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Fos-related antigen 2 (Fra-2) memorizes photoperiod in the rat pineal gland
Neuroscience 132 (2005) 511–518
FOS-RELATED ANTIGEN 2 (FRA-2) MEMORIZES PHOTOPERIOD IN THE RAT PINEAL GLAND L. ENGEL,a B. B. P. GUPTA,b V. LORENZKOWSKI,a B. HEINRICH,a I. SCHWERDTLE,a S. GERHOLD,a H. HOLTHUES,a L. VOLLRATHa AND R. SPESSERTa*
In mammals, pineal function is primarily controlled by a circadian oscillator, which is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Activity of the oscillator is synchronized by light acting on the retina. The circadian oscillator controls the pineal through a polyneuronal pathway that terminates in the pineal (for review see Korf et al., 1998). According to the oscillator’s program, noradrenaline (NA) is released from the intrapineal nerve terminals during the night. By binding to ␣- and -adrenoceptors in rat pinealocytes, NA induces the formation of the second messengers cAMP and cGMP. In the rat pineal, adrenergic induction of cAMP and subsequent phosphorylation of the pre-existing transcription factor “cyclic AMP response element binding protein” (CREB) result in the nocturnal activation of several genes. Among these are genes that encode for the transcription factors “inducible cAMP early repressor” (ICER; Stehle et al., 1993) and “Fos-related antigen-2” (Fra-2; Baler and Klein, 1995; Guillaumond et al., 2000; Spessert et al., 2000). Both transcription factors resemble each other not only in undergoing a daily rhythm (Baler and Klein, 1995; Guillaumond et al., 2000, 2002), but also in restricting cAMP-stimulated transcription of pineal genes during the night (ICER: Stehle et al., 1993; Fra-2: Smith et al., 2001). ICER does so by competing with phospho-CREB for “cyclic AMP response elements” (Stehle et al., 1993). Fra-2 forms heterodimeric complexes with members of the Jun family of transcription factors, which regulate gene transcription by binding to activator protein-1 DNA consensus elements (Guillaumond et al., 2000, 2002). In the rat pineal, the target genes of ICER and Fra-2 appear to be different. ICER regulates the aa-nat gene (Foulkes et al., 1996a), which encodes the key enzyme in melatonin formation, N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87] (for review see Klein et al., 1997). Fra-2 down-regulates the dII gene encoding iodothyronine deiodinase type II (DII; Kamiya et al., 1999), which catalyzes the peripheral deiodination of thyroxine (T4) prohormone to the active 3,3=,5-triiodothyronine (T3). The main known function of the SCN–pineal axis is to imprint photoperiodic information on the organism, the pineal contributing to this process by the photoperiod-dependent synthesis and release of the hormone melatonin (for review see Goldman and Darrow, 1983). In the rat, photoperiodic adaptation of pineal melatonin synthesis appears to be mediated by ICER. The amount of ICER protein increases under short photoperiods and decreases under long photoperiods (Foulkes et al., 1996b) and is inversely correlated with the inducibility of the aa-nat gene (Engel et al., 2004).
a
Department of Anatomy, Johannes Gutenberg University, Saarstrae 19-21, D-55099 Mainz, Germany
b Environmental Endocrinology Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793 022, India
Abstract—As the physiological role of fos-related antigen-2 (Fra-2) is largely unknown and since the pineal plays an important role in the photoperiodic control of the body, we have tested the hypothesis that Fra-2 expression is photoperiod-dependent and may be involved in imprinting photoperiod on the pineal gland and the body as a whole. To this end, we have investigated Fra-2 mRNA expression and Fra-2 protein expression under various light/dark (LD) cycles. A clear nocturnal increase occurs for both monitored parameters under all photoperiodic conditions studied. The level of Fra-2 protein expression clearly depends on photoperiod, because the amount of protein at dark onset and during the night negatively correlates with the length of the photoperiod. Further, high-phosphorylated Fra-2 isoforms are abundant under all photoperiods tested, with the exception of LD 20:4. Because Fra-2 phosphorylation depends on cGMP, a depressed cGMP response to adrenergic stimulation under LD 20:4 appears to explain this finding. We conclude that photoperiod is imprinted on Fra-2 in terms of both protein amount and protein phosphorylation in the rat pineal gland. This imprinting becomes fully manifest after about 7 days only, suggesting that a number of altered photoperiodic cycles are required for pineal Fra-2 to “learn” that the photoperiod has changed. Reportedly, Fra-2 limits expression of the enzyme iodothyronine deiodinase type II, which catalyzes the intracellular deiodination of thyroxine prohormone to the active 3,3=,5-triiodothyronine. We have found that the extent of Fra-2 expression inversely correlates with the dII gene response to cAMP; hence the photoperiodic regulation of Fra-2 may affect the body by changing pineal thyroid hormone metabolism. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: circadian, adrenergic agonists, cAMP, cGMP, iodothyronine deiodinase type II, arylalkylamine N-acetyltransferase. *Corresponding author. Tel: ⫹49-6131-3923718; fax: ⫹49-6131-3923719. E-mail address: [email protected] (R. Spessert). Abbreviations: AA-NAT, arylalkylamine N-acetyltransferase; ANOVA, analysis of variance; CREB, cyclic AMP response element binding protein; dbcAMP, dibutyryl cAMP; DII, iodothyronine deiodinase type II; Fra-2, fos-related antigen-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICER, inducible cAMP early repressor; ISO, isoproterenol; LD, light/dark; MAPK, mitogen-activated protein kinase; NA, noradrenaline; NOS I, nitric oxide synthase I; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PHE, phenylephrine; SCN, suprachiasmatic nucleus; sGC, cytosolic/soluble guanylyl cyclase; SNP, sodium nitroprusside; TCA, trichloroacetic acid; T3, 3,3=,5triiodothyronine; T4, thyroxine; 8-br cGMP, 8-bromo-cGMP. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2004.12.014
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The role played by Fra-2 in the photoperiodic adaptation of the pineal is incompletely understood. There is evidence that, compared with 12-h light/dark (LD 12:12), nocturnal Fra-2 mRNA peaks are phase-advanced (LD 18:6) or are phase-delayed (LD 6:18) under altered photoperiods (Guillaumond et al., 2002). Thus, Fra-2 could also play a role in the photoperiodic adaptation of pineal gene expression. In order to clarify the role of Fra-2, we have studied Fra-2 mRNA formation, the amount of Fra-2 protein and the involvement of other possible target genes under different photoperiodic conditions. Our results suggest that Fra-2 transmits photoperiodic and hence seasonal information to the Fra-2 target gene dII.
EXPERIMENTAL PROCEDURES Animals All experiments on animals were performed under an institutionally approved protocol in accordance with NIH guidelines for the care and use of laboratory animals. All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. Male and female Sprague–Dawley rats (body weights: 150 –180 g) were kept under standard laboratory conditions (illumination with fluorescent strip lights, 200 lux at cage level; 20⫾1 °C; water and food ad libitum) with a LD regimen of 12:12. For the study of the effects of different photoperiods, animals were exposed to LD 20:4, LD 16:8, LD 12:12, LD 8:16 and LD 4:20 (dark onset between 09:00 and 10:00 h depending on the LD regimen) for 2 weeks, unless otherwise indicated. Irrespective of the lighting regimen, the rats were kept under bright white light during the daytime and under dim red light during the night. They were killed by decapitation under bright white light during the daytime and under dim red light during the night. The pineals were quickly removed, immediately frozen in liquid N2 and kept at ⫺70 °C until assayed.
Organ culture For the study of the inducibility of cGMP, Fra-2 mRNA, AA-NAT mRNA and DII mRNA, pineals taken from rats at dark onset were placed on plastic meshes in culture dishes containing 1.5 ml BGJb medium (Gibco, Karlsruhe, Germany) with 1 mg/ml bovine serum albumin fraction V, 2 mM glutamine, 0.125 mg/ml CaCO3 and 0.1 mg/ml ascorbic acid. The organs were cultured in the absence or presence of the specified drugs at 37 °C under an atmosphere of 95% O2 and 5% CO2 for 10 min (cGMP) or 3 h (transcripts). The pineals were quickly frozen in liquid N2 at the end of incubation and stored at ⫺70 °C until assayed.
RNA isolation RNA from three to five pineals per sample was isolated by using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the instructions of the manufacturer. The amount of extracted RNA was determined by measuring the optical density at 260 and 280 nm.
Reverse transcription Extracted RNA (1 g) was reverse-transcribed by using 4 U Omniscript reverse transcriptase (Qiagen) in a total volume of 20 l, containing 2.0 l 10⫻ buffer (supplied with the transcriptase), 0.5 mM each deoxynucleotide triphosphate, 10 U ribonuclease inhibitor and 1 M oligo d(T) primer. A sample without the addition of RNA was routinely included as a control. The reverse transcription mixture was incubated at 37 °C for 60 min to promote
cDNA synthesis. The reaction was terminated by heating the samples at 95 °C for 5 min. The cDNA was diluted 1:5 in RNasefree distilled water and aliquots of 5 l were used for the polymerase chain reaction (PCR).
