Regulation of type IV collagen degrading enzyme by cortisol during human skin fibroblast growth

Vol. 130, No. 2, 1985

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

July 31, 1985

Pages 588-595

REGULATION OF TYPE IV COLLAGEN DEGRADING ENZYME BY CORTISOL DURING HUMAN SKIN FIBROBLAST GROWTH Tuula Collagen

Research

and Department

Sale* Unit,

Department

of Clinical

Kajaanintie Received

and Jouko Oikarineno of Medical

Chemistry*,

52 A, SF-90220

Biochemistry"*

University

Oulu

of Oulu,

22, Finland

May 13, 1985

The effects of cortisol on type IV collagenolytic enzyme activity were studied in human skin fibroblast cultures. Cortisol reduced the production of the enzyme, both in normally dividing fibroblasts, and also when the secretion was induced by the administration of the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) to the subconfluent culture. TPA decreased the glucocorticoid receptor activity and augmented the cGMP content of subconfluent fibroblasts. Type IV collagenolytic activity appeared to be correlated positively with the cGMP/cAMP ratio, and negatively with the glucocorticoid receptor activity, and the activity was increased by the addition of dibutyryl cGMP. This suggests that this enzyme may at least partly be under glucocorticoid control during fibroblast growth, and that TPA may to some degree act via the inactivation of glucocorticoid recep@ 1985 Academic Press, Inc. tors.

Glucocorticoid (1,2).

hormones

Changes

in

implicated

in

decreases

during

reach

the

tions

in

tein

specifically in basement

of

cell

phase

(3).

receptors

Type

IV collagen

degrades

type

for

of metastases

(8).

to

In the

tumour

cells

(9,10),

may at least

fibroblasts.

This

correlates 0006-291X/85

correlates

fibroblast

positively

to

present with

the

partly

enzyme

division with

degradation

the cellular

(type

the main

588

as the

cells

to alterathe

degree

of these

type

e.g.

may be a

shown

for-

IV collageof malignant

glucocorticoid

and now we show that

(6)

during

type

potential

has been

propresent

structures

them,

we show that

be under

of

IV collagenase) collagen

cGMP/cAMP ratio

$1.50

CoWright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

may affect

metastatic

activity

(ll),

again

activity

CAMP and cGMP-dependent

go through work

been

their

may be attributed

enzyme (Z),

be able

activity

normal

through

IV collagen

whose

during

(4,5)

growth

recently

since

increases which

degrading

nase,

human skin

(3),

Proteolytic

a cell

changes

ratio

fibroblast

have

growth, and

These

human skin activity

fibroblast

division

cAMP/cGMP

membranes.

prerequisite mation

normal

of the

kinases.

receptor

regulation

cellular

phosphorylation

inhibit

glucocorticoid

the

stationary the

effectively

control

in

to be induced the

induction

and negatively

with

Vol.

130,

BIOCHEMICAL

No. 2, 1985

the

glucocorticoid

phorbol

receptor

13-acetate

increase

in

receptor

activity,

may

be

enzyme

directly

MATERIALS

(TPA),

and,

AND

By

potent

tumour

may

be

with

caused that

moreover,

associated

BIOPHYSICAL

activity. a

activity

AND

an

the

accumulation

use

of

we

a decrease decrease of

COMMUNICATIONS

12-O-tetradecanoyl

promoter, by

the

RESEARCH

in in

show

that

the

glucocorticoid

receptor

activity

cGMP.

