Circadian Dynamics of the Cone-Rod Homeobox (CRX) Transcription Factor in the Rat Pineal Gland and Its Role in Regulation of Arylalkylamine N-Acetyltransferase (AANAT)

Abstract

The cone-rod homeobox (Crx) gene encodes a transcription factor in the retina and pineal gland. Crx deficiency influences the pineal transcriptome, including a reduced expression of arylalkylamine N-acetyltransferase (Aanat), a key enzyme in nocturnal pineal melatonin production. However, previous functional studies on pineal Crx have been performed in melatonin-deficient mice. In this study, we have investigated the role of Crx in the melatonin-proficient rat pineal gland. The current study shows that pineal Crx transcript levels exhibit a circadian rhythm with a peak in the middle of the night, which is transferred into daily changes in CRX protein. The study further shows that the sympathetic innervation of the pineal gland controls the Crx rhythm. By use of adenovirus-mediated short hairpin RNA gene knockdown targeting Crx mRNA in primary rat pinealocyte cell culture, we here show that intact levels of Crx mRNA are required to obtain high levels of Aanat expression, whereas overexpression of Crx induces Aanat transcription in vitro. This regulatory function of Crx is further supported by circadian analysis of Aanat in the pineal gland of the Crx-knockout mouse. Our data indicate that the rhythmic nature of pineal CRX protein may directly modulate the daily profile of Aanat expression by inducing nighttime expression of this enzyme, thus facilitating nocturnal melatonin synthesis in addition to its role in ensuring a correct tissue distribution of Aanat expression.

Homeobox genes in melatonin‐producing pinealocytes: Otx2 and Crx act to promote hormone synthesis in the mature rat pineal gland

“…Aanat expression was markedly elevated by NE treatment (Figure B; P < 0.0001) in agreement with previous reports . In NE‐treated pinealocytes, knocking down Crx significantly reduced Aanat expression (Figure B; P < 0.0001) in accord with data from a virus‐based model system . Similar results were obtained when knocking down Otx2 (Figure B; P < 0.001), and treating the pinealocytes with siRNAs against both Otx2 and Crx simultaneously produced an additional effect reducing Aanat expression with approximately 75% (Figure B; P < 0.0001).…”

“…Otx2 and Crx have been previously shown to exhibit a circadian rhythm with high transcript levels early during nighttime in the rat pineal gland . To mimic nocturnal adrenergic signaling in primary cultures of rat pinealocytes, NE was added to the culture media.…”

“…For each experiment, pineal glands from 12 to 17 rats were dissected. Pinealocyte primary cultures were prepared, and isolated cells were incubated as previously described . To knock down Crx and Otx2 , Stealth siRNA was obtained from Life Technologies (Nærum, Denmark).…”

“…Crx expression is restricted to retinal photoreceptors and pinealocytes of the pineal gland; although it is essential for retinal photoreceptor maturation, it does not influence development of the pineal gland . Pineal Crx expression exhibits a daily rhythm with high levels during nighttime . The role of Crx specifically in regulation of Aanat expression in the rodent pineal gland has been previously demonstrated .…”

“…Pineal Crx expression exhibits a daily rhythm with high levels during nighttime . The role of Crx specifically in regulation of Aanat expression in the rodent pineal gland has been previously demonstrated . Promoter analyses further suggest that Crx may control pineal expression of Asmt , but experimental evidence from mammalian model systems is missing.…”