0013-7227/92/1301-0257$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine Society
Vol. 130, No. 1 Printed
Effects of Glucocorticoids Protooncogene in AtT-20 SONG-CHANG
LIN,
STEWART
Division of Endocrinology/Metabolism, the Arkansas Cancer Research Center, Little Rock, Arkansas 77205
MAcLEOD,
on Expression Cells AND
JAMES
E
of the fos
W. HARDIN
Departments of Medicine and Biochemistry/Molecular University of Arkansas for Medical Sciences,
ABSTRACT. Glucocorticoid regulation of expression of the protooncogene fos has been examined in AtT-20 cells at both the RNA and protein levels. When cells were incubated continuously in the presence of dexamethasone, an early (30 min) rise in the expression of fos mRNA was observed, which declined by 1 h, but rose again after 2 h of hormone treatment. Six hours after hormone treatment, fos mRNA levels had returned to control levels in spite of the continued presence of dexamethasone. Serum treatment resulted in a sustained increase in fos
in U.S.A.
Biology,
and
mRNA levels; however, the glucocorticoid and serum effects were additive. Dexamethasone and/or serum both increased the steady state levels of fos protein. Glucocorticoid treatment of AtT-20 cells results in complex changes in fos expression, but does not affect their viability or growth rate; these results suggest that fos may play a role in mediation or modulation of glucocorticoid effects other than growth. (Endocrinology 130: 257-262, 1992)
of the c-fos protooncogene is induced by a large variety of agents that affect growth in a number of cell types (1). In almost all of these instances, induction of c-fos expression is rapid and followed by down-regulation of gene activity through an autoregulatory mechanism (2). The regulation of fos expression is, in general, at the transcriptional level and involves 5’ ck regulatory elements; for instance, specific DNA elements involved in its regulation by serum and CAMP have been described in several different cell types (3-5). Induction of fos can be correlated with agents that induce progression of cells through the cell cycle or that alter differentiated cell function (6, 7). The steroid hormone estradiol can induce fos activity in the immature rat uterus (8). The protein products of the c-fos and c-jun genes interact through leucine zippers to form the transcriptional activator AP-1(9-12). Recent studies (13-16) have demonstrated that the glucocorticoid receptor and AP-1 can, through protein-protein interactions, modulate each other’s activities by repressing transcriptional activation by one another. We have studied regulation of fos expression by glucocorticoids in the transformed pituitary cell line AtT20/D-l (17). Although transformed, the AtT-20 cell line retains differentiated functions, in that glucocorticoid
administration results in suppression of POMC gene activity without significantly affecting cell growth (18). We hypothesized that a nuclear protooncogene such as fos could be directly regulated by glucocorticoids and play a role in nongrowth-related hormonal effects. In this report we show that administration of dexamethasone to AtT-20 cells induces the expression of fos at both the RNA and protein levels. This induction is more complicated than has been seen in many other cell types, in that two waves of fos gene activity are induced, and the presence of serum modulates the long term effect of the hormone on the expression of fos.
XPRESSION
Materials
and Methods
Cell culture
AtT-20/D-l cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in 50% Ham’s F-12 and 50% Dulbecco’s Modified Eagle’s Medium containing 10% fetal calf serum. Where indicated, cells were grown in the absence of serum, but the medium was supplemented with 1 rig/ml insulin and 1 rig/ml transferrin. Dexamethasone was dissolved in ethanol and added to a final concentration of 10e7M where indicated, while control cells were treated
with the ethanol
vehicle only.
RNA isolation
Received July 24, 1991. Address all correspondence and requests for reprints to: James W. Hardin, Ph.D., Division of Endocrinology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 587, Little Rock, Arkansas 72205.
Cells were harvested by centrifugation and washed twice with Dulbecco’s PBS. RNA was isolated by the guanidine isothiocyanate procedure, followed by lithium chloride precipitation
(19). Poly(A)+
RNA was isolated
by chromatography
257
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on
258
GLUCOCORTICOID
oligo(dT)-cellulose columns, using the method originally described by Aviv and Leder (20). Protein labeling In some experiments cells were labeled with [35S]methionine. Cells were transferred to methionine-free medium and exposed to 50 &i/ml [35S]methionine for 1 h before hormone exposure. Other additions were made as indicated in the figure legends. Protein extracts Cells were harvested and washed, and protein extracts were prepared as described by Sambucetti and Curran (21). Total protein extracts were resolved on 10% polyacrylamide gels before electrophoretic transfer to nitrocellulose filters, followed by Western blot analysis using a specific fos antibody (22). 35SLabeled extracts were precipitated with antibody and resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gels as described by Riabowol et al. (22). In vitro translation Poly(A)+ RNA was used as a template for protein synthesis, using nuclease-treated reticulocyte lysate (Promega, Madison, WI). Translation products were resolved on polyacrylamide gels as described above. Northern blots RNA was resolved on 6.6% formaldehyde-1.0% agarose gels and transferred to GeneScreen-Plus membranes (DuPont-New England Nuclear, Boston, MA) as described by Thomas (23). The RNA was probed with a fos cDNA representing the coding region of the fos gene that had been labeled with 32Pusing the random primer method of Feinberg and Vogelstein (24). In some casesRNA was directly applied to GeneScreen Plus using a slot blot apparatus and probed in a similar manner. In the case of Northern or slot blots of RNA, estimation of changes in levels of expression was performed by both visual inspection of autoradiograms as well as densitometric scanning using a densitometer, followed by integration of peak heights.
REGULATION
OF fos
Endo. 1992 Vol130 l No 1
RNA was isolated from both populations
of cells, and 5
pg poly(A)+ RNA were separated on a 1% agarose-6.6%
formaldehyde gel. The separated RNA was blotted onto GeneScreen Plus and probed with a 32P-labeled cDNA specific to the protooncogene fos. As shown in Fig. 1, dexamethasone treatment resulted in induction of a 2.2kilobase band corresponding to the expected size of fos mRNA. This result indicates that glucocorticoid treatment results in a significant increase in the steady state level of fos mRNA and that the RNA is stable for some time after hormone
treatment.
