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Volume 17, Number 13,
Issue of July 1, 1997
pp. 4933-4941
Copyright ©1997 Society for Neuroscience
Chronic Fos-Related Antigens: Stable Variants of 
FosB Induced
in Brain by Chronic Treatments
Jingshan Chen1,
Max B. Kelz1,
Bruce T. Hope2,
Yusaku Nakabeppu3, and
Eric J. Nestler1
1 Laboratory of Molecular Psychiatry, Departments of
Psychiatry and Pharmacology, Yale University School of Medicine,
Connecticut Mental Health Center, New Haven, Connecticut 06508, 2 National Institute of Mental Health, Bethesda, Maryland
20892, and 3 Kyushu University, Fukuoka 812, Japan
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Fos family transcription factors are believed to play an important
role in the transcriptional responses of the brain to a variety of
stimuli. Previous studies have described 35 and 37 kDa Fos-like
proteins, termed chronic Fos-related antigens (FRAs), that are induced
in brain in a region-specific manner in response to several chronic
perturbations, including chronic electroconvulsive seizures,
psychotropic drug treatments, and lesions. We show in this study that
the chronic FRAs are isoforms of 
FosB, a truncated splice variant of
FosB that accumulate in brain after chronic treatments because of their
stability. FosB cDNA encodes the expression of 33, 35, and 37 kDa
proteins that arise from a single AUG translation start site. The 35 and 37 kDa proteins correspond to the chronic FRAs that are induced in
brain by chronic treatments, whereas the 33 kDa protein corresponds to
a Fos-like protein that is induced in brain by acute treatments,
findings based on migration on one- and two-dimensional Western blots
with anti-FRA and anti-FosB antibodies. Using cells in which FosB or
FosB expression is under the control of a tetracycline-regulated gene
expression system, we show that the 37 kDa FosB protein exhibits a
remarkably long half-life, the 35 kDa FosB protein exhibits an
intermediate half-life, and the 33 kDa FosB protein and all
FosB-derived proteins exhibit relatively short half-lives. Moreover, we
show that the 33 kDa FosB protein is the first to appear after
activation of FosB expression. Finally, FosB proteins are shown
to possess DNA-binding activity and to exert potent transactivating
effects in reporter gene assays. Together, these findings support a
scheme wherein FosB, expressed as a 33 kDa protein, is modified to
form highly stable isoforms of 35 and 37 kDa. As a result, these stable
isoforms gradually accumulate in the brain with repeated treatments to mediate forms of long-lasting neural and behavioral plasticity.
Key words:
seizure;
cocaine;
FosB;
chronic Fos-related antigens;
gene expression;
neural plasticity
INTRODUCTION
Regulation of gene transcription is proposed to be
an important mediator of long-term responses of the brain to chronic
perturbations (Hyman and Nestler, 1996
). One transcription factor that
has received a great deal of attention for its potential role in these
responses is activator protein-1 (AP-1) (Morgan and Curran, 1991,
1995). AP-1 is a dimer composed of various combinations of Fos- and
Jun-like proteins. AP-1 complexes interact with AP-1 sites, with a
consensus sequence of TGA(G/C)TCA, present in the promoter regions of
target genes to increase or decrease the rate of the transcription of these genes.
There is now a large literature reporting that Fos- and Jun-like
proteins are induced in brain, in a region-specific manner, in response
to a wide variety of stimuli. Induction of these proteins, and the
resulting AP-1 complex they form, is both rapid and transient. Consequently, these proteins could mediate some of the short-lived changes in gene expression elicited by these stimuli (Morgan and Curran, 1991). In contrast, work in several laboratories over the last
few years has identified novel Fos-like proteins, termed chronic
Fos-related antigens (FRAs), that are induced in brain in response to
chronic perturbations and are much more long-lived than other Fos-like
proteins induced acutely. These features make the chronic FRAs
attractive candidates to mediate some of the longer-lasting
transcriptional changes involved in the regulation of brain function
(Hope et al., 1992, 1994a).
The chronic FRAs, identified as 35 and 37 kDa proteins, are
induced in specific brain regions in response to chronic, but not
acute, administration of cocaine (Hope et al., 1994b; Nye et al., 1995;
Moratalla et al., 1996), electroconvulsive seizures (ECSs) (Hope et
al., 1994a), morphine (Nye and Nestler, 1996), nicotine (Pich et al.,
1997), antipsychotic drugs (Nye et al., 1995), and antidepressant drugs
(Hope et al., 1994b). The chronic FRAs also are induced after kainate
lesions of the hippocampus and cortex (Pennypacker et al., 1994; Kasof
et al., 1995) or 6-hydoxydopamine lesions of the striatum (Jian et al.,
1993; Doucet et al., 1996). In each case, the chronic FRAs, once
induced, are relatively stable proteins that persist in brain for long
periods. For example, after a course of chronic cocaine or ECS
administration, chronic FRAs remain detectable in striatum and frontal
cortex, respectively, for at least 2 weeks. Similarly, in lesion
paradigms the chronic FRAs remain detectable for several months.
The identity of the chronic FRAs has remained elusive despite
considerable effort. The chronic FRAs are immunochemically related to
FosB, a truncated splice variant of FosB, but show different Mr values compared with FosB-induced acutely
in brain or cultured cells (Hope et al., 1994b; Chen et al., 1995,
Doucet et al., 1996). These findings, coupled with the observation that
levels of FosB mRNA are not elevated at times when the chronic FRAs
are induced (Chen et al., 1995; Pennypacker et al., 1995), have raised
doubt as to whether the chronic FRAs are indeed FosB-like
proteins.
Despite this controversy, recent work has provided definitive evidence
that the chronic FRAs are products of the fosB gene. Induction of the chronic FRAs by repeated cocaine or ECS treatment is
completely abolished in fosB knock-out mice (Hiroi et al., 1996, 1997). However, this finding leaves unanswered the question of
whether the chronic FRAs represent FosB, shortened forms of FosB, or
novel products of the fosB gene. We show here, using an
inducible expression system in vitro, that the chronic FRAs are, in fact, modified forms of FosB that are highly stable
proteins, which could account for their accumulation in brain in
response to chronic perturbations.