Real-time PCR Real-time PCR was carried out in a total volume of 25 l containing 2.5 l dNTP (2.5 mM; Hybaid, Heidelberg, Germany) each, 2.5 l SybrGreen (Molecular Probes; dilution 1:10,000), 0.75 l primer (10 mM) each, 0.1 l (2 U) Thermo-Start DNA polymerase (Abgene, Hamburg, Germany); 2.5 l 10⫻ buffer (supplied with the DNA polymerase) and 5 l sample. Primers with the following sequences were used: for amplification of Fra-2 cDNA (rat Fra-2 cDNA; GenBank accession number: U18913), (forward) 5=-AAG TGT CGG AAC CGT CGA CGT GAG-3=, (reverse) 5=-TTC AAG GAG TCT GAT GAC TGG TCC-3=; for amplification of AA-NAT cDNA (rat AA-NAT cDNA; GenBank accession number: U40803), (forward) 5=-CTC CCT GCC AGT GAG TTC CG-3=, (reverse) 5=-GGT GAG GAA GTG CCG GAT CTC-3=; for amplification of DII cDNA (rat DII cDNA; GenBank accession number: NM031720), (forward) 5=-ATG TGA GGC GAG GAG GTA G-3=, (reverse) 5=-CAC TGC CAG AGG ACA AAG G-3=. PCR amplification and quantification were performed on an i-Cycler (BioRad, Muenchen, Germany) under the following conditions: denaturation for 3 min at 95 °C, followed by 40 cycles of 50 s at 95 °C, 20 s at 60 °C and 20 s at 72 °C. All amplifications were performed in duplicate. The amount of RNA was calculated from the measured threshold cycles by a standard curve. The data were normalized by determination of the amount of glyceraldehyde-3-phosphate dehydrogenase (GADPH) mRNA (rat GAPDH cDNA; GenBank accession number: NM017008). For a comparison of individual experiments, transcript amount was expressed as the percentage of the maximum in a given experiment.
Western blot For Western blot analysis, three rat pineals were resuspended in 15 l RIPA buffer and sonicated for 15 s. The homogenate was then vortexed (20 s), incubated (4 °C, 15 min) and centrifuged at 10,000⫻g (4 °C, 15 min). A sample of 25 g protein of the supernatant was dissolved in 30 l sample buffer (0.15 M Tris– HCl, pH 6.8, containing 4% SDS, 22% glycerol and 0.05% Bromophenol Blue) and prepared by the addition of 10 l mercaptoethanol and heating for 10 min at 90 °C. SDS gel electrophoresis was performed by using 12% acrylamide/bisacrylamide. Electroblotting onto nitrocellulose filters (0.45 m) was carried out in a semi-dry blot system by applying a 3 mA current per cm2 for 1 h. All subsequent incubations were followed by washes in five changes of phosphate-buffered saline (PBS; pH 7.4, containing 0.1% Tween). The membranes were blocked in 10% skim-milk in PBS (pH 7.4; containing 0.05% Tween) for 2 h and incubated with antibodies raised against Fra-2 (Santa Cruz, CA, USA; sc-604, 1:300 dilution; Matsunobu et al., 2004; Milde-Langosch et al., 2004) or against actin (Sigma, Taufkirchen, Germany; A-2066, 1:300 dilution). The proteins were detected by electrochemiluminescence by using goat anti-rabbit horseradish-peroxidase-conjugated secondary antibody (Dianova, Hamburg, Germany; dilution 1:10,000). The nitrocellulose filters were exposed to Hyperfilm (Amersham Life Science, Freiburg, Germany), which was scanned with a densitometer. Results were quantified by means of the PC program Scion Image Beta 4.02 Win (Scion Corporation, USA).
Measurement of cGMP To determine adrenergically induced cGMP accumulation, pineals at the end of the incubation period were immediately sonicated in 40 l of 5% trichloroacetic acid (TCA) and centrifuged at 1500⫻g
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for 10 min. The supernatant was extracted three times with 200 l water-saturated diethyl ether in order to remove the TCA. The residual ether was removed by heating the samples at 70 °C for 5 min. The samples were then reconstituted with 60 l 0.05 M phosphate buffer (pH 7.4). The cyclic GMP content of the samples was determined by using commercially available enzyme-linked immunosorbent assay kits (Alexis, Gruenberg, Germany). The samples were assayed at different dilutions, each dilution being assayed in duplicate. In brief, sample or standard solution, cGMPspecific rabbit antibodies and a cGMP-acetylcholinesterase-conjugate (tracer) at appropriate dilutions, respectively, were incubated for 18 h at 4 °C in microwell plates that had been pre-coated with mouse monoclonal anti-rabbit antibody. After a washing step, the plates were developed with Ellman’s reagent as described by the manufacturer for 90 min in the dark. Plates were read at a wavelength of 405 nm. The concentrations of cGMP were calculated by interpolation from a standard curve and expressed as pmol/pineal.
Statistical analysis For the determination of transcript amount and protein amount, in each experiment the maximum value of mRNA and protein, respectively, was set to 100%. Data were analyzed by using a one-way analysis of variance (ANOVA) followed by a Fisher’s PLSD test. The temporal patterns of Fra-2 mRNA and AA-NAT mRNA were tested for statistical difference by two-way ANOVA. Spearman’s test was used to determine the relationship between the length of the photoperiod and Fra-2 immunoreactivity. A P value of less than 0.05 was considered as significant.
RESULTS Nocturnal Fra-2 expression under various photoperiods To investigate photoperiodic control of the fra-2 gene, the time course of Fra-2 mRNA expression was monitored under various lighting regimens (LD 12:12, LD 4:20, LD 20:4). Since Fra-2 mRNA expression is known to increase only after dark onset, Fra-2 measurement was conducted from dark onset to the early light phase under each of the photoperiods. We found that, irrespective of the lighting regimen, the amount of Fra-2 transcript was low at the onset of darkness and increased during darkness (Fig. 1A–C). The delay between the onset of darkness and the nocturnal rise in transcript amount depended on the lighting regimen. In comparison with LD 12:12 (Fig. 1A), it was higher under LD 4:20 (Fig. 1B) and lower under LD 20:4 (Fig. 1C). The level of Fra-2 transcript peaked 6 h after dark onset under LD 12:12 (Fig. 1A), 15 h after dark onset under LD 4:20 (Fig. 1B) and 3 h after dark onset under LD 20:4 (Fig. 1C). When referred to light onset, Fra-2 transcript amount peaked after 18 h under LD 12:12 (Fig. 1A), 19 h under LD 4:20 (Fig. 1B), and after 23 h under LD 20:4 (Fig. 1C). Accordingly, under each photoperiod, primarily light onset appears to direct the time-point of Fra-2 mRNA peak expression. The duration of elevated Fra-2 mRNA amount increased with the length of the scotophase (Fig. 1). Under each photoperiod studied, the nocturnal time course of Fra-2 transcript production did not significantly differ from that of AA-NAT mRNA (Fig. 1A–C). To determine whether the photoperiod-dependent changes in the time course of nocturnal Fra-2 transcript
Fig. 1. The nocturnal time course of amounts of rat pineal Fra-2 mRNA and AA-NAT mRNA under LD 12:12 (A), LD 4:20 (B) and LD 20:4 (C). When referred to dark onset, increase in nocturnal Fra-2 transcript and AA-NAT mRNA is phase-advanced under LD 20:4 and phase-delayed under LD 4:20 (see horizontal bars above each graph). Furthermore, the duration of elevated Fra-2 mRNA amount increases with the length of the scotophase. Under each photoperiod studied, the nocturnal time course of Fra-2 transcript amount does not significantly differ from that of AA-NAT mRNA. Transcript amounts from the same pineals were normalized to GAPDH mRNA and expressed as a percentage of the maximum value. Each value represents the mean⫾S.E.M. derived from at least four samples.
results in differential Fra-2 protein expression, Fra-2 immunoreactivity was compared under LD 4:20 and LD 20:4 (Fig. 2). Under both photoperiods, Fra-2 immunoreactivity increased after the onset of darkness. The time course of nocturnal Fra-2 immunoreactivity resembled that of the amount of Fra-2 transcript (Fig. 1B, C); it peaked 15 h after dark onset under LD 4:20 and 3 h after dark onset under LD 20:4. However, despite the amount of Fra-2 mRNA
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Fig. 3. Fra-2 immunoreactivity as a function of the length of photoperiod. At the onset of darkness, pineals were taken from rats that had been housed for 14 days under the indicated lighting regimens. (A) Representative Western blot analysis. Size standards are indicated in kDa (right-hand arrows). (B) The band densities were quantified and expressed as percentage of the maximum. Immunoreactivity of Fra-2 was normalized to actin. Note that Fra-2 immunoreactivity negatively correlates with the length of the photoperiod (P⬍0.01; analyzed by Spearman’s test). Each value represents the mean⫾S.E.M. derived from at least three samples. * P⬍0.05; ** P⬍0.01 compared with the value obtained from the pineals of rats kept under LD 4:20. Fig. 2. Nocturnal Fra-2 immunoreactivity and actin immunoreactivity in pineals obtained from rats under LD 20:4 and LD 4:20. The pineals were obtained from rats killed at the indicated time points. (A) Representative Western blot analysis. Size standards are indicated in kDa (right-hand arrows). (B, C) The band densities were quantified and expressed as percentage of the maximum. Immunoreactivity of Fra-2 was normalized to actin. Each value represents the mean⫾S.E.M. derived from four samples. ** P⬍0.01 compared with the value obtained from the pineals at the onset of darkness under the same photoperiod. ⫹⫹ P⬍0.01 compared with the corresponding value under LD 20:4. ° P⬍0.05 compared with the peak value under LD 20:4.
