Rhythmic Diurnal Synthesis and Signaling of Retinoic Acid in the Rat Pineal Gland and Its Action to Rapidly Downregulate ERK Phosphorylation

Vitamin A is important for the circadian timing system; deficiency disrupts daily rhythms in activity and clock gene expression, and reduces the nocturnal peak in melatonin in the pineal gland. However, it is currently unknown how these effects are mediated. Vitamin A primarily acts via the active metabolite, retinoic acid (RA), a transcriptional regulator with emerging non-genomic activities. We investigated whether RA is subject to diurnal variation in synthesis and signaling in the rat pineal gland. Its involvement in two key molecular rhythms in this gland was also examined: kinase activation and induction of Aanat, which encodes the rhythm-generating melatonin synthetic enzyme. We found diurnal changes in expression of several genes required for RA signaling, including a RA receptor and synthetic enzymes. The RA-responsive gene Cyp26a1 was found to change between day and night, suggesting diurnal changes in RA activity. This corresponded to changes in RA synthesis, suggesting rhythmic production of RA. Long-term RA treatment in vitro upregulated Aanat transcription, while short-term treatment had no effect. RA was also found to rapidly downregulate extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, suggesting a rapid non-genomic action which may be involved in driving the molecular rhythm in ERK1/2 activation in this gland. These results demonstrate that there are diurnal changes in RA synthesis and activity in the rat pineal gland which are partially under circadian control. These may be key to the effects of vitamin A on circadian rhythms, therefore providing insight into the molecular link between this nutrient and the circadian system.