The filter
was stripped
and reprobed with a P-actin cDNA, which indicated that the loads of RNA were equivalent (results not shown). Time course of fos induction and serum
by dexamethusone
We next investigated the time course of fos induction in AtT-20 cells after treatment with dexamethasone (Fig. 2). Cells were treated with 10m7M dexamethasone and at the indicated times were harvested and washed, and RNA was isolated. Total RNA was slot blotted onto GeneScreen Plus and probed as described above. All RNA
loads were essentially equivalent, as indicated by reprobing with @actin cDNA (results not shown). As can be seen from Fig. 2, fos mRNA was induced within 0.5 h after dexamethasone treatment of AtT-20 cells grown in serum-free medium. By 1 h, the level of fos had decreased, but by 2-4 h, a second increase in the level of fos mRNA
was observed, which returned to the control level by 6 h -
+
Nuclear run-on assays Nuclear run-on assays were performed by a modification of the method of Diamond and Goodman (25). Nuclei were isolated as described by Stallcup et al. (26). Purified nuclei (0.2 ml) were added to a buffer containing 200 &i [c~-~~P]UTPand incubated at 30 C for 45 min, 50 pg DNAse-I were added, and digestion was continued for 10 min. RNA was purified by proteinase-K digestion, followed by phenol-chloroform extraction and ethanol precipitation. 32P-Labeled RNA (6.5-6.7 x lo6 cpm) was hybridized to nitrocellulose filters containing varying amounts of either parent plasmid (pGEM-2) or pGEM-2 containing fos cDNA. Other molecular biological techniques were described in Sambrook et al. (27).
Results Induction of fos-specific RNA after dexamethasone treatment AtT-20 cells were treated for 2 h with 2 x 10m6 M dexamethasone or with ethanol vehicle only. Poly(A)+
-2.2 kb FIG. 1. Northern analysis of fos mRNA after dexamethasone treatment of AtT-20 cells. AtT-20 cells were treated with either vehicle (-) or 2 x 1Om6M dexamethasone (+) for 2 h before preparation of RNA. Cells were harvested by centrifugation, and poly(A)+ was isolated. RNA was fractionated on agarose-formaldehyde gels, transferred to a nitrocellulose filter, hybridized with a 32P-labeled fos cDNA probe, followed by autoradiography. This experiment was repeated three times with similar results. kb, Kilobases.
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GLUCOCORTICOID
REGULATION
OF fos
259
regulation of fos expression in AtT-20 cells is complex, and this biophasic pattern of expression may result from treatment with a variety of agents in this particular cell line. Nuclear run-on analysis
0 0.5 1 2
4
6
Incubation Time (Hours) FIG. 2. Time course of fos mRNA induction by dexamethasone. AtT20 cells were incubated in serum-free medium in the presence of 1O-7M dexamethasone for the indicated times. At the end of the incubation, cells were harvested, and total RNA was isolated. Five micrograms of RNA were blotted onto GeneScreen Plus and hybridized to a “Plabeledfos cDNA, followed by autoradiography. This experiment is one representative of four replications that yielded similar patterns.
Dex Serum Dex + Serum
0 0.5 1 2 4 6 HOURS FIG. 3. Time course of fos mRNA induction by dexamethasone (Dex) and serum. AtT-20 cells were grown in serum-free medium. At time zero, cells were transferred to medium containing 1O-7 M dexamethasone, 10% fetal calf serum, or both agents. RNA was prepared at the indicated times and slot blotted and hybridized as described in Fig. 2. This experiment was repeated four times with similar results.
Nuclear run-on analysis to determine transcriptional regulation of fos expression by glucocorticoids was performed. The results of this experiment (Fig. 4) indicate that as early as 15 min after addition of dexamethasone to AtT-20 cells, an increase in the transcriptional initiation of the fos gene was observed. Increased transcription was maintained at 30 min. This result indicates that glucocorticoid treatment results in a rapid increase in the transcriptional activity of the cellular fos gene and that at least part of the increase in fos mRNA accumulation and activity is due to increased transcriptional activity. Translation
of induced fos mRNA
We examined whether the fos mRNA induced by dexamethasone was functional in AtT-20 cells. The initial approach to this was in vitro translation of RNA isolated from AtT-20 cells 2 h after dexamethasone treatment. An equal number of 35S-labeled protein counts was applied to two lanes of a polyacrylamide gel. Proteins were resolved, followed by autoradiography. When RNA from dexamethasone-treated cells was used as template, the translation products contained a 55-kDa band, which is the expected size of the unmodified fos protein (Fig. 5). This band was greatly reduced in translation products when control mRNA was used as template. These results Control
after the hormonal treatment. The rapid initial increase in fos mRNA is expected. What was surprising was the decrease at 1 h, followed by the second wave of fos induction 2 and 4 h after treatment. We repeated this experiment, performing the treatment in the presence or absence of serum. As shown in Fig. 3, the addition of serum resulted in a rapid increase in fos mRNA that was sustained for 6 h after treatment. Dexamethasone plus serum resulted in a rapid increase in fos mRNA levels, which remained elevated throughout the treatment period. The pattern with dexamethasone alone was very similar to that we reported in Fig. 2. From this experiment it is seen that the effects of serum and dexamethasone on fos mRNA induction are additive and suggest that in these cells, fos expression is regulated by at least two mechanisms, which can act synergistically with one another. Also, it should be noted that serum alone causes a biphasic response in fos mRNA expression. Therefore, it would appear from this series of experiments, the
Dex, 15 min.
Dex, 30 min.
