The circadian rhythm of pineal melatonin requires the nocturnal increment of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) protein. To date, only limited information is available in the critical issue of how AANAT protein expression is up-regulated exclusively at night regardless of its species-specific mRNA profiles. Here we show that the circadian timing of AANAT protein expression is regulated by rhythmic translation of AANAT mRNA. This rhythmic control is mediated by both a highly conserved IRES (internal ribosome entry site) element within the AANAT 5 untranslated region and its partner hnRNP Q (heterogeneous nuclear ribonucleoprotein Q) with a peak in the middle of the night. Consistent with the enhancing role of hnRNP Q in AANAT IRES activities, knockdown of the hnRNP Q level elicited a dramatic decrease of peak amplitude in the AANAT protein profile parallel to reduced melatonin production in pinealocytes. This translational regulation of AANAT mRNA provides a novel aspect for achieving the circadian rhythmicity of vertebrate melatonin.[Keywords: AANAT; translation; IRES; hnRNP Q; melatonin; circadian rhythm] Supplemental Material is available at http://www.genesdev.org.

The mammalian circadian rhythm is observed not only at the suprachiasmatic nucleus, a master pacemaker, but also throughout the peripheral tissues. Its conserved molecular basis has been thought to consist of intracellular transcriptional feedback loops of key clock genes. However, little is known about posttranscriptional regulation of these genes. In the present study, we investigated the role of the 3-untranslated region (3UTR) of the mouse cryptochrome 1 (mcry1) gene at the posttranscriptional level. Mature mcry1 mRNA has a 610-nucleotide 3UTR and mediates its own degradation. The middle part of the 3UTR contains a destabilizing cis-acting element. The deletion of this element led to a dramatic increase in mRNA stability, and heterogeneous nuclear ribonucleoprotein D (hnRNP D) was identified as an RNA binding protein responsible for this effect. Cytoplasmic hnRNP D levels displayed a pattern that was reciprocal to the mcry1 oscillation. Knockdown of hnRNP D stabilized mcry1 mRNA and resulted in enhancement of the oscillation amplitude and a slight delay of the phase. Our results suggest that hnRNP D plays a role as a fine regulator contributing to the mcry1 mRNA turnover rate and the modulation of circadian rhythm.Circadian rhythms in living organisms are regulated by an endogenous clock. In mammals, it is well known that the circadian clock is located at the suprachiasmatic nucleus (SCN) in the brain. The SCN orchestrates circadian oscillations in various peripheral tissues (21,36,56). Because the circadian rhythm is conserved from the entire organism to the level of the single cell, many efforts have been made to understand the molecular basis of the regulation of circadian timing (15,20).Enormous progress has been made toward understanding the secrets of the rhythm generator, providing a view of the circadian clock mechanism. Each cell of the body contains a circadian core oscillator composed of essential components for normal circadian behavior. The RNA and protein levels of the components, called clock genes, are known to oscillate in a circadian rhythm, based on studies of the expression of genes, including Clock (49); Bmal1 (5); mPer1 and mPer2 (55); mCry1 and mCry12 (22); Rev-erb␣ (34); and Ror␣ (44). These key clock genes are involved in interacting positive and negative transcriptional feedback loops (9,34,37,44).In mammals, cryptochromes are expressed in every organ, and there are two homologues, mcry1 and mcry2. In general, mcry1 is expressed at a high level in the SCN, whereas mcry2 expression in this region is almost negligible (41). The oscillation of mcry1 transcripts persists when mice are kept in a "free-running" state, such as constant darkness (27), as has been observed for other clock genes and as would be expected of a true circadian regulator. mcry1 expression also shows circadian oscillation in other organs, most notably in the liver; however, the phase of its expression in internal organs is delayed or advanced compared to the phase of its expression in the SCN. Both mCRY1 and mCRY2 ...

Sphingosine-1-phosphate (S1P) is a pluripotent lipid mediator that transmits signals through a family of G protein-coupled receptors to control diverse biological processes. Here, we investigated the effects of S1P on the levels of intracellular calcium and cAMP in differentiated rat white adipocytes and two important aspects of adipocyte-specific physiology, lipolysis and leptin production. In adipocytes, S1P signaling pathway was functionally linked to phospholipase C via pertussis-toxin-sensitive G protein. Interestingly, at higher S1P concentration (1-30 microM), it also induced cAMP generation in a concentration-dependent manner, which was pertussis toxin insensitive and was mimicked by dihydro-S1P and sphingosylphosphoryl-choline but not by its related metabolites, ceramide and sphingosine, or by its structural analogs, phyto-S1P and lysophosphatidic acid. Suramin, a known inhibitor of ligand-receptor interactions, reduced S1P-induced cAMP generation by 60% of control, whereas forskolin-induced cAMP increase was not affected by treatment with suramin. The S1P-induced cAMP generation was functionally linked to cAMP response element-binding protein phosphorylation. Finally, S1P significantly reduced insulin-induced mRNA of ob gene and leptin secretion, whereas S1P increased glycerol release from adipocytes. Both effects of S1P were reversed by a selective adenylyl cyclase inhibitor, SQ22536, without significantly affecting basal values. In conclusion, extracellular S1P elicits the elevation of cytosolic Ca2+ and cAMP with a distinct concentration dependency, and S1P-induced cAMP generation may be mediated by S1P-selective receptors rather than intracellular targets, and the activated adenylyl cyclase-cAMP signaling pathways subsequently increase lipolysis and decrease insulin-induced leptin production in rat white adipocytes.

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The rhythmic nocturnal production of melatonin in pineal glands is controlled by the periodic release of norepinephrine from the superior cervical ganglion. Norepinephrine binds to the ␤-adrenergic receptor and stimulates an increase in intracellular cAMP levels, leading to the transcriptional activation of serotonin N-acetyltransferase, which in turn promotes melatonin production. In the present study, we report that bradykinin inhibits basal-and forskolinstimulated adenylyl cyclase activity, norepinephrine-induced cAMP generation, and N-acetyltransferase expression in a calciumdependent manner. These effects were blocked by pretreatment with U73122 (a selective phospholipase C inhibitor), and 1,2-bis(oaminophenoxy)ethane-N,N,N,N-tetraacetic acid (a Ca 2؉ chelator), but not pertussis toxin. The calcium ionophore, ionomycin, inhibited isoproterenol-mediated cAMP generation, similar to bradykinin. Interestingly, the inhibitory effect of bradykinin was evident only during the daytime. At midday, bradykinin inhibited the cAMP level by ϳ50% but markedly stimulated cAMP production (by ϳ50%) at night. Northern blotting and immunoblotting data disclosed circadian expression of calcium-inhibitable adenylyl cyclase type 6. Expression of adenylyl cyclase type 6 was maximal at Zeitgeber Time (ZT) 01 and very low at ZT 13. Our results suggest that bradykinin-induced calcium inhibits melatonin synthesis through the mediation of adenylyl cyclase type 6 expression.