This Article Abstract Full Text (PDF) All Versions of this Article: 148/2/743    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ho, A. K. Articles by Chik, C. L. Search for Related Content PubMed PubMed Citation Articles by Ho, A. K. Articles by Chik, C. L.

The Role of Inducible Repressor Proteins in the Adrenergic Induction of Arylalkylamine-N-Acetyltransferase and Mitogen-Activated Protein Kinase Phosphatase-1 in Rat Pinealocytes

Departments of Physiology (A.K.H., D.L.T., D.M.P., M.T.W.) and Medicine (C.L.C.), 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 Medicine, 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 the role of two inducible repressor proteins, inducible cAMP early repressor (ICER) and Fos-related antigen 2 (Fra-2) in the adrenergic induction of MAPK phosphatase-1 (MKP-1) as compared with their roles in the induction of arylalkylamine-N-acetyltransferase (AA-NAT) in rat pinealocytes. Treatment of pinealocytes with norepinephrine (NE) caused an increase in the mRNA and protein levels of MKP-1 and AA-NAT, as well as in the AA-NAT activity and melatonin production. NE stimulation also caused a simultaneous increase in the mRNA and protein levels of ICER and Fra-2. Transient knockdown of icer using adenovirus expressing small interfering RNA (siRNA) abolished the NE induction of icer expression but had little effect on the NE induction of mkp-1 or aa-nat expression. In contrast, pretreatment with adenovirus overexpressing icer was effective in reducing the NE induction of mkp-1 and aa-nat. The inhibitory effect of overexpressing icer was reversed by cotreatment with siRNA against icer. siRNA against fra-2 also abolished the NE-stimulated expression of fra-2 but had little effect on the NE induction of mkp-1 and aa-nat expression. Proteasomal inhibition, which reduced the NE-stimulated induction of aa-nat, caused a reduction of ICER and Fra-2. Together, these results indicate that whereas overexpression of ICER can suppress the NE induction of aa-nat and mkp-1, the amount of the repressors, ICER and Fra-2, present during NE induction appears insufficient to exert a significant effect in controlling the expression of these genes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
REPRESSOR PROTEINS PLAY a critical role in regulating the temporal profile of gene expression (1). To generate rhythmic gene expression, both gene induction and repression need to be regulated in a temporally defined manner (2). The synthesis of inducible repressor protein in a precise time-dependent manner could limit the duration of gene expression. Existing data indicate that the nocturnal and adrenergic-regulated genes in the rat pineal gland are under this type of coordinated control (2). Whereas several reports have addressed the role of repressor proteins in the adrenergic induction of arylalkylamine N-acetyltransferase (AA-NAT) in the rat pineal gland (3, 4, 5), little is known about their roles in the regulation of other adrenergic-regulated genes.

AA-NAT, the rhythm-generating enzyme for the synthesis of the hormone of darkness, melatonin (MT), is induced at night in response to norepinephrine (NE) stimulation (6). Synthesis of AA-NAT protein reflects adrenergic cAMP aa-nat mRNA levels, which exhibits a more than 100-fold nocturnal increase (7, 8, 9). Analysis of the aa-nat promoter shows that it contains a cAMP response element (CRE) site and an activator protein-1 (AP-1) site (3, 10, 11). Of the known inducible repressors, inducible cAMP early repressor (ICER) can bind to the CRE site, and it has been proposed that a balance between the activator, phosphorylated CRE binding protein (CREB), and the repressor, ICER, controls the transcription of aa-nat (4, 5). Moreover, ICER is induced at night by NE in the pineal gland (3, 4, 5). Another inducible transcriptional repressor in the rat pineal gland is Fos-related antigen 2 (Fra-2), whose expression is also increased at night and induced by NE (10). Although Fra-2 does not appear to be involved in the adrenergic induction of AA-NAT, it is linked to the expression of the genes encoding type II iodothyronine deiodinase and nectadrin (CD24) (12).

