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Endocrinology Vol. 148, No. 4 1465-1472
Copyright © 2007 by The Endocrine Society

Histone H3 Phosphorylation in the Rat Pineal Gland: Adrenergic Regulation and Diurnal Variation

C. L. Chik, T. G. Arnason, W. G. Dukewich, D. M. Price, A. Ranger and A. K. Ho

Departments of Physiology (W.G.D., D.M.P., A.R., A.K.H.) and Medicine (C.L.C., T.G.A.), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Address all correspondence and requests for reprints to: Dr. A. K. Ho, Department of Physiology, 7-26 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7. E-mail: anho{at}ualberta.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, we investigated phosphorylation of Ser10 in histone H3 by norepinephrine (NE) in the rat pineal gland. In whole-animal studies, we demonstrated a marked increase in histone H3 phosphorylation in the rat pineal gland during the first half of the dark period. Exposure to light during this period caused a rapid decline in histone H3 phosphorylation with an estimated t1/2 of less than 15 min, indicating a high level of dephosphorylation activity. Corresponding studies in cultured pineal cells revealed that treatment with NE produced an increase in histone H3 phosphorylation that peaked between 2 and 3 h and declined rapidly by 4 h. The NE-induced histone H3 phosphorylation was blocked by cotreatment with propranolol or KT5720, a protein kinase A inhibitor, but not by prazosin or other kinase inhibitors. Moreover, only treatment with dibutyryl cAMP but not other kinase activators mimicked the effect of NE on histone H3 phosphorylation. The NE-stimulated H3 phosphorylation was markedly increased by cotreatment with a serine/threonine phosphatase inhibitor, tautomycin or okadaic acid, supporting a high level of ongoing histone H3 dephosphorylation activity. Together, our results indicate that histone H3 phosphorylation is a naturally occurring event at night in the rat pineal gland that is driven almost exclusively by a NE->ß-adrenergic->cAMP/protein kinase A signaling mechanism. This transient histone H3 phosphorylation probably reflects the nocturnal activation of multiple adrenergic-regulated genes in the rat pineal gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE RAT PINEAL gland, the nightly release of norepinephrine (NE) from the sympathetic neurons stimulates both {alpha}1- and ß-adrenergic receptors resulting in a 100-fold increase in intracellular cAMP levels (1, 2, 3). The rise in cAMP, in turn, activates cAMP-dependent protein kinase A (PKA), which translocates to the nucleus and phosphorylates cAMP response element-binding protein (CREB), a transcription factor (4). Phosphorylated CREB binds to cAMP response elements in the promoter region of cAMP-regulated genes and causes activation of their transcription (5, 6, 7). This activation of transcription results in a 150-fold increase in the mRNA of aa-nat, which encodes the melatonin rhythm-generating enzyme (8, 9). In addition, dephosphorylation of phosphorylated CREB by protein serine/threonine phosphatases is involved in the inactivation of aa-nat gene transcription (10).

Through stimulation of {alpha}1- and ß-adrenergic receptors, multiple signaling mechanisms, in addition to PKA, are activated in the rat pineal gland in response to NE stimulation. Stimulation of ß-adrenergic receptors alone produces a 7- to 10-fold increase in cAMP and a 2- to 4-fold increase in cGMP accumulation (1, 3). Stimulation of {alpha}-adrenergic receptors, which activates protein kinase C (PKC) (11, 12) and elevates intracellular Ca2+ concentration (13, 14), potentiates the ß-adrenergic-stimulated cyclic nucleotide responses (1, 3). Aside from induction of arylalkylamine-N-acetyltransferase (AA-NAT) activity, NE stimulation also increases the expression of other proteins including MAPK phosphatase-1 (MKP-1) (15, 16) and transcription factors such as Fos-related antigen 2 (Fra-2) and inducible cAMP early repressor (ICER) (17, 18). Activation of additional signaling pathways such as MAPKs via phosphorylation cascades also occur with NE stimulation (19, 20). Some of these additional NE-stimulated cellular events are involved in the transcriptional regulation of aa-nat (2, 5, 21, 22, 23, 24, 25). Another potential signaling event that could regulate transcriptional activity in the rat pineal gland is histone modification.

