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Endocrinology, doi:10.1210/en.2007-0166
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Endocrinology Vol. 148, No. 7 3523-3531
Copyright © 2007 by The Endocrine Society

The Role of Repressor Proteins in the Adrenergic Induction of Type II Iodothyronine Deiodinase in Rat Pinealocytes

C. L. Chik, M. T. Wloka, D. M. Price and A. K. Ho

Departments of Physiology (M.T.W., D.M.P., A.K.H.) 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 Physiology, 7-26 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7. E-mail: anho{at}ualberta.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, we investigated the transcriptional regulation of the adrenergic induction of type II iodothyronine deiodinase (Dio2) in rat pinealocytes. Treatment of pinealocytes with norepinephrine (NE) caused an increase in the mRNA level of Dio2 that peaked around 2 h and declined over the next 5 h. Both ß- and {alpha}1-adrenergic receptors contributed to the NE induction of Dio2 expression through a cAMP/protein kinase A mechanism. In pinealocytes that had been stimulated by NE, inhibition of transcription by actinomycin had no discernible effect on Dio2 expression. In contrast, inhibition of protein synthesis by cycloheximide enhanced the NE induction of Dio2 expression, suggesting the involvement of a repressor protein. Transient transfection of pinealocytes with adenovirus expressing small interfering RNA against Fos-related antigen 2 (Fra2) enhanced the NE induction of Dio2 expression, whereas the effect of overexpression of the full-length transcript of Fra2 was inhibitory. Time-course study indicated that preventing the NE induction of Fra2 enhanced the NE induction of Dio2 after 3 h, and the enhancement persisted beyond 6 h after NE stimulation. In comparison, transient transfection of pinealocytes with small interfering RNA against inducible cAMP early repressor (Icer) had no effect on the NE induction of Dio2 expression, whereas overexpression of the full-length transcript of Icer caused a small reduction of the NE-stimulated Dio2 expression. Together, our results support Fra-2 as an important transcriptional repressor that helps shape the time profile of the adrenergic induction of Dio2 expression in the rat pineal gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IODOTHYRONINE DEIODINASE IS an enzyme that is of importance in the action of thyroid hormone (1, 2). Among the deiodinases, type 2 deiodinase (Dio2) is responsible for the conversion of T4 to T3 and is an important regulator of the local concentration of intracellular T3 (1, 2). In the rat pineal gland, Dio2 activity increases during the hours of darkness (3, 4, 5). This nocturnal increase is comparable with that in pineal arylalkylamine N-acetyltransferase (AA-NAT), the rhythm generating enzyme for the hormone of darkness, melatonin (3, 4, 5). In contrast to studies on the transcriptional regulation of the nocturnal increase in pineal AA-NAT (6, 7, 8, 9), little is known about the regulation of the rhythmic changes in pineal Dio2 activity other than its regulation at the pretranslational level by a ß-adrenergic mechanism (10).

Through stimulation of {alpha}1- and ß-adrenergic receptors, multiple signaling mechanisms, in addition to protein kinase A, are activated in the rat pineal gland in response to norepinephrine (NE) stimulation. Stimulation of ß-adrenergic receptors alone produces a 7- to 10-fold increase in cAMP and a 2- to 4-fold increase in cGMP accumulation (11, 12). Stimulation of {alpha}1-adrenergic receptors, which activates protein kinase C (13, 14) and elevates intracellular Ca2+ concentration (15, 16), potentiates the ß-adrenergic stimulated cyclic nucleotide responses (11, 12). Aside from induction of AA-NAT and Dio2 activity, NE stimulation also increases the expression of transcription repressors such as Fos-related antigen 2 (Fra-2) and inducible cAMP early repressor (ICER) (17, 18).

