Different signaling mechanisms are involved in the norepinephrine-stimulated TORC1 and TORC2 nuclear translocation in rat pinealocytes.

The distribution of transducers of regulated cAMP-response element-binding protein activity (TORC) between the cytoplasm and the nucleus is tightly regulated and represents one of the main mechanisms whereby the cAMP response element activation activities of TORC are controlled. Whereas both cAMP and Ca(2+) pathways can cause translocation of TORC, the relative importance of these two pathways in regulating different TORC within the same cell is unclear. In this study, we determined the mechanism that regulated TORC1 translocation and compared it with that of TORC2 in rat pinealocytes. Stimulation of pinealocytes with norepinephrine (NE), although having no effect on Torc1 transcription, caused rapid dephosphorylation of TORC1. Although NE also caused rapid dephosphorylation of TORC2, pharmacological studies revealed that TORC1 dephosphorylation could be induced by both ß-adrenoceptor/cAMP and α-adrenoceptor/intracellular Ca(2+) pathways contrasting with TORC2 dephosphorylation being induced mainly through the ß-adrenoceptor/cAMP pathway. PhosTag gel indicated a different pattern of TORC1 desphosphorylation resulting from the selective activation of α- or ß-adrenoceptors. Interestingly, only the α-adrenoceptor/intracellular Ca(2+)-mediated dephosphorylation could translocate TORC1 to the nucleus, whereas the ß-adrenoceptor/cAMP-mediated dephosphorylation of TORC1 was ineffective. In comparison, translocation of TORC2 was induced predominantly by the ß-adrenoceptor/cAMP pathway. Studies with different protein phosphatase (PP) inhibitors indicated that the NE-mediated translocation of TORC1 was blocked by cyclosporine A, a PP2B inhibitor, but that of TORC2 was blocked by okadaic acid, a PP2A inhibitor. Together these results highlight different intracellular signaling pathways that are involved in the NE-stimulated dephosphorylation and translocation of TORC1 and TORC2 in rat pinealocytes.

Adrenergic regulation of the distribution of transducer of regulated cAMP-response element-binding protein (TORC2) in rat pinealocytes.

Transducers of regulated cAMP-response element-binding protein (CREB) activity (TORC) are coactivators that can increase CREB transcriptional activity, suggesting that TORC may regulate the transcription of Aanat, a CREB-target gene. In the present study, we focused on the regulation of TORC2 and its role in Aanat transcription in the rat pineal gland. Although there was no endogenous Torc2 mRNA rhythm in the rat pineal gland and treatment of cultured pinealocytes with norepinephrine (NE) had no effect on the mRNA level of Torc2, the phosphorylation state and intracellular distribution of TORC2 protein were regulated by NE. Immunoblot analysis combined with cytosolic/nuclear fractionation or phosphatase treatment showed that TORC2 protein was rapidly dephosphorylated and translocated to the nucleus after NE stimulation in rat pinealocytes. Similar dephosphorylation of TORC2 also occurred nocturnally in the rat pineal gland. The NE-mediated TORC2 dephosphorylation was blocked by cotreatment with propranolol (a ß-adrenergic antagonist) but not prazosin (an α(1)-adrenergic antagonist) and mimicked by dibutyryl cAMP, indicating the participation of the ß-adrenergic receptor/cAMP pathway. Studies with protein phosphatase inhibitors showed that only okadaic acid and calyculin A were effective in blocking the NE-mediated TORC2 dephosphorylation, suggesting the involvement of protein phosphatase 2A in this dephosphorylation. Moreover, TORC2 overexpression had an enhancing effect on NE-stimulated Aanat transcription. Together, these results indicate that NE stimulation causes nuclear translocation of TORC2 by dephosphorylating the protein through a ß-adrenoceptor/cAMP mechanism and that nuclear localization of TORC2 appears to regulate Aanat transcription by NE in the rat pineal gland.

Salt-inducible kinase 1 in the rat pinealocyte: adrenergic regulation and role in arylalkylamine N-acetyltransferase gene transcription.