MATERIALS AND METHODS
Construction of plasmids. FosB and FosB cDNAs in
pcDEBdelta vectors, under the control of a constitutive SR-alpha
promoter (Nakabeppu et al., 1993), were placed under the control of an inducible promoter as follows. The FosB and FosB cDNAs were
subcloned into the pTet splice (Gibco/BRL, Gaithersburg, MD) by
insertion of the SalI-BamHI fragment into the
blunted SalI site of the pTet splice. The new plasmids were
designated as pTetop-FosB and pTetop-FosB. To place expression of
the tetracycline transactivator (tTA) under the control of the
neuron-specific enolase NSE promoter, we cloned the NSE promoter
(~1.8 kb) in pNSE-LacZ (a gift from Dr. J. Gregor Sutcliffe, Scripps) into pTet-tTAk (Gibco/BRL; the k denotes an added
Kozak sequence to enhance translation) in place of the Tetop-minimal cytomegalovirus (CMV) promoter. The new plasmid was designated pNSE-tTAk, in which the HindII-XhoI fragment of
pTet-tTAk was replaced by the HindII-SacI
fragment of pNSE-LacZ.
There are four AUG codons in FosB cDNA (Zerial et al., 1989;
Nakabeppu and Nathans, 1991). To delete the first AUG, we removed the
HindII fragment of pcDEB-FosB, and the plasmid was
religated. The new plasmid was designated p-1AUG. To delete the first
two AUGs, we removed the BstEII-EcoRV fragment of
pcDEBdelta-FosB, and the plasmid was religated and designated
p-1,2AUGs.
The 4xAP-1/RSV-Luc construct (a gift from Dr. Steven Hyman, National
Institute of Mental Health) consists of a promoter region of four
consensus AP-1 sites, in tandem with a minimal RSV promoter, and a
luciferase reporter gene under the control of this promoter.
Transfections. C6 glioma, SH-SY5Y (Biedler et al., 1978),
and CATH.a (Suri et al., 1993) cells were cultured in DMEM with 5%
fetal bovine serum (FBS), DMEM with 10% FBS, and RPMI 1640 with 8%
horse serum plus 4% FBS, respectively. Transient transfections of the
SH-SY5Y and CATH.a cells were performed by the calcium phosphate
method. Approximately 75% confluent cultures in six-well plates were
transfected with 10 µg of plasmid DNA overnight and then washed with
PBS (10 mM sodium phosphate, pH 7.4, 150 mM
NaCl) three times. The transfected cells were incubated in fresh medium for 24 hr, after which the cells were harvested for Western blotting or
gel shift assays as described below. For luciferase reporter gene
assays, the transfected cells were lysed by 1× reporter lysis buffer
(Promega, Madison, WI). Relative luciferase activity, assayed as
described in the luciferase assay protocol of Promega and measured in a
luminometer, was calculated as enzyme activity per microgram of total
protein (determined by Bradford assays).
For stable transfection, C6 glioma cells were transfected overnight,
washed with PBS three times, and reincubated in fresh medium for 24 hr.
The transfected cells were then split and incubated for another 24 hr.
Stable C6 cell lines transfected with constitutive expression plasmids
pcDEB-FosB and pcDEB-FosB using the gene for hygromycin-B
phosphotransferase (the hygromycin-B resistance gene) were selected by
hygromycin (100 µg/ml). Stable C6 cell lines transfected with
inducible expression plasmids pNSE-tTAk plus pTetop-FosB or
pTetop-FosB were selected by the neomycin resistance marker G418
(100 µg/ml) using cotransfection with a plasmid containing the gene
for aminoglycoside phosphotransferase (the neomycin resistance
gene).
In vivo ECS treatment. Male Sprague Dawley rats
(initial weight, 140-260 g; Camm Research Institute, Wayne, NJ) were
used for all experiments. An ECS was administered, as described
previously (Hope et al., 1994a), via ear clip electrodes (45 mA, 0.3 sec). Chronic animals received a single ECS daily for 10 d.
Control and acute animals received daily sham treatments, in which
electrodes were clipped onto the ears of the rats, but no current was
applied. On day 11, acute animals were given an acute ECS, and control and chronic animals were given sham treatment. Animals were killed 2 hr
later. Previous sham treatments were used in the control and acute
animals to reduce the effects of stress (see Campeau et al., 1991;
Sharp et al., 1991). Cerebral cortex was obtained by gross
dissection.
Gel shift assays. Gel shift assays were performed as
described previously (Hope et al., 1992, 1994a). The transfected cells (~5 × 107) were lysed in 300 µl of
electrophoretic mobility shift assay (EMSA) buffer of Korner et al.
(1989): 20 mM HEPES, pH 7.9, 0.4 M NaCl, 20%
glycerol, 5 mM MgCl2, 0.5 mM
EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 10% µg/ml
leupeptin, 0.1 mM p-aminobenzamidine, 1 µg/ml
pepstatin, 0.5 mM phenylmethylsulfonyl fluoride, and 5 mM dithiothreitol. Cerebral cortex was homogenized with
Dounce homogenizers in 20 vol of the EMSA buffer. Crude homogenates
were incubated on ice for 30 min before centrifugation at 15,000 × g for 20 min at 4°C. Aliquots of supernatants
(containing 20 µg of protein) were incubated at room temperature for
20 min with 1 µg of poly(dI-dC)·poly(dI-dC), 40 µg of bovine
serum albumin, 10 mM Tris-HCl, pH 7.9, 10 mM
KCl, 1 mM EDTA, 4% glycerol, and 1 ng of the radioactively
labeled AP-1 probe derived from the AP-1 site of the human
metallothionein II gene (Hope et al., 1992). Then the samples were
electrophoresed at 150 V for 2 hr in a nondenaturing 6%
acrylamide/0.16% N,N
-methylenebisacrylamide gel containing 25 mM Tris-borate buffer, pH 8.3, 1 mM EDTA,
and 1.6% glycerol. The gels were dried and exposed to x-ray film.
