AANAT Transcription

In the rat, the time course of NAT transcription closely matches with the time course of NAT activity (12,54), suggesting that, in the rat, the regulation of NAT activity comprises an important transcriptional component. Several lines of evidence point to a substantial role of pCREB in the transcriptional activation of AANAT 1) the AANAT promotor region includes two CREs, 2) the kinetics of CREB phosphoryla-tion falls into the temporal gap between NE-induced elevation of cAMP and AANAT transcription, 3) activators of PKA induce CREB phosphorylation and AANAT transcription, 4) inhibitors of PKA lead to a reduction in NE-induced CREB phosphorylation, binding of nuclear extracts to CREs, AANAT activity and melatonin biosynthesis with very similar kinetics (Figures 8 and 5) in vivo pCREB levels rise always before AANAT transcript rises. All findings suggest that pCREB is involved in the upregulation of AANAT transcription. This hypothesis could be substantiated in transfection experiments with newly developed CREB antagonists (A-CREB; 1). Such

Figure 8. Timecourse of the amount of melatonin (squares) in the medium and the appearance of AANAT protein (triangles) in dispersed rat pinealocytes treated with NE (1 ^M) for two to twelve hours. The ordinate shows time in hours, the left abscissa AANAT protein in SUMDENS-values and the right abscissa melatonin in pg/ml. The error bars represent mean ± SEM of 3-4 replicates.

Figure 8. Timecourse of the amount of melatonin (squares) in the medium and the appearance of AANAT protein (triangles) in dispersed rat pinealocytes treated with NE (1 ^M) for two to twelve hours. The ordinate shows time in hours, the left abscissa AANAT protein in SUMDENS-values and the right abscissa melatonin in pg/ml. The error bars represent mean ± SEM of 3-4 replicates.

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Figure 9. Rat pinealocytes transfected with either control DNA (empty vector) or an ICER-sense construct (pICERs) showed stimulation of melatonin biosynthesis under NE- (black columns) and ISO- (gray columns), but not under phenylephrine (PHE)- stimulation (white columns). Cells transfected with an ICER antisense construct (pICERas) showed disinhibition of melatonin biosynthesis upon NE- and ISO-treatment. NE, ISO and PHE were applied for six hours. Bars show a representative experiment (of three replicates).

experiments are currently underway in our laboratory (Pfeffer et al., unpublished results). Also, several lines of evidence point to a role of ICER in the transcriptional inhibition of AANAT 1) the AANAT promotor includes two CREs, to one of which ICER binds better than to a perfect CRE (Maronde et al., unpublished observations), 2) the kinetics of ICER protein induction correlates temporally with the decline in AANAT transcription, 3) activators of PKA cause ICER induction and 4) ICER protein plateaus in vivo when AANAT transcript declines. A role of ICER in the regulation of melatonin biosynthesis also gains support from ICER antisense experiments. Rat pinealocytes transfected with either control DNA or an ICER-sense construct showed normal stimulation of melatonin biosynthesis upon stimulation with NE and ISO. However, those cells transfected with an ICER antisense construct (pICERas) inhibiting ICER upregulation showed an elevated ("disinhibited") melatonin biosynthesis upon NE-and ISO-treatment (Figure 9). It can be concluded from these data that ICER is involved in repressing AANAT transcription. Like pCREB (see above) also ICER fluctuates diurnally in both rats (Maronde et al., unpublished observations) and C3H mice (Von Gall et al., unpublished observations). Thus, the CAMP-induced and CRE-dependent transcriptional activity in the rodent pineal organ may be regulated by a changing ratio in the amount of activating (e.g. pCREB) and inhibiting (ICER) transcription factors (see also Figure 10).

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