Proteasomal Proteolysis in the Adrenergic Induction of Arylalkylamine-N-Acetyltransferase in Rat Pinealocytes
http://www.100md.com
《内分泌学杂志》
Departments of Physiology (D.L.T. , D.M.P., A.K.H.) and Medicine (C.L.C), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
Abstract
In this study, we investigated the effect of proteasomal inhibition on the induction of arylalkylamine-N-acetyltransferase (AA-NAT) enzyme in cultured rat pinealocytes, using two proteasome inhibitors, MG132 and clastolactacystin -lactone (c-lact). Addition of c-lact or MG132 3 h after norepinephrine (NE) stimulation produced a significant increase in AA-NAT protein level and enzyme activity. However, when the proteasome inhibitors were added before or together with NE, significant reductions of the NE-induced aa-nat mRNA, protein, and enzyme activity were observed. A similar inhibitory effect of MG132 on aa-nat transcription was observed when cells were stimulated by dibutyryl cAMP, indicating an effect distal to a post-cAMP step. The inhibitory effect of MG132 on adrenergic-induced aa-nat transcription was long lasting because it remained effective after 14 h of washout and appeared specific for aa-nat because the induction of another adrenergic-regulated gene, MAPK phosphatase-1, by NE was not affected. Time-profile studies revealed that the inhibitory effect of MG132 on NE-stimulated aa-nat induction was detected after 1 h, suggesting accumulation of a protein repressor as a possible mechanism of action. This possibility was also supported by the finding that the inhibitory effect of c-lact on NE-induced aa-nat induction was markedly reduced by cycloheximide, a protein synthesis inhibitor. Together, these results support an important role of proteasomal proteolysis in the adrenergic-mediated induction of aa-nat transcription through its effect on a protein repressor.
Introduction
IN THE MAMMALIAN pineal gland, the nightly release of norepinephrine (NE) from the sympathetic neurons stimulates both 1- and 1-adrenergic receptors resulting in a 100-fold increase in intracellular cAMP levels (1, 2, 3). The rise in cAMP, in turn, stimulates cAMP-dependent protein kinase, which translocates to the nucleus and phosphorylates the transcription factor cAMP response element binding protein (4). Phosphorylated cAMP response element binding protein binds to cAMP response elements in the promoter region of cAMP-regulated genes and causes activation of transcription (5, 6, 7). This activation of transcription results in a 150-fold increase in the mRNA of arylalkylamine-N-acetyltransferase (AA-NAT), the rate-controlling enzyme in the production of melatonin (MT) (8, 9).
The ubiquitin-proteasome degradation pathway is known to play a critical role in regulating many cellular processes such as the cell cycle, cell differentiation, modulation of cell surface receptors and ion channels, regulation of the immune and inflammatory responses, DNA repair, and regulation of transcription factors (10). The 26S proteasome is a multimeric protease complex made up of a proteolytic 20S core particle and capped at one or both ends by 19S regulatory particles (11, 12). Although the 26S proteasome is responsible for both ubiquitin-dependent and -independent protein degradation, the 20S proteasome functions only in ubiquitin-independent protein degradation (13). Studies of proteasomal proteolysis on regulation of cellular processes such as hormone synthesis and cytokine signaling mechanism have been facilitated by the use of specific proteasome inhibitors such as clastolactacystin -lactone (c-lact) and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) (14, 15). Selective induction of a tumor marker gene, glutathione S-transferase P1 by c-lact and MG132 has recently been demonstrated in hepatocellular carcinoma cells (16).
In the rat pineal gland, the steady-state level of AA-NAT protein, besides transcriptional and translational control, is also regulated by proteasome-mediated degradation (17, 18). In stimulated pinealocytes, AA-NAT is phosphorylated by cAMP-dependent protein kinase and interacts with the protein 14-3-3 (18). This association helps to protect AA-NAT from proteasomal proteolysis and increases the enzyme activity. However, at the end of the dark phase, AA-NAT becomes dephosphorylated and is targeted for degradation by the proteasome (19). Although the effect of the proteasome on the steady-state levels of AA-NAT protein have been well established (19), the potential role of the proteasome in modulating the transcription and translation of the aa-nat gene has yet to be investigated. In this study, by using two structurally unrelated specific proteasome inhibitors, MG132 and c-lact, we determined the effects of inhibiting the proteasome on adrenergic induction of aa-nat transcription and compared their effects on AA-NAT protein levels, enzyme activity, and MT production in cultured rat pinealocytes. Our results indicate that proteasomes play an important role in the NE-induced aa-nat transcription.
Materials and Methods
Materials
NE, cycloheximide, dibutyryl cAMP (DbcA) were obtained from Sigma Aldrich Co. (St. Louis, MO). c-lact, MG132, and N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin were obtained from Biomol Co. (Plymouth Meeting, PA). [3H]Acetyl-coenzyme (specific activity, 1 mCi/mmol) was from Amersham Biosciences (Piscataway, NJ). [3H]MT was obtained from NEN Life Science Products (Boston, MA). Polyclonal antibodies against MAPK phosphatase-1 (MKP-1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody against glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) antibody was obtained from Ambion Inc. (Austin, TX). Polyclonal antibodies for the RIA of MT were obtained from CID Tech Co. (Mississauga, Ontario, Canada). 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.
Preparation of cultured pinealocytes and drug treatments
All procedures were reviewed and approved by the Health Sciences Animal Policy and Welfare Committee of the University of Alberta (Edmonton, Alberta). Sprague Dawley rats (male; weighing 150 g) were obtained from the University of Alberta animal unit. Pinealocytes were prepared by papain dissociation of freshly dissected rat pineal glands as described previously (20). Cells were suspended in DMEM containing 10% fetal calf serum and maintained before the experiment at 37 C for 24 h in a mixture of 95% air-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). Pinealocyte total RNAs were isolated using Trizol (Invitrogen Co., Carlsbad, CA) according to manufacturer’s instruction. Samples for Western blot analysis were solubilized in 1x sample buffer by boiling for 5 min and stored at –20 C until electrophoresis. The homogenization buffer contained 20 mM Tris-HCl; 2 mM EDTA; 0.5 mM EGTA; 2 mM phenylmethylsulfonyl fluoride; 1 μg/ml each of aprotinin, leupeptin, and pepstatin; 1 mM sodium orthovanadate; and 1 mM sodium fluoride (pH 7.5). Samples for the determination of AA-NAT activity was immediately frozen in dry ice and stored at –75 C. Media were collected for MT determination.
Western blotting
SDS-PAGE was performed according to the procedure of Laemmli (21) using 10 or 12% acrylamide (Mini-Protein II gel system, Bio-Rad Laboratories, Hercules, CA). After electrophoresis, gels were equilibrated for 20 min in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol). Proteins were transferred onto polyvinylidene difluoride membranes (1.5 h, 45 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 1.5 h. The blots were then incubated overnight at 4 C with diluted specific antisera as indicated. After washing three times with the blocking solution, the blots were incubated with diluted horseradish peroxidase-conjugated second antibodies (Bio-Rad) for 1.5 h at room temperature. They were then washed extensively and developed using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).
RT-PCR
First-strand cDNA was synthesized from the isolated RNA using an Omniscript reverse-transcriptase kit (QIAGEN Inc., Valencia, CA) with an oligo-dT primer. For cell extracts, 3 μl of a 1:10 dilution of cDNA was used. PCR was performed in a 29.3-μl reaction mixture containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 100 μM of each dNTP, 0.75 U Taq polymerase (PerkinElmer Cetus, Emeryville, CA), and 1 μM each of the two primers. PCRs were as follows: denaturing for 1 min at 94 C, annealing for 1 min at 63 C, and extension for 1 min at 72 C. Initial denaturing and final extension were both 5 min in duration. Cycle numbers varied between cell preparations, but in general, 23 cycles were used to amplify aa-nat, 25 cycles for mkp-1, and 22 for gapdh mRNAs. All reaction sets included water blanks as negative controls. Amplified products were separated on ethidium bromide-stained 1.5% agarose gels. PCR products were confirmed by sequencing. All primer sequences were designed using rat gene cDNA sequences obtained from BLAST and primers were selected from 3' regions as outlined elsewhere (22). Primers used (23) are as follows: aa-nat, upstream primer, 5'-GGT TCA CTT TGG GAC AAG GA-3'; downstream primer, 5'-GTG GCA CCG TAA GGA ACA TT-3'; mkp-1, upstream primer, 5'-CTG CTT TGA TCA ACG TCT CG-3'; downstream primer, 5'-AAG CTG AAG TTG GGG GAG AT-3'. Sequences of the gapdh primers used were previously described (23).