METHODS

The cell culture reagents were obtained from Nunc Products (Roskilde, Denmark and Gibco Biocult (Paisley, U.K.). C2,3,4,5-3HlProline, 4 Hjdexamethasone [1,2,4and the radioimmunoassay kits for CAMP and cGMP U.K.). Cortisol sodium sucwere purchased from Amersham (Amersham, Bucks., cinate was obtained from Orion OY (Espoo, Finland). 12-O-Tetradecanoyl phorbol 13-acetate and other chemicals were purchased from Sigma Chemical Louis, MO. U.S.A.). co. (St. Locally established human skin fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) foetal calf 50 @g/ml of ascorbic acid, 100 U/ml of penicillin and 100 pg/ml of serum, streptomycin. Cells at the 5th-10th passage were used for the study. For the enzyme activity determinations cell cultures were washed with phosphate-buffered saline to remove traces of serum, and medium was replaced with serum-free DMEM containing 0.25% bovine serum albumin, 0.1% transferrin, TPA, cortisol or ethanol. After an incubation time of up to 24 h the cells were counted and the cell culture medium was collected after centrifugation for 10 min at 600 x g to remove cells and debris, and the supernatant was stored frozen. The IV collagenase activity was assayed from cell culture medium proteins as described previously (101 by using soluble 13Hlproline-labelled type IV procollagen as a substrate (6). Briefly, enzyme samples were activated with trypsin (IO pg/ml) followed by the addition of soyabean trypsin inhibitor (40 pg/ml), N-ethylmaleimide (4 mM), aprotinin (1000 KIlJ/ml) and substrate (3OUO cpm). The reaction was carried out for 18 h at 35°C and was terminated by adding 20 pl of bovine serum albumin (1 mglml) and 100 pl of a solution containing 10% trichloroacetic acid and 5% tannic acid. The mixture was incubated on ice and the undigested material was precipitated and removed by centrifugation at 5000 x g for 15 min. Radioactivity in the supernatant was measured in a scintillation counter. The specific binding of C3Hldexamethasone to cultured human skin fibroblasts was measured using a whole-cell binding assay (3). Briefly, two plates were combined for each determination and the cells were trypsinized, washed and counted and suspended in phosphate-buffered saline. They were then incubated in the presence of C3Hjdexamethasone (1,3-100 nM) for 30 min at 37"C, washed twice and the bound radioactive dexamethasone counted. for estimation of the non-specific binding of L3H]dexamethasone, a parallel set of tubes containing an additional 200-fold excess of unlabelled dexamethawas s ne incubated and washed as above. The specific binding of [ s Hldexamethasone was calculated by subtracting the non-specific binding from the total binding. The cellular content of active receptors was estimated from the Scatchard plots. The cellular cGMP and CAMP contents were determined from the cells by using radioimmunoassay as described elsewhere (3). Student's t-test and one-way variance analysis were used for the statistical treatment of the data. RESULTS When divide

AND

DISCUSSION

confluent by

subculturing

cultures

of at

a

human ratio

skin of

589

1:2,

fibroblasts type

were IV

collagenase

stimulated

to activity

Vol.

130,

No. 2, 1985

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

0

Fig.

1

Type IV collagenase activity during various phases of human skin fibroblast growth and the effects of TPA and cortisol (HC). Confluent cultures of fibroblasts were subcultured in a ratio of 1:2. During cell division (a), the first two days after subculture, or at confluency (b), more than 5 days after subculture, the cells were incubated i% serum-free medium for 24 h and the enz me activity was determined. When the cells were treated with 10- Q M TPA or low5 M cortisol, fresh medium was used 4 h prior to the collection of the enzyme and the serum-free medium was also The bars represent the mean +S.D. supplemented with these agents. of five to ten determinations. The differences were significant as tested by one-way variance analyses (p
increased

dramatically

basal

levels

The

enzyme

IV

procollagen, and

major

cell

activity

phoresis 75 % of

during

after

the

as

first

during

sizes

band.

The

l),

by

and

declined

demonstrated

division

the

characteristic

of

the

proul(IV)

able

activity

to slab

fragments and

did

to

low

gel

type

electro-

of

chains

degrade

(11).

degrade

(11)

proct2(IV)

not

very

previously

was

SDSlpolyacrylamide

to

enzyme

days as

cell

demonstrated

fluorography,

two

(Fig.

induced

original

extra

the

division

25 % and and

laminin

to

one

(data

not

shown). A potent induce

tumour

type

this

IV

enzyme

tumour

cells

(11).

which in

the

however, cultures

dividing

the

in

actively

of

effective

it

for

control in

the

in

(Fig. reducing

observed higher 24-h (Fig.

1). the

the

been

human

that in

The

and

produced

various media

collection

enzyme

that

on the

to

fibroblasts,

to

1).

demonstrated

skin

similar

was

cells

days of

after

the

period

to

tumour

promoter

this

in

activity

by

skin agent was,

confluent

cells. serum-free

significantly

activity

be

was

increasing

to

previously in

to

the

dividing

cortisol

fibroblasts

collagenase most

of

effective

than

Addition

shown

activity exposed

has

enzyme

been

Accordingly,

were

media

less

TPA,

degrading has

collagenase

fibroblasts

(12),

collagen

activity

subculture,

than

promoter

reduced A concentration enzyme

activity 590

collection the

medium accumulation

of (Table

10s5 I).

of of

M was Cortisol

found

normally type

IV to

clearly

be

Vol.

