D. M. Price

Mitogen-activated protein kinase phosphatase-1 (MKP-1): >100-fold nocturnal and norepinephrine-induced changes in the rat pineal gland.

The norepinephrine‐driven increase in mitogen‐activated protein kinase (MAPK) activity is part of the mechanism that regulates arylalkylamine N‐acetyltransferase (AA‐NAT) activity in the rat pineal gland. We now report a marked nocturnal increase in the expression of a MAPK phosphatase, MAP kinase phosphatase‐1 (MKP‐1), that was blocked by maintaining animals in constant light or treatment with propranolol. MKP‐1 expression was regulated by norepinephrine acting through both α‐ and β‐adrenergic receptors. These results establish a nocturnal increase in pineal MKP‐1 expression that is under the control of a photoneural system. Because substrates of MKP‐1 can influence AA‐NAT activity, our findings suggest the involvement of MKP‐1 in the regulation of the nocturnal AA‐NAT signal.

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.

Timing of mitogen-activated protein kinase (MAPK) activation in the rat pineal gland.

Activation of members of the mitogen-activated protein kinase (MAPK) family of signaling cascades is a tightly controlled event in rat pinealocytes. Cell culture studies indicate that whereas the NE-->cGMP activation of p42/44MAPK is rapid and transient, the NE-->cAMP activation of p38MAPK is slower and more sustained. The decline in the p42/44MAPK response is in part due to the induction of MAPK phosphatase-1 by NE. In comparison, p38MAPK activation is tightly coupled to the synthesis and degradation of an upstream element in its activation cascade. Whole animal studies confirm activation of p42/44MAPK occurring during the early part of night and precedes p38MAPK activation. Studies with selective MAPK inhibitors reveal a modulating effect of MAPKs on arylalkylamine-N-acetyltransferse (AA-NAT) activity, with involvement of p42/44MAPK in the induction of AA-NAT and p38MAPK participating in the amplitude and duration of the AA-NAT response. These effects of p42/44MAPK and p38MAPK on AA-NAT activity match their timing of activation. Taken together, our studies on the timing of MAPK activation and regulation of AA-NAT by MAPKs add to the importance of MAPKs in regulating the circadian biology of the 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.

Mitogen‐activated protein kinase phosphatase‐1 (MKP‐1) preferentially dephosphorylates p42/44MAPK but not p38MAPK in rat pinealocytes

We recently reported a diurnal and norepinephrine (NE) ‐induced expression of mitogen‐activated protein kinase (MAPK) phosphatase‐1 (MKP‐1) in the rat pineal gland and postulated that this MKP‐1 expression might impact adrenergic‐regulated arylalkylamine‐N‐acetyltransferase (AA‐NAT) activity via modulation of MAPKs. In this study, we investigated the effect of depletion of MKP‐1 expression by using doxorubicin, a topoisomerase inhibitor that suppresses the expression of MKP‐1 in other cell types and small interfering RNA targeted against Mkp1 in NE‐stimulated pinealocytes. We found that both treatments were effective in inhibiting NE induction of MKP‐1 expression. Moreover, both treatments also resulted in a prolonged activation of p42/44MAPK and an increase in AA‐NAT induction by NE. In contrast, treatment of pinealocytes with PD98059, an inhibitor of MAPK kinase, reduced NE‐stimulated AA‐NAT activity. Interestingly, suppressing MKP‐1 expression had no effect on the time profile of NE‐stimulated p38MAPK activation. These results indicate that MKP‐1 modulates the profile of AA‐NAT activity by selectively shaping the activation profile of p42/44MAPK but not that of p38MAPK.

The role of protein turnover in regulating MKP-1 levels in rat pinealocytes.

We have previously shown that mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is induced at night under the control of a photoneural system in the rat pineal gland. Because of the established roles of MAPKs, glucocorticoids and proteasome activity in regulating MKP-1 expression in other cell types, their relative contributions to MKP-1 regulation were investigated in rat pinealocytes. We found that neither inhibition of MAPKs nor treatment with dexamethasone affected norepinephrine-stimulated MKP-1 expression. In contrast, treatment with proteasome inhibitors increased norepinephrine-stimulated MKP-1 protein levels and abolished the decline in norepinephrine-stimulated MKP-1 protein levels caused by inhibition of transcription or translation, or blockade of alpha-adrenergic receptors. Taken together, our results indicate that in rat pinealocytes, the continuous and rapid turnover of MKP-1 protein allows for its rapid induction but is not sufficient to generate the sustained increase in MKP-1 expression post-adrenergic stimulation.

Histone modifications on the adrenergic induction of type II deiodinase in rat pinealocytes.

Histone modifications have been shown to play an important role in regulating gene expression. In this study, we investigated the impact of histone modifications on the adrenergic-regulated transcription of type 2 deiodinase (Dio2), a CREB-target gene in the rat pinealocyte. Treatment of pinealocytes with inhibitors of aurora C, a histone kinase, resulted in an inhibitory effect on the adrenergic-stimulated histone H3 Ser10 phosphorylation and Dio2 transcription. Given the established link between histone phosphorylation and acetylation, the role of histone acetylation on the adrenergic-induced Dio2 transcription was investigated. Treatment of pinealocytes with histone deacetylase inhibitors inhibited the adrenergic-induced Dio2 transcription. Chromatin immunoprecipitation with antibodies against acetylated Lys14 of H3 showed an increase in DNA recovery of the promoter region of Dio2 following treatment with trichostatin A. Together, our results indicate that, beside activation of CREB, epigenetic factors such as histone modifications also play an important role in regulating Dio2 transcription.

Opposite Effects of Proteasome Inhibitors in the Adrenergic Induction of Arylalkylamine N‐acetyltransferase in Rat Pinealocytes

In the rat pineal gland, the steady‐state level of arylalkylamine N‐acetyltransferase (AANAT) protein is controlled by transcriptional and translational mechanisms as well as by proteasome‐mediated degradation. Studies with proteasome inhibitors, MG132 and clasto‐lactacystin β‐lactone (c‐lact), show two opposite effects of proteasomal inhibition on norepinephrine (NE)‐induction of Aanat. Addition of MG132 or c‐lact following NE stimulation causes an increase in AANAT protein level and enzyme activity without affecting the level of Aanat mRNA. In contrast, addition of inhibitors prior to NE stimulation reduces the NE‐stimulated Aanat mRNA, AANAT protein, and enzyme activity. The inhibitory effect of proteasomal inhibition on adrenergic‐induced Aanat transcription appears specific for Aanat because it has no effect on the adrenergic induction of mitogen‐activated protein kinase phosphatase‐1 (mkp‐1). The effects of the proteasome inhibitors on NE‐stimulated Aanat induction appear to be mediated by accumulation of a protein repressor.