20S proteasome activity assay
Pelleted pinealocytes were frozen in dry ice and stored overnight at –75 C. The following day they were lysed by sonication in 40 μl of lysis buffer (PBS, 1% Triton X-100, 1 mM dithiothreitol). The lysates were centrifuged for 5 min at 12,000 x g and the supernatant was used for the assay. In a 96-well BD Optilux plate (BD Biosciences, Mississauga, Ontario, Canada), 40 μl of the crude cell lysate were combined with 10 μl of assay buffer [25 mM HEPES, 1 mM EDTA (pH 7.6), and 0.03% sodium dodecyl sulfate], 40 μl water, and 10 μl Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin to give a final concentration of 62.5 μM (24). The reaction was placed in a 37 C incubator in a mixture of 95% air-5% CO2 for 2 h. Fluorescence was measured using a Thermo Electron Fluorescent plate reader (380/460 nm filter set). Cell lysate background readings were prepared by adding 20 μM c-lact to lysed cells 30 min before beginning the assay. Proteasome activity was expressed as picomoles of AMC produced per hour per 105 cells.
AA-NAT assay
AA-NAT activity was determined as described previously (25). Briefly, treated pinealocytes were stored frozen in dry ice until homogenization in a reaction mixture of 0.1 M phosphate buffer (pH 6.8) containing 30 nmol [3H]acetyl coenzyme A (specific activity 1 mCi/mmol) and 1 μmol tryptamine hydrochloride in a final volume of 60 μl. The reaction mixture was incubated at 37 C for 1 h. At the end of the incubation period, the reaction was stopped by the addition of 1 ml of methylene chloride. After vortexing, the aqueous phase was removed, and the organic phase was washed three times with 0.1 M phosphate buffer (pH 6.8). The organic phase was transferred to a scintillation vial, evaporated to dryness, and the radioactive acetylated product determined by scintillation counting. AA-NAT activity was expressed as nanomoles per hour per 105 cells.
MT assay
Briefly, medium MT was extracted from 300 μl of medium by vortexing with 1 ml methylene chloride. After centrifugation, the organic phase was collected and evaporated to dryness. The residue was reconstituted in 500 μl of assay buffer [0.01 M phosphate buffer (pH 6.5) containing 0.1% gelatin]. The recovery of medium MT was more than 98%. The extracted MT was assayed by a RIA as described previously (26).
Results and statistical analysis
For quantitation of RT-PCR analyses, gel images were acquired with Kodak 1-D software on a Kodak 2000R imaging station. For analyses of Western blots, exposed films were scanned and band densitometry of acquired images was performed with Kodak 1-D software. Densitometric values were normalized to percent maximal and presented as the mean ± SEM from at least three independent experiments. For RIA or radioenzymatic assays, data were presented as the mean ± SEM from at least three independent experiments. Statistical analysis involved either a paired t test or ANOVA with the Newman-Keuls test. Statistical significance was set at P < 0.05.
Results
Time-dependent effects of proteasomes in adrenergic induction of aa-nat transcription
Treatment of cultured pinealocytes with NE (3 μM) for 6 h caused a large increase in NE-stimulated aa-nat mRNA (Fig. 1A) and protein levels (Fig. 1B). Compared with cells stimulated with NE alone for 6 h, exposure to a proteasome inhibitor, c-lact (10 μM), for the last 3 h of NE treatment caused a further increase in AA-NAT protein (Fig. 1B) and enzyme activity (Fig. 1C) as reported previously (19) but had no significant effect on the level of aa-nat mRNA (Fig. 1A). In contrast, treatment of pinealocytes with c-lact (10 μM) and NE for 3 h followed by an additional 3 h treatment with NE alone caused a reduction in the level of NE-stimulated aa-nat mRNA (Fig. 1A), protein (Fig. 1B), and enzyme activity (Fig. 1C). A similar inhibition of aa-nat transcription was observed when pinealocytes were treated with MG132 (3 μM), a structurally unrelated proteasome inhibitor to c-lact, for the first 3 h of a 6-h treatment with NE (Fig. 2). These results indicate that proteasomal inhibition can significantly reduce NE stimulated aa-nat transcription during the early phase of NE stimulation.
Treatment with MG132 preceding NE stimulation inhibits adrenergic-stimulated aa-nat induction and MT synthesis
To determine the effect of pretreatment with a proteasome inhibitor on adrenergic-stimulated aa-nat transcription, pinealocytes were treated with MG132 (3 μM) for 3 h, followed by washout of MG132 by replacing the cell media before stimulation with NE (3 μM) for another 3 h. Levels of aa-nat mRNA were significantly reduced when the cells were pretreated with MG132 (Fig. 3). To characterize the effects of proteasome inhibitors on MT synthesis, pinealocytes were pretreated with MG132 (3 μM) or c-lact (10 μM) for 3 h, followed by washout of inhibitors by replacing the cell media before stimulation with NE (1 μM) for another 14 h. Pretreatment with MG132 or c-lact caused a significant reduction in NE-stimulated AA-NAT activity and MT production (Fig. 4). These results suggest treatment with MG132 before NE stimulation is effective in inhibiting NE-stimulated aa-nat induction and MT synthesis. Moreover, proteasome inhibitors appear to have a long-lasting effect on MT synthesis.
Effect of proteasomal inhibition on adrenergic-induced transcription is long lasting and appears specific for aa-nat
To characterize the effect of proteasomal inhibition on adrenergic-induced transcription, pinealocytes were pretreated for 3 h with MG132 (3 μM), followed by replacement with new media that did not contain MG132 and incubation for an additional 14 h before stimulation with NE for 3 h. Pretreatment with MG132 followed by 14 h of washout remained effective in reducing NE-stimulated increases in aa-nat mRNA and protein levels (Fig. 5). In contrast, when the expression of mkp-1, another adrenergic-regulated gene in the rat pineal gland was determined, NE was effective in stimulating mkp-1 transcription in pinealocytes subjected to pretreatment with MG132 followed by 14 h of washout (Fig. 5). Of interest, there was also an increase in basal and adrenergic-stimulated mkp-1 transcription (Fig. 5). These results suggest that the effect of MG132 on adrenergic-induced transcription is long lasting because inhibition of aa-nat transcription is still observed after 14 h of washout. In addition, the inhibition of transcription by MG132 appears specific for aa-nat because it has little effect on the expression of another adrenergic-driven gene, mkp-1.
To confirm the long-lasting effects of the proteasome inhibitors, proteasomal activity in cultured pinealocytes was determined after treatment with MG 132 (3 μM) or c-lact (10 μM) for 3 h followed by 14 h of washout. Under this treatment condition, proteasomal activity in pinealocytes was reduced to 45% and 10% of control cells by MG132 and c-lact, respectively (Fig. 6).
To further characterize the effects of proteasomal inhibition on adrenergic-induced mkp-1 transcription, cultured pinealocytes were subjected to treatment with NE for 6 h in combination with c-lact (10 μM) using the identical treatment protocol as described for Fig. 1. Treatment of cultured pinealocytes with NE (3 μM) for 6 h caused a large increase in NE-stimulated mkp-1 mRNA level (Fig. 7). Compared with cells stimulated with NE alone for 6 h, exposure to a proteasome inhibitor, c-lact (10 μM), for the first 3 h or the last 3 h of 6 h treatment with NE had no significant effect on the level of mkp-1 mRNA (Fig. 7). These results indicate that, unlike its inhibitory effect on aa-nat transcription on the early phase of NE stimulation, proteasomal inhibition had no effect on NE stimulated mkp-1 transcription during the early or late phase of NE stimulation.
