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Endocrinology Vol. 147, No. 11 5052-5060
Copyright © 2006 by The Endocrine Society

Differential Expression of Activator Protein-1 Proteins in the Pineal Gland of Syrian Hamster and Rat May Explain Species Diversity in Arylalkylamine N-Acetyltransferase Gene Expression

Natalia Sinitskaya, Anthony Salingre, Paul Klosen, Florent G. Revel, Paul Pévet and Valérie Simonneaux

Institut des Neurosciences Cellulaires et Intégratives, Département de Neurobiologie des Rythmes, Unité Mixte de Recherche-7168/LC2 Centre National de la Recherche Scientifique-Université Louis Pasteur, Insitut Fédératif de Recherche des Neurosciences de Strasbourg, 67084 Strasbourg, France

Address all correspondence and requests for reprints to: Valérie Simonneaux, Institut des Neurosciences Cellulaires et Intégratives, Département de Neurobiologie des Rythmes, UMR-7168/LC2 CNRS-Université Louis Pasteur, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France. E-mail: simonneaux{at}neurochem.u-strasbg.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Species differences have been reported for the nighttime regulation of arylalkylamine N-acetyltransferase (AA-NAT), the melatonin rhythm-generating enzyme. In particular, de novo synthesis of stimulatory transcription factors is required for Aa-nat transcription in the Syrian hamster but not in the rat pineal gland. The present work investigated the contribution of phosphorylated cAMP-responsive element-binding protein, c-FOS, c-JUN, and JUN-B in the regulation of Aa-nat transcription in Syrian hamsters compared with rats. The nighttime pattern of cAMP-responsive element-binding protein phosphorylation and regulation by norepinephrine observed in the Syrian hamster was similar to those reported in the rat. On the contrary, strong divergences in c-FOS, c-JUN, and JUN-B expression were observed between both species. In Syrian hamster, predominant expression of c-FOS and c-JUN was observed at the beginning of night, whereas a predominant expression of c-JUN and JUN-B was observed in the late night in rat. The early peak of c-FOS and c-JUN, known to form a stimulatory transcription dimer, suggests that they are involved in the nighttime stimulation of Aa-nat transcription. Indeed, early-night administration of a protein synthesis inhibitor (cycloheximide) markedly decreased AA-NAT mRNA levels in Syrian hamster. In the rat, high levels of JUN-B and c-JUN, constituting an inhibitory transcription dimer, are probably involved in the late-night inhibition of Aa-nat transcription. Early-night administration of cycloheximide actually increased AA-NAT mRNA levels toward the late night. Therefore, composition and timing of the pineal activator protein-1 complexes differ between rat and Syrian hamster and may be an activator (Syrian hamster) or an inhibitor (rat) of Aa-nat transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PINEAL HORMONE melatonin, synthesized only during the night, displays seasonal variations that confer the hormone a central role in regulation of annual functions, particularly reproduction in seasonal species (1). Melatonin synthesis is under the control of a central circadian clock, located in the suprachiasmatic nuclei, and regulated via sympathetic input. In rodents, abundant release of norepinephrine (NE) at the beginning of the night triggers activation of arylalkylamine N-acetyltransferase (AA-NAT; EC 2.3.1.87) transcription, which leads to a 100- to 150-fold increase in AA-NAT mRNA and is followed by a 50- to 70-fold increase in AA-NAT activity that drives the nocturnal production of melatonin (2, 3). Importantly, differences in the mechanisms leading to AA-NAT enzyme activation have been shown among rodent species (for review see Refs. 4 and 5).

In the rat pineal gland, the molecular mechanisms involved in AA-NAT regulation by NE have been extensively studied. NE binding to ß-adrenergic receptors activates the cAMP/protein kinase A type II (PKAII)/phosphorylated cAMP-responsive element-binding protein (pCREB) signal transduction cascade (6, 7), whereas NE binding to {alpha}-adrenergic receptors increases Ca2+ concentration and activity of protein kinase C, which in turn potentiates the intracellular accumulation of cAMP (8). Binding of pCREB to the cAMP-responsive element (CRE) in the Aa-nat gene promoter induces a large increase in AA-NAT mRNA followed by protein synthesis and enzyme activation. Additionally, this transcriptional mechanism of AA-NAT regulation is accompanied by posttranslational modifications of AA-NAT through a ubiquitin/proteasome degradation, which is also under the control of NE (9).

Although pCREB is the major stimulatory transcription factor involved in Aa-nat gene transcription, other transcription factors were reported to be activated by NE in the rat pineal gland. In particular, Fos-related antigen 2 (Fra-2) mRNA and protein display a robust increase at night (10, 11), and c-fos and jun-B mRNA are transiently increased after the onset of darkness (12, 13). Via leucine zipper dimerization motifs, FOS and JUN proteins form various dimer combinations known as activator protein-1 complex (AP-1), which regulate transcription of genes containing a tetradecanoyl phorbol acetate response element (TRE) binding site. The presence of TRE as well as CRE binding sites have been shown in rat Aa-nat gene promoter (14), but physiological roles for the AP-1 complex and any individual member of the JUN and FOS families have yet to be established in the rat pineal gland (15, 16).