Another adrenergic-regulated gene in the rat pineal gland is mapk phosphatase-1 (mkp-1). We have previously shown that mkp-1 is induced by NE in cultured rat pinealocytes (13, 14), and the nocturnal induction of mkp-1 in the rat pineal gland may serve to limit the magnitude and duration of MAPK activation (13, 15, 16, 17, 18). MKP-1, via its effects on MAPK activation, functions to modulate the time profile of MT synthesis (13, 19, 20). Because both mkp-1 and aa-nat are adrenergic-regulated genes, in this study, we investigated these two genes in parallel to analyze whether the effects of ICER and Fra-2 on adrenergic-stimulated transcription are specific to aa-nat or in general.

The involvement of a repressor protein in regulating the adrenergic-stimulated aa-nat transcription is also demonstrated by our previous study with proteasome inhibitors (21). We have shown that addition of the selective proteasome inhibitors, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal or clasto-lactacystin ß-lactone (c-lact) before aa-nat is induced by NE causes an inhibition of NE-stimulated aa-nat mRNA, protein, and enzyme activity (21). Moreover, this effect of proteasomal inhibition on the adrenergic stimulation of aa-nat transcription is abolished by cycloheximide, a protein synthesis inhibitor (21). However, the identity of this repressor protein remains unknown; by determining the sensitivity of the two repressor proteins, ICER and Fra-2, toward proteasomal regulation and by examining the effects of their depletion in pinealocytes, we can evaluate whether these repressors mediate the suppressive effect of proteasomal inhibition on the induction of AA-NAT.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
NE was obtained from Sigma Aldrich Co. (St. Louis, MO) and c-lact was 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 CRE modulator (CREM), Fra-2 and MKP-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody against glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was obtained from Ambion Inc. (Austin, TX). Polyclonal antibodies for the RIA of MT were obtained from CIDTech Co. (Mississauga, Ontario, Canada). Polyclonal antibodies against AA-NAT_25–200 (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 (males, weighing 150 g) were obtained from the University of Alberta animal unit. Pinealocytes were prepared from freshly dissected rat pineal glands using a papain dissociation system from Worthington Biochemical Corp. (Lakewood, NJ). 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 and 5% CO₂ unless otherwise indicated. Aliquots of pinealocytes were treated for the duration indicated with drugs that had been prepared in concentrated solutions in water or dimethylsulfoxide. Treated cells were collected by centrifugation (2 min, 12,000 x g). Pinealocyte total RNAs were isolated using Trizol. 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 were immediately frozen in dry ice and stored at –75 C. Media were collected for MT determination.

Design and construction of short hairpin RNA (shRNA)-expressing adenovirus
Small interfering RNA (siRNA) targets were selected by submitting full-length rat cDNA sequences to the online shRNA design utility of Invitrogen Corp. (Carlsbad, CA). The siRNA targets that produced significant gene silencing were as follows: ICER, GAG CTC CTA CTA CTG CTT TGC; Fra-2, GCA CTT CAA ACC TTG TCT TCA. Single-stranded shRNA oligonucleotide DNA templates were synthesized by Invitrogen’s oligonucleotide synthesis service, annealed to double-stranded form, and ligated into the BLOCK-iT U6 RNAi entry vector (Invitrogen Corp.). Recombinant plasmids were transformed into TOPO cells and harvested with a GenElute Plasmid Miniprep Kit (Sigma). shRNA-encoding inserts in the BLOCK-iT U6 RNAi entry vector were sequenced using the supplied U6 primer on an Applied Biosystems model 373A sequencer in the Molecular Biology Service Unit of the Department of Biological Sciences, University of Alberta. Sequencing reactions were performed using a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences). Inserts retaining the correct sequence were recombined with GATEWAY LR Clonase (Invitrogen) into the pAd/BLOCK-iT adenoviral vector plasmid (Invitrogen); the resulting plasmids were transformed into DH5 cells and harvested with a GenElute Plasmid Miniprep Kit (Sigma). The recombinant adenoviral plasmids were digested with PacI (New England Biolabs, Ipswich, MA) to liberate the linear adenoviral genomic fragment, which was transfected into 293A HEK cells (bearing the E1 region of the adenoviral genome) with Lipofectamine 2000 (Invitrogen).