Effect of MG132 on the time profile of NE-stimulated aa-nat transcription
To determine the time period required for proteasome inhibitors to reduce adrenergic-induced aa-nat transcription, cells were treated simultaneously with NE and MG132 for 1, 3, and 5 h. Treatment with MG132 for 1 h had no effect on NE-stimulated aa-nat mRNA level (Fig. 8). However, after 1 h of treatment with NE alone, aa-nat mRNA levels continued to increase, whereas treatment with MG132 prevented any further increase in NE-stimulated aa-nat mRNA levels (Fig. 8B). After 5 h of treatment with MG132 and NE, there was a 65% reduction in aa-nat mRNA level, compared with cells stimulated with NE alone. This suggests that a lag time of 1 h is required by the proteasome inhibitor to demonstrate repression of aa-nat transcription.
To confirm the requirement of a 1-h lag time for the inhibition of aa-nat transcription, a second time-course study was performed in which pinealocytes were pretreated with MG132 (3 μM) for 1 h before NE stimulation. Pretreatment with MG132 reduced NE-stimulated aa-nat mRNA levels at 1 and 5 h by 50 and 75%, respectively (Fig. 8B). This result indicates that a 1-h pretreatment is adequate to demonstrate the inhibitory effect of MG132 on aa-nat transcription.
A post-cAMP step mediates the effect of proteasomal inhibition on adrenergic-stimulated aa-nat transcription
To determine the step by which a proteasome inhibitor exerts its effect on adrenergic induction of aa-nat transcription, pinealocytes were pretreated with MG132 (3 μM) for 3 h, followed by 14 h of washout before cells were stimulated for 3 h with NE (3 μM) or DbcA (1 mM), a cell-permeable cAMP analog. Pretreatment with MG132 caused a significant reduction in both aa-nat mRNA and protein levels in cells that were stimulated with either NE or DbcA (Fig. 9). These results indicate that the inhibitory effect of MG132 on aa-nat transcription is mediated at a post-cAMP step.
Protein synthesis is involved in the proteasomal inhibitor effects on NE-stimulated aa-nat transcription
Considering the primary effect of proteasome inhibitors, a lag time for the demonstration of inhibition of aa-nat transcription suggests accumulation of a protein repressor. To examine whether inhibition of aa-nat transcription by proteasome inhibitors was due to accumulation of a protein, pinealocytes were treated with a protein synthesis inhibitor, cycloheximide, in the presence or absence of c-lact for 3 h before the addition of NE. Treatment with cycloheximide (30 μg/ml) had an enhancing effect on NE-stimulated aa-nat mRNA (Fig. 10). The inhibitory effect of c-lact (10 μM) on NE-stimulated aa-nat transcription was abolished by cotreatment with cycloheximide (Fig. 10). In the presence of cycloheximide (30 μg/ml), NE did not cause an increase in AA-NAT protein after 6 h (data not shown). This result suggests that inhibition of aa-nat transcription is due to accumulation of a protein repressor, and the inhibitory effect can be abolished by inhibition of protein synthesis.
Discussion
A major factor regulating rhythmic and light-induced changes in AA-NAT activity is the steady-state level of AA-NAT protein, which represents a balance of synthesis and degradation. Synthesis of AA-NAT protein is a reflection of adrenergic cAMP aa-nat mRNA levels, which exhibits a nearly 100-fold nocturnal increase in the rat pineal gland (9). Based on in vitro studies of rat pineal glands, in addition to transcriptional control, proteasomal proteolysis also plays an important role in the circadian and photic regulation of AA-NAT activity (19). In the absence of cAMP, AA-NAT is destroyed by proteasomal proteolysis (27, 28). Using cultured pinealocytes, we now show that proteasomal proteolysis not only regulates AA-NAT protein but also plays an important role in the adrenergic induction of aa-nat transcription through its effect on transcription repression.
The time-dependent effect of treatment with the proteasome inhibitor on adrenergic-induced transcription of aa-nat is of interest. Inhibition of the proteasome before or concurrent with NE stimulation causes a significant reduction in aa-nat mRNA and protein levels. In contrast, when the proteasome is inhibited after stimulation with NE, it enhances the adrenergic-stimulated AA-NAT protein levels as reported in a previous study (19) without having an effect on aa-nat mRNA. Moreover, similar time-dependent effects of the proteasome are obtained with two structurally unrelated proteasome inhibitors, MG132 and c-lact. Taken together, our results indicate that these time-dependent effects of proteasome inhibitors are mediated through inhibition of transcription of aa-nat mRNA. Whether proteasomes exert an enhancing or inhibitory effect on adrenergic-regulated AA-NAT protein levels are critically dependent on the time of exposure to proteasomes.
Our results also provide evidence on the step that mediates the effects of proteasomes on adrenergic-induced aa-nat transcription. Because inhibition of the proteasome by MG132 causes a similar inhibition of NE- or DbcA-stimulated aa-nat transcription, the proteasome inhibitor is likely acting at a post-cAMP step distal to the adrenergic receptor.
Treatment with the proteasome inhibitor MG132 also has a long-lasting effect on adrenergic-stimulated AA-NAT induction that remains after 14 h of washout. The sustained effect is confirmed using a proteasome activity assay in rat pinealocytes, which shows persistent inhibition of the proteasome activity, with c-lact causing a greater inhibition than MG132. This difference in inhibition of proteasomal activity between MG132 and c-lact could be related to the concentration of the inhibitors used, or alternatively, partial recovery of the inhibition by MG132, a reversible inhibitor (29). Moreover, our results also suggest a slow turnover of proteasomes in rat pinealocytes because de novo synthesis of functional proteasomes in the cell should lead to a reversal of inhibition of adrenergic-induced aa-nat transcription. In other mammalian cells, proteasomal inhibition causes an enhanced expression of proteasome genes and de novo proteasome biogenesis within 6 h of treatment with the inhibitors (30).
It is also of interest to note that this long-lasting effect of proteasomes on aa-nat transcription is not a general effect that applies to all adrenergic-regulated genes because mkp-1, another adrenergic-regulated gene, is not affected. These results exclude MG132 having a toxic effect on pinealocytes that precludes recovery of adrenergic-regulated gene transcription. The selective effect of MG132 on gene transcription also suggests a difference in the adrenergic regulation of aa-nat and mkp-1 (22) or alternatively a difference in their stability and transcription efficiency. A difference in the stability or transcription efficiency between aa-nat and mkp-1 is supported by the finding that treatment with MG132 alone causes a selective increase in basal mkp-1 mRNA and protein levels.
The effect of proteasomes on aa-nat transcription appears to be mediated by the accumulation of a protein repressor. This is suggested by our observation that a lag time of 1 h is required by the proteasome inhibitor to express inhibition of aa-nat transcription. Moreover, the effect of proteasome inhibition on aa-nat transcription can be abolished by treatment with cycloheximide, a protein synthesis inhibitor. In view of the enhancing effects of proteasome inhibitors on adrenergic-stimulated AA-NAT protein levels when the inhibitors are added 3 h after NE treatment, it is possible that the protein repressor is also under proteasomal regulation. At present, the repressor involved is a subject of speculation. One possible candidate for this protein repressor is induced cAMP early repressor (ICER) (31). Previous studies suggest that ICER may be involved in repressing AA-NAT transcription at the end of the night because ICER is expressed diurnally and binds to cAMP response elements sites (32, 33, 34, 35). Moreover, steady-state levels of ICER are believed to be regulated by both protein synthesis and degradation by the proteasome (36). However, our results indicate that the inhibitory effect of proteasomal inhibition on aa-nat transcription does not require continuous stimulation with NE. Another candidate repressor is downstream regulatory element antagonist modulator, a transcription repressor with established circadian rhythmicity in the rat pineal gland (37).
Inducible genes are under the control of both activating and repressive signals (38, 39). A tight balance between repression and activation of transcription likely regulates the amplitude and duration of gene expression. In the rhythmic transcription of aa-nat, our results indicate that inhibiting proteasomes causes a significant reduction in adrenergic regulation of aa-nat transcription. Moreover, a protein repressor under the regulation of proteasomes appears to be responsible for suppressing adrenergic-mediated transcriptional induction of aa-nat.
Footnotes
This work was supported by grants from the Canadian Institutes of Health Research. D.L.T. was supported by a Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council.