being low at dark onset (Fig. 1B, C), under each photoperiod Fra-2 protein was present at dark onset (Fig. 2). The extent of Fra-2 immunoreactivity differed between the photoperiods. At the onset of darkness and at the time-point of maximum expression, Fra-2 immunoreactivity was enhanced under LD 4:20 in comparison with LD 20:4 (Fig. 2). Furthermore, the photoperiod affected the presence of Fra-2 isoforms of higher molecular weight. This was evident from the observation that Fra-2 migrated between 42 kDa and 46 kDa under LD 4:20 and around 42 kDa under LD 20:4. To determine whether the observed differences also occurred in photoperiods present in temperate zones, the Fra-2 signal at the onset of darkness was monitored as a function of the length of the photoperiod (Fig. 3A, B). The
intensity of the Fra-2 signal gradually decreased with the length of the photoperiod. Under all lighting regimens, with the exception of LD 20:4, the upper bands corresponding to Fra-2 isoforms of higher molecular weight were abundant (Fig. 3A). Fra-2 protein expression as a function of the time of adaptation As Fra-2 immunoreactivity was altered 2 weeks after changing the lighting regimens (Figs. 2, 3), we determined the length of time necessary for this effect to occur (Fig. 4A, B). Differences in Fra-2 immunoreactivity were observed three LD-cycles after changes in the lighting regimen from LD 20:4 to LD 4:20 and were fully developed after seven LD-cycles. Thus, the exposure to between three and seven LD cycles appeared to account for the photoperiod-dependent alteration of Fra-2 immunoreactivity. The role of cGMP in Fra-2 expression To investigate the role of cGMP in Fra-2 expression, pineals collected from rats maintained under LD 20:4 were treated in vitro with the - and ␣-adrenergic agonists iso-
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Fig. 4. Fra-2 immunoreactivity as a function of the time of adaptation. Rats exposed to LD 20:4 were adapted for the time indicated to LD 4:20. Note that full adaptation to LD 4:20 requires approximately 1 week. The pineals were taken from the rats at the onset of the dark phase. Immunoreactivity from three pooled pineals was determined by Western blotting. (A) Representative Western blot analysis. Size standards are indicated in kDa (right-hand arrows). (B) The band densities were quantified and expressed as a percentage of the maximum. Immunoreactivity of Fra-2 was normalized to actin. Each value represents the mean⫾S.E.M. derived from three samples. * P⬍0.05; ** P⬍0.01 compared with the value after 9 weeks of adaptation.
proterenol (ISO)⫹phenylephrine (PHE; 10⫺7 M each) and 8-bromo cGMP (8-br cGMP; 10⫺3 M) and the NO-donor sodium nitroprusside (SNP; 10⫺7 M) for 3 h, after which time Fra-2 mRNA, AA-NAT mRNA and Fra-2 immunoreactivity were determined. Adrenergic stimulation with ISO⫹PHE increased Fra-2 mRNA, AA-NAT mRNA and Fra-2 immunoreactivity (Fig. 5). Incubation with 8-br cGMP or SNP did not induce Fra-2 mRNA formation or AA-NAT mRNA formation (Fig. 5A) but resulted in an upward shift of the Fra-2 signal in Western blotting (Fig. 5B). Cyclic GMP inducibility as a function of the length of the photoperiod Higher molecular weight Fra-2 isoforms were present under all lighting regimens studied, except LD 20:4 (see above). To determine whether a decreased effectiveness of the adrenergic stimulus in inducing cGMP accounted for the differential expression of Fra-2 isoforms, pineals were collected from rats maintained under various photoperiods and treated in vitro with ISO (10⫺7 M) and ISO⫹PHE (10⫺7 M each) for 10 min, followed by the measurement of cGMP content. Adrenergic cGMP inducibility was similar under LD 4:20, LD 12:12 and LD 16:8 but the response was
Fig. 5. In vitro effects of ISO⫹PHE (10⫺7 M each), 8-br cGMP (10⫺3 M) and SNP (10⫺7 M) on Fra-2 mRNA (A), AA-NAT mRNA (A) and Fra-2 immunoreactivity (B, C). Note that 8-br cGMP and SNP do not stimulate Fra-2 transcript formation (A) but induce the appearance of higher molecular weight Fra-2 isoforms (B). The rats were exposed to LD 20:4 for at least 2 weeks. The pineals were taken from rats at the onset of dark phase and incubated for 3 h in the presence or absence of the drugs. (A) Transcript amount was normalized to GAPDH mRNA and expressed as a percentage of the maximum value. (B) Representative Western blot analysis. Size standards are indicated in kDa (right-hand arrows). (C) The band densities were quantified and expressed as percentage of the maximum. Immunoreactivity of Fra-2 was normalized to actin. Each value represents the mean⫾S.E.M. derived from at least four samples. ** P⬍0.01 compared with the respective control group.
significantly reduced under LD 20:4 (Fig. 6A). To explore whether photoperiods affected the time course response of cGMP, we measured cGMP after incubating rat pineals from extreme photoperiods (LD 4:20 and LD 20:4) with ISO⫹PHE for different durations (5 min, 10 min, 30 min and 60 min). Irrespective of the photoperiod, the maximum cGMP response was reached after 10 min of incubation.
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Fig. 7. Effects of dbcAMP (10⫺3 M) on amounts of AA-NAT mRNA and DII mRNA in pineals obtained from rats exposed to various photoperiods. Note that the AA-NAT and the DII response to cAMP are increased under LD 20:4 when compared with LD 4:20. The pineals were taken from rats at the onset of the dark phase and incubated for 3 h in the presence or absence of the respective drugs. Transcript amount was normalized to GAPDH mRNA and expressed as a percentage of the maximum value. Each value represents the mean⫾S.E.M. derived from six samples. ** P⬍0.01 compared with the respective control group. ⫹, ⫹⫹ Values differ significantly from the respective group under LD 4:20: P⬍0.05 and 0.01, respectively.
Fra-2 protein amount should affect dII gene expression. To test this possibility, the mRNA response of the gene to dibutyryl cAMP (dbcAMP) was compared between LD 20:4 and LD 4:20 (Fig. 7). As with AA-NAT mRNA, the DII mRNA response to dbcAMP was higher under LD 20:4 than under LD 4:20. Fig. 6. (A) In vitro effects of ISO (10⫺7 M), ISO plus PHE (10⫺7 M each) and SNP (10⫺7 M) on cGMP accumulation in pineals of rats maintained under LD 4:20, LD 12:12, LD 16:8 and LD 20:4. Adrenergic stimulation of cGMP is depressed under LD 20:4. The pineals were taken from the rats at the onset of the dark phase. Each value represents the mean⫾S.E.M. derived from at least four samples. *, ** Values differ significantly from respective control group: P⬍0.05 and 0.01, respectively. ⫹⫹ Value differs significantly from LD 12:12: P⬍0.01. (B) In vitro time course effects of incubation with ISO plus PHE (10⫺7 M each) on cGMP accumulation in pineals of rats maintained under LD 4:20 and LD 20:4. Control values at various time points were between 1.6 and 2.8 pmol/pineal. Each value represents the mean⫾S.E.M. derived from at least four samples. *, ** Values differ significantly from respective control group: P⬍0.05 and 0.01, respectively. ⫹, ⫹⫹ Values differ significantly from the respective group of LD 20:4: P⬍0.05 and 0.01, respectively.
