siRNA-Mediated Downregulation of Gene Expression in Cultured Rat Pineal Cells

Circadian clock gene expression in the suprachiasmatic nucleus (SCN) controls 24 h rhythms in body functions, but clock genes are also expressed in extra‐hypothalamic tissues, including the melatonin‐producing pineal gland. The nocturnal increase in pineal melatonin synthesis is a hallmark in circadian biology, but the role of local clock gene oscillations in the mammalian pineal gland is unknown. The aim of this work is to determine the role of clock genes in endocrine function of the pineal gland with focus on the Aanat transcript encoding the rhythm‐generating enzyme of melatonin synthesis. Using the rat as a model, we here established 24 h expression patterns of clock genes in the pineal gland in vivo. Lesion studies showed that rhythmic clock gene expression in the pineal gland to a large extent depends on the SCN; further, clock gene rhythms could be re‐established in cultured pineal cells synchronized by rhythmic stimulation with norepinephrine in 12 h pulses, suggesting that pineal cells house a slave oscillator controlled by adrenergic signaling in the gland. Histological analyses showed that clock genes are expressed in pinealocytes and colocalize with Aanat transcripts, thus potentially enabling clock gene products to control cellular melatonin production. To test this, cultured pineal cells were transfected using small interfering RNA to knock down clock gene expression. While successful knockdown of Per1 had a minor effect on Aanat, Clock knockdown produced a marked overexpression of Aanat in the pinealocytes. Our study suggests that SCN‐dependent rhythmic Clock gene expression in the pinealocytes regulates the daily profile of Aanat expression.

Section: Methodsmentioning

confidence: 99%

“…Cell culture procedure and siRNA treatment were performed as previously described. 24 We partially reproduced a synchronization protocol previously used in pineal cell cultures. 19,25 Cell cultures were transfected for 24 h with siRNA targeting different clock genes (Table 1).…”

Section: Pinealocyte Cell Culture and Sirna Transfectionmentioning

confidence: 99%

Role and neural regulation of clock genes in the rat pineal gland: Clock modulates amplitude of rhythmic expression of Aanat encoding the melatonin‐producing enzyme

Blancas-Velazquez

Bering

Bille

et al. 2023

“…Pineal cell cultures were prepared as previously described 38 . To knock down gene expression, each culture was treated with 40 nM of small interfering RNA (siRNA) (Table 3).…”

Section: Methodsmentioning

confidence: 99%

“…Pineal cell cultures were prepared as previously described. 38 To knock down gene expression, each culture was treated with 40 nM of small interfering RNA (siRNA) (Table 3). To mimick nighttime conditions and synchronize cells, they were treated with 3 μM NE (Sigma-Aldrich) or 500 μM dibutyryl-cyclic AMP (DBcAMP) (Sigma-Aldrich) for 6 or 9 h.…”

Section: Preparation Of Cell Culturesmentioning

confidence: 99%

The ISL LIM‐homeobox 2 transcription factor is negatively regulated by circadian adrenergic signaling to repress the expression of Aanat in pinealocytes of the rat pineal gland

Mattern,

Blancas‐Velázquez,

Ngo

et al. 2023

Melatonin is synthesized in the pineal gland during nighttime in response to nocturnal increase in the activity of the enzyme aralkylamine N‐acetyltransferase (AANAT), the transcription of which is modulated by several homeodomain transcription factors. Recent work suggests that the homeodomain transcription factor ISL LIM homeobox 2 (ISL2) is expressed in the pineal gland, but its role is currently unknown. With the purpose of identifying the mechanisms that control pineal expression of Isl2 and the possible function of Isl2 in circadian pineal biology, we report that Isl2 is specifically expressed in the pinealocytes of the rat pineal gland. Its expression exhibits a 24 h rhythm with high transcript and protein levels during the day and a trough in the second half of the night. This rhythm persists in darkness, and lesion studies reveal that it requires intact function of the suprachiasmatic nuclei, suggesting intrinsic circadian regulation. In vivo and in vitro experiments show that pineal Isl2 expression is repressed by adrenergic signaling acting via cyclic AMP; further, Isl2 is negatively regulated by the nocturnal transcription factor cone‐rod homeobox. During development, pineal Isl2 expression is detectable from embryonic day 19, preceding Aanat by several days. In vitro knockdown of Isl2 is accompanied by an increase in Aanat transcript levels suggesting that ISL2 represses its daytime expression. Thus, rhythmic expression of ISL2 in pinealocytes is under the control of the suprachiasmatic nucleus acting via adrenergic signaling in the gland to repress nocturnal expression, while ISL2 itself negatively regulates daytime pineal expression of Aanat and thereby suggestively enhances the circadian rhythm in melatonin synthesis.

“…11 To circumvent these challenges, in vitro methods in which homeobox genes are knocked down in primary cultures of melatonin-proficient rat pinealocytes have been employed. 20,50,84 These studies have revealed interesting aspects of Otx2, Lhx4, and Isl2 in regulation of pineal melatonin synthesis. The role of homeobox genes in melatonin synthesis has been examined in knockout mice with a fully developed pineal gland in the case of Crx 12 and Rax.…”

Section: Homeobox Genes and Control Of Melatonin-synthesizing Enzymes...mentioning

confidence: 99%

Homeobox gene‐encoded transcription factors in development and mature circadian function of the rodent pineal gland

Rath

2024

Homeobox genes encode transcription factors that are widely known to control developmental processes. This is also the case in the pineal gland, a neuroendocrine brain structure devoted to nighttime synthesis of the hormone melatonin. Thus, in accordance with high prenatal gene expression, knockout studies have identified a specific set of homeobox genes that are essential for development of the pineal gland. However, as a special feature of the pineal gland, homeobox gene expression persists into adulthood, and gene product abundance exhibits 24 h circadian rhythms. Recent lines of evidence show that some homeobox genes even control expression of enzymes catalyzing melatonin synthesis. We here review current knowledge of homeobox genes in the rodent pineal gland and suggest a model for dual functions of homeobox gene‐encoded transcription factors in developmental and circadian mature neuroendocrine function.