12510 fos Plasmid,
5 ugs
P-Gem
FIG. 4. Nuclear run-on analysis of fos mRNA induction by dexamethasone (Dex). AtT-20 cells were exposed to dexamethasone for the indicated times, nuclei were isolated, and RNA was labeled by incubation in the presence of [cY-~‘P]UTP. Labeled RNA was isolated and hybridized to filters that contained the parent plasmid pGEM-2 or to plasmids that contained a fos cDNA. This experiment was repeated three times with the same pattern of results obtained.
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GLUCOCORTICOID
REGULATION
OF fos
Endo Voll30
l l
1992 No 1
-fos - fos
5. In vitro translation of poly(A)+ RNA. AtT-20 cells were treated as described in Fig. 1. Two micrograms of poly(A)+ RNA isolated from cells treated for 2 h with vehicle (-) or with dexamethasone (+) were translated using a rabbit reticulocyte lysate and [“S]methionine. Total proteins were separated on a 10% polyacrylamide gel, which was dried and exposed for autoradiography. This experiment was repeated twice with identical results. FIG.
indicate that the increased mRNA detected by Northern blotting represents functional RNA that results in increased levels of the fos protein. Immunoprecipitation
of in vivo synthesized fos
This result was verified by labeling protein synthesized in vivo by AtT-20 cells after dexamethasone treatment. One hour before the addition of dexamethasone, cells were treated with [35S]methionine. Protein extracts were prepared, and an equal number of counts was subjected to immunoprecipitation using a polyclonal antibody specific for fos. The immunoprecipitated material was analyzed by autoradiography of polyacrylamide gels. As shown (Fig. 6), several proteins were induced by dexamethasone treatment, but, again, a specific protein labeled fos was present. This protein actually ran as a 62,000 mol wt protein, reflecting in vivo modifications that are known to occur in fos protein maturation. Other proteins were also present in the immunoprecipitates and were increased after dexamethasone treatment. The lower of these bands probably represents p39-jun, which is known to associate with fos.
0 0.5 1 2 Hours After Dex Treatment 6. Dexamethasone (Dex) induction of 5‘34abeled fos protein. AtT-20 cells were grown in serum-free medium. One hour before the indicated times, 50 pCi/ml [YS]methionine were added to the medium. At time zero, 1W7M dexamethasone was added. At the indicated times, cells were harvested. and cell extracts were prepared. The extracts were exposed to a fos-specific polyclonal antibody, followed by protein-ASepharose. After washing of the beads, labeled proteins were released by boiling in SDS sample buffer and subjected to electrophoresis on a 10% SDS-polyacrylamide gel. The gel was dried and exposed for autoradiography. This experiment was repeated twice with identical results. FIG.
with a fos polyclonal antibody. Dexamethasone and serum both induced fos after incubation for 1 or 4 h (Fig. 7). The presence of both agents had an additive effect on the amounts of fos induced, as observed for fos mRNA.
Western blot analysis of fos protein In further studies, induction of the fos protein by dexamethasone and serum was analyzed by Western blot techniques. After 0, 1, or 4 h of dexamethasone treatment, protein extracts were resolved on SDS-polyacrylamide gels, blotted onto nitrocellulose filters, and probed
Discussion The protooncogene fos appears to play important roles in both differentiated and proliferating cells. Its expression is regulated by a number of agents that have multiple functions in the cell cycle and other aspects of signal
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GLUCOCORTICOID
- ios
1
0 +
++
Treatment Time (Hours)
4 +
+ -+
-
+
DEX
+
Serum
FIG. 7. Dexamethasone (DEX) and/or serum induction of fos protein. AtT-20 cells were grown in serum-free medium and at time zero transferred to medium containing 10m7M dexamethasone, 10% fetal calf serum, or both. At 0, 1, or 4 h, protein extracts were prepared and separated on a 10% polyacrylamide gel. The proteins were transblotted onto nitrocellulose and probed with a fos polyclonal antibody, followed by a goat antirabbit antibody, which was coupled to horseradish peroxidase. The filter was reacted with 35 mM 4-chloro-1-napthol-0.1% HzOz. This experiment was replicated three times with similar results.
transduction (1). It has been generally agreed that fos plays a role in cellular regulation by modification of specific gene transcription (1, 5, 6), and this is probably through interactions with other tram-acting factors, such as the jun protooncogene, to form the transcription factor AP-1 (7,9,12). We have been interested in studying the regulation of fos by glucocorticoid hormones in a highly differentiated transformed cell line, the AtT-20 mouse pituitary tumor cell. This cell is not affected in terms of growth by treatment with glucocorticoids; however, the cell maintains differentiated functions, which are modulated by glucocorticoid treatments (17, 18). Our initial hypothesis was that glucocorticoids probably regulate nuclear protooncogene expression and that regulation of the activities of these nuclear protooncogenes will ultimately play an important role in the action of the hormone. As we have demonstrated in this paper, glucocorticoid treatment of AtT-20 cells results in the accumulation of fos mRNA. The time course of accumulation of fos mRNA is different from what has been seen in most other cell types, in that the induction is slower, and there are two waves of accumulation of fos mRNA. The interactions of serum factors with glucocorticoids in regulation of fos expression appear complex. The recent observations that the glucocorticoid receptor can interact with the various components of the AP1 transcription factor complex and that these interactions can modulate the activities of both AP-1 and the hormone receptor make these observations more interesting (13-16). The continued stimulation of fos expression by glucocorticoids could have important modulatory effects on the glucocorticoid receptor and expression of the genes that are regulated by the hormone-receptor
REGULATION
OF fos
261
