(Received for publication, April 27, 1995; and in revised form, July 20, 1995)
From the
Physiological changes in Fos-like immunoreactivity in the rat
pineal gland are shown here to be due primarily to changes in a
42/46-kDa Fos-related antigen (Fra). Studies are presented that
indicate this 42/46-kDa Fra is Fra-2, a poorly understood member of the
Fos family of transcription factors. Both Fra-2 mRNA and protein are
absent during the day and increase robustly at night on a circadian
basis; organ culture studies indicate that regulation is mediated by an
adrenergic cyclic AMP mechanism. AP-1 binding activity changes
in parallel to changes in the level of Fra-2 protein.
Circadian ()changes in mammalian pineal function are
driven by norepinephrine (NE),
which is released from
terminals in the gland in response to signals originating in the master
circadian oscillator in the suprachiasmatic nucleus (SCN; Refs. 1 and
2). NE acts through [Ca
]
and cyclic AMP to control pineal
function(3, 4, 5) , including the circadian
output of melatonin. Large nocturnal increases in melatonin production
are driven by large changes in serotonin N-acetyltransferase
activity (arylalkylamine N-acetyltransferase, EC 2.3.1.87,
NAT)(5, 6) . The molecular basis of signal
transduction involved in regulating the increase in NAT activity is not
well understood, but available data indicate that the increase in NAT
activity and the abundance of other pineal proteins requires de
novo transcription(1, 5, 7, 8, 9, 10) .
An interesting series of investigations on transcriptional events in
the pineal gland was initiated by cytochemical reports of physiological
and pharmacological changes in Fos-like
immunoreactivity(11, 12) . These and subsequent
reports pointed to the involvement of an unidentified member of the Fos
family of transcription factors in pineal signal
transduction(13, 14) . This family of proteins
includes c-Fos(15) , FosB(16) , FosB2(17) ,
Fos-related antigen (Fra)-1(18, 19) , and
Fra-2(19, 20, 21) . All heterodimerize with
members of the Jun family to produce variations on the activator
protein (AP)-1 transcription factor
theme(22, 23, 24, 25, 26) .
In the study presented here, we extended studies on Fos-related
proteins in the pineal gland and have found that expression of Fra-2 is
physiologically regulated on a circadian basis and that regulation
appears to involve an adrenergic cyclic AMP mechanism.
For
Cleveland analysis (V8), immunoprecipitated Fra-42/46 and in vitro translated Fra-2 were first resolved by SDS-PAGE (12% gel).
Fra-containing bands were excised and placed in the wells of a second
18% PAGE gel. Proteins were treated with V8 protease (0.5-5000
ng/lane, 30 min) in the stacking gel(29) . The partial
proteolytic products were detected using
anti-c-Fos antiserum and visualized by enhanced
chemiluminescence.
Figure 3:
Isolation of a candidate cDNA encoding
Fra-35 and/or Fra-42/46. A, RT-PCR was performed on cDNA from
control (CO) or stimulated (NE, 0.1 µM; 3 h)
pineal glands; primers were APEP and QPEP (see ``Experimental
Procedures''). The NE-induced 980-bp PCR product is indicated by
an arrow. DNA size standards (in kilobase pairs; Bio Ventures,
Murfreesboro, TN) were run on the right lane (Std). B, the 980-bp PCR fragment was subcloned and the resulting
plasmid pCRII/FRA was sequenced. The deduced amino
acid sequence of the NE-induced transcript is aligned with the mouse,
human, and chicken Fra-2 proteins. Only deviations from the consensus
amino acid sequence are shown. The underlined residues contain
the epitope(s) recognized by the anti-c-Fos
antiserum. Leucine residues involved in leucine-zipper interactions are
indicated by the diamond symbols. C, a Northern blot
of total pineal RNA was prepared from groups of 2 animals sacrificed at
times (h): 12, 18, 19:30, 21, 22:30, 24, 1:30, 3, 4:30, and 6 (lanes 1-10) and from animals injected (subcutaneously)
with saline (lane 11), ISO (1 mg/kg, lane 12), or PE
(1 mg/kg, lane 13) at 16 h (2 h before death). The blot was
probed with a radiolabeled fra-2-derived PCR fragment
(nucleotides 495-981). The horizontal bar above the
lanes represents the lighting cycle (dark phase = 1900 to 0500
hours). The same blot was stripped and sequentially exposed to
radiolabeled rat c-fos and human glyceraldehyde-3-phosphate
dehydrogenase probes (G3PDH, middle and bottom
panels in C). The position of RNA molecular weight
standards (Life Technologies, Inc.) is indicated on the right of the
blot. Further technical details are given under ``Experimental
Procedures.''
