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(Received for publication, October 19, 1995; and in revised form, February 21,
1996) From the
Transforming growth factor The transforming growth factor In bovine fasciculata adrenal cells (BAC), the
expression and the maintenance of specific differentiated functions are
regulated not only by corticotropin (ACTH) and angiotensin-II (AngII),
the two main hormones that control steroidogenesis, but also by growth
factors, which have been shown to have pleiotropic effects in addition
to their mitogenic action. In bovine and ovine adrenocortical cells, it
has been shown that TGF Synthetic oligonucleotides represent a new tool to
investigate the role of many proteins in cell growth and
differentiation. Ideally, an antisense oligonucleotide is targeted in a
sequence-specific manner to nucleic acids (RNA or DNA) to inhibit the
expression of a specific protein involved in cellular signal
transduction, growth, proliferation, or differentiation(21) .
Antisense oligonucleotide inhibition of cellular protein production has
been used to study the actions of several growth factors including
basic fibroblast growth factor(22, 23) , insulin-like
growth factor-I(24) , insulin-like growth
factor-II(25) , platelet differentiating growth factor, and
TGF In the present study, using a TGF
TGF
Figure 1:
Time course of TGF
To examine the extent of oligonucleotide degradation within cells
and in the medium during the cellular uptake, TGF
Figure 2:
Intracellular stability of the TGF
Figure 3:
Intracellular distribution of the
TGF
In an attempt to determine the
intracellular formation of an oligonucleotide-RNA duplex, the medium
and the cell lysates were submitted to partial S1 nuclease digestion.
This enzyme digested all the non hybridized nucleotides. As expected,
the TGF
Figure 4:
Intracellular hybridization of the
TGF
Next, we
investigated the intracellular compartment in which the hybridized
TGF
Figure 5:
Intracellular distribution of hybridized
TGF
Figure 6:
Effects of TGF
Figure 7:
Effects of TGF
Figure 8:
Effects
of cyclosporine and/or TGF
Figure 9:
Effects of TGF
One of the
mechanism by which exogenous TGF
Figure 10:
Effects of TGF
Figure 11:
Effects of exogenous TGF
Figure 12:
Effects of transfection on
G
Antisense oligonucleotides have been used as specific
inhibitors of target gene expression. The specificity of an antisense
oligonucleotide is due to highly specific hybridization to its
complementary target sequence on the mRNA by Watson-Crick base pairing.
This is obtained by using an oligonucleotide of about 15 bases directed
against a complementary sequence of target
mRNA(21, 36, 37) . One key parameter in the
oligonucleotide antisense approach is its intracellular concentration,
which is the result of two opposite processes: the rate of penetration
of the antisense molecule across cell membrane, and its rate of
degradation in the cells. The uptake is a saturable process thought to
be mediated by both receptor endocytosis and fluid phase
endocytosis(38, 39, 40) . An increased uptake
has been obtained by encapsulation of the oligonucleotides in cationic
liposomes (41) . Using a cationic liposome-mediated
transfection method in cell culture, we demonstrated a rapid, high, and
similar uptake of both TGF In addition, our results revealed marked
difference between TGF The present studies also show that 44 h
after transfection about 44% and 75% of the TGF Although the potential
autocrine role of TGF Taken together our data demonstrate, for the
first time, that constitutive expression of TGF
Volume 271,
Number 18,
Issue of May 3, 1996 pp. 11027-11033
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
1 Protein by Antisense
Oligonucleotide-induced Increase of Adrenal Cell Differentiated
Functions (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 (TGF1) is a potent
inhibitor of several differentiated functions in bovine adrenal
fasciculata cells (BAC). In addition, these cells express and secrete
this factor. To determine whether this peptide plays an autocrine role
in BAC, cells were transfected with 10 µM unmodified sense
(SON) or antisense (AON) oligonucleotide complementary to the
translation initiation region of the TGF1 mRNA in an attempt to
inhibit TGF1 protein synthesis. We investigated first, the
cellular uptake, the stability, and the intracellular distribution of 
P-labeled TGF1 AON and SON; and second, the effects
of both oligonucleotides on BAC specific functions. We have
demonstrated that in BAC, the TGF1 AON uptake reached a plateau
after 8 h of transfection (16% of the radioactivity added) and remained
fairly constant for at least 24 h. In contrast, the uptake of TGF1
SON reached a plateau after 2 h of transfection (8% of the
radioactivity added), remained stable for only 3 h, and then declined.
