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.
168, No. 2, 1990
BIOCHEMICAL
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.
726
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
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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.
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