Abbreviations: AA-NAT, Arylalkylamine-N-acetyltransferase; c-lact.,clastolactacystin -lactone; DbcA, dibutyryl cAMP, GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; ICER, inducible cAMP early repressor; MG132, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; MKP-1, mitogen-activated protein kinase phosphatase-1; MT, melatonin; NE, norepinephrine.
References
Klein DC, Sugden D, Weller JL 1983 Postsynaptic -adrenergic receptors potentiate the -adrenergic stimulation of pineal serotonin N-acetyltransferase. Proc Natl Acad Sci USA 80:599–603
Klein DC, Berg GR, Weller J 1970 Melatonin synthesis: adenosine 3',5'-monophosphate and norepinephrine stimulate N-acetyltransferase. Science 168:979–980
Klein DC, Berg GR 1970 Pineal gland: stimulation of melatonin production by norepinephrine involves cyclic AMP-mediated stimulation of N-acetyltransferase. Adv Biochem Psychopharmacol 3:241–263
Roseboom PH, Klein DC 1995 Norepinephrine stimulation of pineal cyclic AMP response element-binding protein phosphorylation: primary role of a -adrenergic receptor/cyclic AMP mechanism. Mol Pharmacol 47:439–449
Baler R, Covington S, Klein DC 1997 The rat arylalkylamine N-acetyltransferase gene promoter. cAMP activation via a cAMP-responsive element-CCAAT complex. J Biol Chem 272:6979–6985
Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH 1986 Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci USA 83:6682–6686
Bisotto S, Minorgan S, Rehfuss RP 1996 Identification and characterization of a novel transcriptional activation domain in the CREB-binding protein. J Biol Chem 271:17746–17750
Borjigin J, Wang MM, Snyder SH 1995 Diurnal variation in mRNA encoding serotonin N-acetyltransferase in pineal gland. Nature 378:783–785
Roseboom PH, Coon SL, Baler R, McCune SK, Weller JL, Klein DC 1996 Melatonin synthesis: analysis of the more than 150-fold nocturnal increase in serotonin N-acetyltransferase messenger ribonucleic acid in the rat pineal gland. Endocrinology 137:3033–3045
Ciechanover A, Orian A, Schwartz AL 2000 Ubiquitin-mediated proteolysis: biological regulation via destruction. Bioessays 22:442–451
Voges D, Zwickl P, Baumeister W 1999 The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem 68:1015–1068
Groll D, Bajorek M, Kohler A, Moroder L, Rubin DM, Huber R, Glickman MH, Finley 2000 A gated channel into the proteasome core particle. Nat Struct Biol 7:1062–1067
Orlowski M, Wilk S 2003 Ubiquitin-independent proteolytic functions of the proteasome. Arch Biochem Biophys 415:1–5
Kitiphongspattana K, Mathews CE, Leiter EH, Gaskins HR 2005 Proteasome inhibition alters glucose-stimulated (pro)insulin secretion and turnover in pancreatic -cells. J Biol Chem 280:15727–15734
Starace D, Riccioli A, D’Alessio A, Giampietri C, Petrungaro S, Galli R, Filippini A, Ziparo E, De Cesaris P 2005 Characterization of signaling pathways leading to Fas expression induced by TNF-: pivotal role of NF-B. FASEB J 19:473–476
Usami H, Kusano Y, Kumagai T, Osada S, Itoh K, Kobayashi A, Yamamoto M, Uchida K 2005 Selective induction of a tumor marker glutathione S-transferase P1 by proteasome inhibitors. J Biol Chem 280:25267–25276
Zheng W, Zhang Z, Ganguly S, Weller JL, Klein DC, Cole PA 2003 Cellular stabilization of the melatonin rhythm enzyme induced by nonhydrolyzable phosphonate incorporation. Nat Struct Biol 10:1054–1057
Ganguly S, Weller JL, Ho A, Chemineau P, Malpaux B, Klein DC 2005 Melatonin synthesis: 14-3-3-dependent activation and inhibition of arylalkylamine N-acetyltransferase mediated by phosphoserine-205. Proc Natl Acad Sci USA 102:1222–1227
Gastel JA, Roseboom PH, Rinaldi PA, Weller JL, Klein DC 1998 Melatonin production: proteasomal proteolysis in serotonin N-acetyltransferase regulation. Science 279:1358–1360
Buda M, Klein DC 1978 A suspension culture of pinealocytes: regulation of N-acetyltransferase activity. Endocrinology 103:1483–1493
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Price DM, Chik CL, Ho AK 2004 Norepinephrine induction of mitogen-activated protein kinase phosphatase-1 expression in rat pinealocytes: distinct roles of - and -adrenergic receptors. Endocrinology 145:5723–5733
Price DM, Chik CL, Terriff D, Weller J, Humphries A, Carter DA, Klein DC, Ho AK 2004 Mitogen-activated protein kinase phosphatase-1 (MKP-1): >100-fold nocturnal and norepinephrine-induced changes in the rat pineal gland. FEBS Lett 577:220–226
Shibatani T, Ward WF 1995 Sodium dodecyl sulfate (SDS) activation of the 20S proteasome in rat liver. Arch Biochem Biophys 321:160–166
Ho AK, Price L, Mackova M, Chik CL 2001 Potentiation of cyclic AMP and cyclic GMP accumulation by p38 mitogen-activated protein kinase (p38MAPK) inhibitors in rat pinealocytes. Biochem Pharmacol 62:1605–1611
Man JR, Rustaeus S, Price DM, Chik CL, Ho AK 2004 Inhibition of p38 mitogen-activated protein kinase enhances adrenergic-stimulated arylalkylamine N-acetyltransferase activity in rat pinealocytes. Endocrinology 145:1167–1174
Klein DC, Buda MJ, Kapoor CL, Krishna G 1978 Pineal serotonin N-acetyltransferase activity: abrupt decrease in adenosine 3',5'-monophosphate may be signal for "turnoff." Science 199:309–311
Ganguly S, Gastel JA, Weller JL, Schwartz C, Jaffe H, Namboodiri MA, Coon SL, Hickman AB, Rollag M, Obsil T, Beauverger P, Ferry G, Boutin JA, Klein DC 2001 Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis. Proc Natl Acad Sci USA 98:8083–8088
Kisselev AF, Goldberg AL 2001 Proteasome inhibitors: from research tools to drug candidates. Chem Biol 8:739–758
Meiners S, Heyken D, Weller A, Ludwig A, Stangl K, Kloetzel PM, Kruger F 2003 Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of mammalian proteasomes. J Biol Chem 278:21517–21525
Stehle JH, Foulkes NS, Molina CA, Simonneaux V, Pevet P, Sassone-Corsi P 1993 Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland. Nature 365:314–320
Maronde E, Pfeffer M, Olcese J, Molina CA, Schlotter F, Dehghani F, Korf HW, Stehle JH 1999 Transcription factors in neuroendocrine regulation: rhythmic changes in pCREB and ICER levels frame melatonin synthesis. J Neurosci 19:3326–3336
Blendy JA, Kaestner KH, Weinbauer GF, Nieschlag E, Schutz G 1996 Severe impairment of spermatogenesis in mice lacking the CREM gene. Nature 380:162–165
Foulkes NS, Borjigin J, Snyder SH, Sassone-Corsi P 1996 Transcriptional control of circadian hormone synthesis via the CREM feedback loop. Proc Natl Acad Sci USA 93:14140–14145
Kell CA, Dehghani F, Wicht H, Molina CA, Korf HW, Stehle JH 2004 Distribution of transcription factor inducible cyclic AMP early repressor (ICER) in rodent brain and pituitary. J Comp Neurol 478:379–394
Folco EJ, Koren G 1997 Degradation of the inducible cAMP early repressor (ICER) by the ubiquitin-proteasome pathway. Biochem J 328:37–43
Link WA, Ledo F, Torres B, Palczewska M, Madsen TM, Savignac M, Albar JP, Mellstrom B, Naranjo JR 2004 Day-night changes in downstream regulatory antagonist modulator/potassium channel interacting protein activity contribute to circadian gene expression in pineal gland. J Neurosci 24:5346–5355
Foulkes NS, Whitmore D, Sassone-Corsi P 1997 Rhythmic transcription: the molecular basis of circadian melatonin synthesis. Biol Cell 89:487–494
Molina CA, Foulkes NS, Lalli E, Sassone-Corsi P 1993 Inducibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response repressor. Cell 75:875–886(David L. Terriff, Constan)
Abstract
In this study, we investigated the effect of proteasomal inhibition on the induction of arylalkylamine-N-acetyltransferase (AA-NAT) enzyme in cultured rat pinealocytes, using two proteasome inhibitors, MG132 and clastolactacystin -lactone (c-lact). Addition of c-lact or MG132 3 h after norepinephrine (NE) stimulation produced a significant increase in AA-NAT protein level and enzyme activity. However, when the proteasome inhibitors were added before or together with NE, significant reductions of the NE-induced aa-nat mRNA, protein, and enzyme activity were observed. A similar inhibitory effect of MG132 on aa-nat transcription was observed when cells were stimulated by dibutyryl cAMP, indicating an effect distal to a post-cAMP step. The inhibitory effect of MG132 on adrenergic-induced aa-nat transcription was long lasting because it remained effective after 14 h of washout and appeared specific for aa-nat because the induction of another adrenergic-regulated gene, MAPK phosphatase-1, by NE was not affected. Time-profile studies revealed that the inhibitory effect of MG132 on NE-stimulated aa-nat induction was detected after 1 h, suggesting accumulation of a protein repressor as a possible mechanism of action. This possibility was also supported by the finding that the inhibitory effect of c-lact on NE-induced aa-nat induction was markedly reduced by cycloheximide, a protein synthesis inhibitor. Together, these results support an important role of proteasomal proteolysis in the adrenergic-mediated induction of aa-nat transcription through its effect on a protein repressor.