Levels of AP-1 binding were quantified by measuring the optical density
of specific bands using an image analysis system with National
Institutes of Health (NIH) image software.
Western blotting. One-dimensional Western blotting was
performed as described previously (Hope et al., 1994a). Transfected cells and cerebral cortex were homogenized in EMSA buffer as described above for gel shift assays. Aliquots (containing 50 µg of protein) were then applied to a 10% acrylamide/0.27%
N,N-methylenebisacrylamide resolving gel for SDS-PAGE
overnight at 75 V and then electrotransferred to nitrocellulose at 200 mA for 3 hr. The blots were blocked with four 15 min changes of 2%
(for anti-FRA antibody; kindly provided by Dr. Michael Iadarola,
National Institute of Dental Research, NIH) or 0.5% (for anti-FosB
antibodies; see Chen et al., 1995) nonfat dry milk in PBS-Tween (PBS
containing 0.1% Tween 20). The blots were then incubated overnight on
a shaker at 4°C in a 1:4000 dilution of anti-FRA antibody or a 1:1000
dilution of anti-FosB antibodies in blocking buffer with 0.05% sodium
azide. The blots were washed four times for 15 min each in blocking
buffer and incubated for 2 hr in a 1:4000 dilution of goat anti-rabbit
antibody conjugated to horseradish peroxidase (Vector Laboratories,
Burlingame, CA) in blocking buffer. The blots were washed eight times
for 15 min each with PBS-Tween alone, developed with the enhanced chemiluminescence (ECL) system of Amersham (Arlington Heights, IL), and
exposed to Hyperfilm-ECL (Amersham) for 5-60 sec. Levels of FRA
immunoreactivity were quantified either by measuring the optical
density of specific bands using an image analysis system or by
measuring light intensity using the Bio-Rad (Hercules, CA) GS-363
phosphor-imager.
For two-dimensional Western blotting, samples (containing 225 µg of
protein) were separated by isoelectric focusing in tube gels for the
first dimension according to published procedures (Hope et al., 1994b).
The resulting tube gels were then layered across SDS-polyacrylamide
slab gels (10% acrylamide/0.4% bisacrylamide) and electrophoresed in
the second dimension. The proteins in the resulting gels were
transferred onto nitrocellulose membranes, and Western blotting was
performed as described above.
RESULTS
The 35 and 37 kDa chronic FRAs are products of FosB mRNA
As stated in the introductory remarks, the 35 and 37 kDa chronic
FRAs can be recognized by anti-FosB antibodies but can be distinguished
from FosB (~46 kDa) and FosB (~33 kDa) on one-dimensional gels
(Hope et al., 1994b; Chen et al., 1995). One possible explanation is
that the chronic FRAs are proteolytic products of FosB. To test this
possibility, we established stable C6 glioma cell lines constitutively
expressing FosB or FosB and analyzed proteins generated from the
cell lines by Western blotting using a pan-FRA antibody (Fig.
1). Extracts of cerebral cortex from rats treated chronically with ECSs were analyzed for comparison. FosB-transfected cells expressed FRA-immunoreactive proteins in the
Mr range of 46-50 kDa, which is consistent with
the reported Mr of FosB (Zerial et al., 1989;
Nakabeppu and Nathans, 1991). The cells also expressed lower levels of
FRAs in the Mr range of 35 kDa. Although the 35 kDa band comigrated with the 35 kDa chronic FRA, no 37 kDa chronic FRA-like band was generated by FosB cDNA.
Fig. 1.
Western blots showing expression of FosB- and
FosB-derived proteins in stably transfected C6 glioma cells
(left) and in transiently transfected Cath.a cells
(right). Cells constitutively expressing FosB or FosB
cDNA were prepared. Cell extracts of these lines and of nontransfected
cells (vector) were analyzed by Western blotting using the pan-FRA
antibody (see Materials and Methods), which recognizes a domain
conserved in all Fos-like proteins. Aliquots of cerebral cortex from
rats treated acutely or chronically with ECS were analyzed for
comparison. In both types of cells, FosB cDNA encodes the expression of
proteins at 46-50 and 35 kDa. FosB cDNA encodes the expression of
proteins at 37, 35, 33, 29, and 28 kDa. The 35 and 37 kDa
FosB-encoded proteins comigrate with the 35 and 37 kDa chronic FRAs
induced by chronic ECS treatment. The 33 kDa FosB-encoded protein
comigrates with the 33 kDa acute FRA induced by acute ECS treatment.
The 29 and 28 kDa FosB-encoded proteins do not correspond to FRAs
detected in the brain; they are distinct from an ~30 kDa protein seen
in the chronic ECS sample that is constitutive and not regulated by ECS
treatment. Note that a prominent ~42 kDa protein (possibly FRA-1 or
-2) is present in control C6 cells (vector1) and in the
FosB transfectants but is not in the FosB transfectants. Note also
that an ~40 kDa protein (possibly FRA-1 or -2) is induced in cortex
by acute ECS treatment only. The results shown are representative of at
least four separate determinations. All of the bands recognized by the
anti-FRA antibody represent specific labeling, because detection was
abolished by preincubation of the antibody with the immunizing peptide
antigen (see Hope et al., 1994a).
[View Larger Version of this Image (87K GIF file)]
FosB-transfected cells expressed high levels of five FRAs with
Mr values of 37, 35, 33, 29, and 28 kDa (Fig.