Adenoviral transduction of pinealocytes
Titered viral stocks were used to transduce DNAs encoding shRNAs or full-length icer into pinealocytes. A transduction protocol was initially developed using a LacZ transgene-bearing adenovirus; pinealocytes were transduced and assayed for LacZ expression with a FluoReporter lacZ/Galactosidase Quantitation Kit (Molecular Probes, Eugene, OR) using a Fluoroskan Ascent Microplate Fluorometer and Ascent software (Thermo Labsystems, Waltham, MA). siRNA-bearing adenovirus or adenovirus expressing full length icer was combined with the pinealocyte medium at a multiplicity of infection of approximately 100 viral particles per cell. Full length icer was generated from a PCR-cloned product obtained by amplification of NE-stimulated pinealocyte cDNA using nucleotide sequence 58–400 of icer (accession no. S66024). This product contains the entire protein coding region of icer. For control transduction, LacZ transgene-bearing adenovirus was used. Pinealocytes were stimulated with NE 40 h after transduction and after stimulation were analyzed by Western blot, RT-PCR, or enzymatic assay. Generally, cultured pinealocytes tolerate adenoviral transduction well, and efficient gene silencing with negligible deleterious effects was observed over a 2 orders-of-magnitude range of virus concentration.

Western blotting and RT-PCR
Sample preparation and SDS-PAGE were performed using 12% acrylamide according to the procedure of Laemmli (22) and as described in our previous studies (13, 14, 16). 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. PCRs performed and primers used were as previously reported (13, 14, 23).

AA-NAT enzymatic activity and MT RIA
AA-NAT activity was determined using 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 (20). Medium MT was extracted from 300 µl of medium by vortexing with 1 ml of methylene chloride. The extracted MT was assayed by an RIA as described previously (20).

Statistical analysis
For quantitation of RT-PCR analyses, digital images of ethidium bromide-stained gels containing DNA mass standards were acquired using the Kodak Electrophoresis Documentation and Analysis System, and the Kodak 1D software was used to optimize lane and band identification on a Kodak 2000R imaging station (Eastman Kodak Co., Rochester, NY) (24). For analyses of Western blots, exposed films were scanned, and band densitometry of acquired images was performed with Kodak 1D software. Densitometric values were normalized to percentage 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of ICERsi on adrenergic-stimulated MKP-1 and AA-NAT induction
To investigate the role of ICER in adrenergic-stimulated gene expression in rat pinealocytes, we generated recombinant adenovirus expressing siRNA complementary to the endogenous icer transcript (ICERsi). For the detection of ICER protein, a polyclonal antibody raised against full-length CREM (Santa Cruz Biotechnology) was used. This antibody can detect both CREM and ICER from the rat pinealocyte. Based on our previous study (13, 14), two time points were chosen to optimize the detection of the maximal MKP-1 (2 h) and AA-NAT (6 h) response. Figure 1 shows immunoblots of pinealocytes transiently transfected with adenovirus expressing ICERsi. In the transfection (LacZ) control cells, NE stimulation caused a small but observable increase in the level of ICER protein after 2 and 6 h of stimulation. Treatment with ICERsi abolished the NE-stimulated increase in ICER protein level in rat pinealocytes. Stimulation with NE (3 µM) caused an increase in MKP-1 and AA-NAT protein levels after 2 h. However, treatment with ICERsi had no significant effect on the NE-stimulated MKP-1 or AA-NAT protein levels at 2 and 6 h (Fig. 1B). ICERsi treatment also had no effect on the levels of GAPDH in the presence or absence of NE. In parallel with their effects on the protein levels of AA-NAT, there was no difference in the time course response of NE-stimulated AA-NAT activity and MT production between control and ICERsi-treated pinealocytes (Fig. 2).