However, at all time points, the cGMP response to stimulation by ISO⫹PHE was significantly higher in pineals from LD 4:20 than in pineals from LD 20:4 (Fig. 6B). To determine whether the photoperiod affected cGMP response by influencing the NO target cytosolic/soluble guanylyl cyclase (sGC), we tested the effect of SNP (10⫺7 M) on cGMP formation in pineals of rats from various photoperiods. The cGMP response to NO generated by SNP was similar in pineals from all photoperiods used (Fig. 6A) indicating that the sensitivity of sGC to NO was not influenced by photoperiod. Cyclic AMP inducibility of Fra-2 effector gene dII under various photoperiods Fra-2 appears to inhibit dII gene expression (Smith et al., 2001). Therefore, photoperiod-dependent changes in
DISCUSSION The present study shows that photoperiod is imprinted on pineal Fra-2 protein amount. This becomes evident from the observation that, at dark onset and at the time-point of peak expression, the level of nocturnal Fra-2 protein is higher under short photoperiods than under long photoperiods (Figs. 2, 3). As the duration of elevated Fra-2 transcript is correlated with the length of the scotophase (Guillaumond et al., 2002; Fig. 1A–C), photoperiodic changes in the amount of Fra-2 protein may reflect differential Fra-2 de novo formation. Photoperiodic changes in Fra-2 protein amounts become detectable after three “new” LD-cycles and are fully developed only after seven LD-cycles (Fig. 4). The present results do not indicate how soon the altered photoperiod begins to affect Fra-2 gene expression but show that several LD cycles are necessary for the full photoperiodic adaptation of the Fra-2 protein. How can the slowness of Fra-2 adaptation be explained? In the present study, the interesting observation has been made that, despite Fra-2 mRNA formation being virtually non-existing during the light phase (Baler and Klein, 1995) and only starting after the onset of darkness (Baler and Klein, 1995; Guillaumond et al., 2002; this study), Fra-2 protein is clearly demonstrable already at dark onset. This finding implies that Fra-2 protein is only partly degraded during the light phase and that the amount of nocturnal Fra-2 protein depends not only on Fra-2 de novo formation during the particular night under investigation, but also on Fra-2 de novo formation
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during the dark phases of previous cycles. The effect of the previous cycles explains why, after a change in the photoperiod, Fra-2 protein amount alters only gradually at each cycle until full adaptation is reached. Comparable mechanisms appear to be responsible for the slowness of photoperiodic adaptation in nitric oxide synthase I (NOS I) expression (Spessert and Rapp, 2001), hydroxyindole-Omethyltransferase expression (Ribelayga et al., 1999a,b) and aa-nat gene inducibility (Engel et al., 2004). One could argue that the changes in the above pineal parameters develop slowly in order for the retina-SCN-pineal system to “check” whether the alteration of photoperiod is just a single transient event that can be neglected or a more permanent one to which the organism must adapt. Altered photoperiods affect not only the amount of nocturnal Fra-2, but also the relative fraction of Fra-2 isoforms of higher molecular weight. This is evident from the observation that, under photoperiods of between 4 and 16 h, but not of 20 h, Fra-2 isoforms migrating between 43 and 46 kDa are abundant. In the rat pineal (Baler and Klein, 1995) and in other tissues (Gruda et al., 1994; Boss et al., 2001), the presence of higher molecular weight Fra-2 isoforms (Figs. 2, 3) is mainly attributable to posttranslational Fra-2 phosphorylation. Therefore, Fra-2 probably “stores” photoperiodic history in terms of both protein amount and protein phosphorylation. In an attempt to understand the photoperiod-dependent regulation of Fra-2 phosphorylation, we have investigated the role of cGMP in this process. For this purpose, we compared the effects of adrenergic agonists (which simultaneously induces NO-dependent cGMP formation and cAMP formation), the NO-donor SNP (which mimics activation of the enzyme NOS I and subsequently NOdependent cGMP formation), and 8-br cGMP (Fig. 5A). As reported earlier (Baler and Klein, 1995), adrenergic treatment simultaneously induced Fra-2 mRNA formation (Fig. 5A), increased the total amount of Fra-2 protein (Fig. 5B, C) and led to the appearance of higher molecular weight Fra-2 isoforms (Fig. 5B). While Fra-2 mRNA formation (Fig. 5A) and total amount of Fra-2 protein did not change following treatments with SNP and 8-br cGMP (Fig. 5B, C), a clear increase of higher molecular weight Fra-2 isoforms was seen (Fig. 5B). This observation suggests that cGMP mediates adrenergically stimulated Fra-2 phosphorylation only, whereas cAMP may mediate primarily Fra-2 de novo formation. Furthermore, it strongly argues for a role of the NOS I ¡ NO ¡ sGC ¡ cGMP pathway in rat pineal Fra-2 phosphorylation. In the rat pineal, cGMP induces the activation of the mitogen-activated protein kinase (MAPK) pathway (Ho et al., 1999, 2003). Since the latter is known to affect Fra-2 phosphorylation in tissues (Gruda et al., 1994), the MAPK pathway could well mediate cGMP-dependent Fra-2 phosphorylation in the rat pineal. In the present study, the drop in Fra-2 phosphorylation observed under LD 20:4 coincides with the depressed effectiveness of the adrenergic stimulus in inducing cGMP. Therefore, the photoperiod appears to regulate Fra-2 phosphorylation by altering the adrenergic signal transduction pathway that is known to induce cGMP formation. Since adrenergic
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cGMP formation requires NOS I stimulation (Spessert et al., 1993) and since the extent of NOS I expression (Spessert et al., 1995; Spessert and Rapp, 2001) and total NOS activity (Schaad et al., 1994, 1995) decreases under long photoperiods (LD 20:4 and LD 21:3, respectively) compared with LD 12:12, one can hypothesize that differential NOS I expression accounts for photoperiod-dependent changes in cGMP formation and in Fra-2 phosphorylation. Fra-2 does not appear to play a decisive role in aa-nat gene transcription in the rat pineal (Smith et al., 2001). As melatonin formation primarily depends on aa-nat gene transcription in the rat, photoperiod-dependent changes in Fra-2 are unlikely to contribute to the encoding of photoperiodic information in melatonin formation. The effect of photoperiod on aa-nat gene inducibility (Guillaumond et al., 2002; Engel et al., 2004; this study) appears to depend mainly on differential ICER expression (Foulkes et al., 1996a,b). If Fra-2 does not play a role in melatonin formation, what are the functional implications of the present findings? The pineal-specific transgenic knockdown of Fra-2 is associated with increased nocturnal expression of the gene encoding for DII (Smith et al., 2001) indicating that Fra-2 limits nocturnal dII gene expression in the rat pineal. Since cAMP mediates nocturnal induction of DII (Guerrero et al., 1988), one can hypothesize that Fra-2 restricts cAMP inducibility of the gene and that, consequently, photoperiodic regulation of Fra-2 leads to changes in dII gene inducibility. To test this hypothesis, in the present study cAMP inducibility of the pineal dII gene was compared between LD 20:4 and LD 4:20 in vitro. The finding that the DII mRNA response to cAMP is stronger under LD 20:4 than under LD 4:20, i.e. is inversely correlated with Fra-2 availability, suggests a role of pineal Fra-2 in transferring photoperiodic information to the dII gene by changing the effectiveness of cAMP in inducing dII transcription. Up-regulation of dII expression under long photoperiods and down-regulation of dII expression under short photoperiods is evident not only in the rat pineal (this study), but also in other brain areas of various species (Djungarian hamster: Watanabe et al., 2004; Japanese quail: Yoshimura et al., 2003). Therefore, it is tempting to speculate that Fra-2 also transduces photoperiodic history to the dII gene in tissues other than the pineal. DII is known to catalyze the intracellular deiodination of T4 prohormone to the active T3. Therefore, the photoperiod-dependent regulation of the pineal dII gene should influence the concentration of T3 in the blood and cerebrospinal fluid. Thyroid hormones are essential for the maintenance of seasonal reproductive changes in a number of mammals (Nicholls et al., 1988; Prendergast et al., 2002) and i.c.v. infusion of T3 mimics photoperiodically induced testicular growth in birds (Yoshimura et al., 2003). Therefore, “storage” of the photoperiodic history in the pineal in the form of altered Fra-2 amounts and/or phosphorylation might play a role in the transfer of information concerning annual lighting conditions to the hypothalamo– hypophyseal– gonadal axis. In conclusion, the present study provides evidence for a role of Fra-2 as a transducer of photoperiodic history, thereby resembling that of ICER. Whereas Fra-2 seems to
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preserve photoperiodic information relevant to thyroid hormone activation, ICER provides this information with respect to melatonin formation. Acknowledgments—We thank U. Goeringer-Struwe and B. Herte for their excellent technical assistance. We also thank U. Hulick and B. Heerlein for secretarial help. The data contained in this study are part of theses presented by Verena Lorenzkowski, Bettina Heinrich, Isabell Schwerdtle and Sabine Gerhold toward partial fulfillment of their degree of Dr. Med. at the Johannes Gutenberg University, Mainz. This study was financially supported by the Deutsche Forschungsgemeinschaft (Sp 403/2–1).