1). The 35 and 37 kDa FosB-derived proteins migrated at the same
position as the 35 and 37 kDa chronic FRAs. The 33 kDa FosB-derived
protein migrated at the same position as a FRA induced by acute ECS
(Fig. 1) or acute cocaine treatment (see Chen et al., 1995). In
contrast, the 29 and 28 kDa FosB-derived proteins did not correspond
to FRAs detected in the brain.
Whereas a 37 kDa FRA was generated by only FosB cDNA, a 35 kDa FRA
was generated by both FosB and FosB cDNA. To characterize the
protein products from FosB- and FosB-transfected cells further, we
used antibodies selective for the N terminus [anti-FosB(N)] or the C
terminus [anti-FosB(C)] of FosB to distinguish FosB- from
FosB-derived proteins. This is based on the fact that FosB lacks the
C terminus of FosB and is therefore recognized by only the anti-FosB(N)
antibody, whereas FosB is recognized by both antibodies. As shown in
Figure 2, all five of the FosB-derived proteins were
recognized by anti-FosB(N) but not by anti-FosB(C), which confirms that
these proteins were generated from FosB cDNA. In contrast, all of
the FosB-derived proteins were recognized by both anti-FosB(N) and
anti-FosB(C) antibodies. Because the 35 and 37 kDa chronic FRAs are
recognized only by the anti-FosB(N) antibody (Hope et al., 1994b; Chen
et al., 1995), the results suggest that the ~35 kDa proteins in the
FosB-transfected cells are not alternatively spliced FosB products
and are distinct from the chronic FRAs. Rather, these proteins would
seem to be the same as the 35 kDa FRAs induced in the brain by acute
ECS or cocaine treatment (Chen et al., 1995).
Fig. 2.
Western blots showing the expression of FosB- and
FosB-derived proteins in stably transfected C6 glioma cells.
A, Regions of the FosB and FosB proteins against
which the anti-FosB(N), anti-FosB(C), and pan-FRA antibodies are
directed. B, Extracts of stable cell lines (see Fig. 1)
that allow constitutive expression of FosB or FosB cDNA or extracts
of nontransfected cells (Vector), analyzed by Western
blotting using an antibody directed against the N terminus of FosB
[anti-FosB(N)], an antibody directed against the C terminus of FosB
[anti-FosB(C)], or the pan-FRA antibody. Note that all of the
FosB-derived proteins were recognized by the anti-FosB(N) antibody
but not by the anti-FosB(C) antibody, whereas the FosB-derived proteins
were recognized by both antibodies. Note also that none of the several
proteins expressed in control cells and recognized by the pan-FRA
antibody were recognized by the anti-FosB antibodies. The results shown
are representative of three separate determinations.
[View Larger Version of this Image (65K GIF file)]
To confirm that the chronic FRAs are FosB-derived proteins,
two-dimensional Western blotting was used to compare the chronic FRAs
induced in the brain by chronic ECS treatment with the FosB proteins
expressed in cell culture (Fig. 3). Two cell culture systems were analyzed: transiently transfected CATH.a cells, in which
the 35 and 33 kDa FosB proteins predominate, and stably transfected
C6 glioma cells, in which the 37 kDa FosB protein is expressed as
well. The results of these experiments show that the chronic FRAs
correspond precisely with the 35 and 37 kDa FosB proteins. The 35 kDa chronic FRA migrated on two-dimensional gels as two to three widely
spaced bands that correspond to the positions of 35 kDa FosB protein
bands, whereas the 37 kDa chronic FRA corresponds to the position of
the 37 kDa FosB protein. In contrast, the 33 kDa FosB protein
migrated at the same position as a 33 kDa FRA induced in the brain by
acute ECS or cocaine treatment (Chen et al., 1995). Another observation
from these studies, as mentioned above, is that levels of the 37 kDa
FosB protein are barely detectable in the transiently transfected
cells and that, even in the stably transfected cells, only a relatively
small amount of the 37 kDa FosB was detected (see Figs. 1, 2). These results suggest that the accumulation of the 37 kDa protein
in vitro may be slower than the more robust accumulation of
the 37 kDa chronic FRA in the brain after chronic perturbation.
Fig. 3.
Two-dimensional Western blots showing the
expression of chronic FRAs in the brain and of FosB-derived proteins
in cultured cells. A, CATH.a cells transiently
transfected with FosB cDNA. B, Aliquots of cerebral
cortex from rats treated chronically with ECSs, showing the migration
of the 35 and 37 kDa chronic FRAs as demonstrated previously (Hope et
al., 1994a; Chen et al., 1995). C, C6 glioma cells
stably transfected with FosB cDNA. Cell and brain extracts
were analyzed by two-dimensional Western blotting using the pan-FRA
antibody. The figure shows the comigration of the 37 kDa FosB
with the 37 kDa chronic FRA and of the 35 kDa FosB with the 35 kDa
chronic FRA. The 33 kDa FosB comigrates with a 33 kDa FRA induced in
the brain by an acute ECS treatment (see Chen et al., 1995). The
results shown are representative of two to three separate
determinations.
[View Larger Version of this Image (55K GIF file)]
It should be noted that there was one major protein of ~42 kDa,
detected by the anti-FRA antibody, in the untransfected and FosB-transfected cells (Figs. 1, 2). This band was not recognized by
either the anti-FosB(N) or anti-FosB(C) antibody. Interestingly, expression of this endogenous 42 kDa FRA (which could be FRA-2) was
repressed in the FosB-transfected cells. This suggests the possibility that the FosB proteins are functional and serve as negative regulators of the expression of this protein.
The 33, 35, and 37 kDa FosB proteins are isoforms of the same
gene product
Analysis of the FosB cDNA sequence reveals that there are four
AUG start codons in the FosB mRNA, of which the first AUG locates in
the first exon and the other three AUGs locate in the second exon (Fig.