For the RNase protection
assays, pCRII/ARF2 (antisense version of pCRIIFRA) was
linearized with AccI or BamHI to generate partial or
full riboprobes. T7 polymerase (Promega, Madison, WI) was used for
transcription in the presence of [
-
P]CTP
(Amersham; 800 Ci/mmol) and products were gel-purified. After
hybridization (18 h, 45 °C) with 10 µg of total pineal RNA,
RNA:RNA hybrids were digested for 1 h with a mixture of RNase A +
T1 (Boehringer Mannheim). Protected fragments were resolved using a
denaturing PAGE (6% urea gel) and visualized in a PhosphorImager.
Figure 1:
Circadian rhythm and photoneural
regulation of pineal Fra proteins. Animals were sacrificed at the
indicated time of the day. Glands were removed and rapidly frozen on
solid CO. Horizontal bars in A and B represent lighting (light, open bar; dark, shaded
bar). A, proteins in whole pineal extracts were subjected
to immunoblotting with anti-c-Fos
antiserum (top panel). The second antiserum was horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin. The blot was
stripped and re-exposed to an anti-c-Fos
antiserum (second from top panel), and processed as above. Each
lane represents a pool of two glands. The third panel is a
densitometric quantitation of Western blot data (solid
circles, Fra-42/46; open circles, c-Fos). The fourth
panel presents NAT activity in the extracts(46) . B, analysis of pineal glands from animals housed in a 14:10
light/dark lighting cycle (L:D) or in constant darkness for 4
days (D:D). Pineal extracts were analyzed (from top to bottom) for anti-c-Fos
and
anti-c-Fos
immunoreactivities, AP-1 DNA binding
activity (free probe is not shown), and NAT activity (open
circles). C, effect of continuous light exposure at night
and SCGX on Fra-42/46. Groups of 4 pineal glands were harvested at the
indicated time of day, and whole pineal protein extracts were analyzed
by anti-c-Fos
immunoblotting. Animals were
exposed to darkness (L
D) or to light (L
L) at night. Animals
that had been SCGXed 2 weeks earlier were housed in a 14:10 light/dark
lighting cycle (L
D). One group of SCGX animals was injected with
ISO (1 mg/kg) 4 h before death. In these and following figures, the
migration of prestained protein molecular size standards (Amersham
Corp.) is indicated on the right of each blot (kDa). Further technical
details are given under ``Experimental
Procedures.''
The second serum used,
anti-c-Fos, detects c-Fos at lower levels and in a
selective manner. It failed to detect Fra-42/46, but detected c-Fos (62
kDa) at relatively constant levels at all times of the day; this was
confirmed using a mouse monoclonal
anti-c-Fos
.
These observations
indicate that changes in c-Fos do not seem to contribute to the
increase in Fos-like immunoreactivity seen in cytochemical
studies(11) .
To obtain an independent indication of the pineal function, NAT enzyme activity was also measured in the same protein extracts. NAT activity increased at night in parallel with the increase in Fra-42/46 (Fig. 1A, bottom panel).
To determine if the increase in Fra-42/46 was circadian in nature, as is true of the increase in NAT activity, both were measured during a 24-h period after animals had been housed for 4 days in constant darkness (dark/dark) (Fig. 1B). Fra-42/46 increased during the subjective night, the period that corresponds to the night period of the preceding 14:10 lighting cycle. The increase in Fra-42/46 under these circumstances is clear evidence that the day/night rhythm in Fra-42/46 is truly circadian.