View larger version (32K): [in this window] [in a new window]   FIG. 1. Effect of ICERsi on adrenergic-stimulated AA-NAT, MKP-1, and ICER protein levels. Pinealocytes (1 x 105 cells/0.6 ml) were transiently transfected with adenovirus expressing ICERsi or lacZ for 40 h and stimulated with NE (3 µM) for 2 or 6 h. Cells were collected by centrifugation and prepared for immunoblot as described in Materials and Methods. A, Representative immunoblots from three experiments showing the protein levels of AA-NAT, MKP-1, and ICER; GAPDH is included to demonstrate loading consistency. Arrow indicates the ICER-specific band. B, Histograms of densitometric measurements of AA-NAT and MKP-1 protein presented as percentage of maximal OD value. Each value represents the mean ± SEM (n = 3). Con, Control.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Effect of ICERsi on the time course response of NE-stimulated AA-NAT activity and MT production. Pinealocytes (1 x 105 cells/0.6 ml) were transiently transfected with adenovirus expressing ICERsi or lacZ for 40 h and stimulated with NE (3 µM) for the duration indicated. Cells were collected by centrifugation and prepared for measurement of AA-NAT enzymatic activity (A) and media were collected for MT production (B) as described in Materials and Methods. Each value represents the mean ± SEM (n = 3).

View larger version (54K): [in this window] [in a new window]   FIG. 3. Effect of ICERsi and ICERfl alone or in combination on adrenergic induction of AA-NAT, MKP-1, and ICER. Pinealocytes (1 x 105 cells/0.6 ml) were transiently transfected with adenovirus expressing ICERsi or ICERfl alone or in combination for 40 h and stimulated with NE (3 µM) for 3 or 4 h. A, Representative ethidium bromide-stained agarose gels from three experiments showing aa-nat, mkp-1, and icer mRNA levels in pinealocytes treated as indicated for 3 h; gapdh signal is included to demonstrate loading consistency. B, Representative immunoblots from three experiments showing the protein levels of AA-NAT, MKP-1, and ICER in pinealocytes treated as indicated for 4 h; GAPDH signal is included to demonstrate loading consistency. Arrow indicates the ICER-specific band. C, Histograms of densitometric measurements of ICER, AA-NAT, and MKP-1 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; **, P < 0.05, significantly different from treatment with NE+ICERfl. Con, Control.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Effect of ICERfl on the time course response of NE-stimulated AA-NAT activity and MT production. Pinealocytes (1 x 105 cells/0.6 ml) were transiently transfected with adenovirus expressing ICERfl or lacZ for 40 h and stimulated with NE (3 µM) for the duration indicated. Cells were collected by centrifugation and prepared for measurement of AA-NAT enzymatic activity (A), and media were collected for MT production (B) as described in Materials and Methods. Each value represents the mean ± SEM (n = 3).

View larger version (30K): [in this window] [in a new window]   FIG. 5. Effect of Fra-2si on adrenergic induction of AA-NAT, MKP-1, and Fra-2. Pinealocytes (1 x 105 cells/0.6 ml) were transiently transfected with adenovirus expressing Fra-2si for 40 h and stimulated with NE (3 µM) for the duration indicated. A, Representative ethidium bromide-stained agarose gels from three experiments showing aa-nat, mkp-1, and fra-2 mRNA levels in pinealocytes treated as indicated; gapdh signal is included to demonstrate loading consistency. B, Representative immunoblots from three experiments showing the protein levels of AA-NAT, MKP-1, and Fra-2; GAPDH signal is included to demonstrate loading consistency. C, Histograms of densitometric measurements of Fra-2, MKP-1, and AA-NAT 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. Con, Control.

