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Laboratoire Aragó, Unité Mixte de Recherche 7628/Groupement de Recherche 2821, Université Pierre et Marie Curie and Centre National de la Recherche Scientifique, F-66651 Banyuls-Sur-Mer, Cedex, France
Address all correspondence and requests for reprints to: Jack Falcón, Laboratoire Aragó, Unité Mixte de Recherche 7628/Groupement de Recherche 2821, Université Pierre et Marie Curie and Centre National de la Recherche Scientifique, F-66651 Banyuls-Sur-Mer, Cedex, France, B.P. 44, Avenue du Fontaulé, F-66651 Banyuls-Sur-Mer Cedex, France. E-mail: falcon{at}obs-banyuls.fr.
Elevated production of melatonin by the pineal gland at night is a highly conserved feature of vertebrate physiology. Melatonin is a highly lipophilic molecule that is released into the blood immediately after synthesis. Accordingly, the daily rhythm in plasma melatonin concentration reliably reflects pineal organ synthesis. The pattern is independent of the animals’ habitat, i.e. it is the same in diurnally and nocturnally active vertebrates. The melatonin rhythm is a direct reflection of the prevailing photoperiod and, as a consequence, it is modified over the course of a year: long nights result in longer periods of melatonin production (1, 2). This melatonin rhythm is important because it entrains daily and seasonal behavioral and physiological rhythms to the environmental lighting cycle; these include locomotor activity and sleep, body weight and growth, skin or fur quality and renewal, and reproduction (2, 3, 4). In addition, melatonin can play a role in circadian physiology (5). The reliability of the system that ensures this pattern is determined by a cascade of mechanisms that operate at different levels of the brain. The rhythm typically reflects the activity of circadian clocks synchronized by the light/dark cycle. In mammals, the master circadian clocks are located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The cyclic activity of the clocks is synchronized and entrained by photoperiod perceived by a specific set of retinal neurons, which send axons to the SCN through the retino-hypothalamic tract (6). The information from the SCN is conveyed to the pineal gland by a complex neural pathway involving the paraventricular nuclei of the hypothalamus, the intermediolateral column of the spinal cord, and the superior cervical ganglia, which send sympathetic axons to the pineal gland (7, 8). SCN stimulation of this pathway at night causes an increase in noradrenalin release from the sympathetic endings. This triggers a cascade of events that starts with the activation of adrenergic receptors at the surface of the pinealocytes (7, 8). The final target is the arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the synthesis of melatonin from serotonin (8, 9). This enzyme plays a pivotal role in the rhythmic pattern of melatonin production. Its activity increases 10- to 100-fold at night, which increases production of the melatonin precursor N-acetylserotonin. Subsequent methylation of N-acetylserotonin is the final step in the production of melatonin; it is catalyzed by hydroxyindole-O-methyltransferase, the activity of which does not change significantly on a night/day basis. It is of importance that the final melatonin rhythm reflects the AANAT activity rhythm with no apparent phase delay. Vertebrate AANAT is so tightly linked to time keeping that it has been termed the timezyme (9).
AANAT is a cAMP-dependent enzyme, and the nocturnal rise in AANAT activity is driven by an increase in cAMP production (7, 9). In mammals, this increase is a consequence of the nocturnal release of noradrenalin from the catecholaminergic endings. Noradrenalin activates adenyl cyclase activity through stimulation of ß1-adrenergic receptors at the surface of the pinealocytes (7, 8). The subsequent increase in cAMP is potentiated by the concurrent activation of 
1-adrenergic receptors through a calcium-dependent mechanism. The amplitude of the cAMP response varies in magnitude from approximately 2-fold in cow to approximately 100-fold in the rat. The resulting increase in cAMP stimulates protein kinase A (PKA), which catalyzes the phosphorylation of two highly conserved sites found in all vertebrate AANATs (7, 9). This process provides a rapid mechanism through which cAMP controls the amount of AANAT protein and consequently of AANAT activity. The steady-state level of AANAT protein results from the balance between synthesis and degradation. The cAMP-induced AANAT phosphorylation allows its high affinity binding to 14-3-3 proteins; this binding protects phosphoAANAT protein (pAANAT) from degradation (10). Binding also increases the affinity of pAANAT for serotonin. However, the pAANAT/14-3-3 association is reversible; and, pAANAT can be dephosphorylated, which reduces its affinity for 14-3-3. Unprotected AANAT protein is then degraded through proteasomal proteolysis. Besides this "universal" mechanism (11, 12), cAMP may also control aanat expression in some, but not all, species. In rodents, PKA translocates into the nucleus where it mediates phosphorylation of the cAMP-response-element-binding protein (CREB); once phosphorylated, the transcription factor pCREB binds to multiple cAMP-response-elements in the aanat promoter, resulting in the activation of aanat transcription. As a consequence, there is a large increase in aanat expression (8, 9, 13). In other species such as ungulates and primates, aanat is constitutively expressed. Under this situation, AANAT protein is synthesized continuously and the regulation of AANAT activity relies solely on the control of AANAT protein phosphorylation/dephosphorylation (8, 13).