4A). Based on this sequence
information, we hypothesized that the multiple FosB proteins
expressed from FosB cDNA (i.e., the 37, 35, 33, 29, and 28 kDa bands
in Figs. 1, 2) are alternative translation products from different
translation start sites. To test the hypothesis, we deleted the first
AUG, or both the first and second AUGs, from the FosB cDNA and
analyzed the expression patterns of the deletion mutants in transiently transfected CATH.a cells (Fig. 4B). Contrary to our
hypothesis, we found that the 33, 35, and 37 kDa protein bands
disappeared when the first AUG was deleted and that further deletion of
the second AUG did not eliminate more bands, namely, the 28 and 29 kDa
proteins.
Fig. 4.
Analysis of translation start sites for
FosB-derived proteins. A, Genomic map for FosB and
the location of four alternative start (AUG) codons present in the
resulting mRNA. B, Western blot of extracts of CATH.a
cells transiently transfected with full-length FosB cDNA or with
mutants lacking the first (
1AUG) or the first and
second (1,2AUGs) start codons. Note that
deletion of the first AUG completely obliterated the 37, 35, and 33 kDa
FosB proteins, whereas deletion of the first and second AUGs did not abolish the 28-29 kDa FosB proteins, as indicated by the still higher levels of immunoreactivity present in this region of the gel
compared with that in untransfected cells. The results shown are
representative of three separate determinations.
[View Larger Version of this Image (41K GIF file)]
These results indicate that the 33, 35, and 37 kDa FosB proteins are
encoded by the same open reading frame of the FosB mRNA and that
they are isoforms of one another. In contrast, the 28 and 29 kDa
FosB proteins seem to be translated from start codon(s) downstream
of the second AUG, although we cannot rule out the possibility that
deletion of the first AUGs altered the use of the downstream ones.
Because FosB and FosB mRNAs contain identical exons 1 and 2, it
would be expected that FosB mRNA would encode a similar pattern of
protein products, namely products from the first AUG and smaller
products from the downstream AUG. Indeed, deletion studies confirm that
the 35 kDa FosB protein (see Figs. 1, 2) is translated from a
downstream start codon (data not shown).
The 35 and 37 kDa FosB proteins are highly stable
Previous work has shown that the stability of FosB and
FosB mRNAs are similar, with both returning to control levels within a few hours of acute ECS or cocaine treatment (Chen et al., 1995). Moreover, the two mRNAs show a reduced ability to be induced after repeated treatment. This is in contrast to the gradual accumulation of
the chronic FRAs during a course of chronic treatment. One possible
explanation for these findings is that the chronic FRAs, once induced,
are relatively stable proteins. To test this possibility directly, we
compared the stability of FosB and FosB proteins using transfected
cells in which FosB or FosB expression is under the control of the
tetracycline-regulated gene expression system. It was necessary to use
such an inducible expression system, in which the expression of
proteins can be turned on and off, to assess protein stability, in
contrast to a constitutive system in which the expression of proteins
is always on and would therefore interfere with measures of protein
turnover.
In the tetracycline system (Shockett et al., 1995), FosB and FosB
cDNAs were placed under the control of a promoter consisting of the
tetracycline operator (Tetop) and minimal CMV promoter, as shown in
Figure 5A. In the absence of tetracycline,
tTA binds to the promoter and activates the transcription of the
FosB and FosB cDNAs. In the presence of tetracycline, tTA undergoes
a conformational change and cannot bind to the Tetop promoter, so that
transcription of FosB and FosB is inhibited. In this manner, the
expression of FosB and FosB proteins can be activated or repressed,
respectively, by the absence or presence of tetracycline.
Fig. 5.
Analysis of the stability of FosB and FosB
proteins in CATH.a cells transiently transfected with FosB or FosB
cDNA under the control of the tetracycline expression system.
A, Schematic illustration of the tetracycline-regulated
gene expression system, as adapted from Dr. Rene Hen (Columbia
University, Neuroscience Short Course, Society for Neuroscience, 1996).
B, Western blot of FosB and FosB proteins in CATH.a
cells 24 hr after transfection (time 0) and at varying times after the
addition of tetracycline (4 hr-4 d) to turn off expression.
C, Quantitative representation of the time-dependent
reduction in the 35 kDa FosB protein and the 46 kDa FosB protein
after the addition of tetracycline. The results shown are
representative of two separate determinations.
[View Larger Version of this Image (38K GIF file)]
The tetracycline system was first used to encode FosB and FosB
transcription in transiently transfected CATH.a cells. The transfected
cells were cultured in the absence of tetracycline for 24 hr, after
which time tetracycline was added to the culture medium to turn off
transcription of FosB and FosB cDNAs (Fig. 5B). As shown
in Figure 5B, levels of FosB and FosB proteins decreased
in the cells as the proteins were degraded. It was found that the
FosB proteins were more stable than the FosB proteins. Note, for
example, in Figure 5B that levels of all FosB products were
at barely detectable levels 2 d after addition of tetracycline, whereas the FosB proteins remained at much higher levels. This impression is verified by quantitative analysis of the data. For example, Figure 5C shows a comparison of the rate of
disappearance of the 35 kDa FosB protein and the 46 kDa FosB
protein. Note that because only ~10% of the cells were transfected
in these transient transfection experiments, and expression of FosB
was permitted for a relatively short time only (24 hr), no 37 kDa FosB protein was detected.
To overcome this limitation of transient transfections, and thereby to
study the stability of the 37 kDa FosB protein, we made stable C6
glioma cell lines transfected with the FosB and FosB cDNAs under the
control of the tetracycline-regulated promoter. The use of these cell
lines, in which expression of FosB can be turned on and off in
repeated cycles of tetracycline exposure and withdrawal, is shown in
Figure 6. These initial experiments indicated that the
37 kDa FosB protein appears at significant levels only in cells in
which the expression of FosB cDNA is turned on for relatively long
periods.
Fig. 6.