In the pineal gland of the Syrian hamster, a common model for the study of seasonal physiology, mechanisms underlying AA-NAT activation remain unclear. As in the rat, nighttime stimulation of AA-NAT activity and melatonin synthesis depends on release of NE (17, 18, 19). Furthermore, we recently reported that AA-NAT is also regulated via transcriptional mechanisms (20). However, the molecular mechanisms involved in Syrian hamster Aa-nat gene transcription are different from those described for the rat (21). First, Aa-nat gene expression, enzyme activation, and melatonin synthesis are strongly restricted to the nighttime because NE, {alpha}-/ß-adrenergic agonists, and activators of adenylate cyclase are unable to induce AA-NAT activity and melatonin synthesis during the light phase of the daily cycle (22, 23, 24, 25, 26). Second, Aa-nat transcription requires complex and durable mechanisms that involve newly synthesized stimulatory transcription factors, whose nature is still unknown (21).

In the present work, an analysis of nighttime pCREB, c-FOS, c-JUN, and JUN-B expression was performed in both the Syrian hamster and rat pineal gland with the aim of investigating the contribution of these proteins to the regulation of Aa-nat gene transcription in both species.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experiments were performed in accordance with the European Committee Council Directive of November 24, 1986 (86/609/EEC), and the French Department of Agriculture (license no. 67-250). For experiments, 2- to 3-month-old female Syrian hamsters (Mesocricetus auratus) and 3- to 4-month-old male Wistar rats were used. Animals were housed under constant conditions of ambient humidity and temperature with food and water available ad libitum. Syrian hamsters were raised under a long-day photoperiod with 14 h light (200 lux light intensity from 0500 h) and 10 h dark (2 lux dim red light from 1900 h). Wistar rats were raised under 12 h light (200 lux light intensity from 0700 h) and 12 h dark (2 lux dim red light from 1900 h).

Experimental protocols
Analysis of nighttime expression of transcription factors in Syrian hamster and rat pineal gland.
An initial experiment was carried out to determine whether the transcription factors CREB, pCREB, c-FOS, c-JUN, and JUN-B display rhythmic expression in the Syrian hamster pineal gland. The pineal glands were collected at each of the following times: 1300, 1800, 2000, 2100, 2200, 2300, 0000, 0100, and 0200 h (n = 2 per time point) and were processed for Western blot analysis. For comparison, rat pineal glands (n = 3 per time point) were sampled at 1500, 2100, 2200, 0000, and 0200 h and were processed similarly for Western blot analysis. This experiment was repeated twice.

We then used immunohistochemistry to localize c-FOS, c-JUN, CREB, and pCREB proteins and to confirm the rhythmic expression of these proteins in the Syrian hamster pineal gland. Four hamsters were killed at each of the following times: 1400, 1800, 2000, 2200, 0000, 0200, 0400, 0700, and 1000 h. This experiment was repeated once.

Effect of {alpha}/ß-adrenergic ligand administration on transcription factor expression in Syrian hamster and rat pineal gland.
The effect of {alpha}- or ß-adrenergic antagonists on the early-night expression of CREB, pCREB, c-FOS, and c-JUN in the Syrian hamster pineal gland was examined by immunohistochemistry (n = 4 per experimental point). Animals were divided into four equal groups. Three groups were injected at 1900 h (dark onset) with the {alpha}-adrenergic antagonist prazosin (PRAZ, 15 mg/kg; Sigma Chemical Co., St. Louis, MO) or the ß-adrenergic antagonist propranolol (PROP, 15 mg/kg; Sigma) or vehicle (25% dimethylsulfoxide in Ringer solution) and killed at 2200 h, 3 h after injection; a noninjected control group was killed at 2200 h (nighttime control).

The effect of a daytime injection of {alpha}- and ß-adrenergic agonists on the expression of c-FOS, c-JUN, JUN-B, and CREB in the Syrian hamster pineal gland was examined by Western blot (n = 2 per experimental point) and immunohistochemistry (n = 4 per experimental point). Animals were divided into six equal groups. Two groups were injected at 1300 h with a mixture of {alpha}-adrenergic agonist phenylephrine (PHE, 3 mg/kg; Sigma) and ß-adrenergic agonist isoproterenol (ISO, 3 mg/kg; Sigma) and killed at 1400 and 1600 h; two groups were injected at 1300 h with vehicle (Ringer solution) and killed at 1400 and 1600 h; two noninjected control groups were killed either at 1300 h (daytime control) or 2200 h (nighttime control).

The effect of a daytime injection of {alpha}- and ß-adrenergic agonists on the expression of c-FOS, c-JUN, JUN-B, and CREB in the rat pineal gland was examined by Western blot (n = 3 per experimental point). Animals were divided into six equal groups. At 0900 h, three groups were injected with a mixture of {alpha}-adrenergic agonist PHE (3 mg/kg; Sigma) and ß-adrenergic agonist ISO (3 mg/kg; Sigma), and three groups were injected with vehicle (Ringer solution). Animals of each group were killed 2, 4, and 6 h after injection (at 1100, 1300, and 1500 h).