130,

BIOCHEMICAL

No. 2, 1985

Table I Effects dibutyryl-cGMP

AND

of various cortisol on type IV collagenolytic skin fibroblasts (two

BIOPHYSICAL

concentrations enzyme days after

RESEARCH

and activity subculture) Enzyme

Control Cortisol Cortisol Cortisol Cortisol Dibutyryl-cGMP Dibutyryl-c@lP

10S7 10-6 10-5 10e4

COMMUNICATIONS

administration in dividing

of human

activity (X) 100 82 55

M M M M 10-6 10-6

M a M a + Cortisol

10-5

5"; 150 69

M

The serum-free collection medium was supplemented with the various agents and after an incubation time of 24 h the enzyme activity was determined. Each value is the mean of four determinations. a Dibutyryl-cGMP was added every hour during the collection period.

counteracted agents

the were

The were

modes further

pounds

prior

inducing

effect

simultaneously of

of

added

action

of

TPA

to and

TPA

the cortisol

on

enzyme

collection on

medium type

characterized

by

pretreating

the

to

collection

of

the

Pretreatment

a

vaa IPa. c ;\ 'Y

a

enzyme.

activity,

cells

IV

(Figs.

when

both

1 and

2).

collagenase with

one for

18

activity of

h with

I\ Y

l

+

0 I

a a c \

0 I

Fig.

2.

Effects of pretreatment of skin fibroblasts with TPA and cortisol on the accumulation of type IV collagenase during the 24 h collection incubation. Confluent cultures of human skin fibroblasts were subcultured in a ratio of 1:2, and allowed to recover for 6 h. The cells were pre-exposed either to 10m5 M cortisol, 10W8 M TPA or none for 18 h and the enzyme collection medium containing the above supplements was exchanged. Effects of various variants of the pretreatment and of the 24 h collection incubation on type IV collagenase activity are shown. Panel a shows the effect of pretreatment with cortisol on the induct%i of the enzyme by TPA, and panel 4 the effect of the exposure to cortisol on to TPA. 591

the

comcor-

Vol.

130,

No. 2, 1985

tisol

did

2a),

while

the

not

hand,

during

the

cortisol

to activity.

Direct

the

medium

since

treated

cells

(data

not

to

TPA

about

while

with

the

cAMP/cGMP

collagenase

while

to

and

with

the

phases

of

human

every

hour

enzyme during

might

be mediated

Primarily

factor

growth

stimulus

proteins

of

24

while chap those growth

These of

h

TPA

in

that

a of

to

(see

type Fig.

the

two-fold CAMP

concentration in

for

A2 in

turn

growth

not

after IV

collagenase

of

cause

in

of the

affected the

cellular

in

M dibutyryl-

is

to

induce

possible

that

the

by

effects

of

TPA

act

by

and

via

enhancement

of

the

of

certain

acid of

of

cGMP

(13).

clearly phases

Admiresulted

content The

through

prostaglandins

fibroblasts

shown).

various

to

C (13-15)

residues

cGMP

not with

(3) various

able

arachidonic

accumulation

activity

592

an

subconfluent

treatment

glu-

receptors

synthesis

(data

1).

IO-6

speculated

to

of

may medium

by

IV

ratio

during

that

tyrosine

liberation subsequent

Type

control

kinase

lead

and

increase was

are protein

may

(3).

action.

promoters

example,

the

it

and

of

activating This

decrease

glucocorticoid

collagenase,

phosphorylation

phospholipase

leukotrienes.

nistration

pathway

tumour

by

causing,

of

this

(16-18).

mediated

(19),

inactivation IV

to

was Thus

skin and

of

I).

human reduced,

(3)

incubation

corTPA.

correlate

cGMP/cAMP

activity

(Table

of by

to

it

the

Administration

type

in

receptors

activation

in

phorbol

diacylglycerols

growth

and

of through

the

displacing

receptor

action

that

CAMP

negative

with

collection

the

induction

under

and

cultured and

increases

being

activity collage-

enzyme

demonstrated

positively

fibroblasts

division

the

been

cortisol-

IV

shown

of

the

enzyme,

been

(3)

reduce collagenase

enzyme

the

the of

not

the

content

during

addition

type

of

included

the

the

On

of

of

counteracted

dibutyryl-CAMP

24-h

dividing

of

division

growth.