complex. This could lead to changes in the hormonally regulated gene products that result in the final cellular phenotype. Therefore, the regulation of fos and its interactions with the glucocorticoid receptor, in possible conjunction with jun, could play a major role in glucocorticoid action. Cycloheximide fails to either inhibit the induction of fos mRNA or superinduce its expression in cells treated with dexamethasone for either 1 or 4 h (results not shown). These results would indicate that fos is directly regulated by the glucocorticoid receptor in these cells and that the autoregulatory mechanism described in other systems probably does not function in AtT-20 cells (12). The effect of cycloheximide on serum induction of fos has not been studied. Why the regulation of fos by glucocorticoids in AtT20 cell lines appears to be different from that in other well studied systems is not clear at the present time. We are currently trying to understand the role that fos plays in the AtT-20 cell line and why the time course of fos induction by glucocorticoids and/or serum is different from that in other well studied systems. The AtT-20 cell line has been maintained in culture for a long period of time and possesses an abnormal karyotype. It is possible that mutations have occurred in the promoter region of the fos gene that lead to the regulatory patterns we observe. The expression of fos is also influenced by a number of other factors, such as CAMP and Ca++, and these could be affected by glucocoritcoid treatment in this cell line, leading to the results we observed. The relationship of the glucocorticoid receptor and AP-1 interactions, which may regulate the steady state levels of fos RNA in these cells, are of major interest in our laboratory. Acknowledgments
The authors wish to acknowledge Dr. Karl Riabowol for fos Drs. Bob Harrison, Steve Lippman, and Bill Hen&y for useful discussion, and Mrs. Jodi Skelton for secretarial assistance. antibody,
References 1. Curran T 1988 The fos oncogene. In: Reddy EP, Skalka AM, Curran T (eds) The Oncogene Handbook. Elsevier, New York, pp 307-325 2. Sassone-Corsi P, Sisson JC, Verma IM 1988 Transcriptional autoregulation of the proto-oncogene fos. Nature 334:314-319 3. Treisman R 1985 Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5’ element and c-fos 3’ sequences. Cell 42889-902 4. Treisman R 1986 Identification of a protein-binding site that mediates transcriptional response of the c-fos -gene to serum factors. Cell 46:1184-1190 5. Bravo R. Neubert M. Burckhardt J. Almendral J. Wallich R. Muller R 1987 Involvement of common and cell-type specific pathways in c-fos gene control: stable induction by CAMP in macrophages. Cell 48251-260 6. Distel RJ, Ro HS, Rosen BS, Groves DL, Spiegelman BS 1987
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262
7. 8. 9. 10. 11. 12. 13. 14.
15.
16.
GLUCOCORTICOID Nucleoprotein complexes that regulate gene expression in adipocyte differentiation: direct participation of c-{OS.Cell 49835-844 Curran T, Franza BR 1988 Fos and jun: the AP-1 connection. Cell 55:395-397 Loose-Mitchell DS, Chiappetta C, Stance1 GM 1988 Estrogen regulation of c-fos messenger ribonucleic acid. Mol Endocrinol 2946-951 Cbiu R, Boyle WJ, Meek J, Smeal T, Hunter T, Karin M 1988 The c-fos protein interacts with c-jun/AP-1 to stimulate transcription of AP-1 responsive genes. Cell 54541-552 Gentz R, Rauscher III FJ, Abate C, Curran T 1988 Parallel association of fos and jun leucine zippers juxtaposes DNA binding domains. Science 2431695-1699 Rauscher III FJ, Cohen DR, Curran T, Bos TJ, Vogt PK, Bohmasm D, Tijan R, Franka BRY 1988 Fos-associated protein p39 is the product of the jun proto-oncogene. Science 240:1010-1016 Sassone-Corsi P, Lamph W, Kamps M, Verma I 1988 Fos-associated cellular p39 is related to nuclear transcription factor AP-I. Cell 54:553-560 Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR 1990 Transcription factor interactions: selectors of positive or negative regulation from a single DNA element. Science 249:1266-1272 Jonat C, Rahnsdorf HJ, Park KK, Cato SCB, Gebel S, Ponta H, Herrlich P 1990 Antitumor promotion and antiflammation: downmodulation of AP-1 (fos/jun) activity by glucocorticoid hormone. Cell 62:1X%1204 Yang-Yeng HF, Chambard JC, Sun YL, Smeal T, Schmidt TJ, Drouin J, Karin M 1990 Transcriptional interference between c-jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interactions. Cell 62:12051215 Schule R, Rangarajan P, Kliiver S, Ransone LJ, Balado J, Yang N, Verma IM, Evans RM 1990 Functional antagonism between oncoprotein c-jun and the glucocorticoid receptor. Cell 62:1217-
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OF fos
Endo l 1992 Voll30 l No 1
1226 17. Buonassisi V, Sato G, Cohen AI 1962 Hormone-producing cultures of adrenal and pituitary tumor origin. Proc Nat1 Acad Sci USA 48:1184-1188 18. Watanabe N, Orth DN, Toft DO 1973 Glucocorticoid receptors in pituitary tumor cells. I. Cytosol receptors. J Biol Chem 248:76257631 19. Cathala G, Savouret J-F, Mendez B, West BL, Karin M, Martial JA, Baxter JD 1983 Lab methods: a method for isolation of intact, translationally active ribonucleic acid. DNA 2:329-335 20. Aviv N, Leder P 1972 Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acidcellulose. Proc Nat1 Acad Sci USA 6914081412 21. Sambucetti L, Curran T 1986 The fos protein complex is associated with DNA in isolated nuclei and binds to DNA cellulose. Science 2341417-1419 22. Riabowol KT, Vogatka RJ, Ziff EB, Lamb NJ, Feramisco JR 1988 Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells. Mol Cell Biol8:1670-1676 23. Thomas PS 1980 Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Nat1 Acad Sci USA 77:5201-5205 24. Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease restriction fragments to high specific activity. Anal Biochem 132:6-13 25. Diamond DJ, Goodman HM 1985 Regulation of growth hormone messenger RNA synthesis by dexamethasone and triiodothyronine. Transcriptional rate and mRNA stability changes in pituitary tumor cells. J Mol Biol 181:41-62 26. Stallcup MR, Ring J, Yamamoto KR 1978 Synthesis of mouse mammary tumor virus ribonucleic acid in isolated nuclei from cultured mammary tumor cells. Biochemistry 17~1515-1521 27. Sambrook J, Fritech EF, Maniatis T 1990 Molecular Cloning-A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor
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Vol. 130, No. 1 Printed
Effects of Glucocorticoids Protooncogene in AtT-20 SONG-CHANG
LIN,
STEWART
Division of Endocrinology/Metabolism, the Arkansas Cancer Research Center, Little Rock, Arkansas 77205
MAcLEOD,
on Expression Cells AND
JAMES
E
of the fos
W. HARDIN
Departments of Medicine and Biochemistry/Molecular University of Arkansas for Medical Sciences,
ABSTRACT. Glucocorticoid regulation of expression of the protooncogene fos has been examined in AtT-20 cells at both the RNA and protein levels. When cells were incubated continuously in the presence of dexamethasone, an early (30 min) rise in the expression of fos mRNA was observed, which declined by 1 h, but rose again after 2 h of hormone treatment. Six hours after hormone treatment, fos mRNA levels had returned to control levels in spite of the continued presence of dexamethasone. Serum treatment resulted in a sustained increase in fos
in U.S.A.