After 8 h of transfection, followed by 44 h of culture without
oligonucleotides, the intracellular level of TGF1 AON was still
high with about 8% of the radioactivity added, whereas that of
TGF1 SON represented only 1.2%. Moreover, AON was present in the
cytoplasmic and nuclear fractions, and it was hybridized in both
compartments. However, TGF1 SON was present mainly in the
cytoplasmic fraction where it was not hybridized. Neither TGF1 AON
nor SON modified TGF1 mRNA levels; however, TGF1 AON, but not
SON, caused the disappearance of TGF1 immunoreactivity inside the
cells. Finally, the steroidogenic responsiveness of BAC transfected
with TGF1 AON increased about 2-fold, and this was associated with
a 2-fold increase of the mRNA levels of both cytochrome P450
17
-hydroxylase and 3-hydroxysteroid dehydrogenase. Neither
TGF1 SON nor a scrambled oligonucleotide containing the same
number of G nucleotides as TGF1 AON had any effect on these
parameters. Thus, these studies demonstrate that TGF1 has an
autocrine inhibitory effect on BAC differentiated functions, an effect
that can be overcome by TGF1 AON.
(TGF) (
)family of peptides consists of related disulfide-linked
homodimers that have multifunctional regulatory activities in many cell
types and are expressed in many normal and malignant
tissues(1, 2, 3) . Three isoforms, termed
TGF1, TGF2, and TGF3, have been identified in
mammals(4) . Although in many cells the three TGFs display
comparable activities and potencies, marked differences have been noted
in some cases(2, 3) . Cross-linking experiments have
shown that the TGF family of peptides binds to three different
receptors, named type I, II, and III(2) , which have been
cloned(5, 6, 7, 8) . Recent studies
have determined the role of each type of TGF receptor. Type III
receptor, also known as betaglycan, has no direct role in TGF
signaling, but increases the binding of TGF, in particular
TGF2, to type II receptor, enhances cell responsiveness to
TGF, and diminishes the biological differences between TGF
isoforms(9) . Types I and II receptors are transmembrane
serine/threonine kinases. The role of these molecules in signaling has
now been determined, and both types are required for TGF
signaling(10) . TGF binds directly to receptor II. Bound
TGF is then recognized by receptor I, which is recruited into the
complex and becomes phosphorylated by receptor II. Phosphorylation
allows receptor I to propagate the signal to downstream substrates.
Recent results suggest that serine residues in the GS domain (region
preceding the kinase domain) of receptor I, which are phosphorylated by
receptor II, are important for signal transduction by receptor I (11) .1 is a potent inhibitor of basal as well as
ACTH-induced cortisol production(12, 13) . TGF1
exerts its effects at several levels: inhibition of low density
lipoprotein receptors (12) , inhibition of cytochrome P450
17-hydroxylase and 3-hydroxysteroid dehydrogenase (3
HSD) activities, protein and mRNA
contents(14, 15, 16, 17) , and
down-regulation of ACTH receptors in ovine adrenocortical
cells(18) . TGF1 has also been proposed to regulate the
steroidogenic functions in an autocrine loop (19) in BAC cells,
which possess TGF receptors that are regulated by
ACTH(20) . In addition, BAC cells synthesize (19) and
secrete a latent form of TGF-like activity(15) . In BAC,
TGF1 secretion is regulated by specific peptide hormones; ACTH
decreases TGF1 mRNA level, whereas AngII increases TGF1 mRNA
and protein levels. (
)All these data suggest that TGF1
local production could play an autocrine role on BAC differentiated
functions.1 (23) .1
antisense oligodeoxynucleotide complementary to a sequence that
includes the translation start site of the human TGF1 mRNA, we
have inhibited TGF1 synthesis in BAC and demonstrated an autocrine
role for TGF1 on BAC differentiated functions.
Materials
[
-P]ATP
(>4000 Ci/mmol) and [-P]dCTP (>3000
Ci/mmol) were purchased from ICN Biomedicals France (Orsay), and
[
S]methionine (>1000 Ci/mmol) from Amersham
(Les Ulis, France). Synthetic AngII was obtained from Bachem
(Bubendorf, Switzerland), porcine TGF1 from R& Systems
(Minneapolis, MN), cyclosporine (Sandimmun) from Sandoz
(Rueil-Malmaison, France), synthetic unmodified 15-base
deoxyribonucleotides from Eurogentec-France (Angers),
Lipofectamine(TM) Reagent from Life Technologies, Inc. (Cergy
Pontoise, France), and acroleine from Polysciences (Warrington, PA).
Amplify and Hybond-N membrane were purchased from Amersham (Les Ulis,
France), and Protein A-Sepharose CL-4B from Sigma. Human TGF1 cDNA
was donated by Dr. R. Derynck (Genentech Inc., San Francisco,
CA)(26) , bovine P450 17-hydroxylase cDNA by Dr. M. R.