View larger version (23K): [in this window] [in a new window]   FIG. 6. Effect of Fra-2si on the time course response of NE-stimulated AA-NAT activity and MT production. Pinealocytes (1 x 105 cells/0.6 ml) were transiently transfected with adenovirus expressing Fra-2si or lacZ for 40 h and stimulated with NE (3 µM) for the duration indicated. Cells were collected by centrifugation and prepared for measurement of AA-NAT enzymatic activity (A), and media were collected for MT production (B) as described in Materials and Methods. Each value represents the mean ± SEM (n = 3).

View larger version (35K): [in this window] [in a new window]   FIG. 7. Time-dependent effects of NE on the rate of mRNA synthesis of mkp-1 and aa-nat. Pinealocytes (1 x 105 cells/0.3 ml) were cultured for 24 h and stimulated with NE (3 µM) for the duration indicated. A, Representative ethidium bromide-stained agarose gels from three experiments showing mkp-1 and aa-nat mRNA levels in pinealocytes treated as indicated; gapdh signal is included to demonstrate loading consistency. B, Relative mRNA levels of mkp-1 and aa-nat after NE stimulation as determined from densitometric measurements. Maximal OD value was assigned a value of 1. Each value represents the mean ± SEM (n = 3). C, Relative rate of changes of mkp-1 and aa-nat mRNA levels after NE stimulation. Rate of changes of mRNA levels were calculated from the slope between the two time points from values obtained from panel B. The maximal rate of change was assigned a value of 1. Con, Control.

View larger version (38K): [in this window] [in a new window]   FIG. 8. Time-dependent effects of NE on the induction of the protein levels of ICER and Fra-2. Pinealocytes (1 x 105 cells/0.3 ml) were cultured for 24 h and stimulated with NE (3 µM) for the duration indicated. A, Representative immunoblots from three experiments showing the protein levels of ICER and Fra-2; GAPDH signal is included to demonstrate loading consistency. Arrow indicates the ICER-specific band. B, Relative protein levels of ICER and Fra-2 as determined from densitometric measurements. Maximal OD value was assigned a value of 1. Each value represents the mean ± SEM (n = 3). The relative rate of change of mkp-1 and aa-nat mRNA levels after NE stimulation (from Fig. 7C) was included for comparison. Con, Control.

View larger version (25K): [in this window] [in a new window]   FIG. 9. Effect of proteasomal inhibition on NE-stimulated ICER protein levels. Pinealocytes (0.2 x 105 cells/0.3 ml) were cultured for 24 h and stimulated with NE (3 µM) in the presence or absence of graded concentrations of c-lact for 2 h. A, Representative immunoblots from three experiments showing ICER protein levels; GAPDH signal is included to demonstrate loading consistency. Arrow indicates the ICER-specific band. B, Histograms of densitometric measurements of ICER 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. Con, Control.

View larger version (20K): [in this window] [in a new window]   FIG. 10. Effect of proteasomal inhibition on NE-stimulated Fra-2 protein levels. A, Pinealocytes (0.2 x 105 cells/0.3 ml) were cultured for 24 h and stimulated with NE (3 µM) for 6 h in the presence or absence of c-lact (10 µM) for the duration indicated. Representative immunoblots from three experiments showing Fra-2 and AA-NAT protein levels; GAPDH signal is included to demonstrate loading consistency. B, Histograms of densitometric measurements of Fra-2 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. Con, Control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our observations that preventing the NE-stimulated coinduction of ICER has no effect on the NE-stimulated AA-NAT expression during the first 6 h of stimulation is unexpected. This finding differs from a previous report that shows an enhancement of the NE-stimulated AA-NAT induction within the first 6 h of stimulation in pinealocytes when the antisense methodology is used to knock down ICER induction (26). The explanation for this discrepancy is unclear. However, it is unlikely to be due to a lack of efficacy of siRNA transfection in reducing icer mRNA because the same transfection protocol is able to counteract the effect of transfection with ICERfl as well as negate the suppressive effect of ICERfl on NE-stimulated aa-nat mRNA and protein levels.

