated for O-16 hours in the presence or absence of 0.1 uM dexamethasone. Triton X-100 extracts of cells or cytoplasts (4 ug protein) or conditioned medium (100 ~1) were incubated for 30 minutes with or without 1 x lo-' or 1.5 x lOa Ploug units of urokinase, respectively, and then assayed for 6.5 hours on 1125~]fibrin plates. Dexamethasone-induced inhibition of urokinase was calculated as described in Methods. Percentage of control activity remaining (100 - % inhibition) is plotted against time of incubation. Total radioactivity was 130,000 cpm/well. McDonald icoid

and Gelehrter

binding

binding

in anucleated

was reduced

cytochalasin B prior

Incubation addition

(data

observed

glucocorticoid

PA activity

not

shown).

level inhibitor

(6,7) in

the extent

of the intact

Finally,

intact

also cells

production,

1544

cells

on the

of specific the

levels

of the

cytochalasin or

of inhibitor rate

explain

whose protein dexamethasone albeit

result

of intra-

and release

can not

glucocort-

with

the decreased

by cycloheximide,

3).

specific

Nor was it

or on the production

responsiveness;

and induced

cells.

had no effect

in cytoplasts

to a comparable

saturable,

although

to intact

PA activity,

the medium

synthesis

(Figure

relative

to dexamethasone

blocked

HTC cells,

B treatment.

extra-cellular into

(7) have demonstrated

of protein

the

lack

of

synthesis still

to a reduced

was decreased

extent

Vol.

96, No.

BIOCHEMICAL

4, 1980

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BIOPHYSICAL

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4 TIME

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EXTRACELLULAR

6‘ OF INCUBATION

(hours)

Figure 3: Effect of cycloheximide on the time course of dexamethasone induction of urokinase inhibitory activity. Control cells (open squares) or cells treated with 0.25 uM cycloheximide for four hours prior to, and then during dexamethasone induction (closed squares) were incubated for O-6 hours in the presence and absence of 0.1 !.lM dexamethasone. Cycloheximide treatment reduced protein synthesis 56 + 3% (mean f standard deviation, n=8). Triton X-100 extracts of cells (9 ug protein) and conditioned medium (100 ~1) were incubated for 30 minutes with or without 1 x 10s3 Ploug units of urokinase, then assayed for 6 hours on [1251)-fibrin plates. Dexamethasane-induced inhibition of urokinase was calculated as previously described. Total radioactivity was 58,000 cpm/well. We conclude presence nomenon

that

of an intact does not

or hormone-receptor

the

steroidal

nucleus;

appear

Glucocorticoids

activation

of the gene(s)

regulation

to be mediated

complex

elements.

regulation

with probably for

of PA activity

of this by direct

either

membrane-associated interaction

the membrane

regulate

an inhibitor

requires

phe-

of the hormone

or other

PA activity

the

cytoplasmic

by transcriptional

of plasminoqen

activator.

ACKNOWLEDGMENT We gratefully acknowledge the assistance with the enucleation procedure. This research from the National Cancer Institute (CA 22729). predoctoral training grant (GM 97544) from the T.D.G. was the recipient of a Faculty Research Cancer Society.

of Dr. Roderick A. McDonald was supported by a grant P.A.B. was supported by a National Institutes of Health; Award from the American

REFERENCES 1.

11979) in Monographs on Endocrinology Volume Gelehrter, T. D. Glucocorticoid Hormone Action (Baxcr, J.D. and Rousseau, pp. 561-574. Y eds) Springer-Verlaq, Berlin, Heidelberg.

2.

Wiqler, M., Ford, J.P., and Weinstein, 1-B. (1975) in Proteases and Biological Control, (Reich, E., Rifkin, D.B., and Shaw, E., edr Cold Spring Harbor Laboratory. pp. 849-856.

3.

Carlson, S.A., and Gelehrter, Structure 6, 325-331.

T.D.

1545

(1977).

J.

Supramolecular

12: G.G.,

Vol.

96, No.

BIOCHEMICAL

4, 1980

4.

Seifert, S-C., and Gelehrter, USA 75, 6130-6133.

5.

Rousseau,

6.

Wigler, M.H., and Weinstein, Commun. 63, 669-674.

7.

McDonald,

8.

Lowry, O.H., Rosebrough, J. Biol. Chem., 193,

9.

Strickland,

G.G.

(1975).

R.A.,

AND

T.D.

J. Steroid 1-B.

and Gelehrter,

S.,

BIOPHYSICAL

and Kjeldgaard,

W.H.

Biochem.

6, 75-89.

A.L.,

(1976).

COMMUNICATIONS

Natl.

Biochem.

Acad.

Biophys.

Sci.

Res.

in preparation.

and Randall, J. Biol.

(1951).

Chem. 251,

11.

Wijngaards,

12.

Deutsch,

13.

Gelehrter, T-D., 27, 448A.

14.

(1976). in Methods Wigler, M-H., Neugut, A-I., and Weinstein, I.B. in Cell Biology Vol. XIV (Prescott, D.M., ed) pp. 87-93, AZ --Press.

15,

Ivarie, R-D., Physiology

(1979).

D.G.,

and Mertz, McDonald,

Fan, W.J.-W., 85:357-364.

Haemostas.

Thrombos. E.T. R.A.,

(1970).

1.546

41,

Science

and Barouski,

and Tomkins,

Biophys.

5694-5702.

J., Ploug, 278-289.

(1957).

Biochim.

R.J.

10.

G.

N.

Proc.

manuscript

N.J., Farr, 265-275.

and Beers,

(1978).

(1975).

T.D.

RESEARCH

G.M.

24,

590-600.

170, P.

Acta.

1095-1096.

(1979).

(1975).

J.

Clin.

Cell.

Res.