Histone modifications and their contributions to chromatin remodeling are an essential step in the induction of gene transcription (26). Through covalent histone modifications, transcription factors can gain access to chromatin and stimulate gene expression. A number of posttranslational modifications occur on histone tails (27). Among these, phosphorylation of Ser10 in histone H3’s N-terminal tail is the best characterized link between chromatin modification and activation of a signal transduction pathway by mitogen signals (28). In the rat pineal gland, NE stimulation of PKA is one of the earlier and critical events that leads to induction of aa-nat gene transcription (4, 5, 6, 7). Because PKA as well as other signaling pathways activated by NE have been shown to promote histone H3 phosphorylation either directly or through an intermediate kinase in other tissues (29, 30, 31, 32, 33, 34, 35, 36), it is possible that histone H3 phosphorylation also occurs in the rat pineal gland with NE stimulation. Therefore, in this study, we investigated potential changes in the phosphorylation of histone H3 Ser10 after NE treatment of cultured rat pineal cells and the diurnal changes in the phosphorylation of histone H3 in intact rat pineal glands.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Dibutyryl cAMP, dibutyryl cGMP, isoproterenol, NE, phenylephrine, 4ß-phorbol 12-myristate 13-acetate (PMA), prazosin, and propranolol were obtained from Sigma Aldrich Co. (St. Louis, MO). Antibodies against histone H3 (07-690) and Ser10-phosphorylated H3 (07-353) were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Calphostin C, ionomycin, KT5720, KT5823, KN93, tautomycin, okadaic acid, SB203580, SB202474, UO126, and UO124 were obtained from Calbiochem Corp. (San Diego, CA). Polyclonal antibodies against AA-NAT25–200 (AB3314) were a gift from Dr. D. C. Klein (National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD). All other chemicals were of the purest grades available commercially.

Animal handling and pineal gland isolation
This study was reviewed and approved by the Health Sciences Animal Policy and Welfare Committee of the University of Alberta (Edmonton, Alberta, Canada). Sprague Dawley rats (male, weighing 150 g) were obtained from the University of Alberta animal unit. Animals were housed under a lighting regimen providing 12 h of light every 24 h with lights on at 0600 h [zeitgeber time zero (ZT0)]. For pinealocyte cell culture preparation, 12–15 animals were killed 3 h after the onset of light for each preparation, and pineal glands were removed and placed in ice-cold PBS until enzymatic digestion. Cell yield was approximately 7 x 105 cells per gland. To determine the diurnal variation in histone H3 phosphorylation, groups of animals (n = 3) were killed at various time points as indicated. To determine the effect of exposure to light during the dark period, groups of animals were subjected to acute light exposure for various durations at ZT15. Pineal glands were collected, cleaned in ice-cold PBS, flash-frozen on dry ice, and stored at –75 C until preparation for Western blot analysis or RNA extraction. A dim red light was used when animals were killed during the dark period.

Preparation of cultured pinealocytes and drug treatment
Pinealocytes were prepared by papain dissociation of freshly dissected rat pineal glands using a system from Worthington Biochemical Corp. (Lakewood, NJ). Cells were suspended in DMEM containing 10% fetal calf serum and maintained overnight before the experiment at 37 C for 18 h in a mixture of 95% air and 5% CO2. Aliquots of pinealocytes were treated with drugs that had been prepared in concentrated solutions in water or dimethylsulfoxide for the duration indicated. Treated cells were collected by centrifugation (2 min at 12,000 x g). Samples for Western blot analysis were solubilized in 1x sample buffer by boiling for 7 min and stored at –20 C until electrophoresis.