The finding that whereas overexpressing ICER is effective in inhibiting the induction of AA-NAT and MKP-1, abolishing the NE-stimulated ICER production has no effect on the AA-NAT or MKP-1 induction appears contradictory. One possible explanation of the negligible effect of ICERsi on NE induction of AA-NAT and MKP-1 is that an insufficient amount of the repressor protein is present during the active transcriptional phase of NE-stimulated and CRE-driven genes. Based on the gradients of changes of the NE-stimulated aa-nat and mkp-1 mRNA accumulation in the time course study, NE-stimulated transcription of these two genes (as measured by the steepest increment in mRNA levels) is most active within the first 2 h of NE treatment and this preceded the duration required for a significant accumulation of ICER protein (5). After 2 h of NE stimulation, transcription of aa-nat and mkp-1 mRNAs may no longer be the rate-limiting step in the synthesis of the respective protein. Moreover, in the case of AA-NAT, even in the absence of transcription, the known stability of the induced aa-nat mRNA (14) would only lead to a modest decline. In support of this is our previous observation that treatment with actinomycin 2 h after NE stimulation has a minimal effect on the level of aa-nat mRNA when measured 1 h after addition of actinomycin (14).

As for the effectiveness of transfection with ICERfl in inhibiting the NE-stimulated AA-NAT and MKP-1 induction, this is probably related to the high level of ICER protein present in the pinealocyte before NE stimulation. In this case, ICER expression is mediated by the transfected adenovirus. Thus, any stimulated increase in phosphorylated CREB has to compete with the existing repressor for the CRE site on the promoter of AA-NAT or MKP-1. Together, our results indicate that, besides the level of expression, the temporal relationship between the expression of the repressor and the transcriptional activity of the target gene is probably critical in determining the effectiveness of the repressor. This is particularly important in the case of MKP-1 expression because the NE-induced increase in MKP-1 transcriptional activity occurs within 1 h of NE stimulation.

Binding of Fra-2-containing AP-1 complexes to the AP-1 sites in the mkp-1 and aa-nat promoters could reduce transcription because Fra-2 can form heterodimers with CREB family members (27, 28) and regulate the amplitude and/or turning off of aa-nat and mkp-1 transcription. Although there is a nocturnal induction of Fra-2 (10) as well as a circadian rhythm of AP-1 binding activity (29) and expression of AP-1 complex proteins in the pineal gland (10), transfection of pinealocytes with Fra-2si has no effect on NE-stimulated mRNA and protein levels of MKP-1 or AA-NAT in our study. As discussed above for ICER, this lack of effect of Fra-2 knockdown on adrenergic-stimulated gene expression could be related to the timing of high transcriptional activity for the induction of mkp-1 and aa-nat relative to the expression of Fra-2. Nevertheless, our results are consistent with a lack of effect on the urinary output of 6-sulfatoxy MT, a major metabolite of MT in transgenic rats with knockdown of Fra-2 (12).

Although our results suggest that ICER and Fra-2 are unlikely to be involved in regulating the level or rate of initial induction of mkp-1 or aa-nat by NE, they do not negate the possibility that these repressors may participate in regulating the duration or decline of mkp-1 or aa-nat transcription toward the end of stimulation. Certainly, in CREM mutant mice (whereby ICER and CREM are inactivated), the amplitude of aa-nat expression is elevated during the night, and the duration of aa-nat induction is prolonged (4). However, abolishing both CREM and ICER expression in mice is probably not equivalent to selectively knocking down ICER in rat pinealocytes.

Our recent studies indicate that a protein repressor under the regulation of proteasomes also participates in suppressing the adrenergic-mediated transcriptional induction of aa-nat (21). This is based on the observation that treatment with a proteasome inhibitor concurrent with NE stimulation suppressed the NE-stimulated AA-NAT response. Although proteasomes appear to play a role in regulating the level of ICER in other tissues (30, 31), our results do not support the involvement of ICER or Fra-2 in mediating the inhibition of aa-nat transcription by proteasome inhibitors. This is because treatment with a proteasome inhibitor simultaneously with NE stimulation causes a reduction rather than an accumulation of ICER and Fra-2 protein levels. Thus, the ICER and Fra-2 responses are similar to the AA-NAT response toward the proteasome inhibitor, indicating that these inducible repressors are also subject to the same regulatory influence of the proteasome.