Biology,
and
mRNA levels; however, the glucocorticoid and serum effects were additive. Dexamethasone and/or serum both increased the steady state levels of fos protein. Glucocorticoid treatment of AtT-20 cells results in complex changes in fos expression, but does not affect their viability or growth rate; these results suggest that fos may play a role in mediation or modulation of glucocorticoid effects other than growth. (Endocrinology 130: 257-262, 1992)
of the c-fos protooncogene is induced by a large variety of agents that affect growth in a number of cell types (1). In almost all of these instances, induction of c-fos expression is rapid and followed by down-regulation of gene activity through an autoregulatory mechanism (2). The regulation of fos expression is, in general, at the transcriptional level and involves 5’ ck regulatory elements; for instance, specific DNA elements involved in its regulation by serum and CAMP have been described in several different cell types (3-5). Induction of fos can be correlated with agents that induce progression of cells through the cell cycle or that alter differentiated cell function (6, 7). The steroid hormone estradiol can induce fos activity in the immature rat uterus (8). The protein products of the c-fos and c-jun genes interact through leucine zippers to form the transcriptional activator AP-1(9-12). Recent studies (13-16) have demonstrated that the glucocorticoid receptor and AP-1 can, through protein-protein interactions, modulate each other’s activities by repressing transcriptional activation by one another. We have studied regulation of fos expression by glucocorticoids in the transformed pituitary cell line AtT20/D-l (17). Although transformed, the AtT-20 cell line retains differentiated functions, in that glucocorticoid
administration results in suppression of POMC gene activity without significantly affecting cell growth (18). We hypothesized that a nuclear protooncogene such as fos could be directly regulated by glucocorticoids and play a role in nongrowth-related hormonal effects. In this report we show that administration of dexamethasone to AtT-20 cells induces the expression of fos at both the RNA and protein levels. This induction is more complicated than has been seen in many other cell types, in that two waves of fos gene activity are induced, and the presence of serum modulates the long term effect of the hormone on the expression of fos.
XPRESSION
Materials
and Methods
Cell culture
AtT-20/D-l cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in 50% Ham’s F-12 and 50% Dulbecco’s Modified Eagle’s Medium containing 10% fetal calf serum. Where indicated, cells were grown in the absence of serum, but the medium was supplemented with 1 rig/ml insulin and 1 rig/ml transferrin. Dexamethasone was dissolved in ethanol and added to a final concentration of 10e7M where indicated, while control cells were treated
with the ethanol
vehicle only.
RNA isolation
Received July 24, 1991. Address all correspondence and requests for reprints to: James W. Hardin, Ph.D., Division of Endocrinology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 587, Little Rock, Arkansas 72205.
Cells were harvested by centrifugation and washed twice with Dulbecco’s PBS. RNA was isolated by the guanidine isothiocyanate procedure, followed by lithium chloride precipitation
(19). Poly(A)+
RNA was isolated
by chromatography
257
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on
258
GLUCOCORTICOID
oligo(dT)-cellulose columns, using the method originally described by Aviv and Leder (20). Protein labeling In some experiments cells were labeled with [35S]methionine. Cells were transferred to methionine-free medium and exposed to 50 &i/ml [35S]methionine for 1 h before hormone exposure. Other additions were made as indicated in the figure legends. Protein extracts Cells were harvested and washed, and protein extracts were prepared as described by Sambucetti and Curran (21). Total protein extracts were resolved on 10% polyacrylamide gels before electrophoretic transfer to nitrocellulose filters, followed by Western blot analysis using a specific fos antibody (22). 35SLabeled extracts were precipitated with antibody and resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gels as described by Riabowol et al. (22). In vitro translation Poly(A)+ RNA was used as a template for protein synthesis, using nuclease-treated reticulocyte lysate (Promega, Madison, WI). Translation products were resolved on polyacrylamide gels as described above. Northern blots RNA was resolved on 6.6% formaldehyde-1.0% agarose gels and transferred to GeneScreen-Plus membranes (DuPont-New England Nuclear, Boston, MA) as described by Thomas (23). The RNA was probed with a fos cDNA representing the coding region of the fos gene that had been labeled with 32Pusing the random primer method of Feinberg and Vogelstein (24). In some casesRNA was directly applied to GeneScreen Plus using a slot blot apparatus and probed in a similar manner. In the case of Northern or slot blots of RNA, estimation of changes in levels of expression was performed by both visual inspection of autoradiograms as well as densitometric scanning using a densitometer, followed by integration of peak heights.
REGULATION
OF fos
Endo. 1992 Vol130 l No 1
RNA was isolated from both populations
of cells, and 5
pg poly(A)+ RNA were separated on a 1% agarose-6.6%
formaldehyde gel. The separated RNA was blotted onto GeneScreen Plus and probed with a 32P-labeled cDNA specific to the protooncogene fos. As shown in Fig. 1, dexamethasone treatment resulted in induction of a 2.2kilobase band corresponding to the expected size of fos mRNA. This result indicates that glucocorticoid treatment results in a significant increase in the steady state level of fos mRNA and that the RNA is stable for some time after hormone
treatment.