Western blot
SDS-PAGE was performed according to the procedure of Laemmli (37) using 10% acrylamide in the presence of 1 mg/ml SDS (Mini-Protein II gel system; Bio-Rad, Hercules, CA). After electrophoresis, gels were equilibrated for 15 min in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol). Proteins were transferred onto polyvinylidene difluoride membranes (1 h at 100 V) that were then incubated with a blocking solution [5% dried skim milk in 100 mM Tris (pH 7.5) with 140 mM NaCl and 0.01% Tween 20] for a minimum of 1 h. Blots were then incubated overnight at 4 C with diluted specific antisera as indicated. After washing twice with the blocking solution, blots were incubated with diluted horseradish-peroxidase-conjugated second antibodies (Bio-Rad) for 1 h at room temperature. Blots were then washed extensively and developed using enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL).

RT-PCR
Total RNA was isolated from cultured pineal cells using Trizol. First strand cDNA was synthesized using an Omniscript reverse-transcriptase kit (QIAGEN Inc., Valencia, CA) with an oligo-dT primer. PCR performed and primers used were as previously reported (15, 16).

Results presentation and statistical analysis
A representative immunoblot from three independent experiments is shown. Results were quantified using densitometric measurements and analyzed by Kodak 1D imaging software (Eastman Kodak, Rochester, NY) (38). Densitometric values were normalized as indicated and presented as the mean ± SEM from at least three independent experiments. Statistical analysis involved either a paired t test or ANOVA followed by the Newman-Keuls test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Diurnal variation in histone H3 phosphorylation in the rat pineal gland
To investigate whether there is a diurnal difference in histone H3 phosphorylation in the rat pineal gland, the levels of histone H3 phosphorylation in pineal glands collected at different time points from rats housed under a 12-h light, 12-h dark cycle was determined. As shown in Fig. 1Go, there was a rapid induction in the phosphorylated levels of histone H3 from pineal glands collected during the night, with a significant increase observable at ZT13 and a peak increase occurring at ZT15 (lights on at ZT0; lights off at ZT12). Analysis of the time profiles of induction of histone H3 phosphorylation and AA-NAT revealed that the nocturnal induction of histone H3 phosphorylation preceded the increase in mRNA and protein levels of AA-NAT (Fig. 1CGo). Remarkably, phosphorylated histone H3 declined rapidly after achieving a peak level at ZT15 and was back to basal level 4 h before the onset of light. The changes in the phosphorylated levels of histone H3 occurred in the absence of changes in the protein levels of total (phosphorylated and unphosphorylated) histone H3.


Figure 1
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  		FIG. 1. Day/night variation in histone H3 phosphorylation and AA-NAT expression in the rat pineal gland. Pineal glands were collected from rats, housed under a 12-h light, 12-h dark cycle, at the time points indicated. A, Representative immunoblots from three independent experiments showing phosphorylated H3 (p-H3), AA-NAT, and H3. Each lane contains 25 µg protein. B, Representative ethidium-bromide-stained agarose gels from three independent experiments showing the mRNA levels of aa-nat; gapdh is included to demonstrate loading consistency. C, Densitometric measurements of p-H3 protein, aa-nat mRNA, and AA-NAT protein presented as percentage of maximal OD value. Each value represents the mean ± SEM; n = 3.

 
Exposure to light caused a rapid decline in histone H3 phosphorylation in the night pineal gland
To investigate whether exposure to light in the middle of the night could alter histone H3 phosphorylation, animals were maintained in a normal light/dark cycle and then exposed to light at ZT15; pineal glands were obtained at 10-min intervals for the next 30 min. As shown in Fig. 2BGo, acute light exposure caused a rapid decline in histone H3 phosphorylation with an estimated t1/2 of less than 10 min. In contrast, in control animals in the dark, there was only a modest reduction in pineal histone H3 phosphorylation between ZT15 and ZT15.5 (Fig. 2Go). This rate of decline is similar to that observed with AA-NAT protein after similar light exposure (Fig. 2Go).