In summary, adenoviral transfection proves to be an effective tool in the investigation of the effects of modulating gene expression in rat pinealoctyes. Whereas overexpression of ICER can suppress NE-stimulated aa-nat and mkp-1 induction, the amount of ICER and Fra-2 present during NE stimulation appears insufficient to exert a significant effect on the induction of these two genes. Moreover, neither ICER nor Fra-2 appears to be the repressor protein responsible for the proteasomal inhibition-mediated reduction of NE-induced aa-nat transcription.

	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.

First Published Online November 2, 2006

Abbreviations: AA-NAT, Arylalkylamine-N-acetyltransferase; AP-1, activator protein-1; c-lact, clasto-lactacystin ß-lactone; CRE, cAMP response element; CREB, CRE binding protein; CREM, CRE modulator; Fra-2, Fos-related antigen 2; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; ICER, inducible cAMP early repressor; ICERfl, adenovirus overexpressing the full length ICER transcript; MKP-1, MAPK phosphatase-1; MT, melatonin; NE, norepinephrine; shRNA, short hairpin RNA; si, adenovirus expressing siRNA complementary to the specific transcript; siRNA, short interfering RNA.

Received August 22, 2006.

Accepted for publication October 23, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Courey AJ, Jia S 2001 Transcriptional repression: the long and the short of it. Genes Dev 15:2786–2796[Free Full Text]
2. Foulkes NS, Whitmore D, Sassone-Corsi P 1997 Rhythmic transcription: the molecular basis of circadian melatonin synthesis. Biol Cell 89:487–494[CrossRef][Medline]
3. 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[CrossRef][Medline]
4. 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[Abstract/Free Full Text]
5. 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[Abstract/Free Full Text]
6. Klein DC 1985 Photoneural regulation of the mammalian pineal gland. In: Evered D, Clark S, eds. Photoperiodism, melatonin and the pineal. Ciba Foundation Symposium. London: Pitman; 117:38–56
7. Roseboom PH, Klein DC 1995 Norepinephrine stimulation of pineal cyclic AMP response element-binding protein phosphorylation: primary role of a ß-adrenergic receptor/cyclic AMP mechanism. Mol Pharmacol 47:439–449[Abstract]
8. 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[Abstract]
9. Klein DC, Coon SL, Roseboom PH, Weller JL, Bernard M, Gastel JA, Zatz M, Iuvone PM, Rodriguez IR, Begay V, Falcon J, Cahill GM, Cassone VM, Baler R 1997 The melatonin rhythm-generating enzyme: molecular regulation of serotonin N-acetyltransferase in the pineal gland. Recent Prog Horm Res 52:307–358[Medline]
10. Baler R, Klein DC 1995 Circadian expression of transcription factor Fra-2 in the rat pineal gland. J Biol Chem 270:27319–27325[Abstract/Free Full Text]
11. 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[Abstract/Free Full Text]
12. Smith M, Burke Z, Humphries A, Wells T, Klein DC, Carter D, Baler R 2001 Tissue-specific transgenic knockdown of Fos-related antigen 2 (Fra-2) expression mediated by dominant negative Fra-2. Mol Cell Biol 21:3704–3713[Abstract/Free Full Text]
13. 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[CrossRef][Medline]
14. Price DM, Chik CL, Ho AK 2004 Norepinephrine induction of mitogen-activated protein kinase phosphatase-1 expression in rat pinealocytes: distinct roles of - and ß-adrenergic receptors. Endocrinology 145:5723–5733[Abstract/Free Full Text]
