Estrogen induced expression of the c-jun proto-oncogene in the immature and mature rat uterus

Vol. 168, No. 2, 1990 April 30, 1990

ESTROGEN

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 721-726

BIOCHEMICAL

INDUCED EXPRESSION THE IMMATURE AND

David K. Webbl,

OF THE MATURE

C-JUN RAT

Bruce C. Moulton2,

PROTO-ONCOGENE UTERUS

IN

and Sohaib A. Khanl*

Department of Anatomy and Cell Biology1 Department of Obstetrics & Gynecology2 University of Cincinnati College of Medicine Cincinnati, OH 45267 Received

March

9, 1990

SUMMARY: The expression of the proto-oncogene C-A in response to estradiol treatment in immature and mature rat uterine tissue was measured using a cDNA encoding the mouse c-jut proto-oncogene. This probe hybridized to a major RNA band of 2.7 kb and a minor 3.2 kb band. In Northern blots of total RNA from both immature and mature rat uteri, estradiol treatment resulted in at least a 3 fold increase in expression of the 2.7 kb band over control levels by 3 hr post injection. By 12 hr post injection, expression of C-&J mRNA had returned to control levels. A strong induction (~5 fold) of c-j,, mRNA expression was also observed in stroma-myometrial tissue isolated from mature rats approximately 3 hours after treatment with estradiol. The similar kinetics of induction of c-fos and c-&r emphasizes the functional significance of the fos/iun heterodimer in control of uterine cell proliferation. 0 1990 Academic Press,

kc.

The

administration

proliferative

response

hyperplasia.

Estradiol

18-24

of estradiol which

involves

treatment

by enhanced

the uterus estradiol

increased

treatment

with

administration

treatment

DNA polymerase

shows

also induces

an initial

mitotic

indices

increased

(l-3),

activity

mature

hyperemia

increases the mitotic

hours after a single estradiol

preceded

17-b to ovariectomized

rats induces

followed

index

by hypertrophy

of the luminal

and the increase

a dramatic and

epithelial

in epithelial

cells

mitosis

and DNA synthesis (4). In the prepuberal in all major

synthesis

an early stimulation

uterine

of DNA

within

tissues 24-28 15 hours

in the synthesis of all major

(5).

is rat,

hours after Estradiol

classes of RNA

(5-10). In addition the mRNAs differentiation

to a general increase in RNA expression,

of a number

of uterine

and proliferation.

*To whom correspondence

proto-oncogenes

Estradiol

estradiol

which

has been shown

activates the expression

have been implicated to activate

of

in cellular

the expression

of the

should be addressed. 0006-291x/90 721

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.

BIOCHEMICAL

Vol. 168, No. 2, 1990 c-fos -3

c-m

f N-w

I c-mHa,

c-fos proto-oncogene

and c-d

is dramatically

uteri in response to estradiol. hours post estradiol

injection

Recent experiments which

binds to a specific

of genes resulting identified

tetradecanoylphorbol

13-acetate

on both proteins,

homology

If the formation transcriptional treated

with estradiol

we present

evidence

both immature with kinetics RNA

from

similar

(17-20).

factor binding

(TPA)

mediated

This

induction

which

forms a structure

two other members,

protein

reaching

rat

peak levels at 3

that the expression

mediated known

d-B

tissue of mature

induction

of c-d

MATERIALS

mRNA

sequence has been

mRNA

c-fos.

involved

which

Furthermore,

ovariectomized expression

(17-21,24).

(19,25,26).

for c-fos

mediated

in the rat uterus

to that of c-fos.

is increased

of

share significant

sequence with c-jut

proto-oncogene

The

of a region

zipper”

is necessary

in 12-0-

(21-23).

by the presence

In this paper,

in the whole

rats in response to treatment

to those of the proto-oncogene

the stroma-myometrial

recognition

and jvn-D,

in a manner similar

ovariectomized

in the 5’ region of a number

as a “leucine

of the c-h

of c-d

form a heterodimer

of gene transcription

heterodimer

then the expression

should be increased

that there is a significant

is transient,

site, and as also being

is primarily

of a fos/u

and mature

of the

and mature ovariectomized

sequence (TGACTCA)

to bind to the AP-1 recognition

regulation,

The expression

that the fos and iun proteins

heterodimer

also contains

and the ability

in both immature

transcription

transcription

homology

(1 l-16).

(11,12).