Repressor proteins play a critical role in regulating the temporal profile of gene expression (19). To generate rhythmic gene expression, both gene induction and repression needs to be coordinated in a temporally defined manner (20). The synthesis of inducible repressor protein in a precise time-dependent manner could limit the duration of gene expression. Existing data suggest that the nocturnal and adrenergic regulated genes such as Aanat in the rat pineal gland is under this type of coordinated control (21, 22, 23, 24). Of the known inducible repressors in the pineal gland, ICER has been reported to be involved in the transcription of Aanat (17, 21, 22), and Fra-2 is linked to the expression of the genes encoding Dio2, CD24, and NGFI-B (25, 26). Because of the presence of cAMP response element (CRE) and activator protein-1 (AP-1) sites in the promoter of Dio2 in other mammalian species (27, 28), in this study, we investigated the role of ICER and Fra-2 in the transcriptional regulation of Dio2 expression by NE in rat pinealocytes. The effects of these two repressors on the time profile of expression of Aanat by NE has recently been characterized (24); therefore, we included measurement of Aanat expression as a comparison.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Actinomycin, cycloheximide, dibutyryl cAMP, dibutyryl cGMP, ionomycin, isoproterenol, NE, phenylephrine, prazosin, 4ß-phorbol 12-myristate 13-acetate. and propranolol were obtained from Sigma Chemical Co. (St. Louis, MO). Polyclonal antibodies against CRE modulator and Fra-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody against glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was obtained from Ambion Inc. (Austin, TX). All other chemicals were of the purest grades available commercially.

Preparation of cultured pinealocytes and drug treatment
This study was reviewed and approved by the Health Sciences Animal Policy and Welfare Committee of the University of Alberta (Edmonton, Alberta, Canada). Sprague Dawley rats (male; weighing 150 g) were obtained from the University of Alberta animal unit. For pinealocyte cell culture, 12–15 animals were killed 3 h after the onset of light for each preparation. Pinealocytes were prepared by papain dissociation of freshly dissected rat pineal glands using a system from Worthington Biochemical Corp. (Lakewood, NJ). Cell yield was approximately 7 x 105 cells/gland. Cells were suspended in DMEM containing 10% fetal calf serum and maintained overnight before the experiment at 37 C for 18 h in a mixture of 95% air-5% CO2. Aliquots of pinealocytes (1 x 105 cells per 0.3 ml) 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, 12,000 x g). Pinealocyte total RNAs were isolated using Trizol (Invitrogen Inc., Valencia, CA). Samples for immunoblot 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).

Adenoviral transduction of pinealocytes
The procedure used was as previously described (24). The small interfering RNA (siRNA) targets that produced significant gene silencing were as follows: Icer, GAG CTC CTA CTA CTG CTT TGC; Fra2, GCA CTT CAA ACC TTG TCT TCA. Titered viral stocks were used to transduce DNAs encoding short hairpin RNAs or full-length transcript of the repressor protein into pinealocytes. A transduction protocol was initially developed using a LacZ transgene-bearing adenovirus as described previously. siRNA-bearing adenovirus or adenovirus expressing full-length transcript of the repressor protein was combined with the pinealocyte medium at a multiplicity of infection of approximately 100 viral particles per cell. Full-length Icer and Fra2 were generated from a PCR-cloned product obtained by amplification of NE-stimulated pinealocyte cDNA using nucleotide sequence 58–400 of Icer (accession no. S66024) and nucleotide sequence 1–984 of Fra2 (accession no. NM012954). The generated products contain the entire protein coding region of Icer and Fra2, respectively. For control transduction, LacZ transgene-bearing adenovirus was used. Pinealocytes (1 x 105 cells per 0.6 ml) were stimulated with NE 40 h after transduction and after stimulation were analyzed by RT-PCR or Western blot.

RT-PCR
First-strand cDNA was synthesized from the isolated RNA using an Omniscript reverse transcriptase kit (Invitrogen) with an oligo-dT primer. PCR was performed as previously described (29, 30). Briefly, the reaction mixture containing 3 µl of suitably diluted cDNA as template in 30 µl of 10 mM Tris (pH 8.3) buffer containing 50 mM KCl, 1.5 mM MgCl2, 100 µM of each dNTP, 1.25 U Taq polymerase (PerkinElmer, Cetus, Norwalk, CT), and 1 µM each of the two primers. All PCRs were performed 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 Aanat, 29 cycles for Dio2, 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. Primers used were as follows: Dio2, left primer, GAC TCG GTC ATT CTG CTC AAG; right primer, AGG CTG GCA GTT GCC TAG TA. Sequences of the Aanat, Icer, Fra2, and Gapdh primers used were previously described (29, 30, 31).

