©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Repression of Transforming Growth Factor 1 Protein by Antisense Oligonucleotide-induced Increase of Adrenal Cell Differentiated Functions (*)

(Received for publication, October 19, 1995; and in revised form, February 21, 1996)

Christine Le Roy Patrick Leduque (1) Paul M. Dubois (1) José M. Saez (§) Dominique Langlois

From the From INSERM, INRA U 418, Hôpital Debrousse, 69322 Lyon and the Laboratoire d'Histologie, Faculté de Médecine Lyon-Sud, 69600 Oullins, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor beta1 (TGFbeta1) 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 TGFbeta1 mRNA in an attempt to inhibit TGFbeta1 protein synthesis. We investigated first, the cellular uptake, the stability, and the intracellular distribution of P-labeled TGFbeta1 AON and SON; and second, the effects of both oligonucleotides on BAC specific functions. We have demonstrated that in BAC, the TGFbeta1 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 TGFbeta1 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 TGFbeta1 AON was still high with about 8% of the radioactivity added, whereas that of TGFbeta1 SON represented only 1.2%. Moreover, AON was present in the cytoplasmic and nuclear fractions, and it was hybridized in both compartments. However, TGFbeta1 SON was present mainly in the cytoplasmic fraction where it was not hybridized. Neither TGFbeta1 AON nor SON modified TGFbeta1 mRNA levels; however, TGFbeta1 AON, but not SON, caused the disappearance of TGFbeta1 immunoreactivity inside the cells. Finally, the steroidogenic responsiveness of BAC transfected with TGFbeta1 AON increased about 2-fold, and this was associated with a 2-fold increase of the mRNA levels of both cytochrome P450 17alpha-hydroxylase and 3beta-hydroxysteroid dehydrogenase. Neither TGFbeta1 SON nor a scrambled oligonucleotide containing the same number of G nucleotides as TGFbeta1 AON had any effect on these parameters. Thus, these studies demonstrate that TGFbeta1 has an autocrine inhibitory effect on BAC differentiated functions, an effect that can be overcome by TGFbeta1 AON.


INTRODUCTION

The transforming growth factor beta (TGFbeta) (^1)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 TGFbeta1, TGFbeta2, and TGFbeta3, have been identified in mammals(4) . Although in many cells the three TGFbetas display comparable activities and potencies, marked differences have been noted in some cases(2, 3) . Cross-linking experiments have shown that the TGFbeta 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 TGFbeta receptor. Type III receptor, also known as betaglycan, has no direct role in TGFbeta signaling, but increases the binding of TGFbeta, in particular TGFbeta2, to type II receptor, enhances cell responsiveness to TGFbeta, and diminishes the biological differences between TGFbeta 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 TGFbeta signaling(10) . TGFbeta binds directly to receptor II. Bound TGFbeta 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 TGFbeta1 is a potent inhibitor of basal as well as ACTH-induced cortisol production(12, 13) . TGFbeta1 exerts its effects at several levels: inhibition of low density lipoprotein receptors (12) , inhibition of cytochrome P450 17alpha-hydroxylase and 3beta-hydroxysteroid dehydrogenase (3beta HSD) activities, protein and mRNA contents(14, 15, 16, 17) , and down-regulation of ACTH receptors in ovine adrenocortical cells(18) . TGFbeta1 has also been proposed to regulate the steroidogenic functions in an autocrine loop (19) in BAC cells, which possess TGFbeta receptors that are regulated by ACTH(20) . In addition, BAC cells synthesize (19) and secrete a latent form of TGFbeta-like activity(15) . In BAC, TGFbeta1 secretion is regulated by specific peptide hormones; ACTH decreases TGFbeta1 mRNA level, whereas AngII increases TGFbeta1 mRNA and protein levels. (^2)All these data suggest that TGFbeta1 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 TGFbeta1 (23) .

In the present study, using a TGFbeta1 antisense oligodeoxynucleotide complementary to a sequence that includes the translation start site of the human TGFbeta1 mRNA, we have inhibited TGFbeta1 synthesis in BAC and demonstrated an autocrine role for TGFbeta1 on BAC differentiated functions.


EXPERIMENTAL PROCEDURES

Materials

[-P]ATP (>4000 Ci/mmol) and [alpha-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 TGFbeta1 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 TGFbeta1 cDNA was donated by Dr. R. Derynck (Genentech Inc., San Francisco, CA)(26) , bovine P450 17alpha-hydroxylase cDNA by Dr. M. R. Waterman (Vanderbilt University School of Medecine, Nashville, TN)(27) , and human 3beta 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 Galpha(q)/Galpha proteins and polyclonal anti-TGFbeta1 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, 10M vitamin C, and antibiotics without serum.