Western blots showing the expression of FosB
proteins in C6 glioma cells stably transfected with FosB cDNA under
the control of the tetracycline expression system. A,
Western blot analysis, using the pan-FRA antibody, of extracts of
stable C6 glioma cell lines. B, Quantitative
representation of the Western blots. Results from two independent
stable cell lines are shown. One set of cells was harvested after being
cultured for 3 d in the absence of tetracycline to turn FosB
expression on (on); a second set of cells was harvested after being cultured for 3 d in the absence of tetracycline,
followed by 1 wk in the presence of tetracycline to turn FosB
expression off (on 
off); a third set of cells
was harvested after being cultured for 3 d in the absence of
tetracycline, followed by 1 wk in the presence of tetracycline and then
1 wk in the absence of tetracycline to turn FosB expression back on
(on off on). Note the ability to turn FosB
expression repeatedly on and off by use of the tetracycline expression
system. Note also that the 37 kDa FosB protein appears appreciably
only after more prolonged periods of expression (i.e., in the on
off on condition). The results shown are representative
of two separate determinations of six independent cell lines
examined.
[View Larger Version of this Image (30K GIF file)]
To compare the stability of the 33, 35, and 37 kDa FosB proteins, we
cultured the FosB stable cell lines in the absence or presence of
tetracycline for varying periods. The results of this experiment are
shown in Figure 7. When the cells were cultured in the
absence of tetracycline for 11 d, the 37 kDa protein accumulated to a significant level, approximately comparable to that of the 33 and
35 kDa FosB proteins (Fig. 7, lane 1). In contrast,
when the cells were cultured first in the absence of tetracycline for 5 d and then in the presence of tetracycline for 6 d, the 35 and 37 kDa proteins were detectable at low levels, whereas the 33 kDa
protein was not detectable (Fig. 7, lane 2). This
suggests that the 35 and 37 kDa proteins are more stable than the 33 kDa protein. When FosB expression was on for 5 d, then switched
off for 3 d and back on for 3 d, the first new FosB
protein detected was the 33 kDa protein (Fig. 7, lane
3). This suggests that the 33 kDa protein is the native form
of FosB, that is, the first expressed. When FosB expression was
on for 8 d and then turned off for 3 d, the 33 kDa protein
was not detectable, whereas the 35 and 37 kDa proteins remained (Fig.
7, lane 4). This suggests, again, that the 35 and 37 kDa proteins are more stable than the 33 kDa protein and are
presumably derived from the 33 kDa protein via some form of covalent
modification.
Fig. 7.
Western blot showing the relative stability of the
37, 35, and 33 kDa FosB proteins in stable C6 glioma cells
transfected with FosB cDNA under the control of the tetracycline
expression system. Cells were grown for varying times in the absence or
presence of tetracycline to turn FosB expression on (+) or off (),
respectively, and cell extracts were then analyzed by Western blotting
using the pan-FRA antibody. In lane 1, cells were
harvested after being grown in the absence of tetracycline for 11 d. In lane 2, cells were harvested after being grown in
the absence of tetracycline for 5 d, followed by an additional
6 d in the presence of tetracycline. In lane 3,
cells were harvested after being grown in the absence of tetracycline
for 5 d, followed by 3 d in the presence of tetracycline and
another 3 d in the absence of tetracycline. In lane
4, cells were harvested after being grown in the absence of
tetracycline for 8 d, followed by an additional 3 d in the
presence of tetracycline. Control (untransfected) cells
are shown for comparison and illustrate the presence of an endogenous
FRA, possibly FRA-2, of ~42 kDa (see also Fig. 1). The results
demonstrate that the 33 kDa FosB protein is the first to appear
after activation of FosB expression, but this protein is less stable
compared with the 35 and 37 kDa FosB proteins. The results shown are
representative of three separate determinations using two independent,
stable cell lines.
[View Larger Version of this Image (30K GIF file)]
A quantitative analysis of the stability of FosB and FosB proteins
was performed by culturing cells in the absence of tetracycline for
11 d and harvesting them at varying times after the addition of
tetracycline (Fig. 8). In this analysis the 33 kDa
FosB protein and the FosB-derived proteins showed relatively short
half-lives of 9-10 hr. This short half-life is consistent with the
highly transient appearance of these proteins in the brain in
vivo after acute ECS or cocaine treatment (Hope et al., 1994a,b;
Chen et al., 1995). In contrast, the 35 and 37 kDa FosB proteins
showed longer half-lives. The 35 kDa protein decayed with an estimated half-life of 28 hr, and the 37 kDa protein decayed with an estimated half-life of 208 hr. This dramatic stability of the 37 kDa protein of
>8 d in vitro is comparable to the half-life of 7 d
calculated for the chronic FRAs in vivo after chronic
administration of ECSs or cocaine (Hope et al., 1994b).
Fig. 8.
Analysis of the stability of FosB and FosB
proteins in stable C6 glioma cells transfected with FosB or FosB
cDNAs under the control of the tetracycline expression system. Cells
were grown in the absence of tetracycline for 11 d to turn FosB
(A) or FosB (B) expression
on, after which time the cells were cultured in the presence of
tetracycline for varying periods to turn expression off. The results
demonstrate the dramatic stability of the 37 kDa FosB protein
compared with the other FosB and FosB proteins. The results shown
are representative of two separate determinations using two
independent, stable cell lines.
[View Larger Version of this Image (19K GIF file)]
Transcriptional activity of FosB and FosB proteins
To study the function of FosB and FosB proteins, we first
analyzed their DNA-binding activity. As shown in Figure
9A, expression of FosB proteins was
associated with the appearance of high levels of AP-1 binding. The AP-1
complex so formed migrated at the same position as the chronic AP-1
complex induced in the brain by chronic ECS treatment. Expression of
FosB proteins was also associated with high levels of AP-1 binding, but
the AP-1 complex comigrated in this case with the upper, acute AP-1
complex induced in brain by acute ECS treatment (Fig. 9B).
These results demonstrate that the FosB proteins exhibit DNA-binding
activity. Moreover, the FosB-AP-1 complex seems to be identical to
the chronic AP-1 complex induced in brain by chronic ECS or cocaine
treatment (Hope et al., 1994a,b).