Introduction
IN THE MAMMALIAN pineal gland, the nightly release of norepinephrine (NE) from the sympathetic neurons stimulates both 1- and 1-adrenergic receptors resulting in a 100-fold increase in intracellular cAMP levels (1, 2, 3). The rise in cAMP, in turn, stimulates cAMP-dependent protein kinase, which translocates to the nucleus and phosphorylates the transcription factor cAMP response element binding protein (4). Phosphorylated cAMP response element binding protein binds to cAMP response elements in the promoter region of cAMP-regulated genes and causes activation of transcription (5, 6, 7). This activation of transcription results in a 150-fold increase in the mRNA of arylalkylamine-N-acetyltransferase (AA-NAT), the rate-controlling enzyme in the production of melatonin (MT) (8, 9).
The ubiquitin-proteasome degradation pathway is known to play a critical role in regulating many cellular processes such as the cell cycle, cell differentiation, modulation of cell surface receptors and ion channels, regulation of the immune and inflammatory responses, DNA repair, and regulation of transcription factors (10). The 26S proteasome is a multimeric protease complex made up of a proteolytic 20S core particle and capped at one or both ends by 19S regulatory particles (11, 12). Although the 26S proteasome is responsible for both ubiquitin-dependent and -independent protein degradation, the 20S proteasome functions only in ubiquitin-independent protein degradation (13). Studies of proteasomal proteolysis on regulation of cellular processes such as hormone synthesis and cytokine signaling mechanism have been facilitated by the use of specific proteasome inhibitors such as clastolactacystin -lactone (c-lact) and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) (14, 15). Selective induction of a tumor marker gene, glutathione S-transferase P1 by c-lact and MG132 has recently been demonstrated in hepatocellular carcinoma cells (16).
In the rat pineal gland, the steady-state level of AA-NAT protein, besides transcriptional and translational control, is also regulated by proteasome-mediated degradation (17, 18). In stimulated pinealocytes, AA-NAT is phosphorylated by cAMP-dependent protein kinase and interacts with the protein 14-3-3 (18). This association helps to protect AA-NAT from proteasomal proteolysis and increases the enzyme activity. However, at the end of the dark phase, AA-NAT becomes dephosphorylated and is targeted for degradation by the proteasome (19). Although the effect of the proteasome on the steady-state levels of AA-NAT protein have been well established (19), the potential role of the proteasome in modulating the transcription and translation of the aa-nat gene has yet to be investigated. In this study, by using two structurally unrelated specific proteasome inhibitors, MG132 and c-lact, we determined the effects of inhibiting the proteasome on adrenergic induction of aa-nat transcription and compared their effects on AA-NAT protein levels, enzyme activity, and MT production in cultured rat pinealocytes. Our results indicate that proteasomes play an important role in the NE-induced aa-nat transcription.
Materials and Methods
Materials
NE, cycloheximide, dibutyryl cAMP (DbcA) were obtained from Sigma Aldrich Co. (St. Louis, MO). c-lact, MG132, and N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin were obtained from Biomol Co. (Plymouth Meeting, PA). [3H]Acetyl-coenzyme (specific activity, 1 mCi/mmol) was from Amersham Biosciences (Piscataway, NJ). [3H]MT was obtained from NEN Life Science Products (Boston, MA). Polyclonal antibodies against MAPK phosphatase-1 (MKP-1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody against glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) antibody was obtained from Ambion Inc. (Austin, TX). Polyclonal antibodies for the RIA of MT were obtained from CID Tech Co. (Mississauga, Ontario, Canada). 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.
Preparation of cultured pinealocytes and drug treatments
All procedures were reviewed and approved by the Health Sciences Animal Policy and Welfare Committee of the University of Alberta (Edmonton, Alberta). Sprague Dawley rats (male; weighing 150 g) were obtained from the University of Alberta animal unit. Pinealocytes were prepared by papain dissociation of freshly dissected rat pineal glands as described previously (20). Cells were suspended in DMEM containing 10% fetal calf serum and maintained before the experiment at 37 C for 24 h in a mixture of 95% air-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). Pinealocyte total RNAs were isolated using Trizol (Invitrogen Co., Carlsbad, CA) according to manufacturer’s instruction. Samples for Western blot analysis were solubilized in 1x sample buffer by boiling for 5 min and stored at –20 C until electrophoresis. The homogenization buffer contained 20 mM Tris-HCl; 2 mM EDTA; 0.5 mM EGTA; 2 mM phenylmethylsulfonyl fluoride; 1 μg/ml each of aprotinin, leupeptin, and pepstatin; 1 mM sodium orthovanadate; and 1 mM sodium fluoride (pH 7.5). Samples for the determination of AA-NAT activity was immediately frozen in dry ice and stored at –75 C. Media were collected for MT determination.
Western blotting
SDS-PAGE was performed according to the procedure of Laemmli (21) using 10 or 12% acrylamide (Mini-Protein II gel system, Bio-Rad Laboratories, Hercules, CA). After electrophoresis, gels were equilibrated for 20 min in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol). Proteins were transferred onto polyvinylidene difluoride membranes (1.5 h, 45 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 1.5 h. The blots were then incubated overnight at 4 C with diluted specific antisera as indicated. After washing three times with the blocking solution, the blots were incubated with diluted horseradish peroxidase-conjugated second antibodies (Bio-Rad) for 1.5 h at room temperature. They were then washed extensively and developed using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).
RT-PCR
First-strand cDNA was synthesized from the isolated RNA using an Omniscript reverse-transcriptase kit (QIAGEN Inc., Valencia, CA) with an oligo-dT primer. For cell extracts, 3 μl of a 1:10 dilution of cDNA was used. PCR was performed in a 29.3-μl reaction mixture containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 100 μM of each dNTP, 0.75 U Taq polymerase (PerkinElmer Cetus, Emeryville, CA), and 1 μM each of the two primers. PCRs were as follows: denaturing for 1 min at 94 C, annealing for 1 min at 63 C, and extension for 1 min at 72 C. Initial denaturing and final extension were both 5 min in duration. Cycle numbers varied between cell preparations, but in general, 23 cycles were used to amplify aa-nat, 25 cycles for mkp-1, and 22 for gapdh mRNAs. All reaction sets included water blanks as negative controls. Amplified products were separated on ethidium bromide-stained 1.5% agarose gels. PCR products were confirmed by sequencing. All primer sequences were designed using rat gene cDNA sequences obtained from BLAST and primers were selected from 3' regions as outlined elsewhere (22). Primers used (23) are as follows: aa-nat, upstream primer, 5'-GGT TCA CTT TGG GAC AAG GA-3'; downstream primer, 5'-GTG GCA CCG TAA GGA ACA TT-3'; mkp-1, upstream primer, 5'-CTG CTT TGA TCA ACG TCT CG-3'; downstream primer, 5'-AAG CTG AAG TTG GGG GAG AT-3'. Sequences of the gapdh primers used were previously described (23).