Figure 2
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  		FIG. 2. Effect of exposure to light on histone H3 phosphorylation in the night pineal gland. Pineal glands were collected at the time points indicated in the absence or presence of acute light exposure at ZT15 (+light). A, Representative immunoblots from three independent experiments showing phosphorylated H3 (p-H3), AA-NAT, and H3. Each lane contains 25 µg protein. B and C, Densitometric measurements of p-H3 (B) or AA-NAT protein levels (C) presented as percentage of OD value at ZT15. Each value represents the mean ± SEM; n = 3. The arrow indicates time of exposure to light.

 
Activation of histone H3 phosphorylation by treatment with NE
To determine the intracellular mechanisms involved in histone H3 phosphorylation, in vitro studies with cultured pinealocytes were performed. Treatment of pinealocytes with NE (3 µM) caused a gradual increase in histone H3 phosphorylation that was detectable at 30 min, peaked between 2 and 3 h, and returned rapidly to just above the basal level by 4 h (Fig. 3AGo). The time profile of activation of histone H3 phosphorylation preceded the induction of AA-NAT protein by NE. A 2-h NE treatment increased the levels of histone H3 phosphorylation in a concentration-dependent manner (Fig. 3BGo) with an estimated EC50 of 30 nM; a significant increase was detected with 10 nM NE. The time- and concentration-dependent increases in histone H3 phosphorylation occurred in the absence of changes in total histone H3 protein (Fig. 3Go, A and B).


Figure 3
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  		FIG. 3. Effect of NE on histone H3 phosphorylation and AA-NAT induction. Pinealocytes (2 x 105 cells/0.3 ml) were cultured for 18 h and treated with NE (3 µM) for the indicated time periods (A) or NE (1 nM to 10 µM) for 2 h (B). Cells were collected by centrifugation, dissolved in 1x sample buffer and analyzed by Western blotting using an antibody against phosphorylated H3 (p-H3), H3, and AA-NAT as described in Materials and Methods. CON, Control. Top, Representative immunoblots from three independent experiments; bottom, densitometric measurements of p-H3 and AA-NAT protein presented as percentage of maximal OD value. Each value represents the mean ± SEM; n = 3.

 
Receptor characterization of the adrenergic regulation of histone H3 phosphorylation
To determine the subtype of adrenergic receptors involved in the regulation of histone H3 phosphorylation, selective adrenergic agonists and antagonists at concentrations established previously were used in the study (10, 11, 12). At 2 h post treatment, selective activation of ß-adrenergic receptors by isoproterenol (3 µM in the presence of 3 µM prazosin) but not selective activation of {alpha}-adrenergic receptors by phenylephrine (3 µM in the presence of 3 µM propranolol) caused an increase in histone H3 phosphorylation (Fig. 4Go, A and B). Simultaneous activation of both {alpha}- and ß-adrenergic receptors by treatment with isoproterenol (3 µM) plus phenylephrine (3 µM) or NE (3 µM) caused an increase in histone H3 phosphorylation that appeared more pronounced than treatment with isoproterenol alone. In support of the predominant involvement of ß-adrenergic receptors in this induction, cotreatment with propranolol (3 µM) but not prazosin (3 µM) blocked the histone H3 phosphorylation induced by NE (3 µM) (Fig. 4Go, A and B).


Figure 4
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  		FIG. 4. Receptor characterization of NE-induced histone H3 phosphorylation. Pinealocytes (2 x 105 cells/0.3 ml) were cultured for 18 h and treated for 2 h with NE (3 µM), isoproterenol [ISO, 3 µM with prazosin (Praz, 3 µM)], phenylephrine [PE, 3 µM with propranolol (Prop, 3 µM)] or ISO (3 µM) plus PE (3 µM) (A) or NE (3 µM) alone or in the presence of Prop (3 µM) or Praz (3 µM) (B). CON, Control. Top, Representative immunoblots from three independent experiments showing phosphorylated H3 (p-H3) and H3; bottom, histogram of densitometric measurements of p-H3 protein presented as percentage of maximal OD value. Each value represents the mean ± SEM; n = 3. *, P < 0.05, significantly different from control (left panel); *, P < 0.05, significantly different from treatment with NE (right panel).