Western blotting
SDS-PAGE was performed as described in our previous studies (32, 33) using 12% acrylamide (Mini-Protein II gel system; Bio-Rad, 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), which 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).

Results and 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) (34). For analyses of Western blots, exposed films were scanned and band densitometry of acquired images was performed with Kodak 1D software. Densitometric values were initially expressed relative to the values of GAPDH to account for the variability of loading. Using the values relative to GAPDH, a paired t test was used to determine difference between groups and ANOVA with the Newman-Keuls test was used for comparisons within multiple groups. Because of the variability of the densitometric values between experiments, in most studies, the results were expressed as percent of NE response based on the mean ± SEM from at least three independent experiments. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adrenergic induction of Dio2 expression in the rat pinealocyte
Cultured pineal cells were used to investigate the transcriptional control of Dio2 expression by NE. Treatment of pinealocytes with NE (3 µM) caused a rapid induction of Dio2 mRNA that was detectable within 1 h of treatment, peaked between 2 and 3 h, and declined back to near basal levels after 8 h (Fig. 1Go). In the same experiment, NE treatment caused a gradual increase in Aanat mRNA levels that peaked between 3 and 4 h, and there was a slow decline over the next 4 h (Fig. 1Go).


Figure 1
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  		FIG. 1. Time-dependent effects of NE on the induction of Dio2 and Aanat expression. Pinealocytes (1 x 105 cells/0.3 ml) were cultured for 18 h and stimulated with NE (3 µM) for the duration indicated. A, Representative ethidium bromide-stained agarose gels from three experiments showing Dio2 and Aanat mRNA levels in pinealocytes treated as indicated; gapdh signal is included to demonstrate loading consistency. Con, Control. B, Relative mRNA levels of Dio2 and Aanat 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).

 
Receptor characterization of the adrenergic regulation of Dio2 expression
Previous studies indicated that the nocturnal increase in Dio2 activity is primarily regulated by a ß-adrenergic mechanism (3, 10), but other studies also support a role of {alpha}-adrenergic mechanism in its regulation (35, 36). To determine the contributions of the subtypes of adrenergic receptors in the regulation of the mRNA level of Dio2, selective adrenergic agonists and antagonists at concentrations established previously were used in the study (30, 32, 33). At 2 h after treatment, selective activation of ß-adrenergic receptors by isoproterenol (3 µM in the presence of 3 µM of prazosin) but not selective activation of {alpha}1-adrenergic receptors by phenylephrine (3 µM in the presence of 3 µM of propranolol) caused an increase in the mRNA level of Dio2 (Fig. 2Go, A and B). Simultaneous activation of both {alpha}1- and ß-adrenergic receptors by treatment with isoproterenol (3 µM) plus phenylephrine (3 µM) or NE (3 µM) caused an increase in the mRNA level of Dio2 that was more pronounced than treatment with isoproterenol alone (Fig. 2Go, A and B). In support of the predominant involvement of ß-adrenergic receptors in this induction, cotreatment with propranolol (3 µM) markedly reduced the mRNA level of Dio2 induced by NE (3 µM) (Fig. 2Go, A and B). In contrast, cotreatment with prazosin (3 µM) caused a modest inhibition of the Dio2 mRNA level induced by NE (3 µM) (Fig. 2Go, A and B).


Figure 2
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  		FIG. 2. Receptor and signaling mechanisms involved in NE-induced Dio2 and Aanat mRNA levels. Pinealocytes (1 x 105 cells per 0.3 ml) were cultured for 18 h and treated for 2 h with NE (3 µM), isoproterenol [ISO, 3 µM with prazosin (Praz, 3 µM)], phenylephrine [PE, 3 µM with propranolol (Prop, 3 µM)], ISO (3 µM) plus PE (3 µM), or NE (3 µM) alone or in the presence of Prop (3 µM) or Praz (3 µM) (A) or NE (3 µM), dibutyryl cAMP (DBcAMP, 0.5 mM), dibutyryl cGMP (DBcGMP, 0.5 mM), 4ß-phorbol 12-myristate 13-acetate (PMA, 0.1 µM), or ionomycin (ION, 1 µM) (C). Con, Control. A and C, Representative ethidium bromide-stained agarose gels from three experiments showing Dio2 and Aanat mRNA levels in pinealocytes treated as indicated; gapdh signal is included to demonstrate loading consistency; B and D, histograms of densitometric measurements of Dio2 mRNA levels presented as percent of NE OD value. Each value represents the mean ± SEM (n = 3). *, P < 0.05, significantly different from control; **, P < 0.05, significantly different between the two treatment groups.