Oligonucleotides

Antisense, sense, and scrambled unmodified 15-base deoxyribonucleotides corresponding to the translation initiation region of human TGFbeta1 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 TGFbeta1 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 times 10^8 dpm/µg). The transfection medium containing 1 times 10^6 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 times 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 times 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(2), 0.5% Nonidet P-40, 1 mM dithiothreitol). After centrifugation at 800 times 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(2), 0.25 M sucrose and centrifuged (800 times 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 times 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(4), 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(2)). 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 TGFbeta1 cDNA, bovine P450 17alpha-hydroxylase cDNA, and human 3beta HSD were used as probes (1-2 times 10^6 dpm/ml). Labeling of these probes in the presence of [alpha-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 10^4 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.

TGFbeta1 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-TGFbeta1 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(2)O(2), and 2.5% nickel ammonium sulfate. Next, the cultured cell preparations were mounted in PBS-glycerol (1:1). The specificity of the TGFbeta1 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 times 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-alpha(q)/alpha 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.


RESULTS

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 TGFbeta1 AON or SON (final concentration 10 µM) for different times as described under ``Experimental Procedures.'' Fig. 1shows the time-dependent cellular uptake of TGFbeta1 AON (solid line) and TGFbeta1 SON (dotted line). TGFbeta1 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, TGFbeta1 AON intracellular concentration (500 µM) was about 50-fold higher than that in the medium. TGFbeta1 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.


Figure 1: Time course of TGFbeta1 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, TGFbeta1 AON was analyzed by gel electrophoresis. The results of Fig. 1show that, inside the cells, TGFbeta1 AON appeared to be intact for up to 24 h. However, in the culture medium, a progressive degradation of the TGFbeta1 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 TGFbeta1 SON (data not shown).

Intracellular Stability of the TGFbeta1 AON and SON

To determine the intracellular stability of the oligonucleotides, cells were transfected with P-labeled TGFbeta1 AON or SON for 8 h (time at which TGFbeta1 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 TGFbeta1 AON or SON represented 16% and 6%, respectively, of the radioactivity added. However, after 44 h of culture, the intracellular concentration of TGFbeta1 AON was still 8% of the radioactivity added, whereas that of TGFbeta1 SON represented only 1.2%.


Figure 2: Intracellular stability of the TGFbeta1 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 TGFbeta1 AON and from one experiment for TGFbeta1 SON.



Intracellular Distribution and Hybridization of TGFbeta1 AON and SON

In order to determine the intracellular distribution of both TGFbeta1 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 TGFbeta1 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 TGFbeta1 SON were in the cytoplasmic fraction. Indeed, the TGFbeta1 SON present in the nuclear fraction after 44 h of culture without oligonucleotides represented less than 0.2% of the radioactivity added.


Figure 3: Intracellular distribution of the TGFbeta1 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 TGFbeta1 AON and one experiment from TGFbeta1 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 TGFbeta1 AON and SON derived from the culture medium were almost completely degraded (Fig. 4). Similarly, the TGFbeta1 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 TGFbeta1 AON extracted from cells were protected from degradation. These results indicated that TGFbeta1 AON, but not SON, was hybridized inside the cells.


Figure 4: Intracellular hybridization of the TGFbeta1 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 TGFbeta1 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 TGFbeta1 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 TGFbeta1 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 TGFbeta1 AON and SON on BAC TGFbeta1 mRNA and Protein Content

To assess whether the oligonucleotides were able to modify the transcription and/or the translation of TGFbeta1, we investigated their effects on TGFbeta1 mRNA by Northern blot and on cellular TGFbeta1 protein content by immunocytochemistry. The results of Fig. 6showed that neither TGFbeta1 AON nor SON modified the level of the 2.5-kilobase transcript of TGFbeta1 mRNA. In contrast, the results of immunocytochemistry (Fig. 7) showed that all the control cells (A) or cells pretreated with TGFbeta1 SON (C) were immunoreactive. However, the TGFbeta1 immunoreactivity completely disappeared in TGFbeta1 AON treated cells (D). In B, where the TGFbeta1 antibody was saturated with the peptide used to produce this antibody, there was no TGFbeta1 signal, thus showing the specificity of this antibody. Since it has been reported that cyclosporine increased the expression of TGFbeta1(35) , we treated BAC for 44 h with this factor (1 µg/ml) in the absence or presence of TGFbeta1 AON and we examined the TGFbeta1 content by immunocytochemistry (Fig. 8). Although this method is only semi-quantitative, the results of Fig. 8suggest that cyclosporine increased the cellular TGFbeta1 content, and this effect was blunted by TGFbeta1 AON.


Figure 6: Effects of TGFbeta1 AON and SON on TGFbeta1 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. TGFbeta1 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 TGFbeta1 AON and SON on cell TGFbeta1 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 TGFbeta1 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 TGFbeta1 AON on cell TGFbeta1 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 TGFbeta1 AON and SON on BAC Functions

As described in the Introduction, exogenous TGFbeta1 decreases the steroidogenic responsiveness of BAC; thus, we investigated the effects of both oligonucleotides, TGFbeta1 AON and SON, on the steroidogenic responsiveness to AngII of control and cyclosporine treated cells (Fig. 9). In the absence of cyclosporine, TGFbeta1 AON increased the cortisol response to AngII about 2-fold compared to either control cells or TGFbeta1 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 TGFbeta1 AON was 2.3-fold higher than that of cells treated with cyclosporine alone. Again, TGFbeta1 SON had no effect.