the

did

the

previously

glucocorticoid

the

measured

(Fig.

only

since

from

induction

cGMP

with

fibroblast

cell

causes

of

or inhibition

affect the

that

has

correlate

during in

normal

cGMP TPA

skin

activity

cell

consistent

seems

cGMP

The

that

activity,

cocorticoids, negatively

(3). during

dibutyryl-cGMP

activity

not

the

was

of

collected

synthesis

has

it

enzyme

TPA

induction.

secretion

that

cell

activity

ratio

of

the

possible

did

interrupted

increased

administration receptor

in

when

induce

the

on

incubation

medium

in

by

effect

possibility

indicate

COMMUNICATIONS

collagenase

similar

not

mixture

only

IV

TPA-induced

the

changes

a state

is

is the

results

receptor

fibroblasts

the

cortisol

reaction

cortisol

Glucocorticoid

collection

of

reflect

brought

tisol,

the

does

These

activities

was

containing

the

shown).

cortisol

examined

addition

type

RESEARCH

a cumulative

inhibtion

Neither

inhibitors,

had

and

We also

cortisol.

enzyme

of

BIOPHYSICAL

of

TPA

effect

2b).

AND

induction

with

pretreatment

(Fig. by

nase

the

the

the

latter

enzyme

abolish

pretreatment

other

both

BIOCHEMICAL

(Fig.

correlated of

3a), in

alterations

with fibroblast

Vol.

130,

BIOCHEMICAL

No. 2, 1985

,

AND

BIOPHYSICAL

:a)

RESEARCH

COMMUNICATIONS

(
0.5

= 0 0.4 0 L 0 :

0.3

0

E a

;

0.2

T l

0 0 0.

3.

Effect of TPA on cGMP content in human skin fibroblasts. subculture) were incubated % (v/v) foetal calf serum determined by radioimmunoassay, vity (4) using a whole-cell + S.D. of ten (a) or four TPA-treated cell? differed control cells as tested by

Glucocorticoid by

using

receptor

a

receptors

was

plot.

The

decrease

estimated

in

(Fig.

Thus

with being

receptor vity

were not

have

previously

not

augmented shown). due

or This

to

(31,

action

of

on

resulted

CAMP

and

skin

mediate

in fibroblasts.

the

stimulus

partly

the

cortisol

inactivation gene

we enhanced

593

of type

IV

changes (21). by

cGMP,

may during

that growth

IV

the after

profactor

collagenase

TPA, of

the

(22)

type

this

acti-

since by

TPA

show

on

growth

receptors,

activity

a

a synergistic

the

of

by

Here

have

and

expression

for

acts

accumulation

receptor

by

fibroblasts

proliferation,

exerted

possibility

Scatchard

receptors. to

on

cGMP

in

with

alterations

least

glucocorticoid

factor

of

the

at of

cortisol

the

active

accompanied

subconfluent

cell

measured of

of

was

in

on

growth

glucocorticoid

by

human may

effect

abscissa

synthesis

of

accumulation

excludes

of

(20)

effects

interactions

activation the

an

the

demonstrated

increased

directly

modulation

mediating sion

to

also

molecular

The

the

the even

factor

was content

treatment

TPA

been

fibroblasts cellular

activity that

through

Here due

TPA

inactivation

growth

mediated

prevent

on

the

possible via

epidermal

protein.

did

is

The

intercept

after

enzyme

Glucocorticoids

bably

the

cGMP

skin

(3).

receptor

it

degrading

effect

from in

human

assay

glucocorticoid

3bI.

collagen

of

binding

increase

a a. I-

and glucocorticoid receptor activity Subconfluent fibroblasts (3rd day after for 24 h in medium supplemented with 10 and 10m7 M TPA. cGMP content (a) was and glucocorticoid receptor-actibinding assay. The bars represent mean (b) determinations. The values for the Ggnificantly (***) from those of the Student's J-test (p
activity

whole-cell

1 0 L +a E :

0

a :

0L c c : Fig.

IL

I

0 I- rl

**

cortisol but

cGMP

rather (data

observed

cyclic be

not being

nucleotides the

mechanisms

controlled same

divipathway

treatment

of with

Vol.

