Cryptochrome deficiency enhances transcription but reduces protein levels of pineal Aanat

in Journal of Molecular Endocrinology
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Cryptochrome (Cry) 1 and 2 are essential for circadian rhythm generation, not only in the suprachiasmatic nucleus, the site of the mammalian master circadian clock, but also in peripheral organs throughout the body. CRY is also known as a repressor of arylalkylamine-N-acetyltransferase (Aanat) transcription; therefore, Cry deficiency is expected to induce constantly high pineal melatonin content. Nevertheless, we previously found that the content was consistently low in melatonin-proficient Cry1 and Cry2 double-deficient mice (Cry1 / /Cry2 / ) on C3H background. This study aims to clarify the mechanism underlying this discrepancy. In the Cry1 / /Cry2 / pineal, expression levels of Aanat and clock gene Per1 were consistently high with no circadian fluctuation on the first day in constant darkness, demonstrating that CRY acts in vivo as a repressor of the pineal circadian clock and AANAT. In contrast, the enzyme activity and protein levels of AANAT remained low throughout the day, supporting our previous observation of continuously low melatonin. Thus, effects of Cry deficiency on the responses of β-adrenergic receptors were examined in cultured pineal glands. Isoproterenol, a β-adrenergic stimulant, significantly increased melatonin content, although the increase was smaller in Cry1 / /Cry2 / than in WT mice, during both the day and night. However, the increase in cAMP in response to forskolin was similar in both genotypes, indicating that CRY deficiency does not affect the pathway downstream of the β-adrenergic receptor. These results suggest that a lack of circadian adrenergic input due to CRY deficiency decreases β-receptor activity and cAMP levels, resulting in consistently low AANAT levels despite abundant Aanat mRNA.

 

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    Circadian profiles of pineal melatonin content in LD and DD. Pineal melatonin content was examined every 6 h as shown in WT (open circles) and Cry1//Cry2/ (closed circles) mice. Mice were either maintained under a 12:12 h LD cycle (A) or exposed to constant darkness (DD) for 1 day (B). Dark gray area indicates either dark phase of LD cycle or the subjective night phase under DD. Light gray area indicates the subjective day in DD. Data are presented as the mean ± s.e.m. (n = 4-5). *P < 0.05, **P < 0.01 vs Cry1//Cry2/ (post-hoc unpaired t-test followed by two-way ANOVA).

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    Circadian profiles of pineal Per1 and Aanat mRNA levels. Twenty-four-hour patterns of mRNA levels of pineal Per1 (A) and Aanat (B) in wild-type (open circles) and Cry1//Cry2/ (closed circles) mice on the 1st day in DD. Shadowed areas indicate the dark phase. Dark and light gray areas indicate a subjective night and day, respectively. Data are presented as the mean ± s.e.m. (n = 5). *P < 0.05, **P < 0.01 vs Cry1//Cry2/ (post hoc unpaired t-test followed by two-way ANOVA).

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    Circadian rhythm of AANAT protein levels in WT and Cry1//Cry2/ mice. Representative western blot of pineal AANAT in WT and Cry1//Cry2/ mice shown together with that of β-actin (A). Relative level of AANAT in the pooled pineal glands (B). Data are presented as the mean ± s.e.m. of three independent experiments. White and black columns indicate the relative AANAT protein level of WT and Cry1//Cry2/ mice. The values are normalized to the amount of β-actin in each sample.

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    Circadian profile of pineal AANAT enzyme activity. Twenty-four-hour profile of AANAT activity in WT (open circles) and Cry1//Cry2/ (closed circles) mice in DD. Dark and light gray areas indicate subjective night and day, respectively. Data are presented as the mean ± s.e.m. (n = 5–6). **P < 0.01 vs Cry1//Cry2/ (post hoc unpaired t-test followed by two-way ANOVA).

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    Adrenergic stimulant induced melatonin synthesis and AANAT activity in cultured pineal glands. Cultured pineal glands from WT (open columns) and Cry1//Cry2/ (closed columns) mice show a significant increase in melatonin content after the stimulation with isoproterenol (ISO) compared with vehicle controls at the two circadian phases examined. Abscissa indicates the time of tissue sampling. Responses differed significantly between genotypes but not time of day. Data are presented as the mean ± s.e.m., (n = 10–13). *P < 0.05, **P < 0.01 vs vehicle; P < 0.05 vs Cry1⁄Cry2 mice (post hoc unpaired t-test followed by two-way ANOVA) (A). The AANAT activity in the pineal glands of wild-type (open columns) and Cry1⁄Cry2 (closed columns) mice after stimulation with 2 μM of ISO (B) or 10 μM NE (C) for 2 h. Data are presented as the mean ± s.e.m., (n = 4–5). *P < 0.05 vs vehicle in each genotype (post hoc unpaired t-test followed by two-way ANOVA).

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    Intracellular cAMP after forskolin stimulation in cultured pineal glands. Cultured pineal glands sampled at ZT3 from WT (open column) and Cry1//Cry2/ (closed columns) mice demonstrate similar increases in cAMP content after the treatment with forskolin (FSK). Data are presented as the mean ± s.e.m. (n = 7). **P < 0.01 vs vehicle (post hoc unpaired t-test followed by two-way ANOVA).

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