A daily rhythm of activator protein-1 activity in the rat pineal is dependent upon trans-synaptic induction of junB

doi:10.1016/0306-4522(94)90358-1

Neuroscience

Volume 62, Issue 4, October 1994, Pages 1267-1278

https://doi.org/10.1016/0306-4522(94)90358-1 Get rights and content

Abstract

The daily cycle of phenotypic variation in the mammalian pineal provides a unique model for the investigation of the molecular mechanisms that regulate neurotransmitter synthesis. In the rat, a circadian adrenergic mechanism directs a change in serotonin metabolism that results in the nocturnal production of melatonin. Activity of the activator protein-1 transcriptional regulatory complex, as demonstrated by band-shift assays of rat pineal gland extracts, has now been shown to exhibit a rhythm, in vivo, which is temporally correlated with the rhythm of melatonin synthesis. Thus, nocturnal activator protein-1 activity (23.00 h) is markedly elevated, being 8-fold higher than the level of light-phase activity (P < 0.005). The nocturnal activator protein-1 protein complex is induced through a trans-synaptic, β-adrenoceptor-linked mechanism and is characterized by the prolonged participation of JunB as demonstrated using antibodies for specific activator protein-1 proteins. Indeed, JunB appears to be a major component of nocturnal changes in activator protein-1 activity, JunD forming an additional, constitutive component which is not affected by the nocturnal adrenergic signal. The α₁-adrenoreceptor-linked c-Fos protein, which is coordinately induced with JunB, does not form a stable component of nocturnal activator protein-1 activity. In contrast, parallel experiments showed that c-Fos does form a major component of the hippocampal activator protein-1 complex that is induced in rats following kainic acid treatment. In the pineal, a similar, although not identical, pattern of activator protein-1 activation has also been demonstrated in cultured glands following treatment with norepinephrine. Immunoblotting has demonstrated parallel accumulation of JunB and c-Fos protein in pineal nuclear fractions following stimulation both in vivo and in vitro.

The results provide evidence of posttranscriptional selection of neurotransmitter-stimulated activator protein-1 protein complexes, a mechanism which complements the differential induction of fos and jun genes in the pineal, and serves to generate a specific activator protein-1 transcription factor complex. This finding has general implications for the functional interpretation of fos and jun gene induction in neuronal systems. The stable JunB complex demonstrated here may be considered as one component of a timing mechanism which acts to perpetuate synaptic signals and thereby maintain an appropriate period of nocturnal pineal function.