 
Postreceptor mechanisms involved in NE-induced Dio2 expression
To characterize the postreceptor mechanisms involved in NE-stimulated Dio2 expression, agents that activate the key signaling pathways as NE were tested. Treatment of pinealocytes for 2 h with dibutyryl cAMP (0.5 mM), a membrane permeable cAMP analog, mimicked the effects of NE on Dio2 expression (Fig. 2Go, C and D). In contrast, treatment of pinealocytes for 2 h with the membrane-permeable cGMP analog dibutyryl cGMP (0.5 mM), the activator of protein kinase C 4ß-phorbol 12-myristate 13-acetate (0.1 µM), or the Ca2+ ionophore ionomycin (1 µM) did not cause an increase in the mRNA level of Dio2 (Fig. 2Go, C and D). These results suggest that although {alpha}1-adrenergic receptors activate multiple transduction mechanisms such as protein kinase C and elevation of intracellular Ca2+ concentration, their roles in regulating Dio2 expression are mediated through the potentiation of the ß-adrenergic-stimulated cAMP accumulation.

Requirement of transcription for the maintenance of NE induction of Dio2 expression
To investigate the requirement of transcription for the maintenance of NE induction of Dio2 expression, an inhibitor of transcription actinomycin was added to NE-stimulated pinealocytes. Treatment of pinealocytes with actinomycin (15 µg/ml) after pinealocytes had been stimulated by NE (3 µM) for 3 h did not have a discernible effect on the mRNA level of Dio2 after 1 h (Fig. 3Go, A and B). In the same experiment, actinomycin treatment also had no effect on the mRNA level of Aanat stimulated by NE (Fig. 3BGo). These results indicate that like the Aanat transcript, the Dio2 transcript is relatively stable.


Figure 3
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  		FIG. 3. Inhibition of transcription on NE-induced Dio2 and Aanat mRNA levels. Pinealocytes (1 x 105 cells per 0.3 ml) were stimulated with NE (3 µM) for 3 h before treatment with actinomycin (Act, 15 µg/ml) for 30 and 60 min. Con, Control. A, Representative ethidium bromide-stained gels from three separate experiments showing Dio2 and Aanat mRNA levels; gapdh signal is included to demonstrate loading consistency. B, Densitometric measurements of Dio2 (left panel) and Aanat (right panel) mRNAs, presented as percent of NE response at 3 h. Values represent the mean ± SEM (n = 3).

 
Inhibition of protein synthesis on the NE induction of Dio2 expression
To determine whether protein synthesis plays a role in the NE-stimulated Dio2 expression, pinealocytes were treated with the protein synthesis inhibitor cycloheximide. Cotreat-ment of pinealocytes with cycloheximide (30 µg/ml) and NE (3 µM) for 3 h enhanced the NE induction of Dio2 expression (Fig. 4AGo), similar to a previous study performed in pineal organ culture (10). Cycloheximide also enhanced the NE induction of Aanat mRNA level (Fig. 4BGo). These results suggest that synthesis of a repressor protein may be involved in the transcriptional control of Dio2 expression.


Figure 4
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  		FIG. 4. Inhibition of protein synthesis on NE-induced Dio2 and Aanat mRNA levels. Pinealocytes (1 x 105 cells per 0.3 ml) were stimulated with NE (3 µM) in the presence or absence of cycloheximide (Cyc, 30 µg/ml) for 3 h. Con, Control. A, Representative ethidium bromide-stained gels from three separate experiments showing Dio2 and Aanat mRNA levels; gapdh signal is included to demonstrate loading consistency. B, Densitometric measurements of Dio2 (left panel) and Aanat (right panel) mRNAs, presented as fold changes vs. NE response. Values represent the mean ± SEM (n = 3). *, P < 0.05, significantly different from treatment with NE.