REFERENCES Baler B, Klein DC (1995) Circadian expression of transcription factor Fra-2 in the rat pineal gland. J Biol Chem 270:27319 –27325. Boss V, Roback JD, Young AN, Roback LJ, Weisenhorn DM, MedinaFlores R, Wainer BH (2001) Nerve growth factor, but not epidermal growth factor, increases Fra-2 expression and alters Fra-2/JunD binding to AP-1 and CREB binding elements in pheochromocytoma (PC12) cells. J Neurosci 21:18 –26. Engel L, Mathes A, Schwerdtle I, Pogorzelski B, Holthues H, Vollrath L, Spessert R (2004) Rat pineal arylalkylamine N-acetyltransferase: cyclic AMP inducibility of its gene depends on prior entrained photoperiod. Mol Brain Res 123:45–55. Foulkes NS, Borjigin J, Snyder SH, Sassone-Corsi P (1996a) Transcriptional control of circadian hormone synthesis via the CREM feedback loop. Proc Natl Acad Sci USA 93:14140 –14145. Foulkes NS, Duval G, Sassone-Corsi P (1996b) Adaptive inducibility of CREM as transcriptional memory of circadian rhythms. Nature 381:83–85. Goldman BD, Darrow JM (1983) The pineal gland and mammalian photoperiodism. Neuroendocrinology 37:386 –396. Gruda MC, Kovary K, Metz R, Bravo R (1994) Regulation of fra-1 and fra-2 phosphorylation differs during the cell cycle of fibroblasts and phosphorylation in vitro by MAP kinase affects DNA binding activity. Oncogene 9:2537–2547. Guerrero JM, Santana C, Reiter RJ (1988) Effect of isoproterenol and dibutyryl cyclic AMP on thyroxine type-II 5=-deiodinase and Nacetyltransferase activities in rat pineal organ cultures. Neurosci Lett 89:229 –233. Guillaumond F, Becquet D, Bosler O, Francoise-Bellan AM (2002) Adrenergic inducibility of AP-1 binding in the rat pineal gland depends on prior photoperiod. J Neurochem 83:157–166. Guillaumond F, Sage D, Deprez P, Bosler O, Becquet D, Bosler O, Francoise-Bellan AM (2000) Circadian binding activity of AP-1, a regulator of the arylalkylamine N-acetyltransferase gene in the rat pineal gland depends on circadian Fra-2, c-Jun, and JunD expression and is regulated by the clock’s Zeitgebers. J Neurochem 75:1398 –1407. Ho AK, Hashimoto K, Chik CL (1999) 3=,5=-Cyclic guanosine monophosphate activates mitogen-activated protein kinase in rat pinealocytes. J Neurochem 73:598 – 604. Ho AK, Mackova M, Price L, Chik CL (2003) Diurnal variation in p42/44 mitogen activated protein kinase in the rat pineal gland. Mol Cell Endocrinol 208:23–30. Kamiya Y, Murakami M, Araki O, Hosoi Y, Ogiwara T, Mizuma H, Mori M (1999) Pretranslational regulation of rhythmic type II iodothyronine deiodinase expression by -adrenergic mechanism in the rat pineal gland. Endocrinology 140:1272–1278. Klein DC, Coon SL, Roseboom PH, Weller JL, Bernard M, Gastel JA, Zatz M, Iuvone PM, Rodriguez IR, Begay V, Falcon J, Cahill GM,
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(Accepted 16 December 2004) (Available online 16 March 2005)
FOS-RELATED ANTIGEN 2 (FRA-2) MEMORIZES PHOTOPERIOD IN THE RAT PINEAL GLAND L. ENGEL,a B. B. P. GUPTA,b V. LORENZKOWSKI,a B. HEINRICH,a I. SCHWERDTLE,a S. GERHOLD,a H. HOLTHUES,a L. VOLLRATHa AND R. SPESSERTa*
In mammals, pineal function is primarily controlled by a circadian oscillator, which is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Activity of the oscillator is synchronized by light acting on the retina. The circadian oscillator controls the pineal through a polyneuronal pathway that terminates in the pineal (for review see Korf et al., 1998). According to the oscillator’s program, noradrenaline (NA) is released from the intrapineal nerve terminals during the night. By binding to ␣- and -adrenoceptors in rat pinealocytes, NA induces the formation of the second messengers cAMP and cGMP. In the rat pineal, adrenergic induction of cAMP and subsequent phosphorylation of the pre-existing transcription factor “cyclic AMP response element binding protein” (CREB) result in the nocturnal activation of several genes. Among these are genes that encode for the transcription factors “inducible cAMP early repressor” (ICER; Stehle et al., 1993) and “Fos-related antigen-2” (Fra-2; Baler and Klein, 1995; Guillaumond et al., 2000; Spessert et al., 2000). Both transcription factors resemble each other not only in undergoing a daily rhythm (Baler and Klein, 1995; Guillaumond et al., 2000, 2002), but also in restricting cAMP-stimulated transcription of pineal genes during the night (ICER: Stehle et al., 1993; Fra-2: Smith et al., 2001). ICER does so by competing with phospho-CREB for “cyclic AMP response elements” (Stehle et al., 1993). Fra-2 forms heterodimeric complexes with members of the Jun family of transcription factors, which regulate gene transcription by binding to activator protein-1 DNA consensus elements (Guillaumond et al., 2000, 2002). In the rat pineal, the target genes of ICER and Fra-2 appear to be different. ICER regulates the aa-nat gene (Foulkes et al., 1996a), which encodes the key enzyme in melatonin formation, N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87] (for review see Klein et al., 1997). Fra-2 down-regulates the dII gene encoding iodothyronine deiodinase type II (DII; Kamiya et al., 1999), which catalyzes the peripheral deiodination of thyroxine (T4) prohormone to the active 3,3=,5-triiodothyronine (T3). The main known function of the SCN–pineal axis is to imprint photoperiodic information on the organism, the pineal contributing to this process by the photoperiod-dependent synthesis and release of the hormone melatonin (for review see Goldman and Darrow, 1983). In the rat, photoperiodic adaptation of pineal melatonin synthesis appears to be mediated by ICER. The amount of ICER protein increases under short photoperiods and decreases under long photoperiods (Foulkes et al., 1996b) and is inversely correlated with the inducibility of the aa-nat gene (Engel et al., 2004).
a
Department of Anatomy, Johannes Gutenberg University, Saarstrae 19-21, D-55099 Mainz, Germany
b Environmental Endocrinology Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793 022, India
Abstract—As the physiological role of fos-related antigen-2 (Fra-2) is largely unknown and since the pineal plays an important role in the photoperiodic control of the body, we have tested the hypothesis that Fra-2 expression is photoperiod-dependent and may be involved in imprinting photoperiod on the pineal gland and the body as a whole. To this end, we have investigated Fra-2 mRNA expression and Fra-2 protein expression under various light/dark (LD) cycles. A clear nocturnal increase occurs for both monitored parameters under all photoperiodic conditions studied. The level of Fra-2 protein expression clearly depends on photoperiod, because the amount of protein at dark onset and during the night negatively correlates with the length of the photoperiod. Further, high-phosphorylated Fra-2 isoforms are abundant under all photoperiods tested, with the exception of LD 20:4. Because Fra-2 phosphorylation depends on cGMP, a depressed cGMP response to adrenergic stimulation under LD 20:4 appears to explain this finding. We conclude that photoperiod is imprinted on Fra-2 in terms of both protein amount and protein phosphorylation in the rat pineal gland. This imprinting becomes fully manifest after about 7 days only, suggesting that a number of altered photoperiodic cycles are required for pineal Fra-2 to “learn” that the photoperiod has changed. Reportedly, Fra-2 limits expression of the enzyme iodothyronine deiodinase type II, which catalyzes the intracellular deiodination of thyroxine prohormone to the active 3,3=,5-triiodothyronine. We have found that the extent of Fra-2 expression inversely correlates with the dII gene response to cAMP; hence the photoperiodic regulation of Fra-2 may affect the body by changing pineal thyroid hormone metabolism. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: circadian, adrenergic agonists, cAMP, cGMP, iodothyronine deiodinase type II, arylalkylamine N-acetyltransferase. *Corresponding author. Tel: ⫹49-6131-3923718; fax: ⫹49-6131-3923719. E-mail address: [email protected] (R. Spessert). Abbreviations: AA-NAT, arylalkylamine N-acetyltransferase; ANOVA, analysis of variance; CREB, cyclic AMP response element binding protein; dbcAMP, dibutyryl cAMP; DII, iodothyronine deiodinase type II; Fra-2, fos-related antigen-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICER, inducible cAMP early repressor; ISO, isoproterenol; LD, light/dark; MAPK, mitogen-activated protein kinase; NA, noradrenaline; NOS I, nitric oxide synthase I; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PHE, phenylephrine; SCN, suprachiasmatic nucleus; sGC, cytosolic/soluble guanylyl cyclase; SNP, sodium nitroprusside; TCA, trichloroacetic acid; T3, 3,3=,5triiodothyronine; T4, thyroxine; 8-br cGMP, 8-bromo-cGMP. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2004.12.014
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The role played by Fra-2 in the photoperiodic adaptation of the pineal is incompletely understood. There is evidence that, compared with 12-h light/dark (LD 12:12), nocturnal Fra-2 mRNA peaks are phase-advanced (LD 18:6) or are phase-delayed (LD 6:18) under altered photoperiods (Guillaumond et al., 2002). Thus, Fra-2 could also play a role in the photoperiodic adaptation of pineal gene expression. In order to clarify the role of Fra-2, we have studied Fra-2 mRNA formation, the amount of Fra-2 protein and the involvement of other possible target genes under different photoperiodic conditions. Our results suggest that Fra-2 transmits photoperiodic and hence seasonal information to the Fra-2 target gene dII.
EXPERIMENTAL PROCEDURES Animals All experiments on animals were performed under an institutionally approved protocol in accordance with NIH guidelines for the care and use of laboratory animals. All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. Male and female Sprague–Dawley rats (body weights: 150 –180 g) were kept under standard laboratory conditions (illumination with fluorescent strip lights, 200 lux at cage level; 20⫾1 °C; water and food ad libitum) with a LD regimen of 12:12. For the study of the effects of different photoperiods, animals were exposed to LD 20:4, LD 16:8, LD 12:12, LD 8:16 and LD 4:20 (dark onset between 09:00 and 10:00 h depending on the LD regimen) for 2 weeks, unless otherwise indicated. Irrespective of the lighting regimen, the rats were kept under bright white light during the daytime and under dim red light during the night. They were killed by decapitation under bright white light during the daytime and under dim red light during the night. The pineals were quickly removed, immediately frozen in liquid N2 and kept at ⫺70 °C until assayed.