Effect of early-night cycloheximide (CYCLO) administration on Aa-nat gene expression in Syrian hamster and rat pineal gland.
A first set of experiments was performed to check for the inhibition of c-FOS and c-JUN after CYCLO administration in the Syrian hamster pineal gland. Syrian hamsters (n = 4 per experimental point) were injected with CYCLO (20 mg/kg; Sigma) or vehicle (ethanol 25% in Ringer solution) at 2100 h and killed at 2200 h (time of the c-FOS/c-JUN protein peak), and then the pineal glands were processed for Western blot analysis.

A second set of experiments was performed to verify the effect of CYCLO injection at different times of early night on Aa-nat gene expression in the pineal gland of the Syrian hamster. Hamsters were divided into four equal groups (n = 5 per group). One group was injected with vehicle (ethanol 25% in Ringer solution) at 2100 h (1 h before the c-FOS/c-JUN peak); the three other groups were injected with CYCLO (20 mg/kg) at 2100 h (1 h before the c-FOS/c-JUN peak), 2200 h (peak of the c-FOS/c-JUN), or 2300 h (1 h after c-FOS/c-JUN peak). Animals of the four groups were killed at 0200 h, and the brains were processed for detection of the Aa-nat mRNA expression by in situ hybridization.

A third set of experiments was performed to check for the inhibition of JUN-B and c-JUN after CYCLO administration in the rat pineal gland. Rats (n = 3 per experimental point) were injected with CYCLO (20 mg/kg) or vehicle (ethanol 25% in Ringer solution) at 2100 h and killed at 0200 h (time of the JUN-B/c-JUN protein increase), and then the pineal glands were processed for Western blot analysis.

A fourth set of experiments was performed to verify the effect of CYCLO injection on Aa-nat gene expression in the rat pineal gland. Two groups of rats (n = 5 per group) were injected with CYCLO (20 mg/kg) at 2100 h and two groups with vehicle (ethanol 25% in Ringer solution) at 2100 h. The animals were killed at 0200 and 0400 h, and the brains were processed for in situ hybridization.

Western blot
The animals were deeply anesthetized and killed by decapitation. Pineal glands were carefully removed, frozen on dry ice, and then stored at –80 C. Whole-tissue extracts from pineal glands were prepared in Laemmli sample buffer (27). Protein concentration in the tissue extracts was determined by the Zaman-Verwilghen method (28). Total pineal gland proteins (15 µg/lane) were separated on 14% SDS-PAGE mini-gels prepared according to Doucet et al. (29). Proteins resolved by electrophoresis were electrotransferred to polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA) in Towbin buffer (30). Nonspecific protein binding to the polyvinylidene difluoride membrane was blocked by 2% dry skimmed milk in Tris-buffered saline/Tween 20 buffer (20 mM Tris, 154 mM NaCl, 0.05% Tween 20, pH 7.6) for 1 h. Blots were incubated overnight at room temperature with rabbit polyclonal antibodies to c-FOS (sc-52), c-JUN (sc-1694), JUN-B (sc-46) (Santa Cruz Biotechnology, Santa Cruz, CA), CREB (06-863), or pCREB (06-519) (Upstate Biotechnology, Lake Placid, NY), diluted 1:2000. The blots were washed in Tris-buffered high-salt saline (TBHS)/Tween 20 (20 mM Tris, 500 mM NaCl, 0.05% Tween 20, pH 8.6) and incubated for 1 h with peroxidase-conjugated goat antirabbit IgG (Sigma) diluted 1:20,000. The blots were washed extensively again and the signal visualized by chemiluminescence (Super Signal West Femto for c-FOS in the rat and JUN-B in the hamster and Super Signal West Pico for the other transcription factors; Pierce Chemical Co., Rockford, IL). After antibody stripping with 50 mM Tris-HCl, 100 mM ß-mercaptoethanol, and 2% SDS, the same blot was used again for the detection of the other proteins of interest as well as the reference protein CREB in each experiment.

Semiquantitative analysis of protein level.
Image analysis was performed using the ImageJ program derived from the public domain NIH Image program (developed at the National Institutes of Health and available online at http://rsb.info.nih.gov/nih-image/). Values of c-FOS, c-JUN, and JUN-B are normalized by CREB value and finally expressed as percentage of maximal level for each protein.

Immunohistochemistry
The animals were deeply anesthetized, injected with heparin [250 IU by animal; Choay (Sanofi Winthrop Industrie, Notre Dame de Bondeville, France)] directly into the left ventricle and immediately perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The cranium with the brain was removed and postfixed for 12 h in the perfusion fixative. The brain was carefully dissected, postfixed overnight, rinsed with PBS (30 min and then overnight), and finally dehydrated. Polyethylene glycol (Acros Organics, Fair Lawn, NJ) embedding was then performed according to Klosen et al. (31). The tissues were sectioned at 8 µm on a rotary microtome and mounted on SuperFrost Plus slides. The sections were blocked for 1 h with 3% dry skimmed milk in Tris-buffered saline/Tween 20 buffer and incubated overnight at room temperature with the following rabbit polyclonal primary antibodies: c-FOS (sc-52) 1:2000 dilution, c-JUN (sc-1694) 1:500 (Santa Cruz Biotechnology), CREB (06-863) 1:2000, and pCREB (06-519) 1:1000 (Upstate Biotechnology). After washing for 30 min with TBHS/Tween 20, the slides were incubated for 1 h with donkey antirabbit biotinylated secondary antibody (Jackson Immunoresearch Laboratory, West Grove, PA) diluted 1:2000, washed again with TBHS/Tween 20, and incubated 1 h with streptavidin-peroxidase (Roche, Indianapolis, IN) 1:2000. Immunolabeling was developed using DAB (3,3'-diaminobenzindine; Sigma) peroxidase detection.