It is likely that the circadian
rhythm in Fra-42/46 is generated by the SCN oscillator, because the
increase in Fra-42/46 was blocked by two treatments which block SCN
pineal stimulation, i.e. continuous light at night
(L
L) or superior cervical ganglionectomy (SCGX, Fig. 1C). The lack of an increase in Fra-42/46 did not
reflect tissue refractoriness because it was possible to increase
Fra-42/46 in SCGX or in L
L animals by treatment with the
-adrenergic receptor (
-AR) agonist isoproterenol (ISO; Fig. 1C and data not shown).
The robust circadian increase in Fra-42/46 parallels an equally robust increase in AP-1 DNA binding activity (Fig. 1B; (32) ).
Figure 2:
Pineal Fra-42/46 is induced by a -AR
cyclic AMP mechanism. A, NE time course and dose
response. Samples were analyzed by Western blots as detailed in the
legend to Fig. 1A. Upper panel presents
anti-c-Fos
immunoblotting of Fra-35, Fra-42/46,
and c-Fos in protein extracts of glands treated with NE as indicated.
The lower panels represent a densitometric quantitation of the Western
blot data and NAT activity. B, effects of adrenergic agonists,
protagonists and antagonists on Fra-42/46 induction.
Anti-c-Fos
immunoblotting analysis of whole
protein extracts from pineal glands (2 glands/lane) that were treated
with PE (0.1 µM), ISO (0.1 µM), NE (0.1
µM), or Bt
cAMP (1 mM) for 6 h.
Treatment with 5 µM prazosin, 5 µM propranolol, cycloheximide (CHX, 60 µg/ml), or
actinomycin D (ActD, 50 µg/ml) was started 30 min before
stimulation. C, phosphatase treatment affects the migration of
Fra-42/46. Five µl of a pineal nuclear extract prepared from
NE-stimulated glands (0.1 µM, 6 h) essentially as
described(47) , were treated with the indicated amount of calf
intestinal alkaline phosphatase (AP) at 37 °C for 20 min or left
untreated on ice and subjected to anti-c-Fos
immunoblotting. D, Fra-42/46 is a phosphoprotein. Pineal
glands in culture were stimulated with either NE (10 µM, 3
h) or Bt
cAMP (1 mM, 6 h) during incubation with
P-labeled NaO
PO
(2 mCi/ml).
Extracts were immunoprecipitated with
anti-c-Fos
, Fra proteins were identified by
anti-c-Fos
immunoblotting (right
panel) and autoradiography (left panel). Further
technical details are given under ``Experimental
Procedures.''
The
62-kDa c-Fos signal was not increased by adrenergic treatment (data not
shown). The 55-kDa c-Fos signal, a less modified form of c-Fos (37) , was detected by anti-c-Fos
(Fig. 2A) and anti-c-Fos
(data not presented); this signal did not increase in response to
treatment with 0.1 µM NE. However, a higher concentration
of NE (1.0 and 10 µM) caused a 20-fold increase in 55-kDa
c-Fos, with a peak at 3 h; this confirms previous findings in organ
culture(14) .
It is of interest to compare in vivo changes in expression of 42/46 -Fra and c-Fos (Fig. 1A) with those seen in vitro due to NE treatment (Fig. 2A). This indicates that the physiological profile seems to be reproduced by treatment with 0.1 µM NE. Higher doses produce large changes in c-Fos protein not seen in vivo.
NE is known to act on the pineal gland
through - and
-adrenergic receptors (ARs). To determine
whether Fra-42/46 was regulated by NE by one or the other receptor we
used relatively selective AR agonists. Treatment with the selective
-AR agonist isoproterenol increased 42/46-kDa Fra; however, the
-AR agonist phenylephrine was without effect (Fig. 2B).
In addition, the NE response was
blocked by the
-AR antagonist propranolol but not by the
-AR
antagonist prazosin. These findings imply that the
-AR is
essential for adrenergic regulation of Fra-42/46 protein, but that the
-AR is not.
Evidence of -adrenoreceptor regulation points
to cyclic AMP as the second messenger controlling Fra-42/46. This was
supported by the finding that treatment with dibutyryl cyclic AMP
(Bt
cAMP; Fig. 2B) or forskolin (10
µM; data not shown) increased Fra-42/46. The response to
Bt
cAMP was not inhibited by propranolol, providing further
evidence that propranolol blocked effects of NE through selective
interaction with the
-AR.