 
Postreceptor mechanisms involved in NE-induced histone H3 phosphorylation
To characterize the signaling pathways involved in NE-stimulated histone H3 phosphorylation, the effects of protein kinase activators and inhibitors were tested. Treatment of pinealocytes for 2 h with dibutyryl cAMP (0.5 mM), a membrane-permeable cAMP analog, mimicked the effects of NE on histone H3 phosphorylation (Fig. 5AGo). In contrast, treatment of pinealocytes for 2 h with the membrane-permeable cGMP analog dibutyryl cGMP (0.5 mM), the activator of protein kinase C PMA (0.1 µM), or the Ca2+ ionophore ionomycin (1 µM), did not cause an induction of histone H3 phosphorylation (Fig. 5AGo).


Figure 5
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  		FIG. 5. Effects of protein kinase activators and inhibitors on NE-induced histone H3 phosphorylation. Pinealocytes (5 x 105 cells/0.5 ml) were cultured for 18 h and treated for 2 h with NE (3 µM), dibutyryl cAMP (DBcAMP, 0.5 mM), dibutyryl cGMP (DBcGMP, 0.5 mM), PMA (0.1 µM), and ionomycin (ION, 1 µM) (A) or NE (3 µM) in the absence or presence of KT5720 (3 µM), KT5823 (3 µM), calphostin C (Cal-C, 1 µM), or KN93 (10 µM) (B). Top, Representative immunoblots from three independent experiments showing phosphorylated H3 (p-H3) and H3. CON, Control. Bottom, Histogram of densitometric measurements of p-H3 protein presented as percentage of maximal OD value. Each value represents the mean ± SEM; n = 3. *, P < 0.05, significantly different from treatment with NE (right panel).

 
In the presence of different protein kinase inhibitors, histone H3 phosphorylation stimulated by NE (3 µM) was only reduced by cotreatment with a PKA inhibitor, KT 5720 (3 µM). In contrast, cotreatment with protein kinase G (PKG) inhibitor KT5823 (3 µM), PKC inhibitor calphostin C (1 µM), or Ca2+/calmodulin-dependent protein kinase inhibitor KN93 (10 µM) had no effect on NE-induced histone H3 phosphorylation (Fig. 5BGo). Together these results suggest that unlike other tissues and despite the different signaling pathways activated simultaneously by NE (1, 11, 14, 19, 20), histone H3 phosphorylation appears to be regulated exclusively through the cAMP/PKA signaling pathway in rat pinealocytes.

Effects of inhibitors of MAPKs on NE stimulation of histone H3 phosphorylation
In rat pinealocytes, NE, through stimulation of PKA and PKG, has been shown to activate downstream p42/44MAPK and p38MAPK (19, 20), two signaling mechanisms that can induce phosphorylation of histone H3 in other cell types (28, 34). The involvement of these pathways was tested using UO126 (10 µM), a specific inhibitor of p42/44MAPK kinase, and SB203580 (10 µM), a specific inhibitor of p38MAPK, with inclusion of UO124 (10 µM) and SB202474 (10 µM) as the respective inactive controls. Pretreatment of cells with either UO126 or SB203580 had no effect on histone H3 phosphorylation induced by NE (3 µM) (Fig. 6Go), indicating that histone H3 phosphorylation is not a consequence of MAPK activation in rat pinealocytes. The effectiveness of drug treatment was confirmed by the suppressive effect of UO126 and the enhancing effect of SB203580 on the NE induction of the phosphorylated level of p42/44MAPK as demonstrated previously (39).