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Despite characterization of the dynamics and regulation of these genes in the rat pineal gland, their function is enigmatic. The rate-limiting of melatonin synthesis in rodents involves the gating of Aa-nat expression via activating transcription factors such as phosphoCREB [5,6,16,46] and AP-1 [45], and inhibiting transcription factors such as ICER [38,46]. For unknown, perhaps evolutionary, reasons, post-translational regulation of AA-NAT has the main role in taxonomic groups outside the rodentia: in pinealocytes of sheep, cows, Rhesus monkeys and humans, the NA–cAMP–PKA signaling pathway regulates melatonin synthesis via direct phosphorylation of AA-NAT and the formation of a complex between AA-NAT and the 14–3-3 protein, which protects the enzyme from proteasomal degradation [5,6,16,47,52].
In the mammalian pineal gland, information on environmental lighting conditions that is neuronally encoded by the retina is converted into nocturnally elevated synthesis of the hormone melatonin. Evolutionary pressure has changed the morphology of vertebrate pinealocytes, eliminating direct photoreception and the endogenous clock function. Despite these changes, nocturnally elevated melatonin synthesis has remained a reliable indicator of time throughout evolution. In the photo-insensitive mammalian pineal gland this message of darkness depends on the master circadian pacemaker in the hypothalamic suprachiasmatic nuclei. The dramatic change in vertebrate pinealocytes has received little attention; here, we therefore link the known evolutionary morphodynamics and well-investigated biochemical details responsible for rhythmic synthesis of melatonin with recently characterized patterns of gene expression in the pineal gland. We also address the enigmatic function of clockwork molecules in mammalian pinealocytes.
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The rat pineal gland transcriptome exhibits dynamic daily variation that reflects nocturnally restricted hormone production. Here we have used a protein/DNA interaction array to screen for day–night changes in DNA binding activity that are associated with transcriptional rhythms. Overall, 47 of 54 potential consensus binding sequence activities were detected, and of these, 29 (62%) were found to exhibit day:night differences in level. In addition to known, rhythmic pineal DNA binding activities (CRE and AP-1), multiple novel activities were observed including nocturnally elevated AP-2 consensus sequence binding activity. This array result was validated using conventional DNA binding assays, and we have also demonstrated AP-2β and AP-2γ proteins in the pineal gland, in addition to a nocturnally elevated AP-2α isoform. Our results have confirmed the presence of a complex assembly of transcriptional rhythms in the rat pineal gland and have provided details of more factors that contribute to this aspect of circadian neuroendocrine function.
Regulation of cAMP-induced arylalkylamine N-acetyltransferase, Period1, and MKP-1 gene expression by mitogen-activated protein kinases in the rat pineal gland
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In rodent pineal glands, sympathetic innervation, which leads to norepinephrine release, is a key process in the circadian regulation of physiology and certain gene expressions. It has been shown that gene expression of the rate-limiting enzyme in the melatonin synthesis arylalkylamine N-acetyltransferase (Aa-Nat), circadian clock gene Period1, and mitogen-activated protein kinase (MAPK) phosphtase-1 (MKP-1), is controlled mainly by a norepinephrine-beta-adrenergic receptor-cAMP signaling cascade in the rat pineal gland. To further dissect the signaling cascades that regulate those gene expressions, we examined whether MAPKs are involved in cAMP-induced gene expression. Western blot and immunohistochemical analyses showed that one of the three MAPKs, c-Jun N-terminal kinase (JNK), was expressed in the pineal, and was phosphorylated by cAMP analogue stimulation with a peak 20 min after start of the stimulation, in vitro. A specific JNK inhibitor SP600125 (Anthra[1,9-cd]pyrazol-6(2H)-one1,9-pyrazoloanthrone), but not its negative control (N¹-Methyl-1,9-pyrazoloanthrone), significantly reduced cAMP-stimulated Aa-Nat, Period1, and MKP-1 mRNA levels. Although another MAPK, p38^MAPK, has also been shown to be activated by cAMP stimulation, a p38^MAPK inhibitor, SB203580 (4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole, HCl), showed no effect on cAMP-induced Aa-Nat and Period1 mRNA levels; whereas SB203580, but not its negative analogue SB202474 (4-Ethyl-2(p-methoxyphenyl)-5-(4′-pyridyl)-IH-imidazole, DiHCl), significantly reduced cAMP-induced MKP-1 mRNA levels. Taken together, our data suggest that cAMP-induced Aa-Nat and Period1 are likely to be mediated by activation of JNK, whereas MKP-1 may be mediated by both p38^MAPK and JNK activations.
Rat pineal arylalkylamine-N-acetyltransferase: Cyclic AMP inducibility of its gene depends on prior entrained photoperiod