The involvement of other second
messengers was examined by treating glands with a cyclic GMP analog
(dibutyryl cyclic GMP, 1 mM), with
[Ca]
elevating agents (10
µM ionomycin or 20 µM A23187) or with an
activator of protein kinase C (100 nM phorbol 12-myristate
13-acetate). None elevated Fra-42/46 (data not shown).
Accordingly, it appears that cyclic AMP is the essential second
messenger mediating the effects of NE on Fra-42/46.
Examination of
the Western blots in Fig. 1and Fig. 2indicates that
during the period Fra-42/46 abundance is increased, there is a subtle
upward shift in the dominant component of this broad signal, as seen
with other Fra
systems(34, 35, 36, 37, 38) .
This appears to be associated with phosphorylation because (a)
treatment with alkaline phosphatase shifted the 46-kDa component of the
signal toward the 42-kDa component (Fig. 2C), and (b) the 46-kDa and, to a lesser extent, the 42-kDa component
became labeled in glands incubated with BtcAMP and
P-labeled NaO
PO
(Fig. 2D). Phosphorylation of c-Fos was also
observed (Fig. 2D, NE).
Sequence analysis indicated this product encodes a protein which
contains the peptide sequence recognized by
anti-c-Fos and is 98%, 97%, and 93% homologous
to mouse, human, and chick Fra-2,
respectively(19, 20, 21) , indicating that it
is the rat homolog of Fra-2 (Fig. 3B). All known Fra-2
transcripts encode
35-36-kDa proteins with multiple
candidate phosphorylation sites.
A prominent 6.7-kb Fra-2
transcript was strongly expressed during a short period of a light/dark
cycle starting
1.5 h after lights off (Fig. 3C, upper panel, lane 4) and was induced in glands from
rats injected with the
-AR agonist ISO but not in glands from
animals injected with the
-AR agonist PE (Fig. 3C, upper panel, lanes 12 and 13). The Fra-2
transcript was increased during subjective night in animals maintained
in constant darkness for 4 days (data not shown). Changes in the
abundance of this transcript were paralleled by changes in the strength
of a
2.5-kb signal. The above observations are consistent with the
hypothesis that the
6.7-kb fra-2 transcript encodes
either Fra-35 or Fra 42/46, or both.
c-fos mRNA was also
analyzed (Fig. 3C, middle panel). The 2.4-kb
c-fos transcript was expressed throughout the same 24-h
sampling period, and was 2-3-fold higher 1.5 h after the
onset of darkness, as previously reported(13, 14) . We
also were able to confirm that the strength of this signal increased
transiently >10-fold following a 1-h treatment with 10 µM NE (not shown; (14) ). In contrast, treatment with a lower
concentration of NE (0.1 µM) in vitro or with the
-AR agonist ISO (1 mg/kg) in vivo did not alter the
2.4-kb c-fos signal, whereas the 2.5- and 6.7-kb Fra-2 signals
did increase (Fig. 3C). This establishes that the
2.5-kb Fra-2 signal (Fig. 3C) does not reflect
nonspecific detection of c-fos; and, that fra-2 and
c-fos expression are regulated independently.
We confirmed
that fra-2 mRNA is regulated in the pineal gland using an
RNase protection assay (Fig. 4A). Fully protected
fragments were present in samples from BtcAMP-treated
glands, but not control glands. This essentially eliminates the
possibility that the regulated Northern blot signal (Fig. 3C) reflected artifactual detection due to
nonspecific or partial hybridization and confirms that fra-2
mRNA is increased by cyclic AMP. Similarly, fully protected fragments
were detected in samples from glands obtained at night, but not in day
glands (data not shown). The detection of a single RNase protected
product suggests that the
6.7- and
2.5-kb transcripts (Fig. 3C, upper frame) are the result of
differential posttranscriptional processing of a unique fra-2
mRNA species.
Figure 4:
Evidence that Fra-2 encodes Fra-42/46. A, an RNase protection assay was performed using 10 µg of
total RNA extracted from BtcAMP-stimulated pineal glands.