Waterman (Vanderbilt University School of Medecine, Nashville,
TN)(27) , and human 3 HSD cDNA by Dr. F. Labrie and V. Luu
The (Centre Hospitalier Universitaire Laval,
Québec, Canada)(28) . Polyclonal rabbit
antibody (C-11-V) directed against a common C-terminal peptide of
G
/G
proteins and polyclonal
anti-TGF1 rabbit antibody were prepared in our laboratory as
described previously(29, 30) . Goat anti-rabbit
immunoglobulin G (IgG) conjugated to peroxidase was purchased from
Nordic Immunology (Tilburg, The Netherlands).Isolation and Culture of Bovine Adrenocortical
Cells
BAC were prepared by sequential treatment of adrenal
cortical slices with trypsin (0.16%) as described
previously(31) . Then, cells were purified on a discontinuous
Percoll density gradient (d = 1.032, 1.048, and 1.082
g/ml) to eliminate cellular fragments and red blood cells. The purified
fasciculata cells recovered on the Percoll gradient with a density of
1.048 g/ml were collected, washed and cultured in a chemically defined
medium, Ham's F-12/Dulbecco's modified Eagle's medium
(1:1), containing 10 µg/ml transferrin, 10 µg/ml insulin,
10
M vitamin C, and antibiotics without
serum.Oligonucleotides
Antisense, sense, and scrambled
unmodified 15-base deoxyribonucleotides corresponding to the
translation initiation region of human TGF1 mRNA were used:
antisense (AON) (5`-GGA GGG CGG CAT GGG-3`); sense (SON) (5`-CCC ATG
CCG CCC TCC-3`); scrambled (SCR) (5`-AGG TGG GAG GCG GCG -3`).Cell Transfection and Viability Test
On day 2 of
culture, cells were transfected with labeled and/or unlabeled TGF1
AON or SON. To introduce the oligonucleotides into BAC, a cationic
liposome-mediated transfection method was used. Oligonucleotides
dissolved in one volume of antibiotic-free medium were mixed with
Lipofectamine(TM) reagent dissolved in the same volume of
antibiotic-free medium and incubated for 45 min at room temperature.
Thereafter, the oligonucleotide-liposome complexes were diluted with
eight volumes of antibiotic-free medium and then added to cells that
had been washed twice with antibiotic-free medium. In the experiments
reported, the concentration of oligonucleotides and
Lipofectamine(TM) in the transfection medium was 10 µM (50 µg/ml) and 1.25%, respectively. For the viability test
(trypan blue exclusion assay), the number of living cells was assessed
at the end of the experimental period (8 h of transfection followed by
44 h of culture).Oligonucleotide Cellular Uptake and
Degradation
Oligonucleotides were 5`-labeled with
[-P]ATP by use of bacteriophage T4
polynucleotide kinase and further purified by dialysis (specific
activity 8 
10
dpm/µg). The transfection medium
containing 1 10
dpm/ml P
oligonucleotides and 10 µM unlabeled oligonucleotides was
added to the cells. At indicated times, the culture medium was removed
and saved, cells were washed three times with medium, and the cell
washes were also removed and saved. Cells were lysed in Tris-buffered
saline (10 mM Tris-HCl, pH 7.4, containing 150 mM NaCl and 1% sodium dodecyl sulfate) and extracted with
phenol-chloroform-amyl alcohol (25:25:1, v/v/v). After centrifugation
(12,000 g, 15 min, 4 °C), the aqueous phase was
removed and saved. Then the phenol phase was extracted with water and
centrifuged, and the aqueous phase was removed and pooled with the
first one. Aliquots of the three fractions, i.e. combined
aqueous phases corresponding to the intracellular radioactivity, cell
washes, and culture medium, were counted. Oligonucleotide uptake was
calculated as the percentage of the intracellular radioactivity over
the counts recovered in the three fractions. To determine
oligonucleotide degradation, aliquots containing equal amounts of
radioactivity of the combined aqueous phases and of the culture medium
fraction were analyzed by electrophoresis (10% polyacrylamide, 7 M urea gel) and autoradiographed.Oligonucleotide Distribution and
Hybridization
After transfection with 5`-P-labeled
oligonucleotide, a subcellular fractionation of the cells was carried
out. Briefly, after washes, cells were removed from the culture plate
with trypsin (100 g, 10 min, 4 °C) and washed
twice with medium. The cells were lysed for 10 min at 4 °C in
buffer A (10 mM Tris HCl, pH 7.4, 10 mM NaCl, 5
mM MgCl, 0.5% Nonidet P-40, 1 mM dithiothreitol). After centrifugation at 800 g for 10 min, the upper cytoplasmic fraction (combined cytosol and
cell membranes) was removed and saved. The pellet (nuclei) was
dissolved in a small quantity of buffer A and then laid over 1 ml of 25
mM Tris-HCl, pH 7.4, 2.5 mM MgCl, 0.25 M sucrose and centrifuged (800 g for 10 min).