130,

No. 2, 1985

phorbol

tumour

BlOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

promoters.

cocorticoid

receptors

uncontrolled

cell

level

of The

action

under

in

wound

makes high

The

the

migratory

healing,

while

cells

metastatic

excess

that by

induction

the

type

of

inactivation

cGMP

may

lead

of to

glu-

excessive

the

primary

disturbance

being

IV

collagen

degrading

enzyme

glucocorticoids

of

the

enzyme

potential in

of

This of

in during

the

carcinogenesis

metastatic. potential

carcinogenesis,

at

the

factors.

suggest control

fibroblasts. reflect

an

(23),

growth

data

negative

may

by

in

proliferation of

present

Likewise,

cells the

may,

at

least

cultured

normal (24),

human

fibroblast for of be

the

be skin

division

needed,

overproduction partly,

may

example, the

cause

enzyme of

the

sarcomas.

ACKNOWLEDGEMENTS

This work was supported by grants from the Medical Research Council of the Academy of Finland, the National Institutes of Health, U.S.A. (AM 271391, the Finnish Cancer Foundation and Orion Corporation Ltd, Helsinki, Finland. The valuable suggestions and comments of Dr. Kari I. Kivirikko and Dr. Aarne Gikarinen are gratefully acknowledged and also the skilful technical assistance of Miss Heli Auno and Miss Aila Jokinen. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Cutroneo, K-R., Rokowski, R., and Counts, D.F. (1981) Collagen Relat. Res. 1, 557-568. Rousseau, G.G. (1984) Biochem. J. 224, 1-12. (1984) Biochim. Oikarinen, J., Hamalainen, L., and Oikarinen, A. Biophys. Acta 799, 158-165. Kurl, R.N., and Jacob, S.T. (1984) Biochem. Biophys. Res. Commun. 119, 700-705. Grandics, P., Miller, A., Schmidt, T.J., and Litwack, G. (1984) Biochem. Biophys. Res. Commun. 120, 59-65. Salo, T., Liotta, L.A., and Tryggvason, K. (1983) J. Biol. Chem. 258, 3058-3063. Fessler, L.I., Duncan, K.G., Fessler, J.H., Salo, T., and Tryggvason, K. (1984) J. Biol. Chem. 259, 9783-9789. K. (1982) in Tumor Invasion Liotta, L.A., Garbisa, S., and Tryggvason, and Metastasis pp. 319-333, Martinus Nijhoff Publishers, The Hague. Liotta, L.A., Tryggvason, K., Garbisa, S., Hart, I., Foltz, C.M., and Shafie, S. (1980) Nature 284, 67-68. Salo, T., Liotta, L.A., Keski-Oja, J., Turpeenniemi-Hujanen, T., and Tryggvason, K. (1982) Int. J. Cancer 30, 669-673. Salo, T., Turpeenniemi-Hujanen, T., and Tryggvason, K. 3. Biol. Chem. (in press). and Armuth, V. (1981) Biochim. Biophys. Acta 651, 51-63. Berenblum, I., Nishizuka, Y. (1984) Nature 308, 693-698. O'Brian, C.A., Lawrence, D.S., Kaiser, E.T., and Weinstein, B.I. (1984) Biochem. Biophys. Res. Commun. 124, 296-302. Wenner, C.E., Tomei, L.D., and Leister, K.J. (1984) Transplant. Proceed. 16, 381-385. Davis, R.J., and Czech, M.P. (1984) J. Biol. Chem. 259, 8545-8549. Cachet, C., Gill, G.N., Meisenhelder, J., Cooper, J.A., and Hunter, T. (1984) J. Biol. Chem. 259, 2553-2558. Iwashita, S., and Fox, C.F. (1984) J. Biol. Chem. 259, 2559-2567. Kolata, G. (1983) Science 219, 377-378. 594

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130,

20. 21. 22. 23.

24.

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Baker, J.B. Barsh, G.S., Carney, D.H., and Cunningham, D.D. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1382-1886. Janssens, J.PH., Wittevrongel, C., and DeLoecker, W. (1984) Anticancer Res. 4, 263-268. Groner, B., Kennedy, N., Skroch, P., Hynes, N., and Ponta, H. (1984) Biochim. Biophys. Acta 781, l-6. Bishop, J.M. (1983) Annu. Rev. Biochem. 52, 301-354. Seppa, H., Grotendorst, G., Seppa, S., Schiffmann, E., and Martin, G.R. (1983) J. Cell Biol. 96, 382-385.