 
Effect of changes in Fra-2 protein levels on adrenergic induction of Dio2 expression
In transgenic rats with tissue-specific knockdown of Fra-2, pineal Dio2 expression is increased in the night pineal gland, suggesting Fra-2 probably regulates NE induction of Dio2 expression (25). To investigate the role of Fra-2 in adrenergic-stimulated gene expression in rat pinealocytes, we generated recombinant adenovirus overexpressing the full-length Fra2 transcript (Fra2-fl) as well as adenovirus expressing siRNA complementary to the endogenous Fra2 transcript (Fra2-si). Figure 5AGo shows RT-PCR results of pinealocytes transiently transfected with Fra2-fl and/or Fra2-si. As anticipated, transient transfection with Fra2-fl caused a significant increase in the Fra2 mRNA and protein levels at 5 h (Fig. 5Go, A and B). In control pinealocytes, stimulation with NE (3 µM) for 5 h caused an increase in Dio2 and Aanat mRNA levels. However, transient transfection of pinealocytes with Fra2-fl selectively reduced the Dio2 but not the Aanat mRNA levels stimulated by NE for 5 h (Fig. 5Go, A and C). Fra2-fl treatment also had no effect on the mRNA levels of Gapdh in the presence or absence or NE.


Figure 5
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  		FIG. 5. Effect of Fra2-si and Fra2-fl alone or in combination on NE-stimulated Dio2 and Aanat mRNA levels. Pinealocytes (1 x 105 cells per 0.6 ml) were transiently transfected with adenovirus expressing Fra2-si or Fra2-fl alone or in combination for 40 h and stimulated with NE (3 µM) for 5 h. Con, LacZ control. A, Representative ethidium bromide-stained agarose gels from three experiments showing Dio2, Aanat, and Fra2 mRNA levels in pinealocytes treated as indicated; gapdh signal is included to demonstrate loading consistency. B, Representative immunoblots from three experiments showing AA-NAT and Fra-2 protein levels in pinealocytes treated as indicated; GAPDH signal is included to demonstrate loading consistency. C, Histograms of densitometric measurements of Fra2, Dio2, and Aanat mRNA levels presented as fold changes vs. NE response. 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+Fra2-fl.

 
In comparison, treatment of pinealocytes with Fra2-si significantly reduced the induction of Fra-2 mRNA and protein levels by NE (3 µM) after 5 h as well as reversed the effect of Fra2-fl treatment on the mRNA and protein levels of Fra-2 (Fig. 5Go, A and B). Preventing the induction of Fra2 by using siRNA caused a significant increase in the mRNA level of Dio2 stimulated by NE (3 µM) when measured after 5 h. Fra2-si treatment also counteracted the inhibitory effect of Fra2-fl on the NE-stimulated Dio2 mRNA levels (Fig. 5Go, A and C). In contrast, Fra2-si treatment had no observable effect on NE induction of Aanat mRNA level (Fig. 5Go, A and C). These results indicate that the NE-stimulated expression of Dio2 but not that of Aanat is negatively regulated by Fra-2.

Effect of Fra2-si treatment on the time profile of NE induction of Dio2 expression
To characterize the effect of Fra2-si treatment on the profile of NE induction of Dio2 expression, a time-course study was performed. Transient transfection of pinealocytes with Fra2-si had no effect on the Dio2 expression stimulated by NE (3 µM) after 1 h (Fig. 6Go, A and B). However, Fra2-si treatment had an enhancing effect on the induction of Dio2 expression by NE (3 µM) after 3 h, and the effect was more pronounced 5 and 6 h after NE treatment (Fig. 6Go, A and B). In Fra2-si-treated pinealocytes, there was only a minimal decline in the Dio2 mRNA levels between 3 and 5 h of NE treatment. These results indicate that the effect of Fra2-si treatment on the NE induction of Dio2 expression is time dependent, and the main effect is to prolong the duration of activation.