The purified nuclei pellet was dissolved in buffer A. Aliquots of the
cytoplasmic and nuclear fractions were counted. In order to determine
whether the oligonucleotide formed duplexes with cellular RNA, an S1
nuclease protection assay was performed on aliquots of cell lysates,
cytoplasmic, and nuclear fractions. As a control the medium was also
treated with the enzyme. The nucleic acids of each fraction were
precipitated at -20 °C for 20 min and -70 °C for 10
min with cold ethanol (2.2 volumes), 3 M sodium acetate (0.1
volume), pH 5.2, and 10 µg of tRNA. After centrifugation (12,000
g for 30 min at 4 °C), the pellet was washed with
cold ethanol (70%), lyophilized for a short time, and then resuspended
in 70 µl of hybridization buffer (40 mM PIPES, 400 mM NaCl, 1 mM EDTA). Then 30 µl of each aliquot was
incubated without (control) or with 20 units of S1 nuclease 30 min at
37 °C in 200 µl of digestion buffer (280 mM NaCl, 30
mM sodium acetate, 4.5 mM ZnSO
, 20
µg/ml salmon sperm DNA). The nuclease action was stopped by putting
the samples on ice and by addition of 60 µl of termination buffer
(1.5 M sodium acetate, 125 mM EDTA, 75 mM MgCl). Samples were precipitated at -20 °C
for 20 min and -70 °C for 10 min with 20 µg of tRNA and
0.75 ml of ethanol, centrifuged, and washed as described above. Samples
were analyzed by electrophoresis using 10% polyacrylamide, 7 M urea gel. Protected fragments were visualized byautoradiography.RNA Preparation and Northern Blot Analysis
Total
RNA was isolated from cells by the method of Chomczynski and
Sacchi(32) . Samples (10-15 µg of RNA) were separated
by electrophoresis through a 1% agarose gel containing 10%
formaldehyde. RNA was then transferred to Hybond-N membrane.
Prehybridization and hybridization solutions used were described
previously(30) . Labeled human TGF1 cDNA, bovine P450
17-hydroxylase cDNA, and human 3 HSD were used as probes
(1-2 10 dpm/ml). Labeling of these probes in
the presence of [-P]dCTP was performed with
a Megaprime DNA labeling system (Amersham). The blots were washed with
more or less stringency depending on the probes used and then exposed
to photographic film. The relative intensity of hybridization signals
was quantified by using a scanning densitometer (Preference Sebia,
Paris, France). Equal loading of RNA samples was confirmed by scanning
the 28 S RNA negatives.Immunocytochemistry
BAC cells were plated at a
density of 6.0 10 cells/chamber in eight-chamber tissue
culture slides (Plastic Labtek) and transfected with antisense or sense
oligonucleotide as described above. After the transfection medium was
removed, cells were washed two times with fresh medium, cultured during
44 h and subjected to immunocytochemical analysis. For comparative
studies, control cells were always run in the same immunocytochemical
assay to reduce discrepancies related to interassay variability in
staining intensity.1 expression was examined by an indirect
immunocytochemical method as described previously(30) .
Briefly, cells were fixed 30 min at room temperature in 2% acrolein in
10 mM phosphate buffer (pH 7.4), and washed overnight in 100
mM PBS (pH 7.6) at 4 °C. Cells were then permeabilized
with 0.1% Triton X-100 for 30 min, rinsed, and exposed for 1 h to a
1/40 dilution of nonimmune rabbit serum. The polyclonal anti-TGF1
rabbit antibody was used as primary antibody at a dilution of 1/1000
overnight in a humidity chamber at 4 °C. The second antibody to
rabbit IgGs conjugated to peroxidase was used at a dilution of 1/200
for 1 h at room temperature. To localize the antigen-antibody
complexes, cells were incubated for 2 min with 0.05% 3,3`
diaminobenzidine tetrahydrochloride, 0.01% HO,
and 2.5% nickel ammonium sulfate. Next, the cultured cell preparations
were mounted in PBS-glycerol (1:1). The specificity of the TGF1
antibody has been tested previously (30) .Cortisol Production
It was measured in the medium
by a specific radioimmunoassay(33) .Metabolic Labeling
For metabolic labeling, before
the end of the culture, the cells were preincubated for 1 h in
methionine-free medium, after which the medium was replaced by fresh
methionine-free medium containing [S]methionine
(50 µCi/ml) during the last 4 h.Immunoprecipitation
After metabolic labeling,
cells were washed three times with phosphate buffered saline. Cells
were then lysed by addition of 500 µl of ice-cold
immunoprecipitation buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin). After
centrifugation for 10 min at 10,000 g, the
supernatants from the cells extracts were incubated at 4 °C for 2 h
with nonimmune rabbit serum at a final concentration of 5% and then
with 50 µl of a 50% (v/v) suspension of protein A-Sepharose CL-4B.
After 1 h of incubation, the beads were sedimented by centrifugation.