Fig. 9.
Gel shift assays showing the induction of AP-1
DNA-binding activity after the expression of FosB or FosB in stable
C6 glioma cells. Extracts of a stable cell line that allows
constitutive expression of FosB (A) or FosB
(B) cDNA and of cells transfected with vector DNA
only (Vector control) were analyzed for AP-1
DNA-binding activity using gel shift assays. Extracts of cerebral
cortex from rats that were treated acutely or chronically with ECSs, or
with sham treatments, were analyzed for comparison. As shown in
A, the expression of FosB results in the dramatic
induction of an AP-1 complex that comigrates with the AP-1 complex
induced by chronic, but not acute, ECSs. In contrast, as shown in
B, the expression of FosB results in the induction of an
AP-1 complex that comigrates with the AP-1 complex induced in brain by
acute ECS. The results shown are representative of three separate
determinations.
[View Larger Version of this Image (49K GIF file)]
To test whether FosB proteins can regulate the activity of promoters
containing AP-1 sites, we transiently transfected SH-SY5Y cells with
FosB (in the tetracycline expression system) and with the
4×AP-1/RSV-Luc plasmid (a construct that contains four tandem consensus AP-1 sites driving expression of a luciferase reporter gene).
Analysis of luciferase activity (Fig.
10A) showed that AP-1 promoter
activity was upregulated to a small extent by FosB. This
transactivation activity of FosB on the AP-1 promoter was confirmed
in stable FosB-transfected C6 cells, which showed a dramatic
induction of luciferase activity in response to FosB expression
(Fig. 10B). In contrast to FosB, FosB
downregulated the activity of the promoter; this is illustrated in
Figure 10C, which shows a decrease in luciferase activity in
stable FosB-transfected C6 cells after FosB expression. Tetracycline
itself exerted no effect on AP-1 promoter activity in SH-SY5Y or C6
cells that lack FosB and FosB plasmids, which excludes the
possibility of a tetracycline artifact (data not shown).
Fig. 10.
Analysis of the transactivating properties of
FosB and FosB proteins in SH-SY5Y and C6 glioma cells.
A, Results from SH-SY5Y cells transiently transfected
with FosB cDNA under the control of the tetracycline expression
system. B, C, Results from C6 glioma cells stably transfected with FosB and FosB, respectively, under the
control of the tetracycline expression system. Both types of cells were
also transiently transfected with 4×AP-1/RSV-Luc, which contains a
promoter of four tandem consensus AP-1 sites driving the expression of
a luciferase reporter gene. The results demonstrate the ability of
FosB to upregulate AP-1 promoter activity in both cell lines. In
contrast, FosB downregulates AP-1 promoter activity in the C6 cells.
The results shown are representative of three separate
determinations.
[View Larger Version of this Image (19K GIF file)]
DISCUSSION
The major finding of this study is that the 35 and 37 kDa
chronic FRAs, induced in the brain by a variety of chronic treatments (see introductory remarks), are the stable products of FosB. We
detected five proteins generated from FosB cDNA, of which the 33, 35, and 37 kDa proteins are isoforms. The 33 kDa protein is the first
to appear after induction of FosB expression, with the other forms
presumably generated by covalent modification. The 35 and particularly
the 37 kDa isoforms are more stable than the 33 kDa isoform, which
provides the probable mechanism for the accumulation of the 35 and 37 kDa chronic FRAs in the brain after repeated perturbations.
Accumulation of the 37 kDa FosB occurs very slowly in
vitro, but the stability of this protein is highest among the
FosB isoforms, which could explain how both the 35 and 37 kDa
proteins reach similar levels in vivo after chronic
treatments. Together, these findings provide direct support for our
earlier working hypothesis (Hope et al. 1994b) that proposed the slow
accumulation of stable chronic FRAs by repeated treatments.
Recently, fosB knock-out mice were developed (Brown et al.,
1996). Induction of the 35 and 37 kDa chronic FRAs was completely absent after repeated ECS or cocaine treatment in the / mutant mice, as opposed to the robust induction seen in +/+ wild-type mice
(Hiroi et al., 1996, 1997). Although the knock-out disrupts both FosB
and FosB mRNAs, information from the knock-out mice provides strong
confirmation of the conclusions of the current study that the chronic
FRAs are FosB proteins.
Previous work has shown that FosB and FosB mRNAs have similar
stability, as revealed by RNase protection analysis (Chen et al.,
1995). Both mRNAs are induced rapidly and transiently by acute cocaine
or ECS treatment, which is consistent with the properties of
fosB as an immediate early gene. However, in addition to the unstable protein products of the gene (i.e., the 33 kDa FosB and all
FosB proteins), FosB mRNA gives rise to products (i.e., the 35 and
37 kDa proteins) that are relatively stable. Indeed, the 37 kDa protein
exhibits an estimated half-life that is far longer than that of other
immediate early gene products. This explains how the 35 and 37 kDa
proteins accumulate in the brain after chronic treatments when levels
of FosB mRNA are low. Moreover, these results suggest that the
fosB gene is an atypical immediate early gene, in that it
generates unstable protein products in response to a single acute
stimulation and stable products in response to repeated stimulations.
The stable products can be viewed in two ways from a functional point
of view. They could exert the same transcriptional effect as the
unstable products and thereby play a role in saving "energy" for
the cell as it responds in the same way to repeated stimulations.
Alternatively, they could exert the opposite transcriptional effect as
the unstable products and thereby play a negative feedback role in
opposing further responses to the stimulations. Of course, these
possibilities are not mutually exclusive, in that the chronic FRAs
could play both roles, depending on the various target genes in
question and the Jun family member with which they form functional AP-1 complexes.
