Dexamethasone inhibition and phorbol myristate acetate stimulation of plasminogen activator in human embryonic lung cells

BIOCHEMICAL

Vol.99,No.2,1981

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 379-384

March31,1981

DEXAMETHASONE INHIBITION

AND PHORBOL MYRISTATE ACETATE STIMULATION

ACTIVATOR

Infectious

Disease

and Harvard

Received

January

IN HUMAN EMBRYONIC LUNG CELLSI.

Jaken2, Christiana

Susan

19,

OF PLASMINOGEN

Geffen,

Unit, Medical

and Paul

Massachusetts School,

H. Black3v4

General

Boston,

Hospital,

MA 02114

1981

SUMMARY Treatment of human embryonic lung cells with dexamethasone resulted in a decrease in plasminogen activator activity measured in the fibrinolytic assay. The decrease in activity could at least partially be explained by the presence of an inhibitory substance(s) based on the following observations of lysates of dexamethasone-treated vs. control cells: a) an increase in specific activity following subcellular fractionation; b) an increase in fibrinolytic activity following separation by gel electrophoresis; c) an increase in fibrinolytic activity following mild acid-treatment; and d) a decrease in urokinase-directed fibrinolytic activity in mixing experiments. Phorbol myristate acetate increased plasminogen activator activity without affecting the level of inhibitory substance. INTRODUCTION Plasminogen

activator

tically

in several

lyzes

the conversion

plasmin.

Thus,

cell

(PA) types

of the

controlled

is

a serine

upon malignant serum zymogen,

release

protease

which

is

transformation. plasminogen,

of PA is thought

increased This

drama-

enzyme

to the active

to be an important

cata-

protease, factor

in

1

This work supported

2

Present

address:

Interdisciplinary Public Health,

3

Present

address:

Department of Microbiology and Hubert H. Humphrey Cancer Research Center, Boston University School of Medicine, 80 E. Concord Street, Boston, MA 02118.

4

To whom all correspondence should be addressed: Department of Microbiology, Boston University School Concord Street, L-504D, Boston, MA 02118.

was supported by NIH grant CA 10126 & Contract by National Research Service Award CA 05913 Programs in Health, 665 Huntington Avenue,

CP 43222. Harvard Boston,

SJ was

School of MA 02115.

Dr. Paul H. Black, of Medicine, 80 East

Abbreviations used: Plasminogen activator, PA; phorbol myristate acetate, PMA; human embryonic lung cells, HEL; urokinase, UK; sodium dodecyl sulfatepolyacrylamide gel electrophoresis, NaDodSOq-PAGE.

0006-291X/81/060379-06$01.00/0 379

Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 99, No. 2,198l

BIOCHEMICAL

regulation

of localized

nant

may be related

cells For

cells. controlling This (HEL)

report

describes

and contrasts which

the

it

important

enzymatic

the regulatory

features

in these

the

cells

For -this

promoter,

PA in several

MATERIALS

of these factors

in human embryonic

(8,13,14).

tumor

in PA by dexamethasone

properties

activity.

of PA activity

of PA by the potent

in malig-

to understand

the modulation

in detail

COMMUNICATIONS

production

and metastatic

has become

of this

RESEARCH

The increased

invasive

has been shown to induce

decrease

BIOPHYSICAL

events.

we have studied

induction

(PMA) (which

to

reasons

expression

3T3 cells,

and the

fibrinolytic

the

cells

studied

these

AND

phorbol cell

types

as characterized

with purpose

lung

those

of

we have

myristate

acetate

in culture

(l-6)),

by Rifkin

(7).

AND METHODS

Cell culture. HEL cells were used between passages 13 and 18. The cells were plated at 1 x 106 cells/60 mm dish in Dulbecco's modified Eagle's medium containing .JO% fetal calf serum and antibiotics. The cells were allowed to attach for 24 hours. On Preparation of samples. the following day, fresh medium and the dexamethasone or PMA were added. At the appropriate times, cell lysates were collected (after rinsing 2 times) in 0.5 ml 0.1 M Tris (pH 8.1) containing 0.1% Triton X-100. The samples were centrifuged for 5 minutes at 800 x g and the supernatants were collected and frozen for later assays of PA and protein. For subcellular fractionation, homogenates were The prepared and nuclei were removed by centrifugation as described (8). postnuclear supernatants were then centrifuged at 100,000 x g for 60 minutes. Lysates were acidified to pH 2.7 with glycine as described by Loskutoff and They were then incubated at 370 for 60 minutes. Edgington. activity values

PA assays. PA was assayed by measuring plasminogen-dependent fibrinolytic as described (8). Each sample was assayed at least in triplicate and the obtained represent the mean with an error of no more than 15%.