The supernatants were collected and submitted to immunoprecipitation
with anti-/ IgG (1/50, v/v) at 4
°C for 2 h, followed by incubation with protein A-Sepharose CL-4B.
After centrifugation, the beads were washed four times with ice-cold
immunoprecipitation buffer and once with 0.1% SDS. The radiolabeled
proteins in the final pellet were analyzed by electrophoresis using
7.5% polyacrylamide gels. Gels were fixed, soaked in Amplify, dried,
and the labeled proteins were revealed by fluorography.Western Blot Analysis
After appropriate
treatments, cells were lysed and submitted to Western blot analysis as
described previously (34) except 10% polyacrylamide gels were
without urea.Statistical Analysis
Statistical analysis were
performed with Student's t test for comparison of two
groups. Differences were considered significant when p <
0.05.
Cell Viability
Exposure of BAC to AON or SON or
SCR oligonucleotides did not affect cell viability: 89.5% ±
1.0%, 87.4% ± 3.4%, 90.2% ± 2.7%, respectively, versus 91.3% ± 0.4% in control cells (n = 3).Oligonucleotide Uptake and Degradation
To
investigate the kinetics of oligonucleotide uptake and degradation,
cells were transfected with P-labeled TGF1 AON or SON
(final concentration 10 µM) for different times as
described under ``Experimental Procedures.'' Fig. 1shows the time-dependent cellular uptake of TGF1 AON (solid line) and TGF1 SON (dotted line).
TGF1 AON cellular uptake started within the first minutes of
transfection, increased progressively to reach a plateau at 8 h, and
remained stable for the next 24 h. At this time, 16% of the
radioactivity were inside the cells. Taking into account the cellular
volume and the specific activity, TGF1 AON intracellular
concentration (500 µM) was about 50-fold higher than that
in the medium. TGF1 SON cellular uptake was also rapid. Uptake
reached a plateau at 2 h, remained stable for only 3 h, and then
declined. At the plateau, 8% of the radioactivity was inside the cells.
1
AON and SON uptake and degradation. Cells were transfected with P-labeled AON (solid line) or SON (dotted
line) for 2.5, 5, 10, 15, 20, 30, and 45 min and 1, 1.30, 2, 3, 8,
12, and 24 h. For each time point, cells were lysed and extracted with
phenol-chloroform. Oligonucleotide uptake was determined by counting
the aqueous phase as described under ``Experimental
Procedures.'' Results are expressed as the percentage of total
radioactivity (top). Aliquots of AON cell lysate and medium
containing the same radioactivity were taken at different time points
and subjected to urea-PAGE and autoradiography (bottom).
1 AON was
analyzed by gel electrophoresis. The results of Fig. 1show
that, inside the cells, TGF1 AON appeared to be intact for up to
24 h. However, in the culture medium, a progressive degradation of the
TGF1 AON was observed. The oligonucleotide was first transformed
into another compound with higher mobility. This in turn was converted
into another compound with faster mobility after 3 h. A similar pattern
of degradation, but much more rapid, was observed with TGF1 SON
(data not shown).Intracellular Stability of the TGF
To determine the intracellular stability of the
oligonucleotides, cells were transfected with 1 AON and
SONP-labeled
TGF1 AON or SON for 8 h (time at which TGF1 AON uptake was
maximum), the medium was then removed and replaced with fresh medium
without oligonucleotides, and the culture continued for another 44 h (Fig. 2). As shown, after 8 h of transfection, the intracellular
concentration of TGF1 AON or SON represented 16% and 6%,
respectively, of the radioactivity added. However, after 44 h of
culture, the intracellular concentration of TGF1 AON was still 8%
of the radioactivity added, whereas that of TGF1 SON represented
only 1.2%.
1
AON and SON. Cells were transfected with P-labeled AON or
SON for 8 h. Several wells were harvested, while the medium of the
other wells was removed and replaced with fresh medium without
oligonucleotides. The culture was continued for another 44 h. Aliquots
of cell extracts at 8 h and 8 h followed by 44 h of culture were taken
and counted. The results are expressed as percent of total
radioactivity. Results are the mean ± S.D. of duplicate
measurements from two separate experiments for the TGF1 AON and
from one experiment for TGF1 SON.
Intracellular Distribution and Hybridization of TGF
In order to determine the intracellular distribution
of both TGF1
AON and SON1 AON and SON, subcellular fractionation of the cells
was performed after 8 h of transfection and 44 h after the
oligonucleotides' removal (Fig. 3). After 8 h of
transfection 60% and 40% of the intracellular TGF1 AON were in the
cytoplasmic and nuclear fractions, respectively. This distribution was
the opposite after 44 h of culture. In contrast, at both times more
than 85% of the intracellular TGF1 SON were in the cytoplasmic
fraction. Indeed, the TGF1 SON present in the nuclear fraction
after 44 h of culture without oligonucleotides represented less than
0.2% of the radioactivity added.