Figure 6
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  		FIG. 6. Effects of MAPK inhibitors on NE-induced histone H3 phosphorylation. Pinealocytes (5 x 105 cells/0.5 ml) were cultured for 18 h and treated for 2 h with NE (3 µM) in the absence or presence of SB203580 (10 µM), SB202474 (10 µM), UO126 (10 µM), or UO124 (10 µM). CON, Control. A, Representative immunoblots from three experiments showing phosphorylated H3 (p-H3), H3, p-p38MAPK, and p-p42/44MAPK; B, histogram of densitometric measurements of p-H3 protein presented as percentage of NE response. Each value represents the mean ± SEM; n = 3.

 
Continuous stimulation is required to maintain the NE-induced histone H3 phosphorylation
In view of the rapid decline in histone H3 phosphorylation 3 h after NE stimulation (Fig. 3Go), we investigated the requirement of continuous adrenergic stimulation in the maintenance of NE-induced histone H3 phosphorylation. Pinealocytes were treated with NE (3 µM) for 2 h to induce histone H3 phosphorylation before the addition of propranolol or prazosin. Blocking ß-adrenergic receptors with propranolol (3 µM) caused a rapid reduction in NE-induced H3 phosphorylation with an estimated t1/2 of around 15 min (Fig. 7Go, A and B). In contrast, blocking {alpha}-adrenergic receptors with prazosin (3 µM) had little effect on NE-induced histone H3 phosphorylation. These results suggest that in the absence of continuous ß-adrenergic stimulation, phosphorylated histone is rapidly dephosphorylated in the rat pinealocyte.


Figure 7
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  		FIG. 7. Effect of adrenergic receptor blockade on NE-induced histone H3 phosphorylation. Pinealocytes (5 x 105 cells/0.5 ml) were cultured for 18 h and treated for 2 h with NE (3 µM) before treatment with propranolol (Prop, 3 µM) or prazosin (Praz, 3 µM) for 15 and 30 min. CON, Control. Top, Representative immunoblots from three experiments showing phosphorylated H3 (p-H3) and H3; bottom, densitometric measurements of phosphorylated H3 (p-H3) protein presented as percentage of maximal OD value. Each value represents the mean ± SEM; n = 3. Arrow indicates time of addition of Prop or Praz.

 
Phosphatase inhibitors enhance NE-induced histone H3 phosphorylation
To confirm the importance of dephosphorylation in regulating the level of histone H3 phosphorylation in the rat pineal gland, the effect of inhibition of protein serine/threonine phosphatases was determined. Treatment of pinealocytes with two phosphatase inhibitors, tautomycin (1 µM) or okadaic acid (0.3 µM), on its own had no effect on histone H3 phosphorylation. However, both tautomycin and okadaic acid caused a marked enhancement of the NE (3 µM) induction of histone H3 phosphorylation (Fig. 8Go, A and B), indicating the presence of a high level of dephosphorylation activity in the rat pineal gland. These results suggest that in the basal conditions, there is little histone phosphorylation without NE stimulation.


Figure 8
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  		FIG. 8. Effects of phosphatase inhibitors on NE-induced histone H3 phosphorylation. Pinealocytes (5 x 105 cells/0.5 ml) were cultured for 18 h and treated for 2 h with NE (3 µM) in the absence or presence of tautomycin (Tauto, 1 µM) or okadaic acid (Oka, 0.3 µM). Con, Control. A, Representative immunoblots from three experiments showing phosphorylated H3 (p-H3) and H3; B, densitometric measurements of p-H3 protein presented as percentage of maximal OD value. Each value represents the mean ± SEM; n = 3. *, P < 0.05, significantly different from treatment with NE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Covalent modification of histone tails, in particular phosphorylation of Ser10 in histone H3, is crucial for chromosome condensation and cell-cycle progression as well as gene transcription in response to cell signaling events (26). In this study, we focus on the regulation of histone H3 Ser10 phosphorylation and the intracellular mechanisms involved in a neuroendocrine organ during the process of hormone synthesis. We found that histone H3 phosphorylation is a natural event that occurs every night in the rat pineal gland. Moreover, NE induces H3 phosphorylation almost exclusively through a ß-adrenergic receptor->PKA mechanism even though NE activates multiple signaling mechanisms in the rat pineal gland.