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The nocturnal biosynthesis of melatonin in the rat pineal depends on strongly enhanced expression of the enzyme N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87]. AA-NAT transcription is stimulated during darkness by adrenergic inputs to the pineal from the suprachiasmatic nucleus (SCN). Nocturnal activation of the AA-NAT promotor following stimulation of pinealocyte adrenoceptors involves cAMP-dependent stimulation of protein kinase A (PKA). The nocturnal rise in AA-NAT depends on the lighting conditions. As compared with light/dark (LD) 12:12, the delay between dark onset and the nocturnal rise in AA-NAT is shortened under long photoperiods and prolonged under short photoperiods. Here, we report that the rapidity of nocturnal AA-NAT induction depends on cAMP inducibility of the gene. Accordingly, cAMP produces a strong AA-NAT response in pineals obtained from rats housed under long photoperiods and a weak AA-NAT response under short photoperiods. Changes in AA-NAT inducibility are fully developed not earlier than after seven cycles. This observation suggests that long-term changes in the photoperiod are necessary to achieve full adjustment of cAMP inducibility of the gene. A direct relationship was found between cAMP-dependent AA-NAT inducibility and the pineal protein kinase A (PKA) activity. As compared to LD 12:12, PKA activity was increased under LD 20:4 and attenuated under LD 4:20. On the basis of the present findings, we suggest that the photoperiod determines the effectiveness of nocturnal AA-NAT induction by long-term modulation of the intrapineal pathway that transmits the cAMP signal to the AA-NAT gene.
Rhythmic melatonin secretion does not correlate with the expression of arylalkylamine N-acetyltransferase, inducible cyclic AMP early repressor, Period1 or Cryptochrome1 mRNA in the sheep pineal
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The pineal gland, through nocturnal melatonin, acts as a neuroendocrine transducer of daily and seasonal time. Melatonin synthesis is driven by rhythmic activation of the rate-limiting enzyme, arylalkylamine N-acetyltransferase (AA-NAT). In ungulates, AA-NAT mRNA is constitutively high throughout the 24-h cycle, and melatonin production is primarily controlled through effects on AA-NAT enzyme activity; this is in contrast to dominant transcriptional control in rodents. To determine whether there has been a selective loss of circadian control of AA-NAT mRNA expression in the sheep pineal, we measured the expression of other genes known to be rhythmic in rodents (inducible cAMP early repressor ICER, the circadian clock genes Period1 and Cryptochrome1, as well as AA-NAT). We first assayed gene expression in pineal glands collected from Soay sheep adapted to short days (Light: dark, 8-h: 16-h), and killed at 4-h intervals through 24-h. We found no evidence for rhythmic expression of ICER, AA-NAT or Cryptochrome1 under these conditions, whilst Period1 showed a low amplitude rhythm of expression, with higher values during the dark period. In a second group of animals, lights out was delayed by 8-h during the final 24-h sampling period, a manipulation that causes an immediate shortening of the period of melatonin secretion. This did not significantly affect the expression of ICER, AA-NAT or Cryptochrome1 in the pineal, whilst a slight suppressive effect on overall Per1 levels was observed. The attenuated response to photoperiod change appears to be specific to the ovine pineal, as the first long day induced rapid changes of Period1 and ICER expression in the hypothalamic suprachiasmatic nuclei and pituitary pars tuberalis, respectively.
Overall, our data suggest a general reduction of circadian control of transcript abundance in the ovine pineal gland, consistent with a marked evolutionary divergence in the mechanism regulating melatonin production between terrestrial ruminants and fossorial rodents.
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In mammals, photic entrainment of circadian rhythms likely involves light- and clock-dependent expression of immediate early genes, including fos-like and jun-like genes, in the rat suprachiasmatic nucleus. Using an electrophoretic mobility shift assay, we evaluated whether the photic regulation of DNA-binding activity and composition of activating protein-1 (AP-1) complexes in the suprachiasmatic nucleus is also dependent on circadian phase. Phase-dependent light inducibility in the expression of fra-2 and c-fos genes and in immunoreactive Fra-2 and c-Fos protein expression was also evaluated, by in situ hybridization and immunocytochemistry. Light’s effects on AP-1 DNA-binding differed both qualitatively and quantitatively according to the circadian phase at which light was applied. This phase dependence accounted for by both compartmentalization of proteins involved in constitutive AP-1 complexes within the nucleus or cytoplasm and control of the extent to which the expression of specific complexes was induced. It was then shown that the mechanisms by which the circadian clock gates the photic induction of AP-1 components differed according to the nature of the protein.

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A daily rhythm of activator protein-1 activity in the rat pineal is dependent upon trans-synaptic induction of junB

Abstract

Mol. Brain Res.

Eur. J. Pharmac. mol. Pharmac.

J. biol. Chem.

Cell

J. biol. Chem.

Neuron

Trends pharmac. Sci.

Biochem. Pharmac.

Neuron

Mol. cell. Endocr.

Neuron

Neuron

Neuron

Dimerization and DNA binding alter phosphorylation of Fos and Jun

A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells

Nucleic Acids Res.

Light regulates expression of a Fos-related protein in rat suprachiasmatic nuclei

The pineal gland: a neurochemical transducer

Science

Growth factors and depolarization activate distinct programs of early response gene expression: dissociation of fos and jun induction

Genes Dev.

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

Analyt. Biochem.

The fos oncogene

Isolation and characterization of the c-fos (rat) cDNA and analysis of post-translational modification in vitro

Oncogene

The small subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxyttricha nova and Stylonychia pustulata

J. Mol. Biol. Evol.