15. Ho AK, Price DM, Terriff D, Chik CL 2006 Timing of mitogen-activated protein kinase (MAPK) activation in the rat pineal gland. Mol Cell Endocrinol 252:34–39[CrossRef][Medline]
16. Ho AK, Chik CL 2000 Adrenergic regulation of mitogen-activated protein kinase in rat pinealocytes: opposing effects of protein kinase A and protein kinase G. Endocrinology 141:4496–4502[Abstract/Free Full Text]
17. Ho AK, Mackova M, Price L, Chik CL 2003 Diurnal variation of p42/44 mitogen-activated protein kinase in the rat pineal gland. Mol Cell Endocrinol 208:23–30[CrossRef][Medline]
18. Chik CL, Mackova M, Price D, Ho AK 2004 Adrenergic regulation and diurnal rhythm of p38 mitogen-activated protein kinase phosphorylation in the rat pineal gland. Endocrinology 145:5194–5201[Abstract/Free Full Text]
19. Ho AK, Mackova M, Cho C, Chik CL 2003 Regulation of 90-kilodalton ribosomal S6 kinase phosphorylation in the rat pineal gland. Endocrinology 144:3344–3350[Abstract/Free Full Text]
20. Man JR, Rustaeus S, Price D, 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[Abstract/Free Full Text]
21. Terriff D, Chik CL, Price DM, Ho AK 2005 Proteasomal proteolysis in the adrenergic induction of arylalkylamine-N-acetyltransferase in rat pinealocytes. Endocrinology 146:4795–4803[Abstract/Free Full Text]
22. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
23. Chik CL, Liu QY, Li B, Klein DC, Zylka M, Kim DS, Chin H, Karpinski E, Ho AK 1997 _1D L-type Ca²⁺-channel currents: inhibition by a ß-adrenergic agonist and pituitary adenylate cyclase-activating polypeptide (PACAP) in rat pinealocytes. J Neurochem 68:1078–1087[Medline]
24. Pizzonia J 2001 Electrophoresis gel image processing and analysis using the KODAK 1D software. Biotechniques 30:1316–1320[Medline]
25. 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[CrossRef][Medline]
26. Pfeffer M, Maronde E, Korf HW, Stehle JH 2000 Antisense experiments reveal molecular details on mechanisms of ICER suppressing cAMP-inducible genes in rat pinealocytes. J Pineal Res 29:24–33[CrossRef][Medline]
27. Wisdom R, Verma IM 1993 Transformation by Fos proteins requires a C-terminal transactivation domain. Mol Cell Biol 13:7429–7438[Abstract/Free Full Text]
28. Hai T, Curran T 1991 Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc Natl Acad Sci USA 88:3720–3724[Abstract/Free Full Text]
29. Guillaumond F, Sage D, Deprez P, Bosler O, Becquet D, Francois-Bellan AM 2000 Circadian binding activity of AP-1, a regulator of the aryalkylamine N-acetyltransferase gene in the rat pineal gland, depends on circadian Fra-2, c-Jun, and Jun-D expression and is regulated by the clock’s zeitgebers. J Neurochem 75:1398–1407[CrossRef][Medline]
30. Folco EJ, Koren G 1997 Degradation of the inducible cAMP early repressor (ICER) by the ubiquitin-proteasome pathway. Biochem J 328:37–43
31. Yehia G, Schlotter F, Razavi R, Alessandrini A, Molina CA 2001 Mitogen-activated protein kinase phosphorylates and targets inducible cAMP early repressor to ubiquitin-mediated destruction. J Biol Chem 276:35272–35279[Abstract/Free Full Text]

This article has been cited by other articles:

				C. L. Chik, M. T. Wloka, D. M. Price, and A. K. Ho The Role of Repressor Proteins in the Adrenergic Induction of Type II Iodothyronine Deiodinase in Rat Pinealocytes Endocrinology, July 1, 2007; 148(7): 3523 - 3531. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 148/2/743    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ho, A. K. Articles by Chik, C. L. Search for Related Content PubMed PubMed Citation Articles by Ho, A. K. Articles by Chik, C. L.