1 AON and SON. Cells were transfected with P-labeled AON or SON for 8 h or 8 h followed by 44 h of
culture and subcellular fractionation was carried out as described
under ``Experimental Procedures.'' Aliquots of the
cytoplasmic and nuclear fractions were counted. The results are
expressed as percent of intracellular radioactivity. Results are the
mean ± S.E. of triplicate measurements from three separate
experiments for the TGF1 AON and one experiment from TGF1
SON.
1 AON and SON derived from the culture medium were almost
completely degraded (Fig. 4). Similarly, the TGF1 SON
extracted from cells was also degraded. In contrast, after 8 h of
transfection, as well as after 44 h of culture without
oligonucleotides, about 40% of the TGF1 AON extracted from cells
were protected from degradation. These results indicated that TGF1
AON, but not SON, was hybridized inside the cells.
1 AON and SON. Cells were incubated with P-labeled
AON or SON for 8 h or 8 h followed by 44 h of culture. Aliquots of
medium and cell lysates were precipitated by ethanol to recover the
nucleic acids. For each condition, aliquots containing equal amounts of
radioactivity were incubated for 30 min at 37 °C in the absence or
the presence of 20 units of S1 nuclease. All samples were analyzed by
urea-PAGE and autoradiography. The intensity of the signal after S1
nuclease digestion is expressed as the percentage of the signal without
S1 nuclease digestion (100%). Top, diagram; bottom,
autoradiography of one representative
experiment.
1 AON was located. Subcellular fractionations were performed
after 8 h of transfection and after an additional 44 h of culture
without oligonucleotides. The extracts from cytoplasmic and nuclear
fractions were submitted to partial S1 nuclease digestion (Fig. 5). After 8 h of transfection, the hybridization of the
TGF1 AON was more marked in the cytoplasmic (about 33%) than in
the nuclear fraction (about 16%), whereas after 44 h of culture,
although the percentage of hybridization increased in both compartments
(44% and 75% for the cytoplasmic and the nuclear fractions,
respectively), the ratio was reversed.
1 AON. Cells were incubated with P-labeled AON for
8 h or 8 h followed by 44 h of culture. Subcellular fractionation was
performed and aliquots of culture medium, cytoplasmic and nuclear
fractions were subjected or not to partial S1 nuclease digestion.
Aliquots were then analyzed by urea-PAGE and autoradiography. Results
are expressed as described in the legend of Fig. 4. Top, diagram; bottom, autoradiography of one
representative experiment.
Effects of TGF
To assess whether the oligonucleotides were
able to modify the transcription and/or the translation of TGF1 AON and SON on BAC TGF1 mRNA
and Protein Content1,
we investigated their effects on TGF1 mRNA by Northern blot and on
cellular TGF1 protein content by immunocytochemistry. The results
of Fig. 6showed that neither TGF1 AON nor SON modified the
level of the 2.5-kilobase transcript of TGF1 mRNA. In contrast,
the results of immunocytochemistry (Fig. 7) showed that all the
control cells (A) or cells pretreated with TGF1 SON (C) were immunoreactive. However, the TGF1
immunoreactivity completely disappeared in TGF1 AON treated cells (D). In B, where the TGF1 antibody was saturated
with the peptide used to produce this antibody, there was no TGF1
signal, thus showing the specificity of this antibody. Since it has
been reported that cyclosporine increased the expression of
TGF1(35) , we treated BAC for 44 h with this factor (1
µg/ml) in the absence or presence of TGF1 AON and we examined
the TGF1 content by immunocytochemistry (Fig. 8). Although
this method is only semi-quantitative, the results of Fig. 8suggest that cyclosporine increased the cellular TGF1
content, and this effect was blunted by TGF1 AON.
1 AON and SON on
TGF1 mRNA. Cells were incubated for 8 h without (control cells) or
with AON or SON (10 µM). The medium was removed, replaced
by fresh medium without oligonucleotides, and the culture continued for
44 h. TGF1 mRNA was extracted and analyzed by Northern blot. A
representative Northern blot of one of the six experiments performed is
shown.
1 AON and SON on cell
TGF1 protein content. Cells were incubated for 8 h without
(control cells) or with AON or SON (10 µM). The medium was
removed, replaced by fresh medium without oligonucleotides, and the
culture continued for 44 h. Immunocytochemical staining was performed
using a specific TGF1 antibody as described under
``Experimental Procedures.'' A, control cells; B, control cells incubated with the antibody saturated with
the peptide (10 µg/ml) used to produce this antibody; C,
cells transfected with SON; D, cells transfected with
AON.
1 AON on cell TGF1 protein content.
Cells were incubated for 8 h without (A and C) or
with AON (B and D). The medium was replaced by fresh
medium without (A and B) or with (C and D) 1 µg/ml cyclosporine and the culture continued for 44
h. Immunocytochemical staining was performed as described in Fig. 7.