Organ culture For the study of the inducibility of cGMP, Fra-2 mRNA, AA-NAT mRNA and DII mRNA, pineals taken from rats at dark onset were placed on plastic meshes in culture dishes containing 1.5 ml BGJb medium (Gibco, Karlsruhe, Germany) with 1 mg/ml bovine serum albumin fraction V, 2 mM glutamine, 0.125 mg/ml CaCO3 and 0.1 mg/ml ascorbic acid. The organs were cultured in the absence or presence of the specified drugs at 37 °C under an atmosphere of 95% O2 and 5% CO2 for 10 min (cGMP) or 3 h (transcripts). The pineals were quickly frozen in liquid N2 at the end of incubation and stored at ⫺70 °C until assayed.
RNA isolation RNA from three to five pineals per sample was isolated by using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the instructions of the manufacturer. The amount of extracted RNA was determined by measuring the optical density at 260 and 280 nm.
Reverse transcription Extracted RNA (1 g) was reverse-transcribed by using 4 U Omniscript reverse transcriptase (Qiagen) in a total volume of 20 l, containing 2.0 l 10⫻ buffer (supplied with the transcriptase), 0.5 mM each deoxynucleotide triphosphate, 10 U ribonuclease inhibitor and 1 M oligo d(T) primer. A sample without the addition of RNA was routinely included as a control. The reverse transcription mixture was incubated at 37 °C for 60 min to promote
cDNA synthesis. The reaction was terminated by heating the samples at 95 °C for 5 min. The cDNA was diluted 1:5 in RNasefree distilled water and aliquots of 5 l were used for the polymerase chain reaction (PCR).
Real-time PCR Real-time PCR was carried out in a total volume of 25 l containing 2.5 l dNTP (2.5 mM; Hybaid, Heidelberg, Germany) each, 2.5 l SybrGreen (Molecular Probes; dilution 1:10,000), 0.75 l primer (10 mM) each, 0.1 l (2 U) Thermo-Start DNA polymerase (Abgene, Hamburg, Germany); 2.5 l 10⫻ buffer (supplied with the DNA polymerase) and 5 l sample. Primers with the following sequences were used: for amplification of Fra-2 cDNA (rat Fra-2 cDNA; GenBank accession number: U18913), (forward) 5=-AAG TGT CGG AAC CGT CGA CGT GAG-3=, (reverse) 5=-TTC AAG GAG TCT GAT GAC TGG TCC-3=; for amplification of AA-NAT cDNA (rat AA-NAT cDNA; GenBank accession number: U40803), (forward) 5=-CTC CCT GCC AGT GAG TTC CG-3=, (reverse) 5=-GGT GAG GAA GTG CCG GAT CTC-3=; for amplification of DII cDNA (rat DII cDNA; GenBank accession number: NM031720), (forward) 5=-ATG TGA GGC GAG GAG GTA G-3=, (reverse) 5=-CAC TGC CAG AGG ACA AAG G-3=. PCR amplification and quantification were performed on an i-Cycler (BioRad, Muenchen, Germany) under the following conditions: denaturation for 3 min at 95 °C, followed by 40 cycles of 50 s at 95 °C, 20 s at 60 °C and 20 s at 72 °C. All amplifications were performed in duplicate. The amount of RNA was calculated from the measured threshold cycles by a standard curve. The data were normalized by determination of the amount of glyceraldehyde-3-phosphate dehydrogenase (GADPH) mRNA (rat GAPDH cDNA; GenBank accession number: NM017008). For a comparison of individual experiments, transcript amount was expressed as the percentage of the maximum in a given experiment.
Western blot For Western blot analysis, three rat pineals were resuspended in 15 l RIPA buffer and sonicated for 15 s. The homogenate was then vortexed (20 s), incubated (4 °C, 15 min) and centrifuged at 10,000⫻g (4 °C, 15 min). A sample of 25 g protein of the supernatant was dissolved in 30 l sample buffer (0.15 M Tris– HCl, pH 6.8, containing 4% SDS, 22% glycerol and 0.05% Bromophenol Blue) and prepared by the addition of 10 l mercaptoethanol and heating for 10 min at 90 °C. SDS gel electrophoresis was performed by using 12% acrylamide/bisacrylamide. Electroblotting onto nitrocellulose filters (0.45 m) was carried out in a semi-dry blot system by applying a 3 mA current per cm2 for 1 h. All subsequent incubations were followed by washes in five changes of phosphate-buffered saline (PBS; pH 7.4, containing 0.1% Tween). The membranes were blocked in 10% skim-milk in PBS (pH 7.4; containing 0.05% Tween) for 2 h and incubated with antibodies raised against Fra-2 (Santa Cruz, CA, USA; sc-604, 1:300 dilution; Matsunobu et al., 2004; Milde-Langosch et al., 2004) or against actin (Sigma, Taufkirchen, Germany; A-2066, 1:300 dilution). The proteins were detected by electrochemiluminescence by using goat anti-rabbit horseradish-peroxidase-conjugated secondary antibody (Dianova, Hamburg, Germany; dilution 1:10,000). The nitrocellulose filters were exposed to Hyperfilm (Amersham Life Science, Freiburg, Germany), which was scanned with a densitometer. Results were quantified by means of the PC program Scion Image Beta 4.02 Win (Scion Corporation, USA).
Measurement of cGMP To determine adrenergically induced cGMP accumulation, pineals at the end of the incubation period were immediately sonicated in 40 l of 5% trichloroacetic acid (TCA) and centrifuged at 1500⫻g
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for 10 min. The supernatant was extracted three times with 200 l water-saturated diethyl ether in order to remove the TCA. The residual ether was removed by heating the samples at 70 °C for 5 min. The samples were then reconstituted with 60 l 0.05 M phosphate buffer (pH 7.4). The cyclic GMP content of the samples was determined by using commercially available enzyme-linked immunosorbent assay kits (Alexis, Gruenberg, Germany). The samples were assayed at different dilutions, each dilution being assayed in duplicate. In brief, sample or standard solution, cGMPspecific rabbit antibodies and a cGMP-acetylcholinesterase-conjugate (tracer) at appropriate dilutions, respectively, were incubated for 18 h at 4 °C in microwell plates that had been pre-coated with mouse monoclonal anti-rabbit antibody. After a washing step, the plates were developed with Ellman’s reagent as described by the manufacturer for 90 min in the dark. Plates were read at a wavelength of 405 nm. The concentrations of cGMP were calculated by interpolation from a standard curve and expressed as pmol/pineal.
Statistical analysis For the determination of transcript amount and protein amount, in each experiment the maximum value of mRNA and protein, respectively, was set to 100%. Data were analyzed by using a one-way analysis of variance (ANOVA) followed by a Fisher’s PLSD test. The temporal patterns of Fra-2 mRNA and AA-NAT mRNA were tested for statistical difference by two-way ANOVA. Spearman’s test was used to determine the relationship between the length of the photoperiod and Fra-2 immunoreactivity. A P value of less than 0.05 was considered as significant.
RESULTS Nocturnal Fra-2 expression under various photoperiods To investigate photoperiodic control of the fra-2 gene, the time course of Fra-2 mRNA expression was monitored under various lighting regimens (LD 12:12, LD 4:20, LD 20:4). Since Fra-2 mRNA expression is known to increase only after dark onset, Fra-2 measurement was conducted from dark onset to the early light phase under each of the photoperiods. We found that, irrespective of the lighting regimen, the amount of Fra-2 transcript was low at the onset of darkness and increased during darkness (Fig. 1A–C). The delay between the onset of darkness and the nocturnal rise in transcript amount depended on the lighting regimen. In comparison with LD 12:12 (Fig. 1A), it was higher under LD 4:20 (Fig. 1B) and lower under LD 20:4 (Fig. 1C). The level of Fra-2 transcript peaked 6 h after dark onset under LD 12:12 (Fig. 1A), 15 h after dark onset under LD 4:20 (Fig. 1B) and 3 h after dark onset under LD 20:4 (Fig. 1C). When referred to light onset, Fra-2 transcript amount peaked after 18 h under LD 12:12 (Fig. 1A), 19 h under LD 4:20 (Fig. 1B), and after 23 h under LD 20:4 (Fig. 1C). Accordingly, under each photoperiod, primarily light onset appears to direct the time-point of Fra-2 mRNA peak expression. The duration of elevated Fra-2 mRNA amount increased with the length of the scotophase (Fig. 1). Under each photoperiod studied, the nocturnal time course of Fra-2 transcript production did not significantly differ from that of AA-NAT mRNA (Fig. 1A–C). To determine whether the photoperiod-dependent changes in the time course of nocturnal Fra-2 transcript
Fig. 1. The nocturnal time course of amounts of rat pineal Fra-2 mRNA and AA-NAT mRNA under LD 12:12 (A), LD 4:20 (B) and LD 20:4 (C). When referred to dark onset, increase in nocturnal Fra-2 transcript and AA-NAT mRNA is phase-advanced under LD 20:4 and phase-delayed under LD 4:20 (see horizontal bars above each graph). Furthermore, the duration of elevated Fra-2 mRNA amount increases with the length of the scotophase. Under each photoperiod studied, the nocturnal time course of Fra-2 transcript amount does not significantly differ from that of AA-NAT mRNA. Transcript amounts from the same pineals were normalized to GAPDH mRNA and expressed as a percentage of the maximum value. Each value represents the mean⫾S.E.M. derived from at least four samples.