By monitoring the phosphorylation state of histone H3, we showed in the present study that there is a marked increase in histone H3 phosphorylation during the first half of the dark period in the rat pineal gland. Parallel studies performed in cultured pineal cells revealed that NE treatment, while having no effect on histone H3 protein, increases the level of histone H3 phosphorylation in a time- and concentration-dependent manner. The induction phase is rapid and detectable within 30 min. Once the induction reached its peak level, a rapid decline follows. This time profile of NE induction of histone H3 phosphorylation precedes that of aa-nat induction and is in line with its potential role in regulating the transcription of aa-nat and other adrenergic-induced genes in the rat pineal gland as shown by microarray technology (40, 41). Considering that the pineal is stimulated by the release of NE from the sympathetic nerves at night (1, 2), these results suggest that the adrenergic-stimulated histone H3 phosphorylation in the rat pineal gland is probably driven by the endogenous circadian clock in the suprachiasmatic nucleus (42, 43), similar to other cellular processes in the pineal that have a diurnal rhythm.

It is of interest to note that nighttime light exposure has previously been shown to cause phosphorylation of Ser10 in the tail of histone H3 in the mouse suprachiasmatic nucleus (44). Moreover, the effect of light on histone H3 phosphorylation parallels the induction of c-fos, with both events occurring in the same suprachiasmatic neurons. However, in the same study, no histone H3 phosphorylation was observed in mouse pineal glands collected during daytime or nighttime. The absence of histone H3 phosphorylation in the previous study could be related to the early and transient nature of H3 phosphorylation that occurs at night and the infrequent sampling in that study (44). Interestingly, the early induction of histone H3 phosphorylation during the dark period is consistent with its role in gene transcription. In addition to aa-nat, there are multiple NE->ß-adrenergic->cAMP/PKA-regulated genes with diverse functions that have been characterized to date in the rat pineal gland. These include mkp-1, icer, fra-2, and the genes that encode methionine adenosyltransferase, the orphan nuclear receptor NGFI-B and the oligopeptide transporter PepT1 (15, 17, 18, 45, 46, 47). Although the transcription of the specific adrenergic-stimulated gene(s) that is linked to histone H3 phosphorylation remains to be determined, it is probable that this PKA-mediated histone modification may impact on the transcriptional regulation of multiple PKA-regulated genes.

Another interesting observation in the present study is the effect of light exposure during the dark period on the nocturnal induction of histone H3 phosphorylation. Acute light exposure causes a rapid decline in histone H3 phosphorylation with a t1/2 of less than 10 min. The rate of decline is similar to that observed with AA-NAT protein after the same treatment or in NE-stimulated pinealocytes after ß-adrenergic blockade. These results indicate that there is a high level of histone H3 dephosphorylation activity in the rat pineal gland and that continuous stimulation is required to maintain a sustained increase in the phosphorylated level of histone H3 in this tissue. However, the acute effect of light at night on histone H3 phosphorylation in the pineal gland is likely dissociated from the entraining effect of light on the clock in the suprachiasmatic nucleus because the level of histone H3 phosphorylation is already suppressed toward the end of night when light has an entraining effect.

