(Received for publication, October 19, 1995; and in revised form, February 21, 1996)
From the
Transforming growth factor 1 (TGF
1) 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 TGF
1 mRNA in an attempt to
inhibit TGF
1 protein synthesis. We investigated first, the
cellular uptake, the stability, and the intracellular distribution of
P-labeled TGF
1 AON and SON; and second, the effects
of both oligonucleotides on BAC specific functions. We have
demonstrated that in BAC, the TGF
1 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 TGF
1
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 TGF
1 AON was still
high with about 8% of the radioactivity added, whereas that of
TGF
1 SON represented only 1.2%. Moreover, AON was present in the
cytoplasmic and nuclear fractions, and it was hybridized in both
compartments. However, TGF
1 SON was present mainly in the
cytoplasmic fraction where it was not hybridized. Neither TGF
1 AON
nor SON modified TGF
1 mRNA levels; however, TGF
1 AON, but not
SON, caused the disappearance of TGF
1 immunoreactivity inside the
cells. Finally, the steroidogenic responsiveness of BAC transfected
with TGF
1 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
TGF
1 SON nor a scrambled oligonucleotide containing the same
number of G nucleotides as TGF
1 AON had any effect on these
parameters. Thus, these studies demonstrate that TGF
1 has an
autocrine inhibitory effect on BAC differentiated functions, an effect
that can be overcome by TGF
1 AON.
The transforming growth factor (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
TGF
1, TGF
2, and TGF
3, have been identified in
mammals(4) . Although in many cells the three TGF
s 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
TGF
2, 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) .
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 TGF1 is a potent inhibitor of basal as well as
ACTH-induced cortisol production(12, 13) . TGF
1
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) . TGF
1 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,
TGF
1 secretion is regulated by specific peptide hormones; ACTH
decreases TGF
1 mRNA level, whereas AngII increases TGF
1 mRNA
and protein levels. (
)All these data suggest that TGF
1
local production could play an autocrine role on BAC differentiated
functions.
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
TGF1 (23) .
In the present study, using a TGF1
antisense oligodeoxynucleotide complementary to a sequence that
includes the translation start site of the human TGF
1 mRNA, we
have inhibited TGF
1 synthesis in BAC and demonstrated an autocrine
role for TGF
1 on BAC differentiated functions.
TGF1 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-TGF
1
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% H
O
,
and 2.5% nickel ammonium sulfate. Next, the cultured cell preparations
were mounted in PBS-glycerol (1:1). The specificity of the TGF
1
antibody has been tested previously (30) .
Figure 1:
Time course of TGF1
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).
To examine the extent of oligonucleotide degradation within cells
and in the medium during the cellular uptake, TGF1 AON was
analyzed by gel electrophoresis. The results of Fig. 1show
that, inside the cells, TGF
1 AON appeared to be intact for up to
24 h. However, in the culture medium, a progressive degradation of the
TGF
1 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 TGF
1 SON
(data not shown).
Figure 2:
Intracellular stability of the TGF1
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 TGF
1 AON and
from one experiment for TGF
1 SON.
Figure 3:
Intracellular distribution of the
TGF1 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 TGF
1 AON and one experiment from TGF
1
SON.
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 TGF1 AON and SON derived from the culture medium were almost
completely degraded (Fig. 4). Similarly, the TGF
1 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 TGF
1 AON extracted from cells
were protected from degradation. These results indicated that TGF
1
AON, but not SON, was hybridized inside the cells.
Figure 4:
Intracellular hybridization of the
TGF1 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.
Next, we
investigated the intracellular compartment in which the hybridized
TGF1 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
TGF
1 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.
Figure 5:
Intracellular distribution of hybridized
TGF1 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.
Figure 6:
Effects of TGF1 AON and SON on
TGF
1 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. TGF
1 mRNA was extracted and analyzed by Northern blot. A
representative Northern blot of one of the six experiments performed is
shown.
Figure 7:
Effects of TGF1 AON and SON on cell
TGF
1 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 TGF
1 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.
Figure 8:
Effects
of cyclosporine and/or TGF1 AON on cell TGF
1 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.
Figure 9:
Effects of TGF1 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).
One of the
mechanism by which exogenous TGF1 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 TGF
1 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.
Figure 10:
Effects of TGF1 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.
Figure 11:
Effects of exogenous TGF1 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 TGF
1. The medium was removed, and the cells washed and
then stimulated for 2 h with AngII 10
M. 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).
Figure 12:
Effects of transfection on
G/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.
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 TGF1 AON and SON during the first 2 h of
transfection. Thereafter, the kinetics of TGF
1 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 TGF
1
AON and SON. Although, as indicated above, the intracellular level of
both TGF
1 AON and SON were different after the first hours of
transfection, both appeared intact in the cells. However, degradation
products of both TGF
1 AON and SON appeared in the culture medium.
This process was more rapid and marked for TGF
1 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.
In addition, our results revealed marked
difference between TGF1 AON and SON concerning their stability and
cellular distribution. First, after 8 h of transfection followed by 44
h of culture, intracellular TGF
1 AON concentration was still high
(8%), whereas that of SON was only 1.2%. Second, at any time most of
the intracellular TGF
1 SON was located in the cytoplasm, and was
not hybridized. However, TGF
1 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,
TGF
1 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) .
The present studies also show that 44 h
after transfection about 44% and 75% of the TGF1 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, TGF
1 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 TGF
1 mRNA was not modified by TGF
1 AON. In
contrast, TGF
1 AON causes complete inhibition of TGF
1 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 TGF
1 mRNA(49, 50) . The first hypothesis is
unlikely because no decrease of TGF
1 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 TGF
1 AON blocked the synthesis of
TGF
1 protein is by translation arrest.
Although the potential
autocrine role of TGF1 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
TGF
1 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 TGF
1 on BAC was obtained by
showing that TGF
1 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 TGF
1 in these cells ( (14, 15, 16, 17) and the present
data). Moreover, the effects of TGF
1 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
TGF
1; and third, they did not modify the normal production of
unrelated proteins.
Taken together our data demonstrate, for the
first time, that constitutive expression of TGF1 by BAC has an
autocrine inhibitory effect on the differentiated functions of these
cells. Moreover, since TGF
1 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.