results in differential Fra-2 protein expression, Fra-2 immunoreactivity was compared under LD 4:20 and LD 20:4 (Fig. 2). Under both photoperiods, Fra-2 immunoreactivity increased after the onset of darkness. The time course of nocturnal Fra-2 immunoreactivity resembled that of the amount of Fra-2 transcript (Fig. 1B, C); it peaked 15 h after dark onset under LD 4:20 and 3 h after dark onset under LD 20:4. However, despite the amount of Fra-2 mRNA
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Fig. 3. Fra-2 immunoreactivity as a function of the length of photoperiod. At the onset of darkness, pineals were taken from rats that had been housed for 14 days under the indicated lighting regimens. (A) Representative Western blot analysis. Size standards are indicated in kDa (right-hand arrows). (B) The band densities were quantified and expressed as percentage of the maximum. Immunoreactivity of Fra-2 was normalized to actin. Note that Fra-2 immunoreactivity negatively correlates with the length of the photoperiod (P⬍0.01; analyzed by Spearman’s test). Each value represents the mean⫾S.E.M. derived from at least three samples. * P⬍0.05; ** P⬍0.01 compared with the value obtained from the pineals of rats kept under LD 4:20. Fig. 2. Nocturnal Fra-2 immunoreactivity and actin immunoreactivity in pineals obtained from rats under LD 20:4 and LD 4:20. The pineals were obtained from rats killed at the indicated time points. (A) Representative Western blot analysis. Size standards are indicated in kDa (right-hand arrows). (B, C) The band densities were quantified and expressed as percentage of the maximum. Immunoreactivity of Fra-2 was normalized to actin. Each value represents the mean⫾S.E.M. derived from four samples. ** P⬍0.01 compared with the value obtained from the pineals at the onset of darkness under the same photoperiod. ⫹⫹ P⬍0.01 compared with the corresponding value under LD 20:4. ° P⬍0.05 compared with the peak value under LD 20:4.
being low at dark onset (Fig. 1B, C), under each photoperiod Fra-2 protein was present at dark onset (Fig. 2). The extent of Fra-2 immunoreactivity differed between the photoperiods. At the onset of darkness and at the time-point of maximum expression, Fra-2 immunoreactivity was enhanced under LD 4:20 in comparison with LD 20:4 (Fig. 2). Furthermore, the photoperiod affected the presence of Fra-2 isoforms of higher molecular weight. This was evident from the observation that Fra-2 migrated between 42 kDa and 46 kDa under LD 4:20 and around 42 kDa under LD 20:4. To determine whether the observed differences also occurred in photoperiods present in temperate zones, the Fra-2 signal at the onset of darkness was monitored as a function of the length of the photoperiod (Fig. 3A, B). The
intensity of the Fra-2 signal gradually decreased with the length of the photoperiod. Under all lighting regimens, with the exception of LD 20:4, the upper bands corresponding to Fra-2 isoforms of higher molecular weight were abundant (Fig. 3A). Fra-2 protein expression as a function of the time of adaptation As Fra-2 immunoreactivity was altered 2 weeks after changing the lighting regimens (Figs. 2, 3), we determined the length of time necessary for this effect to occur (Fig. 4A, B). Differences in Fra-2 immunoreactivity were observed three LD-cycles after changes in the lighting regimen from LD 20:4 to LD 4:20 and were fully developed after seven LD-cycles. Thus, the exposure to between three and seven LD cycles appeared to account for the photoperiod-dependent alteration of Fra-2 immunoreactivity. The role of cGMP in Fra-2 expression To investigate the role of cGMP in Fra-2 expression, pineals collected from rats maintained under LD 20:4 were treated in vitro with the - and ␣-adrenergic agonists iso-
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Fig. 4. Fra-2 immunoreactivity as a function of the time of adaptation. Rats exposed to LD 20:4 were adapted for the time indicated to LD 4:20. Note that full adaptation to LD 4:20 requires approximately 1 week. The pineals were taken from the rats at the onset of the dark phase. Immunoreactivity from three pooled pineals was determined by Western blotting. (A) Representative Western blot analysis. Size standards are indicated in kDa (right-hand arrows). (B) The band densities were quantified and expressed as a percentage of the maximum. Immunoreactivity of Fra-2 was normalized to actin. Each value represents the mean⫾S.E.M. derived from three samples. * P⬍0.05; ** P⬍0.01 compared with the value after 9 weeks of adaptation.
proterenol (ISO)⫹phenylephrine (PHE; 10⫺7 M each) and 8-bromo cGMP (8-br cGMP; 10⫺3 M) and the NO-donor sodium nitroprusside (SNP; 10⫺7 M) for 3 h, after which time Fra-2 mRNA, AA-NAT mRNA and Fra-2 immunoreactivity were determined. Adrenergic stimulation with ISO⫹PHE increased Fra-2 mRNA, AA-NAT mRNA and Fra-2 immunoreactivity (Fig. 5). Incubation with 8-br cGMP or SNP did not induce Fra-2 mRNA formation or AA-NAT mRNA formation (Fig. 5A) but resulted in an upward shift of the Fra-2 signal in Western blotting (Fig. 5B). Cyclic GMP inducibility as a function of the length of the photoperiod Higher molecular weight Fra-2 isoforms were present under all lighting regimens studied, except LD 20:4 (see above). To determine whether a decreased effectiveness of the adrenergic stimulus in inducing cGMP accounted for the differential expression of Fra-2 isoforms, pineals were collected from rats maintained under various photoperiods and treated in vitro with ISO (10⫺7 M) and ISO⫹PHE (10⫺7 M each) for 10 min, followed by the measurement of cGMP content. Adrenergic cGMP inducibility was similar under LD 4:20, LD 12:12 and LD 16:8 but the response was
Fig. 5. In vitro effects of ISO⫹PHE (10⫺7 M each), 8-br cGMP (10⫺3 M) and SNP (10⫺7 M) on Fra-2 mRNA (A), AA-NAT mRNA (A) and Fra-2 immunoreactivity (B, C). Note that 8-br cGMP and SNP do not stimulate Fra-2 transcript formation (A) but induce the appearance of higher molecular weight Fra-2 isoforms (B). The rats were exposed to LD 20:4 for at least 2 weeks. The pineals were taken from rats at the onset of dark phase and incubated for 3 h in the presence or absence of the drugs. (A) Transcript amount was normalized to GAPDH mRNA and expressed as a percentage of the maximum value. (B) Representative Western blot analysis. Size standards are indicated in kDa (right-hand arrows). (C) The band densities were quantified and expressed as percentage of the maximum. Immunoreactivity of Fra-2 was normalized to actin. Each value represents the mean⫾S.E.M. derived from at least four samples. ** P⬍0.01 compared with the respective control group.
significantly reduced under LD 20:4 (Fig. 6A). To explore whether photoperiods affected the time course response of cGMP, we measured cGMP after incubating rat pineals from extreme photoperiods (LD 4:20 and LD 20:4) with ISO⫹PHE for different durations (5 min, 10 min, 30 min and 60 min). Irrespective of the photoperiod, the maximum cGMP response was reached after 10 min of incubation.
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Fig. 7. Effects of dbcAMP (10⫺3 M) on amounts of AA-NAT mRNA and DII mRNA in pineals obtained from rats exposed to various photoperiods. Note that the AA-NAT and the DII response to cAMP are increased under LD 20:4 when compared with LD 4:20. The pineals were taken from rats at the onset of the dark phase and incubated for 3 h in the presence or absence of the respective drugs. Transcript amount was normalized to GAPDH mRNA and expressed as a percentage of the maximum value. Each value represents the mean⫾S.E.M. derived from six samples. ** P⬍0.01 compared with the respective control group. ⫹, ⫹⫹ Values differ significantly from the respective group under LD 4:20: P⬍0.05 and 0.01, respectively.
Fra-2 protein amount should affect dII gene expression. To test this possibility, the mRNA response of the gene to dibutyryl cAMP (dbcAMP) was compared between LD 20:4 and LD 4:20 (Fig. 7). As with AA-NAT mRNA, the DII mRNA response to dbcAMP was higher under LD 20:4 than under LD 4:20. Fig. 6. (A) In vitro effects of ISO (10⫺7 M), ISO plus PHE (10⫺7 M each) and SNP (10⫺7 M) on cGMP accumulation in pineals of rats maintained under LD 4:20, LD 12:12, LD 16:8 and LD 20:4. Adrenergic stimulation of cGMP is depressed under LD 20:4. The pineals were taken from the rats at the onset of the dark phase. Each value represents the mean⫾S.E.M. derived from at least four samples. *, ** Values differ significantly from respective control group: P⬍0.05 and 0.01, respectively. ⫹⫹ Value differs significantly from LD 12:12: P⬍0.01. (B) In vitro time course effects of incubation with ISO plus PHE (10⫺7 M each) on cGMP accumulation in pineals of rats maintained under LD 4:20 and LD 20:4. Control values at various time points were between 1.6 and 2.8 pmol/pineal. Each value represents the mean⫾S.E.M. derived from at least four samples. *, ** Values differ significantly from respective control group: P⬍0.05 and 0.01, respectively. ⫹, ⫹⫹ Values differ significantly from the respective group of LD 20:4: P⬍0.05 and 0.01, respectively.