Investigations into the signal transduction mechanisms involved in NE-induced histone H3 phosphorylation show involvement of ß-adrenergic receptors because only propranolol, the ß-adrenergic antagonist, but not prazosin, the {alpha}-adrenergic antagonist, can block the NE-induced histone H3 phosphorylation. Downstream from the adrenergic receptors, our results indicate that histone H3 phosphorylation is primarily mediated by PKA. This conclusion is based on our observations that only dibutyryl cAMP can mimic the effect of NE on histone H3 phosphorylation, and KT5720, a PKA inhibitor, is effective in inhibiting NE-induced histone H3 phosphorylation. Consistent with our results, PKA has been shown to promote histone H3 phosphorylation in other cell types (29, 32). Our results also indicate that other signaling mechanisms including PKG, PKC, and Ca2+/calmodulin do not participate in the NE-induced phosphorylation of histone H3 even though PKC has been reported to mediate histone H3 phosphorylation in other tissues (31, 33). Together, these results suggest that although multiple signaling pathways are activated by NE, histone H3 phosphorylation in the rat pineal gland appears to be exclusively regulated through the PKA pathway.

In other cell types, histone H3 phosphorylation can occur downstream of the p38MAPK and/or p42/44MAPK pathways (28, 34), two additional signaling pathways activated by NE in rat pinealocytes (19, 20). Comparison of the time profiles showed a similarity between the appearance of NE-induced histone H3 phosphorylation and the activation of p38MAPK by NE. Moreover, PKA is involved in both histone H3 phosphorylation and activation of p38MAPK (20). However, pretreatment with a specific inhibitor of p38MAPK, SB203580, has no effect on NE-induced histone H3 phosphorylation, indicating that histone H3 phosphorylation is not a consequence of p38MAPK activation in rat pinealocytes in contrast to findings in other cell type (34). p42/44MAPK also does not appear to be linked to histone H3 phosphorylation in this system because pretreatment with UO126, an inhibitor of MAPK kinase, likewise has no effect on NE-induced histone H3 phosphorylation.

In regard to the intermediate mechanism involved in NE-induced histone H3 phosphorylation, PKA can act either directly or through intermediate kinase in its induction. Potential intermediate kinases include ribosomal S6 kinase 2 (RSK-2), mitogen- and stress-activated protein kinase 1 (MSK-1), or aurora kinase (28, 30, 35, 36). Ribosomal S6 kinase 2 and mitogen- and stress-activated protein kinase 1, mediators of p42/44MAPK and p38MAPK, can phosphorylate histone H3 in vitro (28, 35). However, our results do not support a role of p42/44MAPK or p38MAPK in the NE-induced histone H3 phosphorylation. Whether aurora kinase is involved in the histone H3 phosphorylation in rat pinealocytes remains to be determined.

Our results also indicate that the phosphorylation state of histone H3 in rat pinealocytes appears to be subjected to a high level of dephosphorylation activity under basal conditions. This observation is based on the finding that although inhibition of protein serine/threonine phosphatase by tautomycin or okadaic acid alone cannot induce histone H3 phosphorylation on its own, both inhibitors are effective in augmenting the NE induction of histone phosphorylation by up to 4-fold. This result indicates a high level of tonic phosphatase activity causing ongoing histone H3 dephosphorylation and could account for the rapid decline in the phosphorylated H3 levels after light exposure in the intact pineal gland at night.

In summary, we demonstrate in the present study that histone H3 Ser10 phosphorylation is a naturally occurring event with an increase during the early dark period. This histone H3 phosphorylation is under adrenergic control upon activation of ß-adrenergic receptors. Among the multiple signaling pathways activated by NE, PKA appears to be exclusively involved in the NE induction of histone H3 phosphorylation. Moreover, the presence of a high level of phosphatase activity in the rat pineal gland ensures a rapid decline in histone H3 phosphorylation in the absence of stimulation. This transient PKA-mediated histone H3 phosphorylation is probably of general importance to the transcriptional activation of multiple adrenergic->cAMP/PKA-regulated genes.


   Footnotes
 
This work was supported by grants from the Canadian Institutes of Health Research.

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 21, 2006

Abbreviations: AA-NAT, Arylalkylamine-N-acetyltransferase; CREB, cAMP response element-binding protein; H3, histone H3; NE, norepinephrine; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PMA, 4ß-phorbol 12-myristate 13-acetate; ZT, zeitgeber time.

Received October 31, 2006.

Accepted for publication December 11, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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