DNA recognition

of the jun/fos

family

increased

have demonstrated

formation

The iun

B proto-oncogenes

This increase in expression

in increased

as the AP-1

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

uterus of

estradiol

17-8

we have isolated total

rats and present evidence

in this tissue.

AND METHODS

Animals: The mature rats used in this study were ovariectomized Sprague-Dawley female rats (150-174 g, Zivic-Miller or Harlan). The immature rats were ovariectomized Sprague-Dawley female rats (40-45 g, Zivic-Miller). The animals were given animal chow and water ad libitum in animal facilities illuminated between the hours of 0500 and 1900. Animals were injected SC with 2 pg of estradiol 17-P dissolved in 0.5 ml of 5Oh ethanol and 95% saline in the periscapular region. Animals were sacrificed by cervical dislocation. Uteri were quickly excised, trimmed of extraneous connective tissue, and frozen between two blocks of dry ice. For the preparation of luminal epithelial cell extracts and isolated stroma-myometrial tissue, the uterine horns were slit longitudinally and placed in cold phosphate-buffered saline containing 1 mg/ml heparin (Sigma) and 10 mM ribonucleosidevanadyl complexes (Sigma) in a round bottom tube containing five 5 mm glass balls and were mixed with a Vortex mixer for 2 min at 4°C (27). Immediately after preparation, the epithelial extract was transferred to 6M guanidine thiocyanate (Sigma) for isolation of total RNA. The stroma-myometrial tissue was frozen between two blocks of dry ice prior to total RNA isolation. Total RNA Isolation: Total RNA was isolated from whole uteri, epithelial cells, and stromamyometrial tissue by the method of Chomczynski and Sacchi (28). Tissues were homogenized 722

Vol.

BlOCHEMlCAL

168, No. 2, 1990

AND BlOPHYSiCAL

RESEARCH COMMUNICATIONS

in an RNA homogenization buffer containing 4M guanidine thiocyanate (Sigma), 25mM sodium The homogenate was extracted with citrate (pH 7), 0.5% sarcosyl, and 2-mercaptoethanol. phenol and chloroform. The aqueous phase was precipitated with l/10 volume of 2M sodium acetate (pH 4) and 1 volume of isopropanol. The concentration of total RNA was determined by measuring the optical density at Ass,. The concentrations were confirmed by comparing the strength of the florescence of ethidium bromide stained 28s and 18s ribosomal RNA bands on an agarose-formaldehyde gel. Northern Blot Analvsis: 25 or 50 pg samples of total RNA from each time point were electrophoresed on formaldehyde-agarose gels and then transferred to nitrocellulose paper weight marker RNA (Bethesda Research (Schleicher & Schuell) by capillary blot. Molecular Laboratories) was stained with ethidium bromide and photographed to measure RNA migration. Filters were prehybridized in a buffer containing 40% formamide, 4x SSC, 0.04 M sodium phosphate, 1.3x Denhardt’s solution, 0.1% SDS, 1 pg/ml poly-riboadenylic acid, and 0.2 mg/ml of denatured salmon sperm DNA at 42°C for at least 3 hr. [s2P] labeled DNA probes were prepared by nick translation of a 1.5 kb & 1 fragment of a mouse c-jun cDNA (obtained from I. M. Verma, Salk Institute) or a constitutively expressed cDNA probe designated 1A (obtained from C. R. Lyttle, University of Pennsylvania). Specific activity of the probes ranged from 9~10~ to 5x10*. Denatured probe was added to the prehybridization buffer at a final concentration of approximately 1~10~ dpm/ml of buffer and incubated overnight at 42°C. Filters were autoradiographed after washing twice in 2x SSC and 0.1% SDS at room temperature for 10 minutes, and twice in 0.1x SSC and 0.1% SDS at 55-6OaC for 20 minutes. Where indicated in the text, the intensity of the RNA bands was measured using a Hoefer Scientific Instruments GS300 scanning densitometer. RESULTS In order to determine uterus of immature

the pattern

of expression

analyzed

by Northern

a probe,

Hybridization

Northern

uteri of both mature and immature

of c-u

peaked

as early

as 30 minutes

transient.