Figure 6
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  		FIG. 6. Time-course effect of Fra2-si on NE-stimulated Dio2 mRNA levels. Pinealocytes (1 x 105 cells per 0.6 ml) were transiently transfected with adenovirus expressing Fra2-si for 40 h and stimulated with NE (3 µM) for the duration indicated. Con, LacZ control. A, Representative ethidium bromide-stained agarose gels from three experiments showing Dio2 and Fra2 mRNA levels in pinealocytes treated as indicated; gapdh signal is included to demonstrate loading consistency. B, Densitometric measurements of Dio2 mRNA levels presented as percent of NE response at 3 h. Each value represents the mean ± SEM (n = 3). *, P < 0.05, significantly different from treatment with NE.

 
Effect of changes in ICER protein levels on NE induction of Dio2 expression
Figure 7Go shows RT-PCR results of pinealocytes transiently transfected with adenovirus overexpressing the full-length Icer transcript (Icer-fl) or expressing siRNA complementary to the endogenous Icer transcript (Icer-si). Transient transfection with Icer-fl caused a significant increase in the Icer mRNA level (Fig. 7Go, A and C). Stimulation with NE (3 µM) for 5 h caused an increase in Dio2 and Aanat mRNA levels. Transient expression of Icer-fl in pinealocytes caused a reduction in the Dio2 mRNA levels stimulated by NE (3 µM) after 5 h (Fig. 7Go, A and C). Icer-fl treatment also reduced the NE-stimulated Aanat mRNA levels as reported previously (24). However, abolishing the NE-stimulated Icer mRNA increase had no effect on the mRNA level of Dio2 or Aanat stimulated by NE (3 µM) (Fig. 7Go, A and B). The efficacy of the Icer-si treatment was demonstrated by its ability to block the NE induction of Icer as well as reverse the inhibitory effect of Icer-fl on the NE-stimulated Dio2 (Fig. 7Go, A and B) and Aanat mRNA levels (24). These results indicate that whereas overexpressing Icer causes a reduction in the mRNA levels of Dio2 and Aanat, siRNA treatment against Icer, which abolished the NE-stimulated increase in Icer, has no observable effect on the NE induction of Dio2 and Aanat expression.


Figure 7
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  		FIG. 7. Effect of Icer-si and Icer-fl alone or in combination on NE-stimulated Dio2 and Aanat mRNA levels. Pinealocytes (1 x 105 cells per 0.6 ml) were transiently transfected with adenovirus expressing Icer-si or Icer-fl alone or in combination for 40 h and stimulated with NE (3 µM) for 5 h. Con, LacZ control. A, Representative ethidium bromide-stained agarose gels from three experiments showing Dio2, Aanat, and Icer mRNA levels in pinealocytes treated as indicated; gapdh signal is included to demonstrate loading consistency. B, Histograms of densitometric measurements of Icer, Dio2, and Aanat mRNA levels presented as fold changes vs. NE response. 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+Icer-fl.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inducible genes are under the control of both activating and repressive signals (19, 20), and coordination between these signals likely governs the rhythmic expression of inducible genes such as Dio2 in the rat pineal gland. In this study, we investigated the contributions of the subtypes of adrenergic receptors and the role of repressor proteins in the transcriptional control of Dio2 expression. It was found that although the NE induction of Dio2 expression is primarily regulated by a ß-adrenergic/cAMP mechanism, there is also contribution from the {alpha}1-adrenergic receptor, but this effect is mediated through the potentiating action on cAMP accumulation. In the case of regulation by repressor proteins, by using siRNA to knock down the expression of Fra2 or Icer after NE stimulation, we found that preventing the induction of Fra2 causes an increase in the amplitude and duration of the NE-stimulated Dio2 expression, whereas preventing the induction of Icer did not have a significant effect on the NE-stimulated Dio2 expression.

Previous studies indicated that the nocturnal increase in Dio2 expression as well as Dio2 activity is regulated by a ß-adrenergic/cAMP mechanism (3, 10), but other studies also support a role of {alpha}-adrenergic mechanism in its regulation (35, 36). Using pineal cell culture, we established the contribution of {alpha}1-adrenergic receptors to the NE induction of Dio2 expression. This is based on the observations that {alpha}1-adrenergic blockade with prazosin causes a reduction in the NE induction of Dio2 expression, and treatment with the {alpha}1-adrenergic agonist phenylephrine has a potentiating effect on the ß-adrenergic-stimulated Dio2 expression. However, of the signaling mechanisms activated by NE, only the membrane-permeable cAMP analog, dibutyryl cAMP, can mimic the effect of NE on Dio2 expression. Other signaling mechanisms activated by NE, including protein kinase G, protein kinase C, and elevation of intracellular Ca2+ concentration, do not appear to participate in the adrenergic induction of Dio2 expression. These results support the conclusion that contribution of {alpha}1-adrenergic receptors to the NE induction of Dio2 expression is likely through its potentiating effect on the ß-adrenergic-stimulated cAMP accumulation rather than directly through other signaling mechanisms activated by {alpha}1-adrenergic receptors.