The amount of inhibition of K-directed inhibition assays. was calculated from % hydrolysis (UK + 1Ysate Y during a % hydrolysis (UK alone) 1 hour incubation at 370. The amount of cell protein in each sample was 2.5Ag and under the conditions used contributed
Urokinase (UK) fibrinolytic activity

Cell

Sodium lysates

dodecyl sulfate-polyacrylamide gel electrophoresis were prepared for electrophoresis as described

Miscellaneous. was estimated using

(NaDodSOq-PAGE). (13).

Dexamethasone and PMA were purchased from the method of Lowry (10). UK was obtained

Sigma. Protein from Abbott.

RESULTS Dexamethasone-treatment Previous

studies

by

results Rifkin

(7)

in an inhibitor have

380

documented

of the fibrinolytic the

assay.

dexamethasone-induced

BIOCHEMICAL

Vol. 99, No. 2,198l

decrease

in fibrinolytic

dependent

decrease

laboratories with

in

presence

substances

attributed

to removal

tory

inhibitory

following

following

mild

activity

acid-treatment

which

(9);

mixing

experiments

d)

with

in the extracts

of testing

whether

explained

by the

pellet in

1.

increase

in recovered

samples

also

is

only

consistent

during

electrophoresis.

medium

from

electrophoresis * Because the lytic activities to inhibitory

activity of an acid-

fibrinolytic to the

activity

in

presence

of

was employed

as a means

in PA in HEL cells

and dexamethasone-treated increase

in specific

was a 2.7-fold This

been

one PA species vs. applied with

large

reported

increase increase for

inhibitor

the

( -6OK activity

separation

Similar WI-38 substantial

fibrinolytic of both substances

of inhibi-

fibrinolytic

methods

was

in fibrinolytic

to the destruction

could

be

cells

is

(9).

results cells

of have

381

activity

cells

and

is

shown

an inhibitory

in Table

no activity

The

for

large

from

PA

conditioned

was observed after

2.

vs. control substance

reported

was

of PA from

was observed.

was observed

assay is a two-stage PA (or UK) and plasmin, as potential inhibitors

activity

The recovery

been

in the

specific

in dexamethasone-treated

in which

activity

in

cells

Daltons)

activity

in specific

endothelial

and dexamethasone-treated

transformed and

in

a) an which

to separation

decrease

of a soluble

in control

sample

there cultures.

has

In each

inhibitory

of evidence:

was attributed

to the small

cultures,

to the removal

NaDodSOq-PAGE

which

of

fractionation

UK-directed

of control

In contrast

centrifugation

lines

increase

Each of these

fractionation

from control

attributed

extracts

have been associated

b) an increase

was attributed in

Several

of an inhibitor.

dexamethasone-treated

with

an

the dexamethasone-related

Subcellular shown in Table

of several

dose-

report.

presence

subcellular

c)

(9,12).

induction

The

(9);

a similar

that

was attributed

a decrease cell

inhibitors

which

which

PA (11);

inhibitor

in PA activity

inhibitor

the

labile

in

following

of a soluble

from

as described

on the basis

COMMUNICATIONS

We obtained

substance(s)*.

NaDodSOq-PAGE

substances

RESEARCH

HEL cells.

decreases

has been postulated in fibrinolytic

in

PA activity

of

increase

activity

activity

have reported

the

AND BIOPHYSICAL

before

electrophoresis

assay depending on the in this report we will of either activity.

proteorefer

Vol. 99, No. 2,198l

BIOCHEMICAL

TABLE

1:

AND

BIOPHYSICAL

Subcellular

RESEARCH

distribution

of

COMMUNICATIONS

PA

PA

Sample

% hydrolysis/flgl Control Post-nuclear

supernatant

(30)

5.4

x g pellet

33.9

(0)

14.4

100,000

x g supernatant

26.2

(0)

1.9

Post-nuclear

supernatants

cells

broken

by

then

centrifuged

pellet

and

were

Dounce at

These

results

100,000

increase

were

refer

to

73.3

(30)

(18)

76.0

(0)

(0)

27.0

(0)

out

The

supernatant

postnuclear to

collect

% increase

of lysates activity

samples

TABLE

2:

in

to the presence

and was separated

fibrinolytic

dexamethasone-treated

(178)

by centrifuging

60 minutes

the

attributed

acid-treatment

in

x g for

PMAtreated

nuclei

from was

a 100,000

x g

fraction.

activity

of mild

prepared

homogenization.

supernatant

the fibrinolytic effect

29.3

100,000

1 Numbers in parentheses acid treatment.

(11).

Dexamethasonetreated

activity

of a substance

out during

is shown

acid-treatment

of

PA from

Sample

Control

indicating

% Recovered

% hydrolysis

Total applied

Total recovered

1486

276

19

10-9

M dexamethasone-treated

995

375

38

10-8

M dexamethasone-treated

285

219

77

6725

1413

21

PMA-treated Lysates The eluted

from

cell

fibrinolytic from

cultures activities

the

gels

were

were of

collected the

total

measured.

382

and lysate

prepared and

for the

electrophoresis.