The filter
was stripped
and reprobed with a P-actin cDNA, which indicated that the loads of RNA were equivalent (results not shown). Time course of fos induction and serum
by dexamethusone
We next investigated the time course of fos induction in AtT-20 cells after treatment with dexamethasone (Fig. 2). Cells were treated with 10m7M dexamethasone and at the indicated times were harvested and washed, and RNA was isolated. Total RNA was slot blotted onto GeneScreen Plus and probed as described above. All RNA
loads were essentially equivalent, as indicated by reprobing with @actin cDNA (results not shown). As can be seen from Fig. 2, fos mRNA was induced within 0.5 h after dexamethasone treatment of AtT-20 cells grown in serum-free medium. By 1 h, the level of fos had decreased, but by 2-4 h, a second increase in the level of fos mRNA
was observed, which returned to the control level by 6 h -
+
Nuclear run-on assays Nuclear run-on assays were performed by a modification of the method of Diamond and Goodman (25). Nuclei were isolated as described by Stallcup et al. (26). Purified nuclei (0.2 ml) were added to a buffer containing 200 &i [c~-~~P]UTPand incubated at 30 C for 45 min, 50 pg DNAse-I were added, and digestion was continued for 10 min. RNA was purified by proteinase-K digestion, followed by phenol-chloroform extraction and ethanol precipitation. 32P-Labeled RNA (6.5-6.7 x lo6 cpm) was hybridized to nitrocellulose filters containing varying amounts of either parent plasmid (pGEM-2) or pGEM-2 containing fos cDNA. Other molecular biological techniques were described in Sambrook et al. (27).
Results Induction of fos-specific RNA after dexamethasone treatment AtT-20 cells were treated for 2 h with 2 x 10m6 M dexamethasone or with ethanol vehicle only. Poly(A)+
-2.2 kb FIG. 1. Northern analysis of fos mRNA after dexamethasone treatment of AtT-20 cells. AtT-20 cells were treated with either vehicle (-) or 2 x 1Om6M dexamethasone (+) for 2 h before preparation of RNA. Cells were harvested by centrifugation, and poly(A)+ was isolated. RNA was fractionated on agarose-formaldehyde gels, transferred to a nitrocellulose filter, hybridized with a 32P-labeled fos cDNA probe, followed by autoradiography. This experiment was repeated three times with similar results. kb, Kilobases.
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regulation of fos expression in AtT-20 cells is complex, and this biophasic pattern of expression may result from treatment with a variety of agents in this particular cell line. Nuclear run-on analysis
0 0.5 1 2
4
6
Incubation Time (Hours) FIG. 2. Time course of fos mRNA induction by dexamethasone. AtT20 cells were incubated in serum-free medium in the presence of 1O-7M dexamethasone for the indicated times. At the end of the incubation, cells were harvested, and total RNA was isolated. Five micrograms of RNA were blotted onto GeneScreen Plus and hybridized to a “Plabeledfos cDNA, followed by autoradiography. This experiment is one representative of four replications that yielded similar patterns.
Dex Serum Dex + Serum
0 0.5 1 2 4 6 HOURS FIG. 3. Time course of fos mRNA induction by dexamethasone (Dex) and serum. AtT-20 cells were grown in serum-free medium. At time zero, cells were transferred to medium containing 1O-7 M dexamethasone, 10% fetal calf serum, or both agents. RNA was prepared at the indicated times and slot blotted and hybridized as described in Fig. 2. This experiment was repeated four times with similar results.
Nuclear run-on analysis to determine transcriptional regulation of fos expression by glucocorticoids was performed. The results of this experiment (Fig. 4) indicate that as early as 15 min after addition of dexamethasone to AtT-20 cells, an increase in the transcriptional initiation of the fos gene was observed. Increased transcription was maintained at 30 min. This result indicates that glucocorticoid treatment results in a rapid increase in the transcriptional activity of the cellular fos gene and that at least part of the increase in fos mRNA accumulation and activity is due to increased transcriptional activity. Translation
of induced fos mRNA
We examined whether the fos mRNA induced by dexamethasone was functional in AtT-20 cells. The initial approach to this was in vitro translation of RNA isolated from AtT-20 cells 2 h after dexamethasone treatment. An equal number of 35S-labeled protein counts was applied to two lanes of a polyacrylamide gel. Proteins were resolved, followed by autoradiography. When RNA from dexamethasone-treated cells was used as template, the translation products contained a 55-kDa band, which is the expected size of the unmodified fos protein (Fig. 5). This band was greatly reduced in translation products when control mRNA was used as template. These results Control
after the hormonal treatment. The rapid initial increase in fos mRNA is expected. What was surprising was the decrease at 1 h, followed by the second wave of fos induction 2 and 4 h after treatment. We repeated this experiment, performing the treatment in the presence or absence of serum. As shown in Fig. 3, the addition of serum resulted in a rapid increase in fos mRNA that was sustained for 6 h after treatment. Dexamethasone plus serum resulted in a rapid increase in fos mRNA levels, which remained elevated throughout the treatment period. The pattern with dexamethasone alone was very similar to that we reported in Fig. 2. From this experiment it is seen that the effects of serum and dexamethasone on fos mRNA induction are additive and suggest that in these cells, fos expression is regulated by at least two mechanisms, which can act synergistically with one another. Also, it should be noted that serum alone causes a biphasic response in fos mRNA expression. Therefore, it would appear from this series of experiments, the
Dex, 15 min.
Dex, 30 min.