20S proteasome activity assay
Pelleted pinealocytes were frozen in dry ice and stored overnight at –75 C. The following day they were lysed by sonication in 40 μl of lysis buffer (PBS, 1% Triton X-100, 1 mM dithiothreitol). The lysates were centrifuged for 5 min at 12,000 x g and the supernatant was used for the assay. In a 96-well BD Optilux plate (BD Biosciences, Mississauga, Ontario, Canada), 40 μl of the crude cell lysate were combined with 10 μl of assay buffer [25 mM HEPES, 1 mM EDTA (pH 7.6), and 0.03% sodium dodecyl sulfate], 40 μl water, and 10 μl Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin to give a final concentration of 62.5 μM (24). The reaction was placed in a 37 C incubator in a mixture of 95% air-5% CO2 for 2 h. Fluorescence was measured using a Thermo Electron Fluorescent plate reader (380/460 nm filter set). Cell lysate background readings were prepared by adding 20 μM c-lact to lysed cells 30 min before beginning the assay. Proteasome activity was expressed as picomoles of AMC produced per hour per 105 cells.
AA-NAT assay
AA-NAT activity was determined as described previously (25). Briefly, treated pinealocytes were stored frozen in dry ice until homogenization in a reaction mixture of 0.1 M phosphate buffer (pH 6.8) containing 30 nmol [3H]acetyl coenzyme A (specific activity 1 mCi/mmol) and 1 μmol tryptamine hydrochloride in a final volume of 60 μl. The reaction mixture was incubated at 37 C for 1 h. At the end of the incubation period, the reaction was stopped by the addition of 1 ml of methylene chloride. After vortexing, the aqueous phase was removed, and the organic phase was washed three times with 0.1 M phosphate buffer (pH 6.8). The organic phase was transferred to a scintillation vial, evaporated to dryness, and the radioactive acetylated product determined by scintillation counting. AA-NAT activity was expressed as nanomoles per hour per 105 cells.
MT assay
Briefly, medium MT was extracted from 300 μl of medium by vortexing with 1 ml methylene chloride. After centrifugation, the organic phase was collected and evaporated to dryness. The residue was reconstituted in 500 μl of assay buffer [0.01 M phosphate buffer (pH 6.5) containing 0.1% gelatin]. The recovery of medium MT was more than 98%. The extracted MT was assayed by a RIA as described previously (26).
Results and statistical analysis
For quantitation of RT-PCR analyses, gel images were acquired with Kodak 1-D software on a Kodak 2000R imaging station. For analyses of Western blots, exposed films were scanned and band densitometry of acquired images was performed with Kodak 1-D software. Densitometric values were normalized to percent maximal and presented as the mean ± SEM from at least three independent experiments. For RIA or radioenzymatic assays, data were presented as the mean ± SEM from at least three independent experiments. Statistical analysis involved either a paired t test or ANOVA with the Newman-Keuls test. Statistical significance was set at P < 0.05.
Results
Time-dependent effects of proteasomes in adrenergic induction of aa-nat transcription
Treatment of cultured pinealocytes with NE (3 μM) for 6 h caused a large increase in NE-stimulated aa-nat mRNA (Fig. 1A) and protein levels (Fig. 1B). Compared with cells stimulated with NE alone for 6 h, exposure to a proteasome inhibitor, c-lact (10 μM), for the last 3 h of NE treatment caused a further increase in AA-NAT protein (Fig. 1B) and enzyme activity (Fig. 1C) as reported previously (19) but had no significant effect on the level of aa-nat mRNA (Fig. 1A). In contrast, treatment of pinealocytes with c-lact (10 μM) and NE for 3 h followed by an additional 3 h treatment with NE alone caused a reduction in the level of NE-stimulated aa-nat mRNA (Fig. 1A), protein (Fig. 1B), and enzyme activity (Fig. 1C). A similar inhibition of aa-nat transcription was observed when pinealocytes were treated with MG132 (3 μM), a structurally unrelated proteasome inhibitor to c-lact, for the first 3 h of a 6-h treatment with NE (Fig. 2). These results indicate that proteasomal inhibition can significantly reduce NE stimulated aa-nat transcription during the early phase of NE stimulation.
Treatment with MG132 preceding NE stimulation inhibits adrenergic-stimulated aa-nat induction and MT synthesis
To determine the effect of pretreatment with a proteasome inhibitor on adrenergic-stimulated aa-nat transcription, pinealocytes were treated with MG132 (3 μM) for 3 h, followed by washout of MG132 by replacing the cell media before stimulation with NE (3 μM) for another 3 h. Levels of aa-nat mRNA were significantly reduced when the cells were pretreated with MG132 (Fig. 3). To characterize the effects of proteasome inhibitors on MT synthesis, pinealocytes were pretreated with MG132 (3 μM) or c-lact (10 μM) for 3 h, followed by washout of inhibitors by replacing the cell media before stimulation with NE (1 μM) for another 14 h. Pretreatment with MG132 or c-lact caused a significant reduction in NE-stimulated AA-NAT activity and MT production (Fig. 4). These results suggest treatment with MG132 before NE stimulation is effective in inhibiting NE-stimulated aa-nat induction and MT synthesis. Moreover, proteasome inhibitors appear to have a long-lasting effect on MT synthesis.
Effect of proteasomal inhibition on adrenergic-induced transcription is long lasting and appears specific for aa-nat
To characterize the effect of proteasomal inhibition on adrenergic-induced transcription, pinealocytes were pretreated for 3 h with MG132 (3 μM), followed by replacement with new media that did not contain MG132 and incubation for an additional 14 h before stimulation with NE for 3 h. Pretreatment with MG132 followed by 14 h of washout remained effective in reducing NE-stimulated increases in aa-nat mRNA and protein levels (Fig. 5). In contrast, when the expression of mkp-1, another adrenergic-regulated gene in the rat pineal gland was determined, NE was effective in stimulating mkp-1 transcription in pinealocytes subjected to pretreatment with MG132 followed by 14 h of washout (Fig. 5). Of interest, there was also an increase in basal and adrenergic-stimulated mkp-1 transcription (Fig. 5). These results suggest that the effect of MG132 on adrenergic-induced transcription is long lasting because inhibition of aa-nat transcription is still observed after 14 h of washout. In addition, the inhibition of transcription by MG132 appears specific for aa-nat because it has little effect on the expression of another adrenergic-driven gene, mkp-1.
To confirm the long-lasting effects of the proteasome inhibitors, proteasomal activity in cultured pinealocytes was determined after treatment with MG 132 (3 μM) or c-lact (10 μM) for 3 h followed by 14 h of washout. Under this treatment condition, proteasomal activity in pinealocytes was reduced to 45% and 10% of control cells by MG132 and c-lact, respectively (Fig. 6).
To further characterize the effects of proteasomal inhibition on adrenergic-induced mkp-1 transcription, cultured pinealocytes were subjected to treatment with NE for 6 h in combination with c-lact (10 μM) using the identical treatment protocol as described for Fig. 1. Treatment of cultured pinealocytes with NE (3 μM) for 6 h caused a large increase in NE-stimulated mkp-1 mRNA level (Fig. 7). Compared with cells stimulated with NE alone for 6 h, exposure to a proteasome inhibitor, c-lact (10 μM), for the first 3 h or the last 3 h of 6 h treatment with NE had no significant effect on the level of mkp-1 mRNA (Fig. 7). These results indicate that, unlike its inhibitory effect on aa-nat transcription on the early phase of NE stimulation, proteasomal inhibition had no effect on NE stimulated mkp-1 transcription during the early or late phase of NE stimulation.