Effects of TGF
As described in the Introduction, exogenous TGF1 AON and SON on BAC
Functions1
decreases the steroidogenic responsiveness of BAC; thus, we
investigated the effects of both oligonucleotides, TGF1 AON and
SON, on the steroidogenic responsiveness to AngII of control and
cyclosporine treated cells (Fig. 9). In the absence of
cyclosporine, TGF1 AON increased the cortisol response to AngII
about 2-fold compared to either control cells or TGF1 SON-treated
cells. Cyclosporine alone decreased the steroidogenic responsiveness of
BAC by about 50%, compared to cells not treated with cyclosporine.
However, the cortisol production of cells treated with cyclosporine and
TGF1 AON was 2.3-fold higher than that of cells treated with
cyclosporine alone. Again, TGF1 SON had no effect.
1 AON and SON on
cortisol production. Cells were incubated for 8 h without (control
cells) or with AON or SON (10 µM), the culture was then
continued for 44 h in the absence or the presence of 1 µg/ml
cyclosporine. The medium was removed, the cells were washed, then
stimulated for 2 h with AngII 10
M.
Results, expressed as ng/10 cells, are the mean ±
S.E. of three experiments. Different letters represent a
significant difference (p <
0.05).
1 decreases the steroidogenic
capacity of BAC is by decreasing the mRNA levels of P450
17-hydroxylase and 3 HSD(14, 16) . The
results of Fig. 10clearly show that TGF1 AON, but not SON,
increased P450 17-hydroxylase and 3 HSD mRNA levels (2- and
1.7-fold, respectively), which encode two key enzymes in the
steroidogenic pathway.
1 AON and SON on P450
17-hydroxylase and 3 HSD mRNA levels. Cells were incubated
for 8 h without (control cells) or with AON or SON (10
µM). The medium was removed, replaced by fresh medium
without oligonucleotides, and the culture continued for 44 h. P450
17-hydroxylase and 3 HSD mRNA were extracted and analyzed by
Northern blot. Top, mean ± S.E. of three to six
experiments. Different letters represent a significant
difference (p < 0.05). Bottom, Northern blot of
one representative experiment.
Control Experiments to Demonstrate the Specificity of
TGF
To prove that all the TGF1 AON Effects1 AON
effects on BAC functions were specific, some additional controls were
performed. First, cells were transfected with a scrambled
oligonucleotide (SCR) containing the same number of G nucleotides (10
of 15) as TGF1 AON but in a scrambled order. The results (Fig. 11) showed that, in contrast to TGF1 AON, neither SON
nor SCR modified the cortisol secretion and the mRNA levels of P450
17-hydroxylase. Second, none of the transfected oligonucleotides
did change the sensitivity of the cells to the inhibitory effects of
TGF1, since exogenous TGF1 caused similar inhibition of both
cortisol secretion and P450 17-hydroxylase mRNA levels, in control
and in transfected cells (Fig. 11). Finally, to prove that
transfection did not produce a general inhibition of protein synthesis,
we investigated the effects of transfection with the three
oligonucleotides on the rate of synthesis and on the steady-state
levels of G/G proteins, which are
not affected by exogenous TGF1(34) . The results showed
that neither the rate of synthesis (Fig. 12A) nor the
steady-state levels (Fig. 12B) of
G/G proteins were affected in
transfected cells regardless of the nucleotide used.
1 on BAC
steroidogenic responses. Cells were incubated for 8 h without (control
cells, CNT) or with AON, SON, or SCR (10 µM), and the
culture was then continued for 44 h in the absence or the presence of 2
ng/ml of TGF1. The medium was removed, and the cells washed and
then stimulated for 2 h with AngII 10M. A, cortisol production was determined by RIA. Results,
expressed as ng/10 cells, are the mean ± S.D. of
duplicate measurements from two separate experiments. B, in
the same two experiments P450 17-hydroxylase mRNA were analyzed by
Northern blot (one representative
autoradiography).
/G synthesis and steady-state
levels. Cells were incubated for 8 h without (control cells, CNT) or with AON, SON or SCR (10 µM), the culture
was then continued for 44 h. [S]Methionine (50
µCi/ml) was added during the last 4 h of incubation. A,
immunoprecipitation of radiolabeled
G/G. B, Western blot from
the cell lysates using G/G antibody.
1 AON and SON during the first 2 h of
transfection. Thereafter, the kinetics of TGF1 SON and AON were
different. Indeed, whereas the intracellular concentration of SON,
after a short lag period, declined, the concentration of AON continued
to increase reaching a plateau at 8 h (16% of the radioactivity added)
and remained stable for at least 24 h. This uptake is several times
higher than that observed in others studies in which no cationic
liposomes were
used(42, 43, 44, 45) . These kinetic
studies allowed us to determine the optimal time of transfection (8 h)
and to investigate the stability and the distribution of both TGF1
AON and SON. Although, as indicated above, the intracellular level of
both TGF1 AON and SON were different after the first hours of
transfection, both appeared intact in the cells. However, degradation
products of both TGF1 AON and SON appeared in the culture medium.