Semiquantitative analysis of immunolabeled pineal tissue was performed on pineal sections corresponding to one section for each of the four hamsters’ pineal glands, all sections being treated exactly the same way. Image analysis was performed using ImageJ. The total integrated density (TID) of immunoreactive areas was measured and given as TID per square micrometer.

In situ hybridization
The animals were deeply anesthetized and killed by decapitation. Whole brains with the pineal gland were carefully removed, frozen on dry ice, and stored at –80 C. Coronal brain sections (16 µm) were cut in a cryostat at the level of the pineal gland and mounted onto gelatin-coated slides. The slides were stored at –80 C until hybridization. We used a mixture of antisense oligonucleotide synthetic probes for NAT (oligo 1, 251–300; oligo 2, 491–537; oligo 3, 774–823 of rat NM_012818; Invitrogen, Carlsbad, CA). The oligonucleotide mix was labeled by terminal deoxynucleotide transferase (Roche) with [35S]deoxy-ATP (1250 Li/mmol; PerkinElmer NEN Life Science Products Radiochemicals, Norwalk, CT) according to the manufacturer’s protocol. Sections were fixed, acetylated, dehydrated in graded ethanol baths, and hybridized overnight at 37 C, with 106 cpm labeled probe per slide. Posthybridization consisted of five 15-min washes in 1x sodium saline citrate at 55 C and two 30-min washes in 1x sodium saline citrate at room temperature. After dehydration in graded ethanol baths, the slides were exposed to BioMax Film (Kodak, Rochester, NY) for 5 d.

Semiquantitative analysis of the autoradiographs was performed with the computerized analysis system Biocom-program RAG 200 (Biocom, Les Ulis, France). The specific labeling was determined as the difference between labeling in the pineal gland and nonspecific labeling in the cortex of the brain.

Data analysis
Statistical analyses for proteins and AA-NAT mRNA level were performed using one-way ANOVA followed by Tukey’s multicomparison test. The differences were considered significant for P < 0.01 and P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of nighttime expression of transcription factors in the pineal gland
To visualize rhythmic expression of transcription factors in the Syrian hamster pineal gland, the levels of CREB, pCREB, c-FOS, c-JUN, and JUN-B proteins were analyzed by Western blot at different times throughout the day (Fig. 1Go, A and B). A constant level of CREB protein, a known constitutive transcription factor, was observed throughout the daily cycle in the Syrian hamster pineal gland. Therefore, CREB protein was used as an internal control for protein deposition in Western blot analysis. Surprisingly, no reproducible daily variation of pCREB could be observed in the pineal glands (data not shown), most probably as a consequence of tissue sampling. Irrespective of time, cutting of sympathetic afferents during pineal removal leads to release of large amounts of endogenous NE, which could induce rapid CREB phosphorylation. Using the c-FOS antibody, an early peak of c-FOS protein was observed as soon as 2100 h with a maximum at 2200 h (3 h after dark onset) with a decrease later in the night. Expression of c-JUN, a potential c-FOS partner in the AP-1 complex, showed a very similar pattern. c-JUN protein expression was highest between 2100 and 2300 h. As expected, a basal level of c-JUN protein was detected during the day and the second part of the night because of a positive autoregulatory loop (32). Expression of JUN-B, another putative c-FOS and c-JUN partner, was very low at all times in the Syrian hamster pineal gland, even though JUN-B was readily detectable in control baby hamster kidney (BHK-21) cells (data not shown). Therefore, JUN-B expression in the Syrian hamster pineal gland was analyzed with a more sensitive chemiluminescence substrate. Low levels of JUN-B were observed with a peak coinciding with c-FOS and c-JUN (3 h after dark onset).


Figure 1
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  		FIG. 1. Pattern of CREB, c-FOS, c-JUN, and JUN-B expression in the pineal gland of Syrian hamster and rat at early night. Hamsters raised under a 14-h light, 10-h dark cycle and rats raised under a 12-h light, 12-h dark cycle, all with lights off at 1900 h, were killed at the indicated time points. Pineal glands of Syrian hamsters (A and B) and rats (C and D) were processed for protein detection by Western blot and semiquantitative analysis of c-FOS, c-JUN, and JUN-B levels. Values of c-FOS, c-JUN, and JUN-B are normalized by CREB value and finally expressed as percentage of maximal level for each protein. Each point of semiquantitative analysis is the mean of two hamsters or three rats. *, Super Signal West Femto chemiluminescence substrate was used for signal visualization.