60K Dalton

1.

control

gels

PA Total

in Table

thus

polyacrylamide

masked

(11).

of

was 30% vs. 178%, respectively,

Recovery

which

electrophoresis

on PA activity

following

following

species

The The and

an

BIOCHEMICAL

Vol. 99, No. 2,198l

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS

x PMA-treated x

Figure

increase

1.

in

The effect of with PMA (100 collected at surements of

the

level

samples.

Endothelial

inhibitor

of

demonstrated

cells

through

control

have

mixing

protein

Finally,

experiments

with

have been

of hepatoma

tissue

fibrinolytic

was an increase

the

presence

cell

lysates

of

cells

for

cells

(Fig.

1).

inhibitor,

or both,

control

PMA-treated

cell

was made

with

activity

recovery of

substantial from

the

from gels,

100,000

in either

NaDodSOq-PAGE

control was the

The amount of acid-stimulation

change

in the

vs.

the

or PMA-treated same for in cell

was 38 + 7% and 54 + 7%, respectively no significant

and acid-treatment.

x g pellet

level

control lysates

383

cells

resulted

in only

samples

27% vs.

to

and PMA-treated

accompanying

was not

The recovery

samples

These

between

in specific

supernatant 1).

could

subcellular

The increase

(Table

to PMA,

increase

respect

and PMA-treated of control

this

a comparison

post-nuclear

(mean + SE, n = 6). of inhibitor

the same

were exposed

Because

PA, decreased

fractionation,

was

(12).

by increased

samples

inhibitor

dexamethasone-treated

be explained and

an

and UK. Using

When HEL cells

2 hours

an acid-labile

from dexamethasone-treated

reported

culture

dexamethasone-treated

to contain

from control

activity.

in PA within

in

reported

lysates

of UK. Lysates results

PMA stimulates

been

in each assay,

Similar cultures

inhibitor

also

(9).

in 78% inhibition

inhibition.

there

an acid-labile

fibrinolysis

amount of cell resulted

of

PMA on intracellular PA levels. Cells were treated rig/ml) as described in Methods. Cell lysates were several times following addition of PMA for meafibrinolytic activity.

results

(Table

2).

samples indicate

PMA-treatment.

Vol. 99, No. 2,198l

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

CONCLUSIONS Treatment inhibitory

of HEL cells substance

procedures. caused

The

treated

of

in or

samples These

which

to control were

PA in HEL cells for

increased activity

the

weight

The regulation

because not

by four

that

levels

of an

independent

assay

dexamethasone-treatment

removal restore

undertaken

of

activity

in mitogen-stimulated

open

did

initially

of

inhibitory

activities

an inhibitor with

of 75K:49K and growing

species

to compare

In contrast

also activity

which

with

in

lysates

cells does

or

two molecular

activity

is large (13,14).

not

change

of PA in HEL and 3T3 cells,

the regulatory

of

we have 3T3 cells

acid-treatment. weight

in quiescent

In contrast, upon

therefore,

inhibition appears

species, cells HEL cells

features found

:: 4. ;: k 9. 10. 11.

The

::: 14.

have one

through

Quigley, J.P. (1979) Cell 17, 131-141. I.B. (1976) Nature 276, 232-233. Wigler, M., and Weinstein, and Reich, E. (1978) Cell 15, 385-392. Wilson, E.L., Wilson, E.L., and Reich, E. (1979) Cancer Res. 39, 1579-1586. Goldfarb, J.H., and Quigley, J.P. (1978) Cancer Res. 38, 4601-4609. Christman, J.K., Copp. R.P., Pednnan, L., and Whalen, C.E. (1978) Cancer Res. 38, 3854-3860. Rifkin, D.B. (1978) J. Cell Physiol. 97, 421-428. Jaken, S., and Black, P.H. (1979) Proc. Natl. Acad. Sci. 76, 246-250. T.S. (1977) Proc. Natl. Acad. Sci. 74, Loskutoff, D.J., and Edgington, 3903-3907. Lowry, O.H., Rosebrough, J., Farr, A.L., and Randall, R.J. (1951) J. Biol. CheM. 193, 265-275. Roblin, R.O., Young, P.L., and Bell, T.E. (1978) Biochem. Biophys. Res. Jaken, Jaken,

S., S.,

and Black, and Black,

P.H. P.H.

T.D. (1978) Proc. Natl. Sci. 75, 6130-6133. (1981) J. Cell Biol., in press. (1981) J. Cell Biol., in press.

384

PA

and small

or stimulation. to occur

by

75K and

mechanisms.

COMM.82, 165-172. Seifert, J., and Gelehrter,

no

either

REFERENCES

1.

by

of dexamethasone-

to HEL cells,

centrifugation

is associated

The ratio

to increased

levels.

presence

in 313 cells

molecular

levels

to 3T3 cells.

specific

49K Daltons.

distinct

enzyme

leads

be demonstrated

remains

acid-treatment

studies

evidence

dexamethasone

could

possibility

a decrease

centrifugation

with