However, at all time points, the cGMP response to stimulation by ISO⫹PHE was significantly higher in pineals from LD 4:20 than in pineals from LD 20:4 (Fig. 6B). To determine whether the photoperiod affected cGMP response by influencing the NO target cytosolic/soluble guanylyl cyclase (sGC), we tested the effect of SNP (10⫺7 M) on cGMP formation in pineals of rats from various photoperiods. The cGMP response to NO generated by SNP was similar in pineals from all photoperiods used (Fig. 6A) indicating that the sensitivity of sGC to NO was not influenced by photoperiod. Cyclic AMP inducibility of Fra-2 effector gene dII under various photoperiods Fra-2 appears to inhibit dII gene expression (Smith et al., 2001). Therefore, photoperiod-dependent changes in
DISCUSSION The present study shows that photoperiod is imprinted on pineal Fra-2 protein amount. This becomes evident from the observation that, at dark onset and at the time-point of peak expression, the level of nocturnal Fra-2 protein is higher under short photoperiods than under long photoperiods (Figs. 2, 3). As the duration of elevated Fra-2 transcript is correlated with the length of the scotophase (Guillaumond et al., 2002; Fig. 1A–C), photoperiodic changes in the amount of Fra-2 protein may reflect differential Fra-2 de novo formation. Photoperiodic changes in Fra-2 protein amounts become detectable after three “new” LD-cycles and are fully developed only after seven LD-cycles (Fig. 4). The present results do not indicate how soon the altered photoperiod begins to affect Fra-2 gene expression but show that several LD cycles are necessary for the full photoperiodic adaptation of the Fra-2 protein. How can the slowness of Fra-2 adaptation be explained? In the present study, the interesting observation has been made that, despite Fra-2 mRNA formation being virtually non-existing during the light phase (Baler and Klein, 1995) and only starting after the onset of darkness (Baler and Klein, 1995; Guillaumond et al., 2002; this study), Fra-2 protein is clearly demonstrable already at dark onset. This finding implies that Fra-2 protein is only partly degraded during the light phase and that the amount of nocturnal Fra-2 protein depends not only on Fra-2 de novo formation during the particular night under investigation, but also on Fra-2 de novo formation
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during the dark phases of previous cycles. The effect of the previous cycles explains why, after a change in the photoperiod, Fra-2 protein amount alters only gradually at each cycle until full adaptation is reached. Comparable mechanisms appear to be responsible for the slowness of photoperiodic adaptation in nitric oxide synthase I (NOS I) expression (Spessert and Rapp, 2001), hydroxyindole-Omethyltransferase expression (Ribelayga et al., 1999a,b) and aa-nat gene inducibility (Engel et al., 2004). One could argue that the changes in the above pineal parameters develop slowly in order for the retina-SCN-pineal system to “check” whether the alteration of photoperiod is just a single transient event that can be neglected or a more permanent one to which the organism must adapt. Altered photoperiods affect not only the amount of nocturnal Fra-2, but also the relative fraction of Fra-2 isoforms of higher molecular weight. This is evident from the observation that, under photoperiods of between 4 and 16 h, but not of 20 h, Fra-2 isoforms migrating between 43 and 46 kDa are abundant. In the rat pineal (Baler and Klein, 1995) and in other tissues (Gruda et al., 1994; Boss et al., 2001), the presence of higher molecular weight Fra-2 isoforms (Figs. 2, 3) is mainly attributable to posttranslational Fra-2 phosphorylation. Therefore, Fra-2 probably “stores” photoperiodic history in terms of both protein amount and protein phosphorylation. In an attempt to understand the photoperiod-dependent regulation of Fra-2 phosphorylation, we have investigated the role of cGMP in this process. For this purpose, we compared the effects of adrenergic agonists (which simultaneously induces NO-dependent cGMP formation and cAMP formation), the NO-donor SNP (which mimics activation of the enzyme NOS I and subsequently NOdependent cGMP formation), and 8-br cGMP (Fig. 5A). As reported earlier (Baler and Klein, 1995), adrenergic treatment simultaneously induced Fra-2 mRNA formation (Fig. 5A), increased the total amount of Fra-2 protein (Fig. 5B, C) and led to the appearance of higher molecular weight Fra-2 isoforms (Fig. 5B). While Fra-2 mRNA formation (Fig. 5A) and total amount of Fra-2 protein did not change following treatments with SNP and 8-br cGMP (Fig. 5B, C), a clear increase of higher molecular weight Fra-2 isoforms was seen (Fig. 5B). This observation suggests that cGMP mediates adrenergically stimulated Fra-2 phosphorylation only, whereas cAMP may mediate primarily Fra-2 de novo formation. Furthermore, it strongly argues for a role of the NOS I ¡ NO ¡ sGC ¡ cGMP pathway in rat pineal Fra-2 phosphorylation. In the rat pineal, cGMP induces the activation of the mitogen-activated protein kinase (MAPK) pathway (Ho et al., 1999, 2003). Since the latter is known to affect Fra-2 phosphorylation in tissues (Gruda et al., 1994), the MAPK pathway could well mediate cGMP-dependent Fra-2 phosphorylation in the rat pineal. In the present study, the drop in Fra-2 phosphorylation observed under LD 20:4 coincides with the depressed effectiveness of the adrenergic stimulus in inducing cGMP. Therefore, the photoperiod appears to regulate Fra-2 phosphorylation by altering the adrenergic signal transduction pathway that is known to induce cGMP formation. Since adrenergic
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cGMP formation requires NOS I stimulation (Spessert et al., 1993) and since the extent of NOS I expression (Spessert et al., 1995; Spessert and Rapp, 2001) and total NOS activity (Schaad et al., 1994, 1995) decreases under long photoperiods (LD 20:4 and LD 21:3, respectively) compared with LD 12:12, one can hypothesize that differential NOS I expression accounts for photoperiod-dependent changes in cGMP formation and in Fra-2 phosphorylation. Fra-2 does not appear to play a decisive role in aa-nat gene transcription in the rat pineal (Smith et al., 2001). As melatonin formation primarily depends on aa-nat gene transcription in the rat, photoperiod-dependent changes in Fra-2 are unlikely to contribute to the encoding of photoperiodic information in melatonin formation. The effect of photoperiod on aa-nat gene inducibility (Guillaumond et al., 2002; Engel et al., 2004; this study) appears to depend mainly on differential ICER expression (Foulkes et al., 1996a,b). If Fra-2 does not play a role in melatonin formation, what are the functional implications of the present findings? The pineal-specific transgenic knockdown of Fra-2 is associated with increased nocturnal expression of the gene encoding for DII (Smith et al., 2001) indicating that Fra-2 limits nocturnal dII gene expression in the rat pineal. Since cAMP mediates nocturnal induction of DII (Guerrero et al., 1988), one can hypothesize that Fra-2 restricts cAMP inducibility of the gene and that, consequently, photoperiodic regulation of Fra-2 leads to changes in dII gene inducibility. To test this hypothesis, in the present study cAMP inducibility of the pineal dII gene was compared between LD 20:4 and LD 4:20 in vitro. The finding that the DII mRNA response to cAMP is stronger under LD 20:4 than under LD 4:20, i.e. is inversely correlated with Fra-2 availability, suggests a role of pineal Fra-2 in transferring photoperiodic information to the dII gene by changing the effectiveness of cAMP in inducing dII transcription. Up-regulation of dII expression under long photoperiods and down-regulation of dII expression under short photoperiods is evident not only in the rat pineal (this study), but also in other brain areas of various species (Djungarian hamster: Watanabe et al., 2004; Japanese quail: Yoshimura et al., 2003). Therefore, it is tempting to speculate that Fra-2 also transduces photoperiodic history to the dII gene in tissues other than the pineal. DII is known to catalyze the intracellular deiodination of T4 prohormone to the active T3. Therefore, the photoperiod-dependent regulation of the pineal dII gene should influence the concentration of T3 in the blood and cerebrospinal fluid. Thyroid hormones are essential for the maintenance of seasonal reproductive changes in a number of mammals (Nicholls et al., 1988; Prendergast et al., 2002) and i.c.v. infusion of T3 mimics photoperiodically induced testicular growth in birds (Yoshimura et al., 2003). Therefore, “storage” of the photoperiodic history in the pineal in the form of altered Fra-2 amounts and/or phosphorylation might play a role in the transfer of information concerning annual lighting conditions to the hypothalamo– hypophyseal– gonadal axis. In conclusion, the present study provides evidence for a role of Fra-2 as a transducer of photoperiodic history, thereby resembling that of ICER. Whereas Fra-2 seems to
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preserve photoperiodic information relevant to thyroid hormone activation, ICER provides this information with respect to melatonin formation. Acknowledgments—We thank U. Goeringer-Struwe and B. Herte for their excellent technical assistance. We also thank U. Hulick and B. Heerlein for secretarial help. The data contained in this study are part of theses presented by Verena Lorenzkowski, Bettina Heinrich, Isabell Schwerdtle and Sabine Gerhold toward partial fulfillment of their degree of Dr. Med. at the Johannes Gutenberg University, Mainz. This study was financially supported by the Deutsche Forschungsgemeinschaft (Sp 403/2–1).
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(Accepted 16 December 2004) (Available online 16 March 2005)