 
For comparison, the daily pattern of CREB, c-FOS, c-JUN, and JUN-B expression in the rat pineal gland was analyzed by Western blot (Fig. 1Go, C and D). Again, a constant level of CREB was observed throughout the day and night. c-JUN protein was expressed in a constant manner with a faint increase at 0000 and 0200 h. In contrast, JUN-B protein level increased considerably between 0000 and 0200 h, that is 5–7 h after dark onset. c-FOS protein was hardly detected at any time points in the rat pineal gland, although it was readily detectable in control rat glioma C6 cells (data not shown). Therefore, c-FOS expression was further analyzed using the sensitive chemiluminescence substrate. Under these conditions, c-FOS expression could be observed with the highest levels occurring 5–7 h after dark onset, similar to the JUN-B profile.

To analyze the dynamics of CREB phosphorylation and examine the localization of pCREB, c-FOS, and c-JUN transcription factors in the Syrian hamster pineal gland, additional experiments were performed using immunohistochemistry. All of the tested transcription factors were expressed in a large majority of pineal cells with nuclear localization (Fig. 2AGo). Semiquantitative analysis of the immunodetected signal for CREB, pCREB, c-FOS, and c-JUN is represented in Fig. 2BGo. In contrast to the Western blot approach, tissue fixation by paraformaldehyde perfusion allowed the detection of a day/night difference in pineal pCREB levels. The level of pCREB was almost undetectable during daytime, rose dramatically 3 h after dark onset, and stayed elevated until the end of the night. In accordance with the Western blot experiment, the level of CREB did not vary significantly throughout the daily cycle. The early expression of c-FOS and c-JUN proteins was confirmed with both proteins peaking at 2200 h and decreasing thereafter.


Figure 2
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  		FIG. 2. Pattern of CREB, pCREB, c-FOS, and c-JUN expression in the pineal gland of Syrian hamster. Hamsters raised under a 14-h light, 10-h dark cycle, with lights off at 1900 h, were perfused with paraformaldehyde at the indicated time points. Pineal glands were processed for protein detection by immunohistochemistry. A, Photographs of CREB, pCREB, c-FOS, and c-JUN immunoreaction on pineal sections of Syrian hamster at 1400 and 2200 h. Scale bar, 50 µm. B, Semiquantitative analysis of CREB, pCREB, c-FOS, and c-JUN levels in the pineal gland of Syrian hamster at different time points. Values are expressed as the TID of immunolabeled pineal tissue divided by the quantification area in square micrometers (TID/µm2). Each point is the mean ± SEM of four animals. *, P < 0.05 compared with daytime points.

 
Effect of {alpha}/ß-adrenergic ligand administration on transcription factor expression in the pineal gland
To determine whether adrenergic receptors were involved in early-night expression of the studied transcription factors in the Syrian hamster pineal gland, selective {alpha}- or ß-adrenergic antagonists were administered at the onset of night, and the expression of CREB, pCREB, c-FOS, and c-JUN proteins was examined (Fig. 3Go). As expected, CREB expression was not altered by the adrenergic antagonists. In contrast, CREB phosphorylation was strongly inhibited by either the ß-adrenergic antagonist PROP or the {alpha}-adrenergic antagonist PRAZ. Expression of c-FOS and c-JUN proteins at the beginning of the night was also strongly inhibited by PRAZ or PROP administration. Vehicle injection did not alter the levels of these transcription factors. Administration of the {alpha}-adrenergic agonist PHE and the ß-adrenergic agonist ISO at midday had no stimulatory effect on c-FOS, c-JUN, and CREB protein expression 1 or 3 h after injection of the agonists (data not shown).


Figure 3
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  		FIG. 3. Effect of acute {alpha}- and ß-adrenergic antagonist injection on CREB, pCREB, c-FOS, and c-JUN expression in the Syrian hamster pineal gland. Hamsters raised under a 14-h light, 10-h dark cycle with lights off at 1900 h were injected with the {alpha}-adrenergic antagonist PRAZ (PRA, 15 mg/kg) or a ß-adrenergic antagonist PROP (PRO, 15 mg/kg) or Ringer (vehicle) at 1900 h and killed at 2200 h. Noninjected hamsters were killed at 2200 h as a nighttime control (22 h). Pineal glands were processed for protein detection by immunohistochemistry and semiquantitative analysis of CREB, pCREB, c-FOS, and c-JUN levels was performed. Values are expressed as the TID of immunolabeled pineal tissue divided by the quantification area in square micrometers (TID/µm2). Each point is the mean ± SEM of four animals. *, P < 0.05 compared with vehicle-injected animals.

 
For comparison, the effect of daytime administration of the {alpha}-adrenergic agonist PHE and the ß-adrenergic agonist ISO on CREB, c-FOS, c-JUN, and JUN-B protein expression in the rat pineal gland was examined (Fig. 4Go). CREB and c-JUN expression was not altered by the adrenergic agonists. In contrast, c-FOS and JUN-B were strongly induced as soon as 2 h after adrenergic agonist injection. Expression of both c-FOS and JUN-B was completely diminished 4 and 6 h later. Vehicle injection did not alter the levels of studied transcription factors.