The mechanism responsible for the stability of the 35 and 37 kDa
FosB proteins remains unknown. For some immediate early genes, such
as c-fos and myc, the encoded proteins contain
several potential PEST (P, proline; E, glutamatic acid; S, serine; and T, threonine) sequences, which are correlated with fast protein degradation (Rogers et al., 1986). Computer analysis of FosB and FosB amino acid sequences by the PEST-FIND program (Rechsteiner and
Rogers, 1996) revealed four potential PEST sequences in FosB and two
potential PEST sequences in FosB (Table 1). The fact that the 35 and 37 kDa FosB proteins are more stable than FosB proteins may be attributable to the smaller number of PEST sequences in
their amino acid sequences. However, the PEST sequences alone cannot
explain the stability of these proteins, because the 33 kDa FosB
protein also contains only two PEST sequences. Rather, it would seem
that the 33 kDa protein is modified in some way to result in the 35 and
37 kDa proteins and that this modification is important for
stabilization of these proteins. A potential role for phosphorylation
in generating the 35 and 37 kDa isoforms is currently under
investigation.
Accumulation of FosB isoforms would be expected to have an important
impact on brain function, because we show that these proteins exhibit
AP-1 DNA-binding activity and can regulate the activity of a promoter
containing AP-1 sites in a reporter gene assay. Such an important role
for FosB proteins is supported by studies of fosB
knock-out mice, which exhibit impaired behavioral plasticity to chronic
ECS and cocaine treatments (Hiroi et al., 1996; Hiroi, Brown, Haile,
Hong, Greenberg, and Nestler, unpublished observations). Numerous
neural genes are known to contain AP-1 sites in their promoter regions
(see Morgan and Curran, 1991; Chen et al., 1995). Work is now needed to
identify which of these putative target genes are regulated by the
stable FosB isoforms that are induced in specific brain regions by
chronic treatments and that are ultimately responsible for the
resulting behavioral plasticity. Although further research is needed to
delineate the precise mechanisms involved, the long-lasting nature of
the stable FosB proteins make them attractive candidate molecules to
mediate some of the long-term adaptive changes in the brain associated with a variety of chronic perturbations.
FOOTNOTES
Received Feb. 7, 1997; revised April 3, 1997; accepted April 11, 1997.
This work was supported by United States Public Health Service Grants
DA07359, MH51399, MH25642, and DA00203 (E.J.N.) and a gift from Johnson
and Johnson (B.T.H.).
Correspondence should be addressed to Dr. Eric J. Nestler, Laboratory
of Molecular Psychiatry, Departments of Psychiatry and Pharmacology,
Yale University School of Medicine, Connecticut Mental Health Center,
34 Park Street, New Haven, CT 06508.
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M. Werme, C. Messer, L. Olson, L. Gilden, P. Thoren, E. J. Nestler, and S. Brene
Delta FosB Regulates Wheel Running
J. Neurosci.,
September 15, 2002;
22(18):
8133 - 8138.
[Abstract]
[Full Text]
[PDF]
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E. J. Nestler, M. Barrot, and D. W. Self
Delta FosB: A sustained molecular switch for addiction
PNAS,
September 25, 2001;
98(20):
11042 - 11046.
[Abstract]
[Full Text]
[PDF]
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J. Chen, Y. Zhang, M. B. Kelz, C. Steffen, E. S. Ang, L. Zeng, and E. J. Nestler
Induction of Cyclin-Dependent Kinase 5 in the Hippocampus by Chronic Electroconvulsive Seizures: Role of {Delta}FosB
J. Neurosci.,
December 15, 2000;
20(24):
8965 - 8971.
[Abstract]
[Full Text]
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M. B. Kelz, J. R. Kuszak, Y. Yang, W. Ma, C. Steffen, K. Al-Ghoul, Y.-J. Zhang, J. Chen, E. J. Nestler, and A. Spector
{Delta}FosB-Induced Cataract
Invest. Ophthalmol. Vis. Sci.,
October 1, 2000;
41(11):
3523 - 3538.
[Abstract]
[Full Text]
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B. B. Nankova, M. Rivkin, M. Kelz, E. J. Nestler, and E. L. Sabban
Fos-Related Antigen 2: Potential Mediator of the Transcriptional Activation in Rat Adrenal Medulla Evoked by Repeated Immobilization Stress
J. Neurosci.,
August 1, 2000;
20(15):
5647 - 5653.
[Abstract]
[Full Text]
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P.-Q. Yuan and H. Yang
Hypothyroidism induces Fos-like immunoreactivity in ventral medullary neurons that synthesize TRH
Am J Physiol Endocrinol Metab,
November 1, 1999;
277(5):
E927 - E936.
[Abstract]
[Full Text]
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N. Hiroi, G. J. Marek, J. R. Brown, H. Ye, F. Saudou, V. A. Vaidya, R. S. Duman, M. E. Greenberg, and E. J. Nestler
Essential Role of the fosB Gene in Molecular, Cellular, and Behavioral Actions of Chronic Electroconvulsive Seizures
J. Neurosci.,
September 1, 1998;
18(17):
6952 - 6962.
[Abstract]
[Full Text]
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J. Chen, M. B. Kelz, G. Zeng, N. Sakai, C. Steffen, P. E. Shockett, M. R. Picciotto, R. S. Duman, and E. J. Nestler
Transgenic Animals with Inducible, Targeted Gene Expression in Brain
Mol. Pharmacol.,
September 1, 1998;
54(3):
495 - 503.
[Abstract]
[Full Text]
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E. J. Nestler and G. K. Aghajanian
Molecular and Cellular Basis of Addiction
Science,
October 3, 1997;
278(5335):
58 - 63.
[Abstract]
[Full Text]
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N. Hiroi, J. R. Brown, C. N. Haile, H. Ye, M. E. Greenberg, and E. J. Nestler
FosB mutant mice: Loss of chronic cocaine induction of Fos-related proteins and heightened sensitivity to cocaine's psychomotor and rewarding effects
PNAS,
September 16, 1997;
94(19):
10397 - 10402.
[Abstract]
[Full Text]
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