12510 fos Plasmid,
5 ugs
P-Gem
FIG. 4. Nuclear run-on analysis of fos mRNA induction by dexamethasone (Dex). AtT-20 cells were exposed to dexamethasone for the indicated times, nuclei were isolated, and RNA was labeled by incubation in the presence of [cY-~‘P]UTP. Labeled RNA was isolated and hybridized to filters that contained the parent plasmid pGEM-2 or to plasmids that contained a fos cDNA. This experiment was repeated three times with the same pattern of results obtained.
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-fos - fos
5. In vitro translation of poly(A)+ RNA. AtT-20 cells were treated as described in Fig. 1. Two micrograms of poly(A)+ RNA isolated from cells treated for 2 h with vehicle (-) or with dexamethasone (+) were translated using a rabbit reticulocyte lysate and [“S]methionine. Total proteins were separated on a 10% polyacrylamide gel, which was dried and exposed for autoradiography. This experiment was repeated twice with identical results. FIG.
indicate that the increased mRNA detected by Northern blotting represents functional RNA that results in increased levels of the fos protein. Immunoprecipitation
of in vivo synthesized fos
This result was verified by labeling protein synthesized in vivo by AtT-20 cells after dexamethasone treatment. One hour before the addition of dexamethasone, cells were treated with [35S]methionine. Protein extracts were prepared, and an equal number of counts was subjected to immunoprecipitation using a polyclonal antibody specific for fos. The immunoprecipitated material was analyzed by autoradiography of polyacrylamide gels. As shown (Fig. 6), several proteins were induced by dexamethasone treatment, but, again, a specific protein labeled fos was present. This protein actually ran as a 62,000 mol wt protein, reflecting in vivo modifications that are known to occur in fos protein maturation. Other proteins were also present in the immunoprecipitates and were increased after dexamethasone treatment. The lower of these bands probably represents p39-jun, which is known to associate with fos.
0 0.5 1 2 Hours After Dex Treatment 6. Dexamethasone (Dex) induction of 5‘34abeled fos protein. AtT-20 cells were grown in serum-free medium. One hour before the indicated times, 50 pCi/ml [YS]methionine were added to the medium. At time zero, 1W7M dexamethasone was added. At the indicated times, cells were harvested. and cell extracts were prepared. The extracts were exposed to a fos-specific polyclonal antibody, followed by protein-ASepharose. After washing of the beads, labeled proteins were released by boiling in SDS sample buffer and subjected to electrophoresis on a 10% SDS-polyacrylamide gel. The gel was dried and exposed for autoradiography. This experiment was repeated twice with identical results. FIG.
with a fos polyclonal antibody. Dexamethasone and serum both induced fos after incubation for 1 or 4 h (Fig. 7). The presence of both agents had an additive effect on the amounts of fos induced, as observed for fos mRNA.
Western blot analysis of fos protein In further studies, induction of the fos protein by dexamethasone and serum was analyzed by Western blot techniques. After 0, 1, or 4 h of dexamethasone treatment, protein extracts were resolved on SDS-polyacrylamide gels, blotted onto nitrocellulose filters, and probed
Discussion The protooncogene fos appears to play important roles in both differentiated and proliferating cells. Its expression is regulated by a number of agents that have multiple functions in the cell cycle and other aspects of signal
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GLUCOCORTICOID
- ios
1
0 +
++
Treatment Time (Hours)
4 +
+ -+
-
+
DEX
+
Serum
FIG. 7. Dexamethasone (DEX) and/or serum induction of fos protein. AtT-20 cells were grown in serum-free medium and at time zero transferred to medium containing 10m7M dexamethasone, 10% fetal calf serum, or both. At 0, 1, or 4 h, protein extracts were prepared and separated on a 10% polyacrylamide gel. The proteins were transblotted onto nitrocellulose and probed with a fos polyclonal antibody, followed by a goat antirabbit antibody, which was coupled to horseradish peroxidase. The filter was reacted with 35 mM 4-chloro-1-napthol-0.1% HzOz. This experiment was replicated three times with similar results.
transduction (1). It has been generally agreed that fos plays a role in cellular regulation by modification of specific gene transcription (1, 5, 6), and this is probably through interactions with other tram-acting factors, such as the jun protooncogene, to form the transcription factor AP-1 (7,9,12). We have been interested in studying the regulation of fos by glucocorticoid hormones in a highly differentiated transformed cell line, the AtT-20 mouse pituitary tumor cell. This cell is not affected in terms of growth by treatment with glucocorticoids; however, the cell maintains differentiated functions, which are modulated by glucocorticoid treatments (17, 18). Our initial hypothesis was that glucocorticoids probably regulate nuclear protooncogene expression and that regulation of the activities of these nuclear protooncogenes will ultimately play an important role in the action of the hormone. As we have demonstrated in this paper, glucocorticoid treatment of AtT-20 cells results in the accumulation of fos mRNA. The time course of accumulation of fos mRNA is different from what has been seen in most other cell types, in that the induction is slower, and there are two waves of accumulation of fos mRNA. The interactions of serum factors with glucocorticoids in regulation of fos expression appear complex. The recent observations that the glucocorticoid receptor can interact with the various components of the AP1 transcription factor complex and that these interactions can modulate the activities of both AP-1 and the hormone receptor make these observations more interesting (13-16). The continued stimulation of fos expression by glucocorticoids could have important modulatory effects on the glucocorticoid receptor and expression of the genes that are regulated by the hormone-receptor