Whereas most attention has been focused on the mechanisms of dark induction of AANAT activity and melatonin secretion, little attention has been paid to how this process is limited and terminated. This is crucial because to be a reliable indicator of circadian time the system must be turned on as well as turned off with great precision. Morning light inhibits noradrenalin release at the sympathetic endings, and AANAT activity is dramatically and acutely inhibited with a half-life of 3.5 min in the rat (9). However, melatonin production declines before light onset, suggesting that one or more repressing factors enter into the game late at night. Negative regulation may occur at any step of the noradrenalin/adrenergic/cAMP/PKA/CREB/AANAT signaling pathway. This pathway activates a number of other genes in the mammalian pinealocyte among which some have been suspected of encoding inhibitors of aanat expression. These include inducible cAMP early repressor (ICER) (14, 15) and Fra-2 (fos-related antigen 2) (16, 17). Interest in ICER arose because it can bind to the cAMP-response-elements of the aanat promoter, thus competing with pCREB. Interest in the transcription factor Fra-2 was stimulated because it can form heterodimers with Jun family members and bind to activating protein 1 binding sites, which occur in the aanat promoter. However, the importance of these transcription factors in controlling expression of aanat has been questioned, because the aanat response remains nearly normal if either of these genes is silenced in rat (18), although some stimulation of AANAT expression was seen in ICER-deficient mice (19). Dephosphorylation of pCREB contributes to reduce aanat expression at the end of the night, probably as a result of a noradrenalin-induced translocation of serine/threonine phosphatase 1 into the pinealocytes nuclei (20). However, the decline in aanat expression at the end of the night cannot explain by itself the arrest of AANAT activity and melatonin secretion; additionally, regulation of aanat expression is seen in some species but not in others. The key might be cAMP because in all vertebrate species investigated AANAT protein stability depends highly on it. As night progresses, noradrenalin release from the sympathetic nerve fibers decreases, and the membrane-bound adrenergic receptors desensitize (13). This is one factor that might contribute to reducing cAMP content.
An increasing rate in cAMP degradation occurring as the night progresses is another possibility. This was explored by Kim et al. (21) in the current issue of Endocrinology, who extended the observation that there is a day/night variation in the level of the enzyme that inactivates cAMP phosphodiesterase (PDE). The authors first showed that the rhythm in PDE activity is due essentially to the 4B2 variant (one of the 20 known PDE4 isoforms), which they found highly expressed in the pineal gland compared with other tissues. It is the major contribution of this study that PDE4B2 mRNA, protein amount, and activity are elevated during the second half of the night and that these increases are controlled by the noradrenalin/ß-adrenergic/cAMP/PKA pathway, probably involving phosphorylation of CREB. The kinetics of this response is in agreement with the conclusion that a noradrenalin-induced elevation of cAMP results in the activation of AANAT early at night and of PDE4B2 later at night. This facilitates the control of the intensity of the response to cAMP and the rate at which cAMP is destroyed once the noradrenergic stimulation is terminated. The study by Kim et al. (21) provides impressive evidence of how factors that activate the pineal gland also induce in parallel the expression of a system, which negatively modulates this activation, thereby enhancing the precision and reliability of the pineal gland as an indicator of time.
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Received January 18, 2007.
Accepted for publication January 25, 2007.
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