Expression

expression

control

onto

constitutively estradiol

The expression

I fragment

rats (figure

that the expression

post injection. of c-a

though The

the gel (figure

1). Densitometric

injection.

expressed in uterine

probe

recognizes

tissue and, as reported

(29). 723

was

cDNA

tracing

(figure

as

whole

In whole

whole

at least 3

could be detected

of c-&r uteri

of the

2). Expression

mRNA

was

had declined

to

uteri of immature

levels by 12 hours post estradiol

1A in order to confirm This

treatment

in expression

from mature and immature

3).

mRNA

from a mouse c-u

and mature

levels by 6 hours post estradiol

probe designated

of c-d

of the 2.7 kb band increased

increase

to control

in the whole

RNA was isolated at 0,

an increase in expression

in both immature

had returned

c-u

band and a 3.2 kb minor band

and mature rats in response to estradiol

blots of total RNA

constitutive loaded

revealed

at 3 hours post injection,

above control

Northern

total cellular

with this probe detected a 2.7 kb major

autoradiogram

rats, c-&r

with estradiol.

blot analysis using a 1.5 kb &

fold in both immature

slightly

of the proto-oncogene

and mature rats treated with estradiol,

0.5, 1, 3, 6, and 12 hours after injection

in the whole

AND DISCUSSION

injection.

uteri were also probed

with a

that equal amounts of RNA were a 2 kb mRNA

earlier,

species which

is

did not change in response to

BIOCHEMICAL

Vol. 168, No, 2, 1990

A

123456 I I I

4.4kb

-

3.2kb 2.7kb 2.4kb

-

I

I

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I

02

Effect

of Estrodiol

o-o

Mature

0

2

on Uterine

Expression 0-O

4

6

of c-jun Immature

6

10

12

HOWS

Figure 1: (A) Autoradiograph of Northern blot of immature rat uterine total RNA probed with [ssP] labeled mouse c-h cDNA probe. 50 pg of total RNA were loaded in each lane. Lane 1: non-estradiol treated control; Lane 2: 0.5 hr post estradiol injection; Lane 3: 1 hr post estradiol injection; Lane 4: 3 hr post estradiol injection; Lane 5: 6 hr post estradiol injection; Lane 6: 12 hr post estradiol injection. (B) Autoradiograph of Northern blot of mature rat uterine total RNA probed with ts2P] labeled mouse c-iyn probe. 50 pg of total RNA were loaded in each lane. Lane 1: non-estradiol treated control; Lane 2: 0.5 hr post estradiol injection; Lane 3: 1 hr post estradiol injection; Lane 4: 3 hr post estradiol injection; Lane 5: 6 hr post estradiol injection. Positions of RNA size markers are indicated at the sides of the autoradiographs. Figure 2: Densitometric scan3 of the 2.7 kb c-&r band from the Northern blots in figure 1. 0-m immature rats. O-O: mature rats. The c-h mRNA levels are expressed in relative units based on control samples present on the same blot.

In two

additional

ovariectomized

experiments,

rat uteri

myometrial

tissue (27,28).

myometrial

were

demonstrated

a strong induction

with

non-estradiol

data indicated

seperated

Northern

tissue of rats which

control

the epithelial

activated

by estradiol

rats and in the stroma-myometrial similar

to those reported

was isolated

blot analysis of total RNA

from

from

the stroma-

the uterine

stroma-

3 or 3.5 hours with estradiol

of c-h

in estradiol

mRNA

of c-d

expression 4).

mRNA

injection

we have demonstrated 17-p with

RNA

tissue of mature

had been treated for either

levels at 3 and 3.5 hours post estradiol In this report,

and total

treated rats (figure

that the expression

and stroma-myometrial

Densitometric was increased

respectively

tissue of mature

for the c-fos proto-oncogene 124

treated rats compared

scans of the Northern

6.9 and 5.7 fold over control

of mRNA

encoding

in the uteri of both mature rats.

blot

(data not shown).

that the expression

the same kinetics

17-8

The kinetics

c-d

is

and immature

of this induction

are

in the same system and represents

one

Vol.

168, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

c- jun-

A

2kb -

B 0

3

2kb -

0

4

Figure 3: (A) Autoradiograph of Northern Blot of immature rat uterine total RNA from figure 1A re-probed with tsaP] labeled 1A cDNA. (B) Autoradiograph of Northern blot of mature rat uterine total RNA from figure 1B re-probed with [s2P] labeled 1A cDNA. The size of the bands are indicated at the sides of the autoradiographs. Figure 4: Autoradiograph of Northern blot of 25 pg of total RNA from the stroma-myometrial tissue of mature ovariectomized rats treated with estradiol 17-P. Lane 1: non-estradiol treated control; Lane 2: 3.5 hr post estradiol injection; Lane 3: non-estradiol treated control; Lane 4: 3 hr post estradiol injection.

of the earliest markers of estrogen action in the uterus (11,12). have been shown

to form a heterodimer

the co-activation important that

other

proliferative

regulatory members

of the expression step in estrogen of the iun

which

can act as a transcriptional

of the c-fos induced

family,

and c-u

uterine

U-B

response of the uterus to estradiol

Since the iun and fos proteins regulatory

proto-oncogenes

growth

and differentiation.

and a-D,

may also play

and is the focus of future

factor,

may be an It is likely a role

in the

research.

Acknowledgments: This research was supported by NIH grant # HD22918 and HD10721. We thank Dr. I. M. Verma (Salk Institute) and Dr. C. R. Lyttle (University of Pennsylvania) for providing the c-u and 1A cDNAs. The authors wish to thank Barbara Burch for preparation of this manuscript. REFERENCES 1. 2. 3. 4. 5. 6. 7.

Martin L., Finn C., A., and Trinder, G. (1973) J. Endocrinol. 56, 133-144. Clark, B., F. (1973) J. Endocrinol. 56, 341-342. Clark, B., F. (1973) J. Endocrinol. a, 527-528. Kaye, A., M., Sheratzky, D., and Lindner, H., R. (1972) Biochim. Biophys. Acta 2&l, 475-486. Kirkland, J., LaPointe, L., Justin, E., and Stancel, G. (1979) Biol. Reprod. 2l, 269-272. Billing, J., R., Barbiroli, B., and Smellie, R., M., S. (1969) Biochem. J. 112, 563-569. Luck, D., N., and Hamilton, T., H. (1972) Proc. Natl. Acad. Sci. USA 69, 157-161. 72s

Vol. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Knowler, J., T., and Smellie, R., M., S. (1971) Biochem. J. 125, 60.5-614. Knowler, J., T., and Smellie, R., M., S. (1973) Biochem. J. 131, 689-697. Mohla, S., DeSombre, E., R., and Jensen, E., V. (1972) Biochem. Biophys. Res. Commun. 4&, 661-667. Loose-Mitchell, D., Chiappetta, C., and Stancel, G. (1988) Mol. Endocrinology 2, 946-951. Weisz, A., and Bresciani, F. (1988) Mol. Endocrinology 2, 816-824. Murphy, L., Murphy, L., and Friesen, H. (1987) Endocrinology 120, 1882-1888. Travers, M., and Knowler, J. (1987) FEB 211, 27-30. Mukku, V., and Stancel, G. (1985) J. Biol. Chem. 260, 9820-9824. Lingham, R., Stancel, G., M., and Loose-Mitchell, D., S. (1988) Mol. Endocrinol. 2, 230-235. Kouzarides, T., and Ziff, E. (1988) Nature 336, 646-651. Holazonetis, T., Georgopoulos, K., Greenberg, M., and Leder, P. (1988) Cell 55, 917-924. Nokabeppu, Y., Ryder, K., and Nathans, D. (1988) Cell s., 907-915. Sassone-Corsi, P., Ransone, L., Lamph, W., and Verma. I. (1988) Nature 336, 692-695. Neuberg, M., Schuerman, M., Hunter, J., and Muller, R. (1989) Nature 338, 589-590. Lee, W., Mitchell, P., and Tijan, R. (1987) Cell 49, 741-752. Rauscher, F., Sambucetti, L., Curran, T., Distel, R., and Spiegalman, B. (1988) Cell 52, 471-480. Schuerman, M., Hunter, J., Jenuwein, T., Ryseck, R., Bravo, R., and Muller, R. (1989) Cell 56, 507-516. Schutte, J., Viallet, J., Nau, M., Segal, S., Fedorko, and Minna, J. (1989) Cell 59, 987-997. Chiu, R., Angel, P., and Karin, M. (1989) Cell 3, 979-986. Fagg, B., Martin, L., Rogers, L., Clark, B., and Quarmby, V.E. (1979) J. Reprod. Fert. 57, 335-339. Chomczynski, P., and Sacchi, E. (1987) Anal. Biochem. lf&?, 156-159. Hsu, C., J., Komm, B., S., Lyttle, C., R., and Frankel, (1988) Endocrinology 122, 631-639.

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