Effect of MG132 on the time profile of NE-stimulated aa-nat transcription
To determine the time period required for proteasome inhibitors to reduce adrenergic-induced aa-nat transcription, cells were treated simultaneously with NE and MG132 for 1, 3, and 5 h. Treatment with MG132 for 1 h had no effect on NE-stimulated aa-nat mRNA level (Fig. 8). However, after 1 h of treatment with NE alone, aa-nat mRNA levels continued to increase, whereas treatment with MG132 prevented any further increase in NE-stimulated aa-nat mRNA levels (Fig. 8B). After 5 h of treatment with MG132 and NE, there was a 65% reduction in aa-nat mRNA level, compared with cells stimulated with NE alone. This suggests that a lag time of 1 h is required by the proteasome inhibitor to demonstrate repression of aa-nat transcription.
To confirm the requirement of a 1-h lag time for the inhibition of aa-nat transcription, a second time-course study was performed in which pinealocytes were pretreated with MG132 (3 μM) for 1 h before NE stimulation. Pretreatment with MG132 reduced NE-stimulated aa-nat mRNA levels at 1 and 5 h by 50 and 75%, respectively (Fig. 8B). This result indicates that a 1-h pretreatment is adequate to demonstrate the inhibitory effect of MG132 on aa-nat transcription.
A post-cAMP step mediates the effect of proteasomal inhibition on adrenergic-stimulated aa-nat transcription
To determine the step by which a proteasome inhibitor exerts its effect on adrenergic induction of aa-nat transcription, pinealocytes were pretreated with MG132 (3 μM) for 3 h, followed by 14 h of washout before cells were stimulated for 3 h with NE (3 μM) or DbcA (1 mM), a cell-permeable cAMP analog. Pretreatment with MG132 caused a significant reduction in both aa-nat mRNA and protein levels in cells that were stimulated with either NE or DbcA (Fig. 9). These results indicate that the inhibitory effect of MG132 on aa-nat transcription is mediated at a post-cAMP step.
Protein synthesis is involved in the proteasomal inhibitor effects on NE-stimulated aa-nat transcription
Considering the primary effect of proteasome inhibitors, a lag time for the demonstration of inhibition of aa-nat transcription suggests accumulation of a protein repressor. To examine whether inhibition of aa-nat transcription by proteasome inhibitors was due to accumulation of a protein, pinealocytes were treated with a protein synthesis inhibitor, cycloheximide, in the presence or absence of c-lact for 3 h before the addition of NE. Treatment with cycloheximide (30 μg/ml) had an enhancing effect on NE-stimulated aa-nat mRNA (Fig. 10). The inhibitory effect of c-lact (10 μM) on NE-stimulated aa-nat transcription was abolished by cotreatment with cycloheximide (Fig. 10). In the presence of cycloheximide (30 μg/ml), NE did not cause an increase in AA-NAT protein after 6 h (data not shown). This result suggests that inhibition of aa-nat transcription is due to accumulation of a protein repressor, and the inhibitory effect can be abolished by inhibition of protein synthesis.
Discussion
A major factor regulating rhythmic and light-induced changes in AA-NAT activity is the steady-state level of AA-NAT protein, which represents a balance of synthesis and degradation. Synthesis of AA-NAT protein is a reflection of adrenergic cAMP aa-nat mRNA levels, which exhibits a nearly 100-fold nocturnal increase in the rat pineal gland (9). Based on in vitro studies of rat pineal glands, in addition to transcriptional control, proteasomal proteolysis also plays an important role in the circadian and photic regulation of AA-NAT activity (19). In the absence of cAMP, AA-NAT is destroyed by proteasomal proteolysis (27, 28). Using cultured pinealocytes, we now show that proteasomal proteolysis not only regulates AA-NAT protein but also plays an important role in the adrenergic induction of aa-nat transcription through its effect on transcription repression.
The time-dependent effect of treatment with the proteasome inhibitor on adrenergic-induced transcription of aa-nat is of interest. Inhibition of the proteasome before or concurrent with NE stimulation causes a significant reduction in aa-nat mRNA and protein levels. In contrast, when the proteasome is inhibited after stimulation with NE, it enhances the adrenergic-stimulated AA-NAT protein levels as reported in a previous study (19) without having an effect on aa-nat mRNA. Moreover, similar time-dependent effects of the proteasome are obtained with two structurally unrelated proteasome inhibitors, MG132 and c-lact. Taken together, our results indicate that these time-dependent effects of proteasome inhibitors are mediated through inhibition of transcription of aa-nat mRNA. Whether proteasomes exert an enhancing or inhibitory effect on adrenergic-regulated AA-NAT protein levels are critically dependent on the time of exposure to proteasomes.
Our results also provide evidence on the step that mediates the effects of proteasomes on adrenergic-induced aa-nat transcription. Because inhibition of the proteasome by MG132 causes a similar inhibition of NE- or DbcA-stimulated aa-nat transcription, the proteasome inhibitor is likely acting at a post-cAMP step distal to the adrenergic receptor.
Treatment with the proteasome inhibitor MG132 also has a long-lasting effect on adrenergic-stimulated AA-NAT induction that remains after 14 h of washout. The sustained effect is confirmed using a proteasome activity assay in rat pinealocytes, which shows persistent inhibition of the proteasome activity, with c-lact causing a greater inhibition than MG132. This difference in inhibition of proteasomal activity between MG132 and c-lact could be related to the concentration of the inhibitors used, or alternatively, partial recovery of the inhibition by MG132, a reversible inhibitor (29). Moreover, our results also suggest a slow turnover of proteasomes in rat pinealocytes because de novo synthesis of functional proteasomes in the cell should lead to a reversal of inhibition of adrenergic-induced aa-nat transcription. In other mammalian cells, proteasomal inhibition causes an enhanced expression of proteasome genes and de novo proteasome biogenesis within 6 h of treatment with the inhibitors (30).
It is also of interest to note that this long-lasting effect of proteasomes on aa-nat transcription is not a general effect that applies to all adrenergic-regulated genes because mkp-1, another adrenergic-regulated gene, is not affected. These results exclude MG132 having a toxic effect on pinealocytes that precludes recovery of adrenergic-regulated gene transcription. The selective effect of MG132 on gene transcription also suggests a difference in the adrenergic regulation of aa-nat and mkp-1 (22) or alternatively a difference in their stability and transcription efficiency. A difference in the stability or transcription efficiency between aa-nat and mkp-1 is supported by the finding that treatment with MG132 alone causes a selective increase in basal mkp-1 mRNA and protein levels.
The effect of proteasomes on aa-nat transcription appears to be mediated by the accumulation of a protein repressor. This is suggested by our observation that a lag time of 1 h is required by the proteasome inhibitor to express inhibition of aa-nat transcription. Moreover, the effect of proteasome inhibition on aa-nat transcription can be abolished by treatment with cycloheximide, a protein synthesis inhibitor. In view of the enhancing effects of proteasome inhibitors on adrenergic-stimulated AA-NAT protein levels when the inhibitors are added 3 h after NE treatment, it is possible that the protein repressor is also under proteasomal regulation. At present, the repressor involved is a subject of speculation. One possible candidate for this protein repressor is induced cAMP early repressor (ICER) (31). Previous studies suggest that ICER may be involved in repressing AA-NAT transcription at the end of the night because ICER is expressed diurnally and binds to cAMP response elements sites (32, 33, 34, 35). Moreover, steady-state levels of ICER are believed to be regulated by both protein synthesis and degradation by the proteasome (36). However, our results indicate that the inhibitory effect of proteasomal inhibition on aa-nat transcription does not require continuous stimulation with NE. Another candidate repressor is downstream regulatory element antagonist modulator, a transcription repressor with established circadian rhythmicity in the rat pineal gland (37).
Inducible genes are under the control of both activating and repressive signals (38, 39). A tight balance between repression and activation of transcription likely regulates the amplitude and duration of gene expression. In the rhythmic transcription of aa-nat, our results indicate that inhibiting proteasomes causes a significant reduction in adrenergic regulation of aa-nat transcription. Moreover, a protein repressor under the regulation of proteasomes appears to be responsible for suppressing adrenergic-mediated transcriptional induction of aa-nat.
Footnotes
This work was supported by grants from the Canadian Institutes of Health Research. D.L.T. was supported by a Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council.
Abbreviations: AA-NAT, Arylalkylamine-N-acetyltransferase; c-lact.,clastolactacystin -lactone; DbcA, dibutyryl cAMP, GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; ICER, inducible cAMP early repressor; MG132, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; MKP-1, mitogen-activated protein kinase phosphatase-1; MT, melatonin; NE, norepinephrine.