The recognition of the basic leucine zipper domain in the regulation of transcriptional activity of cAMP response element-binding protein by salt-inducible kinase (SIK) prompted our investigation of the regulatory role of this kinase in the induction of Aa-nat and other cAMP-regulated genes in the rat pineal gland. Here we report Sik1 expression was induced by norepinephrine (NE) in rat pinealocytes primarily through activation of beta-adrenergic receptors, with a minor contribution from activation of alpha-adrenergic receptors. Treatments with dibutyryl cAMP, and to a lesser extent, agents that elevate intracellular Ca(2+) mimicked the effect of NE on Sik1 expression. In parallel to the results of the pineal cell culture studies, a marked nocturnal induction of Sik1 transcription was found in whole-animal studies. Knockdown of Sik1 by short hairpin RNA amplified the NE-stimulated Aa-nat transcription and other adrenergic-regulated genes, including Mapk phosphatase 1, inducible cAMP repressor, and type 2 iodothyronine deiodinase in a time-dependent manner. In contrast, overexpressing Sik1 had an inhibitory effect on the NE induction of Aa-nat and other adrenergic-regulated genes. Together, our results indicate that the adrenergic induction of Sik1 in the rat pineal gland is primarily through the beta-adrenergic receptor --> protein kinase A pathway. SIK1 appears to function as part of an endogenous repressive mechanism that regulates the peak and indirectly the duration of expression of Aa-nat and other cAMP-regulated genes. These findings support a role for SIK1 in framing the temporal expression profile of Aa-nat and other adrenergic-regulated genes in the rat pineal gland.

Nocturnal activation of aurora C in rat pineal gland: its role in the norepinephrine-induced phosphorylation of histone H3 and gene expression.

We have shown previously that Ser10 phosphorylation of histone H3 occurs in rat pinealocytes after stimulation with norepinephrine (NE) and that histone modifications such as acetylation appear to play an important role in pineal gene transcription. Here we report the nocturnal phosphorylation of a Ser10 histone H3 kinase, Aurora C, in the rat pineal gland. The time profile of this phosphorylation parallels the increase in the level of phospho-Ser10 histone H3. Studies with cultured pinealocytes indicate that Aurora C phosphorylation is induced by NE and this induction can be blocked by cotreatment with propranolol or KT5720, a protein kinase A inhibitor. Moreover, only treatment with dibutyryl cAMP, but not other kinase activators, mimics the effect of NE on Aurora C phosphorylation. These results indicate that Aurora C is phosphorylated primarily by a beta-adrenergic/protein kinase A-mediated mechanism. Treatment with an Aurora C inhibitor reduces the NE-induced histone H3 phosphorylation and suppresses the NE-stimulated induction of arylalkylamine N-acetyltransferase (AA-NAT), the rhythm-controlling enzyme of melatonin synthesis, and melatonin production. The effects of Aurora C inhibitors on adrenergic-induced genes in rat pinealocytes are gene specific: inhibitory for Aa-nat and inducible cAMP repressor but stimulatory for c-fos. Together our results support a role for the NE-stimulated phosphorylation of Aurora C and the subsequent remodeling of chromatin in NE-stimulated Aa-nat transcription. This phenomenon suggests that activation of this mitotic kinase can be induced by extracellular signals to participate in the transcriptional induction of a subset of genes in the rat pineal gland.

Differential effects of vocalization type, singer and listener on ZENK immediate early gene response in black-capped chickadees (Poecile atricapillus).

Here we examined immediate early gene (ZENK) induction to vocalizations in the ascending auditory pathway of black-capped chickadees (Poecile atricapillus) to assess the impact that the sex of the producer and perceiver has on ZENK induction. We manipulated the playback by both the vocal type (song/call) and sex of producer (male/female), and then presented these stimuli classes to either male or female black-capped chickadees. Neural response to the stimulus was quantified by the amount of protein of the IEG ZENK (also known as zif-268, egr-1, ngf-Ia and krox-24) in the caudal medial nidopallium (NCM) and caudomedial mesopallium (CMM). Overall, there was more ZENK induction in CMM and the dorsal parts of the caudal medial nidopallium (NCMd) than in the ventral parts of the caudal medial nidopallium (NCMv) and males had more ZENK induction than females. CMM had the most complex responding of ZENK induction to stimuli such that vocalization type, sex of producer, and sex of perceiver all affected ZENK induction. The silence controls had the least ZENK induction compared to any other group.