Figure 4
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  		FIG. 4. Effect of acute {alpha}- and ß-adrenergic agonist injection on CREB, c-FOS, c-JUN, and JUN-B expression in the rat pineal gland. Rats raised under a 12-h light, 12-h dark cycle with lights off at 1900 h were injected with a mixture of {alpha}-adrenergic agonist, PHE, and ß-adrenergic agonist, ISO, 3 mg/kg of each agonist (Ag) or Ringer as a vehicle (Veh) at 0900 h and killed 2, 4, and 6 h after injection. Pineal glands were processed for JUN-B, c-FOS, c-JUN, and CREB detection by Western blot. Three rats were used for each point. *, Super Signal West Femto chemiluminescence substrate was used for signal visualization.

 
Effect of early-night CYCLO administration on Aa-nat gene expression in the Syrian hamster and rat pineal gland
To test the hypothesis that c-FOS and c-JUN transcription factors synthesized de novo at the beginning of night are necessary for Aa-nat gene transcription in the Syrian hamster pineal gland, the effect of a protein synthesis inhibitor, CYCLO, was tested. We first verified that CYCLO injection at 2100 h inhibited the early-night (2200 h) rise of c-FOS and c-JUN synthesis (Fig. 5AGo). Together with c-FOS/c-JUN inhibition, CYCLO injection at 2100 h markedly reduced the level of Aa-nat mRNA 5 h after injection as compared with vehicle-injected animals (Fig. 5BGo).


Figure 5
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  		FIG. 5. Effect of a single injection of CYCLO given early in the night on Aa-nat mRNA levels in the Syrian hamster and rat pineal gland. Hamsters raised under a 14-h light, 10-h dark cycle with lights off at 1900 h were injected with CYCLO (20 mg/kg) or vehicle (ethanol 25% in Ringer solution) at 2100 h. A, c-FOS, c-JUN, JUN-B, and CREB detection in pineal gland of hamsters injected with CYCLO (2 ) or vehicle (3 ) killed at 2200 h. Noninjected hamsters killed at 1300 and 2200 h were used as daytime (lane 1) and nighttime (lane 4) controls; c-FOS, c-JUN and CREB were detected by Western blot. B, Detection of Aa-nat mRNA expression by in situ hybridization in the pineal gland of hamsters injected at 2100 h with vehicle (light bar) or CYCLO (dark bar) and killed at 0200 h. Rats raised under a 12-h light, 12-h dark cycle with lights off at 1900 h were injected with CYCLO (20 mg/kg) or vehicle (ethanol 25% in Ringer solution) at 2100 h. C, JUN-B, c-JUN, and CREB detection in pineal gland of rats injected with CYCLO (lane 2) or vehicle (lane 3). Rats were killed at 0200 h. Noninjected rats killed at 1300 and 0200 h were used as daytime (lane 1) and nighttime (lane 4) controls. JUN-B, c-JUN, and CREB were detected by Western blot. D, Levels of Aa-nat mRNA in the pineal gland of rats injected at 2100 h with CYCLO (dark bars) or vehicle (light bars) and then killed at 0200 or 0400 h. The levels of Aa-nat mRNA (B and D) are expressed as percentage of vehicle-injected animals at 0200 h. Each data point is the mean ± SEM of five animals. *, P < 0.05 compared with vehicle-injected animals.

 
For comparison, the effect of a protein synthesis inhibitor, CYCLO, on the late night c-JUN and JUN-B expression and Aa-nat gene transcription in the rat pineal gland was examined. Similarly to the Syrian hamster, CYCLO injection at 2100 h inhibited the late-night (0200 h) rise of c-JUN and JUN-B synthesis (Fig. 5CGo). But in contrast to the Syrian hamster, early-night administration of CYCLO in the rat did not reduce the level of Aa-nat mRNA at 0200 h compared with vehicle-injected animals. On the contrary, the level of Aa-nat mRNA observed at 0400 h was significantly increased after CYCLO injection compared with vehicle-injected rats (Fig. 5DGo).

To evaluate the importance of c-FOS/c-JUN on Aa-nat gene transcription in the Syrian hamster pineal gland, the effect of CYCLO was tested at different time points at the beginning of night (Fig. 6Go). CYCLO injection reduced the level of Aa-nat mRNA at all indicated time points but more strongly when it was injected at 2100 h (1 h before the c-FOS/c-JUN peak) and 2200 h (during the c-FOS/c-JUN peak) than at 2300 h (1 h after the c-FOS/c-JUN peak). These results emphasize the importance of early-night c-FOS and c-JUN proteins for Aa-nat gene transcription in the Syrian hamster pineal gland.


Figure 6
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  		FIG. 6. Effect of single injections of CYCLO given at different nighttime points on Aa-nat mRNA levels in the Syrian hamster pineal gland. Hamsters raised under a 14-h light, 10-h dark cycle, with lights off at 1900 h, were injected with vehicle (ethanol 25% in Ringer solution, light bar) at 2100 h or CYCLO (20 mg/kg, dark bars) at 2100, 2200, or 2300 h. All the animals were killed at 0200 h. Aa-nat mRNA was detected by in situ hybridization and the levels of Aa-nat mRNA are expressed as percentage of vehicle-injected animals. Each data point is the mean ± SEM of five animals. **, P < 0.01 compared with vehicle-injected animals; *, P < 0.05 compared with vehicle-injected animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have indicated that the mechanisms involved in the regulation of pineal AA-NAT, the melatonin rhythm-generating enzyme, are different in the Syrian hamster from those described for the rat (22, 23, 24, 25, 26). Recently, we have reported that newly synthesized stimulatory transcription factors are required for Aa-nat gene expression and melatonin synthesis in the Syrian hamster (21) but not in the rat (2). In the present work, the contribution of pCREB, c-FOS, c-JUN, and JUN-B proteins to the regulation of Aa-nat gene transcription in the Syrian hamster compared with the rat was investigated.