Full and partial length riboprobes were generated form pCRII/ARF2
(equivalent to pCRII/FRA
, except for the orientation
of the insert) by linearization with BamHI or AccI
respectively, and T7 RNA polymerase-driven transcription (right
panel, Fra-2 coding region is represented by a shaded
bar). Control hybridization reactions contained 10 µg of
transfer (t) RNA. The migration of the undigested probes is
presented in the last two lanes on the right.
Radiolabeled HindIII-digested
-DNA fragments (New England
Biolabs, Beverly, MA) were run in parallel to estimate the molecular
weight of the RNase digestion products (Std). B, in vivo and in vitro expression of the 980-bp Fra-2
open reading frame monitored by anti-c-Fos
immunoblotting. The 980-bp PCR fragment was subcloned into pCR3
(pCR3/FRA) and transfected into Cos7 cells using Lipofectamine(TM)
(Life Technologies, Inc.; lane 3). Mock-transfected cells are
shown in lane 4. Messenger RNA was synthesized from
pCRII/FRA
with T7 RNA polymerase and used to program a
rabbit reticulocyte lysate (lane 5). A control translation
reaction was programmed with luciferase RNA (lane 6). In
addition, samples containing endogenous Fra-42/46 and Fra-35 from
control (lane 1) or NE-stimulated pineal glands in culture
(0.1 µM, 6 h; lane 2) were run for comparison. C, V8 protease partial digestion patterns of endogenous and
recombinant Fra proteins. Further technical details are given under
``Experimental Procedures.''
Expression of the fra-2
gene results in accumulation of 6.7- and 2.5-kb fra-2 mRNA
transcripts. The predicted size of the protein encoded by fra-2 is 35 kDa. We suspect that a 35-kDa protein is a
precursor that is subject to extensive posttranslational modification,
including phosphorylation; such modification results in the appearance
of multiple isoforms. This is in agreement with the findings that
expression of human fra-2 in 208F cells generates a
43-kDa protein(41) , that there is similar
posttranslational processing of c-Fos(31, 37) , mouse
Fra-1, and mouse Fra-2(38) , and with our transfection studies.
Based on this, the terms Fra-2
, Fra-2
, and
Fra-2
will be used in the future to identify Fra-2
isoforms with masses of 35, 42, and 46 kDa, respectively. It is
possible that each isoform has distinctly different functional
characteristics, based on current thinking about the functional
importance of transcription factor phosphorylation and the observation
that phosphorylation of Fra-2 appears to correlate with enhanced AP-1
DNA binding activity in another system(38) .
It is interesting that changes in Fra-2 isoforms appear to be associated with changes in AP-1 DNA binding activity (Fig. 1B). The possibility that the increase in Fra-2 in turn increases AP-1 binding provides an interesting hypothetical mechanism through which Fra-2 could influence gene expression, i.e. through interaction with AP-1 or related (e.g. CRE-like) regulatory elements.
The precise function of Fra-2 in transcription has not been established in other systems. Whereas FosB, v-fos, and c-Fos are strong transcriptional activators, Fra-1, Fra-2, and FosB2 are not. This difference resides in functionally different trans-activation domains (41) . In the case of Fra-2, efficient AP-1 binding combined with weak activation points to a possible negative role. This negative role is consistent with the report that Fra-2 can suppress c-Jun(42) . However, Jun D is not suppressed by Fra-2. Accordingly, it is possible that Fra-2 isoforms may have a complex role in the pineal gland because c-Jun and junD display remarkably different patterns of nocturnal expression in this tissue(14) .
If a Fra-2 isoform plays a negative role in the pineal gland, one can envision a dual negative feedback system controlling transcription under the regulation of cyclic AMP. Cyclic AMP-dependent induction of the inducible cyclic AMP early repressor (10) and of Fra-2 could coordinately inhibit expression of both CRE- and AP-1-regulated genes. Alternatively, it is possible that cross-talk exists between these systems, because of the similarity of the consensus binding sites (AP-1 site, TGAGTCA; CRE site, TGACGTCA; (43, 44, 45) ).
The discovery that Fra-2 expression can be physiologically regulated
by a transmitter cyclic AMP mechanism has broad implications
because of the central role cyclic AMP plays in cellular regulation.
Accordingly, the results of studies of Fra-2 isoforms in the pineal
gland should be helpful in understanding their physiological function
in neural and non-neural tissues.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U18913[GenBank].