References
Klein DC, Sugden D, Weller JL 1983 Postsynaptic -adrenergic receptors potentiate the -adrenergic stimulation of pineal serotonin N-acetyltransferase. Proc Natl Acad Sci USA 80:599–603
Klein DC, Berg GR, Weller J 1970 Melatonin synthesis: adenosine 3',5'-monophosphate and norepinephrine stimulate N-acetyltransferase. Science 168:979–980
Klein DC, Berg GR 1970 Pineal gland: stimulation of melatonin production by norepinephrine involves cyclic AMP-mediated stimulation of N-acetyltransferase. Adv Biochem Psychopharmacol 3:241–263
Roseboom PH, Klein DC 1995 Norepinephrine stimulation of pineal cyclic AMP response element-binding protein phosphorylation: primary role of a -adrenergic receptor/cyclic AMP mechanism. Mol Pharmacol 47:439–449
Baler R, Covington S, Klein DC 1997 The rat arylalkylamine N-acetyltransferase gene promoter. cAMP activation via a cAMP-responsive element-CCAAT complex. J Biol Chem 272:6979–6985
Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH 1986 Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci USA 83:6682–6686
Bisotto S, Minorgan S, Rehfuss RP 1996 Identification and characterization of a novel transcriptional activation domain in the CREB-binding protein. J Biol Chem 271:17746–17750
Borjigin J, Wang MM, Snyder SH 1995 Diurnal variation in mRNA encoding serotonin N-acetyltransferase in pineal gland. Nature 378:783–785
Roseboom PH, Coon SL, Baler R, McCune SK, Weller JL, Klein DC 1996 Melatonin synthesis: analysis of the more than 150-fold nocturnal increase in serotonin N-acetyltransferase messenger ribonucleic acid in the rat pineal gland. Endocrinology 137:3033–3045
Ciechanover A, Orian A, Schwartz AL 2000 Ubiquitin-mediated proteolysis: biological regulation via destruction. Bioessays 22:442–451
Voges D, Zwickl P, Baumeister W 1999 The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem 68:1015–1068
Groll D, Bajorek M, Kohler A, Moroder L, Rubin DM, Huber R, Glickman MH, Finley 2000 A gated channel into the proteasome core particle. Nat Struct Biol 7:1062–1067
Orlowski M, Wilk S 2003 Ubiquitin-independent proteolytic functions of the proteasome. Arch Biochem Biophys 415:1–5
Kitiphongspattana K, Mathews CE, Leiter EH, Gaskins HR 2005 Proteasome inhibition alters glucose-stimulated (pro)insulin secretion and turnover in pancreatic -cells. J Biol Chem 280:15727–15734
Starace D, Riccioli A, D’Alessio A, Giampietri C, Petrungaro S, Galli R, Filippini A, Ziparo E, De Cesaris P 2005 Characterization of signaling pathways leading to Fas expression induced by TNF-: pivotal role of NF-B. FASEB J 19:473–476
Usami H, Kusano Y, Kumagai T, Osada S, Itoh K, Kobayashi A, Yamamoto M, Uchida K 2005 Selective induction of a tumor marker glutathione S-transferase P1 by proteasome inhibitors. J Biol Chem 280:25267–25276
Zheng W, Zhang Z, Ganguly S, Weller JL, Klein DC, Cole PA 2003 Cellular stabilization of the melatonin rhythm enzyme induced by nonhydrolyzable phosphonate incorporation. Nat Struct Biol 10:1054–1057
Ganguly S, Weller JL, Ho A, Chemineau P, Malpaux B, Klein DC 2005 Melatonin synthesis: 14-3-3-dependent activation and inhibition of arylalkylamine N-acetyltransferase mediated by phosphoserine-205. Proc Natl Acad Sci USA 102:1222–1227
Gastel JA, Roseboom PH, Rinaldi PA, Weller JL, Klein DC 1998 Melatonin production: proteasomal proteolysis in serotonin N-acetyltransferase regulation. Science 279:1358–1360
Buda M, Klein DC 1978 A suspension culture of pinealocytes: regulation of N-acetyltransferase activity. Endocrinology 103:1483–1493
Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Price DM, Chik CL, Ho AK 2004 Norepinephrine induction of mitogen-activated protein kinase phosphatase-1 expression in rat pinealocytes: distinct roles of - and -adrenergic receptors. Endocrinology 145:5723–5733
Price DM, Chik CL, Terriff D, Weller J, Humphries A, Carter DA, Klein DC, Ho AK 2004 Mitogen-activated protein kinase phosphatase-1 (MKP-1): >100-fold nocturnal and norepinephrine-induced changes in the rat pineal gland. FEBS Lett 577:220–226
Shibatani T, Ward WF 1995 Sodium dodecyl sulfate (SDS) activation of the 20S proteasome in rat liver. Arch Biochem Biophys 321:160–166
Ho AK, Price L, Mackova M, Chik CL 2001 Potentiation of cyclic AMP and cyclic GMP accumulation by p38 mitogen-activated protein kinase (p38MAPK) inhibitors in rat pinealocytes. Biochem Pharmacol 62:1605–1611
Man JR, Rustaeus S, Price DM, Chik CL, Ho AK 2004 Inhibition of p38 mitogen-activated protein kinase enhances adrenergic-stimulated arylalkylamine N-acetyltransferase activity in rat pinealocytes. Endocrinology 145:1167–1174
Klein DC, Buda MJ, Kapoor CL, Krishna G 1978 Pineal serotonin N-acetyltransferase activity: abrupt decrease in adenosine 3',5'-monophosphate may be signal for "turnoff." Science 199:309–311
Ganguly S, Gastel JA, Weller JL, Schwartz C, Jaffe H, Namboodiri MA, Coon SL, Hickman AB, Rollag M, Obsil T, Beauverger P, Ferry G, Boutin JA, Klein DC 2001 Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis. Proc Natl Acad Sci USA 98:8083–8088
Kisselev AF, Goldberg AL 2001 Proteasome inhibitors: from research tools to drug candidates. Chem Biol 8:739–758
Meiners S, Heyken D, Weller A, Ludwig A, Stangl K, Kloetzel PM, Kruger F 2003 Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of mammalian proteasomes. J Biol Chem 278:21517–21525
Stehle JH, Foulkes NS, Molina CA, Simonneaux V, Pevet P, Sassone-Corsi P 1993 Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland. Nature 365:314–320
Maronde E, Pfeffer M, Olcese J, Molina CA, Schlotter F, Dehghani F, Korf HW, Stehle JH 1999 Transcription factors in neuroendocrine regulation: rhythmic changes in pCREB and ICER levels frame melatonin synthesis. J Neurosci 19:3326–3336
Blendy JA, Kaestner KH, Weinbauer GF, Nieschlag E, Schutz G 1996 Severe impairment of spermatogenesis in mice lacking the CREM gene. Nature 380:162–165
Foulkes NS, Borjigin J, Snyder SH, Sassone-Corsi P 1996 Transcriptional control of circadian hormone synthesis via the CREM feedback loop. Proc Natl Acad Sci USA 93:14140–14145
Kell CA, Dehghani F, Wicht H, Molina CA, Korf HW, Stehle JH 2004 Distribution of transcription factor inducible cyclic AMP early repressor (ICER) in rodent brain and pituitary. J Comp Neurol 478:379–394
Folco EJ, Koren G 1997 Degradation of the inducible cAMP early repressor (ICER) by the ubiquitin-proteasome pathway. Biochem J 328:37–43
Link WA, Ledo F, Torres B, Palczewska M, Madsen TM, Savignac M, Albar JP, Mellstrom B, Naranjo JR 2004 Day-night changes in downstream regulatory antagonist modulator/potassium channel interacting protein activity contribute to circadian gene expression in pineal gland. J Neurosci 24:5346–5355
Foulkes NS, Whitmore D, Sassone-Corsi P 1997 Rhythmic transcription: the molecular basis of circadian melatonin synthesis. Biol Cell 89:487–494
Molina CA, Foulkes NS, Lalli E, Sassone-Corsi P 1993 Inducibility and negative autoregulation of CREM: an alternative promoter directs the expression of ICER, an early response repressor. Cell 75:875–886(David L. Terriff, Constan)