Because no data were available on the transcription factors involved in Aa-nat regulation in the Syrian hamster, the expression and phosphorylation of the transcription factor CREB were examined first. CREB levels in pineal cells were constant, whereas pCREB levels displayed a marked increase during the night. CREB phosphorylation depended on NE because application of ß- or {alpha}-adrenergic antagonists at the beginning of night strongly reduced pCREB levels. These findings show that the NE input to the pineal gland of the Syrian hamster induces rapid CREB phosphorylation soon after night onset. Our data, together with previous observations, indicate that the molecular mechanisms of NE-dependent CREB phosphorylation are similar between the two rodent species. Thus, in the pineal gland of both species, stimulation of adrenergic receptors at the beginning of night leads to adenylate cyclase activation, cAMP accumulation, and finally CREB phosphorylation (Refs. 23 , 34 , 35 and this study) suggesting that NE-induced CREB phosphorylation is conservative in the rodent pineal gland.

Even though pCREB appears crucial, newly synthesized transcription factors may be involved in the regulation of Aa-nat transcription in rodents. Indeed, besides the CRE binding site, the rat Aa-nat gene promoter contains additional sites for other transcription factors. Among these, the TRE site for AP-1 complex binding seems important for the modulation of Aa-nat transcription (14). AP-1 proteins have been characterized as highly dynamic complexes that can form 18 different homodimers (among JUN proteins) and heterodimers (between FOS and JUN proteins) in response to different extracellular stimuli (for review see Ref. 36). Additionally, the composition of AP-1 can change in a time-dependent manner during continuous stimulation. Thus, in serum-stimulated fibroblasts, AP-1 mediates selective control of transcriptional activity of early and late genes by different cooperative recruitment of individual FOS and JUN proteins (37). To evaluate the contribution of AP-1 to the differential regulation of Aa-nat gene expression in Syrian hamster and rat pineal glands, we analyzed the nighttime expression of several members of the AP-1 complex, c-FOS, c-JUN, and JUN-B.

In the Syrian hamster, we showed that nighttime NE stimulation of pinealocytes results in a marked early and synchronous rise of c-FOS and c-JUN protein expression indicating c-FOS/c-JUN AP-1 complex formation at this time. Importantly, the c-FOS/c-JUN heterodimer is much more stable than any other homodimers and displays a high affinity for TRE binding sites (38). Although we observed a faint increase in JUN-B expression, we do not consider this to have a major influence on AP-1 formation between c-FOS and c-JUN proteins at the beginning of the night. Importantly, AP-1 complexes made with c-JUN proteins form stimulatory transcription factors, whereas hetero- and homodimers composed with JUN-B are negative regulators (32, 39). Therefore, the early and synchronous appearance of c-FOS and c-JUN before Aa-nat mRNA (Fig. 7AGo) indicates that the c-FOS/c-JUN complex is probably a stimulatory transcription factor for Aa-nat expression. Indeed, in vivo injection of the protein synthesis inhibitor CYCLO in the early night, which fully inhibited c-FOS/cJUN protein expression but did not affect CREB phosphorylation (unpublished data), markedly decreased Aa-nat gene transcription. Moreover, inhibition of Aa-nat gene transcription was smaller when CYCLO was injected after the peak of c-FOS/c-JUN expression. These results support our hypothesis that the c-FOS/c-JUN AP-1 complex formed at the beginning of the night is involved in the nighttime Aa-nat transcription in the Syrian hamster. In this study, we analyzed the expression of only four transcription factors (pCREB, c-FOS, c-JUN, and JUN-B) among many (40, 41). Obviously, CYCLO injection at the early night may also inhibit the synthesis of other stimulatory transcription factors, not studied in this work, that may be important for Aa-nat transcription. Additionally, the expression of the Aa-nat gene at each time point will depend on the concurrence of different stimulatory (pCREB, c-FOS, c-JUN, etc.) and inhibitory (ICER, Fra-2, JUN-B, JUN-D, etc.) factors. Additional molecular analysis will be necessary to establish whether and how the Syrian hamster Aa-nat promoter is regulated by the c-FOS/c-JUN complex.


Figure 7
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  		FIG. 7. Models for the differential involvement of AP-1 transcription factors in Syrian hamster and rat Aa-nat gene expression. A, In the Syrian hamster pineal gland, c-FOS and c-JUN proteins are expressed concomitantly at early night and may form a stimulatory heterodimer AP-1 complex that amplifies Aa-nat transcriptional activation. B, In the rat pineal gland, JUN-B is expressed late in the night and may form an inhibitory homodimer AP-1 with c-JUN proteins that represses Aa-nat gene transcription at the end of the night.