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complex. This could lead to changes in the hormonally regulated gene products that result in the final cellular phenotype. Therefore, the regulation of fos and its interactions with the glucocorticoid receptor, in possible conjunction with jun, could play a major role in glucocorticoid action. Cycloheximide fails to either inhibit the induction of fos mRNA or superinduce its expression in cells treated with dexamethasone for either 1 or 4 h (results not shown). These results would indicate that fos is directly regulated by the glucocorticoid receptor in these cells and that the autoregulatory mechanism described in other systems probably does not function in AtT-20 cells (12). The effect of cycloheximide on serum induction of fos has not been studied. Why the regulation of fos by glucocorticoids in AtT20 cell lines appears to be different from that in other well studied systems is not clear at the present time. We are currently trying to understand the role that fos plays in the AtT-20 cell line and why the time course of fos induction by glucocorticoids and/or serum is different from that in other well studied systems. The AtT-20 cell line has been maintained in culture for a long period of time and possesses an abnormal karyotype. It is possible that mutations have occurred in the promoter region of the fos gene that lead to the regulatory patterns we observe. The expression of fos is also influenced by a number of other factors, such as CAMP and Ca++, and these could be affected by glucocoritcoid treatment in this cell line, leading to the results we observed. The relationship of the glucocorticoid receptor and AP-1 interactions, which may regulate the steady state levels of fos RNA in these cells, are of major interest in our laboratory. Acknowledgments
The authors wish to acknowledge Dr. Karl Riabowol for fos Drs. Bob Harrison, Steve Lippman, and Bill Hen&y for useful discussion, and Mrs. Jodi Skelton for secretarial assistance. antibody,
References 1. Curran T 1988 The fos oncogene. In: Reddy EP, Skalka AM, Curran T (eds) The Oncogene Handbook. Elsevier, New York, pp 307-325 2. Sassone-Corsi P, Sisson JC, Verma IM 1988 Transcriptional autoregulation of the proto-oncogene fos. Nature 334:314-319 3. Treisman R 1985 Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5’ element and c-fos 3’ sequences. Cell 42889-902 4. Treisman R 1986 Identification of a protein-binding site that mediates transcriptional response of the c-fos -gene to serum factors. Cell 46:1184-1190 5. Bravo R. Neubert M. Burckhardt J. Almendral J. Wallich R. Muller R 1987 Involvement of common and cell-type specific pathways in c-fos gene control: stable induction by CAMP in macrophages. Cell 48251-260 6. Distel RJ, Ro HS, Rosen BS, Groves DL, Spiegelman BS 1987
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262
7. 8. 9. 10. 11. 12. 13. 14.
15.
16.
GLUCOCORTICOID Nucleoprotein complexes that regulate gene expression in adipocyte differentiation: direct participation of c-{OS.Cell 49835-844 Curran T, Franza BR 1988 Fos and jun: the AP-1 connection. Cell 55:395-397 Loose-Mitchell DS, Chiappetta C, Stance1 GM 1988 Estrogen regulation of c-fos messenger ribonucleic acid. Mol Endocrinol 2946-951 Cbiu R, Boyle WJ, Meek J, Smeal T, Hunter T, Karin M 1988 The c-fos protein interacts with c-jun/AP-1 to stimulate transcription of AP-1 responsive genes. Cell 54541-552 Gentz R, Rauscher III FJ, Abate C, Curran T 1988 Parallel association of fos and jun leucine zippers juxtaposes DNA binding domains. Science 2431695-1699 Rauscher III FJ, Cohen DR, Curran T, Bos TJ, Vogt PK, Bohmasm D, Tijan R, Franka BRY 1988 Fos-associated protein p39 is the product of the jun proto-oncogene. Science 240:1010-1016 Sassone-Corsi P, Lamph W, Kamps M, Verma I 1988 Fos-associated cellular p39 is related to nuclear transcription factor AP-I. Cell 54:553-560 Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR 1990 Transcription factor interactions: selectors of positive or negative regulation from a single DNA element. Science 249:1266-1272 Jonat C, Rahnsdorf HJ, Park KK, Cato SCB, Gebel S, Ponta H, Herrlich P 1990 Antitumor promotion and antiflammation: downmodulation of AP-1 (fos/jun) activity by glucocorticoid hormone. Cell 62:1X%1204 Yang-Yeng HF, Chambard JC, Sun YL, Smeal T, Schmidt TJ, Drouin J, Karin M 1990 Transcriptional interference between c-jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interactions. Cell 62:12051215 Schule R, Rangarajan P, Kliiver S, Ransone LJ, Balado J, Yang N, Verma IM, Evans RM 1990 Functional antagonism between oncoprotein c-jun and the glucocorticoid receptor. Cell 62:1217-
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1226 17. Buonassisi V, Sato G, Cohen AI 1962 Hormone-producing cultures of adrenal and pituitary tumor origin. Proc Nat1 Acad Sci USA 48:1184-1188 18. Watanabe N, Orth DN, Toft DO 1973 Glucocorticoid receptors in pituitary tumor cells. I. Cytosol receptors. J Biol Chem 248:76257631 19. Cathala G, Savouret J-F, Mendez B, West BL, Karin M, Martial JA, Baxter JD 1983 Lab methods: a method for isolation of intact, translationally active ribonucleic acid. DNA 2:329-335 20. Aviv N, Leder P 1972 Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acidcellulose. Proc Nat1 Acad Sci USA 6914081412 21. Sambucetti L, Curran T 1986 The fos protein complex is associated with DNA in isolated nuclei and binds to DNA cellulose. Science 2341417-1419 22. Riabowol KT, Vogatka RJ, Ziff EB, Lamb NJ, Feramisco JR 1988 Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells. Mol Cell Biol8:1670-1676 23. Thomas PS 1980 Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Nat1 Acad Sci USA 77:5201-5205 24. Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease restriction fragments to high specific activity. Anal Biochem 132:6-13 25. Diamond DJ, Goodman HM 1985 Regulation of growth hormone messenger RNA synthesis by dexamethasone and triiodothyronine. Transcriptional rate and mRNA stability changes in pituitary tumor cells. J Mol Biol 181:41-62 26. Stallcup MR, Ring J, Yamamoto KR 1978 Synthesis of mouse mammary tumor virus ribonucleic acid in isolated nuclei from cultured mammary tumor cells. Biochemistry 17~1515-1521 27. Sambrook J, Fritech EF, Maniatis T 1990 Molecular Cloning-A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor
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