This process was more rapid and marked for TGF1 SON than for AON.
Whether the degradation occurred inside or outside the cells was not
determined in the present study. However, on the one hand, our culture
did not contain serum thought to have DNase activity(36) . On
the other hand, after transfection of the cells for 8 h followed by
extensive washings, oligonucleotide degradation products appeared in
the fresh medium during the next 44 h of culture (data not shown).
Thus, it is likely that the degradation of both oligonucleotides takes
place inside the cells.1 AON and SON concerning their stability and
cellular distribution. First, after 8 h of transfection followed by 44
h of culture, intracellular TGF1 AON concentration was still high
(8%), whereas that of SON was only 1.2%. Second, at any time most of
the intracellular TGF1 SON was located in the cytoplasm, and was
not hybridized. However, TGF1 AON was predominant in the cytoplasm
after 8 h of transfection; it became prevalent in the nucleus at the
end of the experimental period. In addition, in both compartments,
TGF1 AON was hybridized. This hybridization was particularly
intense in the nucleus, and it was higher after 8 h of transfection
followed by 44 h of culture (without oligonucleotides) than immediately
after transfection. These results agree with other data showing that
c-Myb (40) and prorenin (41) antisense oligonucleotides
were preferentially accumulated in the nucleus. However, these results
differ from those of Temsamani et al.(44) showing
preferential cytoplasmic localization of several antisense
oligonucleotides. Although the exact mechanism of oligonucleotide
transfer from cytoplasm to nucleus is not completely understood, a
passive diffusion through the nuclear pores has been
postulated(40) .1 AON present in
the cytoplasm and nucleus, respectively, were resistant to S1 nuclease
digestion. Since the target sequence is present in both primary
transcript and mRNA, TGF1 AON could hybridize to both and
interfere with pre-mRNA maturation and/or nucleocytoplasmic transport (36, 37, 46) but not with transcription,
since the level of TGF1 mRNA was not modified by TGF1 AON. In
contrast, TGF1 AON causes complete inhibition of TGF1 protein
production. This inhibition could be the result of either degradation
of RNA by RNase H, which selectively cleaves the RNA at DNA-RNA
heteroduplexes(47, 48) , or inhibition of the
translation by AON hybridization to the translation initiation site of
the TGF1 mRNA(49, 50) . The first hypothesis is
unlikely because no decrease of TGF1 mRNA was observed. Although
recent data show that c-Myb AON was not associated with ribosomes or
endoplasmic reticulum(40) , our results strongly suggest that
the main mechanism by which TGF1 AON blocked the synthesis of
TGF1 protein is by translation arrest.1 has been suggested in several cell
types(1, 2, 3) , only in two models, rat
vascular smooth muscle cells (23) and human colon carcinoma
cell line(51) , has this been proven by using the antisense
approach. Our results show that the biological consequences of
TGF1 protein synthesis inhibition in control as well as in
cyclosporine treated cells were a significant increase of cortisol
production in response to AngII and ACTH (data not shown). Another
demonstration of the autocrine role of TGF1 on BAC was obtained by
showing that TGF1 AON, but neither SON nor SCR, increased about 2-
and 1.7-fold the mRNA levels of P450 17-hydroxylase and 3
HSD, respectively, an effect that was opposite to that induced by
exogenous TGF1 in these cells ( (14, 15, 16, 17) and the present
data). Moreover, the effects of TGF1 AON on steroidogenic
responses of viable BAC were specific. First, they were not mimicked by
SON or SCR; second, they could be reversed by addition of exogenous
TGF1; and third, they did not modify the normal production of
unrelated proteins.1 by BAC has an
autocrine inhibitory effect on the differentiated functions of these
cells. Moreover, since TGF1 is expressed by many cell types, it is
likely that this factor might also play an autocrine role in other
models. Finally, these studies illustrate and confirm that antisense
technology should find widespread application for investigating the
exact role of many regulatory proteins on cell growth and
differentiation.
),
transforming growth factor ; BAC, bovine adrenal fasciculata
cell(s); ACTH, adrenocorticotropin hormone; AngII, angiotensin-II;
3 HSD, 3-hydroxysteroid dehydrogenase; AON, antisense
oligonucleotide; SON, sense oligonucleotide; SCR, scrambled
oligonucleotide; PBS, phosphate-buffered saline; PIPES,
1,4-piperazinediethanesulfonic acid.)
We thank Dr. P. Durand for a critical reading of the
manuscript, Dr. Starletta Williams for reviewing the English
manuscript, and J. Bois for secretarial help.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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