Figure 9: Effects of TGFbeta1 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 10M. Results, expressed as ng/10^6 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 TGFbeta1 decreases the steroidogenic capacity of BAC is by decreasing the mRNA levels of P450 17alpha-hydroxylase and 3beta HSD(14, 16) . The results of Fig. 10clearly show that TGFbeta1 AON, but not SON, increased P450 17alpha-hydroxylase and 3beta HSD mRNA levels (2- and 1.7-fold, respectively), which encode two key enzymes in the steroidogenic pathway.


Figure 10: Effects of TGFbeta1 AON and SON on P450 17alpha-hydroxylase and 3beta 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 17alpha-hydroxylase and 3beta 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 TGFbeta1 AON Effects

To prove that all the TGFbeta1 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 TGFbeta1 AON but in a scrambled order. The results (Fig. 11) showed that, in contrast to TGFbeta1 AON, neither SON nor SCR modified the cortisol secretion and the mRNA levels of P450 17alpha-hydroxylase. Second, none of the transfected oligonucleotides did change the sensitivity of the cells to the inhibitory effects of TGFbeta1, since exogenous TGFbeta1 caused similar inhibition of both cortisol secretion and P450 17alpha-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 Galpha(q)/Galpha proteins, which are not affected by exogenous TGFbeta1(34) . The results showed that neither the rate of synthesis (Fig. 12A) nor the steady-state levels (Fig. 12B) of Galpha(q)/Galpha proteins were affected in transfected cells regardless of the nucleotide used.


Figure 11: Effects of exogenous TGFbeta1 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 TGFbeta1. 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/10^6 cells, are the mean ± S.D. of duplicate measurements from two separate experiments. B, in the same two experiments P450 17alpha-hydroxylase mRNA were analyzed by Northern blot (one representative autoradiography).




Figure 12: Effects of transfection on Galpha(q)/Galpha 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 Galpha(q)/Galpha. B, Western blot from the cell lysates using Galpha(q)/Galpha antibody.




DISCUSSION

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 TGFbeta1 AON and SON during the first 2 h of transfection. Thereafter, the kinetics of TGFbeta1 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 TGFbeta1 AON and SON. Although, as indicated above, the intracellular level of both TGFbeta1 AON and SON were different after the first hours of transfection, both appeared intact in the cells. However, degradation products of both TGFbeta1 AON and SON appeared in the culture medium. This process was more rapid and marked for TGFbeta1 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 TGFbeta1 AON and SON concerning their stability and cellular distribution. First, after 8 h of transfection followed by 44 h of culture, intracellular TGFbeta1 AON concentration was still high (8%), whereas that of SON was only 1.2%. Second, at any time most of the intracellular TGFbeta1 SON was located in the cytoplasm, and was not hybridized. However, TGFbeta1 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, TGFbeta1 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 TGFbeta1 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, TGFbeta1 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 TGFbeta1 mRNA was not modified by TGFbeta1 AON. In contrast, TGFbeta1 AON causes complete inhibition of TGFbeta1 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 TGFbeta1 mRNA(49, 50) . The first hypothesis is unlikely because no decrease of TGFbeta1 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 TGFbeta1 AON blocked the synthesis of TGFbeta1 protein is by translation arrest.

Although the potential autocrine role of TGFbeta1 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 TGFbeta1 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 TGFbeta1 on BAC was obtained by showing that TGFbeta1 AON, but neither SON nor SCR, increased about 2- and 1.7-fold the mRNA levels of P450 17alpha-hydroxylase and 3beta HSD, respectively, an effect that was opposite to that induced by exogenous TGFbeta1 in these cells ( (14, 15, 16, 17) and the present data). Moreover, the effects of TGFbeta1 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 TGFbeta1; 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 TGFbeta1 by BAC has an autocrine inhibitory effect on the differentiated functions of these cells. Moreover, since TGFbeta1 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.


FOOTNOTES

*
This work was supported by grants from INSERM, University Claude Bernard (Lyon), and Fondation pour la Recherche Médicale Française. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: INSERM-INRA U418, Hôpital Debrousse, 69322 Lyon Cedex 05, France. Tel.: 33-78-25-18-08; Fax: 33-78-25-61-68.

(^1)
The abbreviations used are: TGFbeta, transforming growth factor beta; BAC, bovine adrenal fasciculata cell(s); ACTH, adrenocorticotropin hormone; AngII, angiotensin-II; 3beta HSD, 3beta-hydroxysteroid dehydrogenase; AON, antisense oligonucleotide; SON, sense oligonucleotide; SCR, scrambled oligonucleotide; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid.

(^2)
C. Le Roy, P. Leduque, P. M. Dubois, J. M. Saez, and D. Langlois, unpublished results.


ACKNOWLEDGEMENTS

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.


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