 
In the rat pineal gland, the dynamics and composition of AP-1 complexes were found to be completely different. First, our data together with previous observations (11) indicate that NE stimulation of rat pineal glands leads to delayed AP-1 expression, with some AP-1 components being expressed during the second part of the night coincidentally with Aa-nat mRNA (Fig. 7BGo). Second, we observed predominant expression of c-JUN and JUN-B proteins compared with c-FOS, suggesting a different composition of the AP-1 complex between the two rodent species. In the rat pineal gland, therefore, the AP-1 complex may be composed of at least c-JUN and JUN-B. Because of the temporal pattern and the proteins involved, it is likely that the AP-1 complex observed here is a negative regulator of Aa-nat transcription. This hypothesis is strengthened by the findings that injection of the protein synthesis inhibitor CYCLO at the beginning of the night, which inhibited c-JUN/JUN-B protein expression, induces a significant increase of Aa-nat mRNA level at the end of the night but not earlier. c-JUN and JUN-B may therefore be part of an AP-1 family of inhibitory transcription factors already described in the rat pineal gland such as Fra-2 and JUN-D proteins (10, 11).

In the Syrian hamster pineal gland, we demonstrated that c-FOS and c-JUN protein expression is not endogenous but depends on nighttime NE stimulation. We showed that both ß- and {alpha}-adrenergic receptors are involved in c-FOS protein expression at the beginning of the night. Similarly to c-FOS, c-JUN protein regulation in the Syrian hamster pineal gland was also found to involve ß- and {alpha}-adrenergic receptors. However, daytime injection of adrenergic agonists failed to induced c-FOS and c-JUN expression, showing a time-restricted ability of NE to activate these transcription factors. This is in agreement with our previous study showing an inability of NE to induce Aa-nat gene expression at daytime (21). Because induction of c-fos gene expression by adrenergic agonist is also blocked at daytime (unpublished data) and the c-fos gene promoter contains an E-box binding site (42), it is possible that the time restriction of c-FOS induction is due to local clock gene activity. In the rat retina, it has been proposed that Aa-nat gene expression is gated by an endogenous circadian clock (43) and circadian oscillation of c-FOS protein in photoreceptors (44) is coincidental with nighttime melatonin production. Altogether, these results suggest that c-FOS and c-JUN expression may be somehow coupled to a pineal circadian clock, whose mechanisms and function are yet to be discovered.

In the rat pineal, as expected, daytime adrenergic stimulation leads to an increase of c-FOS and JUN-B levels as it does for Aa-nat expression and melatonin production (2). Surprisingly, c-FOS and JUN-B induction was rapid and transient with a maximum 2 h after the injection, which is in contrast to their late induction at night. Early reports have shown that c-fos and jun-B mRNA are expressed early at night, as soon as 2 h after dark onset (13). The delay between c-fos/jun-B mRNA and proteins during the night may be due to protein degradation at the beginning of the night. In the rat, the AP-1 complex is thought to be a repressor of NE-regulated genes such as Aa-nat. We hypothesize that the early-night content of the rat pineal negative regulator AP-1 is reduced by posttranslational mechanisms to allow a full Aa-nat gene transcription. This hypothesis is supported by the recent observation that inhibition of proteasomal proteolysis before or concurrent with NE stimulation causes a significant reduction in Aa-nat mRNA and protein level in cultured rat pinealocytes (33). In the late night, AP-1 level is increased to allow a reduction in Aa-nat gene expression and melatonin synthesis. In the pineal gland of the Syrian hamster where c-fos gene expression at early night (unpublished data) is directly followed by c-FOS protein accumulation, it is probable that such a posttranslational modulation does not occur.

In conclusion, the present study reports a NE-driven nighttime stimulation of CREB phosphorylation and subsequent expression of a c-FOS/c-JUN stimulatory AP-1 complex in the Syrian hamster pineal gland. We propose that the molecular mechanisms involved in NE-dependent CREB phosphorylation are conserved in the Syrian hamster and rat pineal gland. However, the regulation and function of the AP-1 complexes are different between the two species. In the Syrian hamster, early-night synthesis of c-FOS and c-JUN proteins appears critical for the stimulation of Aa-nat transcription, whereas in the rat, late-night synthesis of JUN-B proteins may be involved in the down-regulation of Aa-nat toward the end of the night.


   Acknowledgments
 
We thank Daniel Bonn for taking care of the animals and Dr. David Hicks for English correction of the manuscript.


   Footnotes
 
This work was supported by a NATO fellowship.

Disclosure summary: The authors have nothing to disclose.

First Published Online August 3, 2006

Abbreviations: AA-NAT, Arylalkylamine N-acetyltransferase; AP-1, activator protein-1; CRE, cAMP-responsive element; CREB, cAMP-responsive element-binding protein; CYCLO, cycloheximide; Fra-2, Fos-related antigen 2; ISO, isoproterenol; NE, norepinephrine; pCREB, phosphorylated CREB; PHE, phenylephrine; PKAII, protein kinase A type II; PRAZ, prazosin; PROP, propranolol; TBHS, Tris-buffered high-salt saline; TID, total integrated density; TRE, tetradecanoyl phorbol acetate response element.

Received April 20, 2006.

Accepted for publication July 21, 2006.


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 Introduction
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 Results
 Discussion
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