By inhibiting protein synthesis with cycloheximide, we found an increase in the NE induction of Dio2 expression, suggesting the involvement of repressor proteins in its regulation. Of the two repressors examined, overexpression of either Fra-2 or ICER causes an inhibition of the adrenergic induction of Dio2 expression. Mechanistically this inhibition could result from Fra-2 forming heterodimers with members of the cAMP response element binding protein (CREB) family (37, 38) or ICER competing with phosphorylated CREB for the CRE site on the promoter of Dio2. However, our studies using siRNAs indicated that only preventing the NE induction of Fra2 but not Icer has an effect on the NE-stimulated Dio2 expression.

Our results with pinealocytes transfected with siRNA against Fra2 also indicate that the effect of Fra-2 on the NE induction of Dio2 expression in the rat pineal gland is time dependent. Fra2-si treatment has no effect on Dio2 expression when measured within 1 h of NE stimulation. This could simply reflect the low basal expression of Fra-2 protein present before and during the first hour of induction by NE. In support of this is our previous study that showed a significant accumulation of Fra-2 protein was observed after only 3 h of NE stimulation (24). In contrast, when Dio2 expression is measured after 3 h of NE stimulation, Fra2-si treatment becomes effective and enhances the NE induction of the mRNA level of Dio2. The reciprocal relationship between Fra-2 and Dio2 expression is consistent with the observation that the extent of Fra2 expression is inversely correlated with the Dio2 response to cAMP in rats subjected to different photoperiods (39).

In our study, we also found that preventing the NE induction of Fra2 but not Icer by siRNA has an enhancing effect on the NE induction of Dio2 expression. This suggests that Fra-2 but not ICER is the probable repressor protein that helps shape the adrenergic induction of Dio2 in the rat pineal gland. Considering that the induction of both Fra2 and Icer are primarily under adrenergic cAMP regulation and that overexpression of either repressors can suppress the NE induction of Dio2 expression, we anticipate that ICER is also involved in the adrenergic regulation of Dio2 expression. Therefore, a lack of effect of ICER on the NE induction of Dio2 expression is somewhat unexpected. We excluded that the lack of effect of Icer-si treatment on Dio2 expression is secondary to insufficient Icer silencing because the identical treatment is effective in reversing the effects of treatment with Icer-fl on the level of Icer expressed as well as the NE induction of Dio2 expression. Instead, our results suggest that under NE stimulation, the amount of endogenous ICER protein induced may not be sufficient to compete with phosphorylated CREB for the CRE promoter to cause a reduction in Dio2 transcription. However, with overexpression of exogenous ICER, this repressor is effective in suppressing the NE induction of Dio2 expression.

Our results therefore support the Fra-2 protein induced by NE probably functions as a major negative signal for Dio2 transcription and highlight the importance of the AP-1 complex in the sustained Dio2 expression. Whereas activation of the CRE element on the Dio2 promoter is required for the induction of its transcription, the AP-1 site appears more important than the CRE site in limiting the duration of the response. In support of this, mutation of the AP-1 site up-regulates the human Dio2 promoter activity (40), and in transgenic rats with knockdown of Fra-2, the nocturnal Dio2 expression is increased (25).

By comparing the effect of Fra-2 on the expression profile of Dio2 vs. Aanat, we established that the effect of Fra-2 is also gene specific. Although the cAMP-CRE mechanism is involved in the induction of Dio2 and Aanat and both CRE and AP-1 sites are present in the Dio2 and Aanat promoters (9, 27, 28), only Dio2 expression is negatively regulated by Fra-2. Neither overexpression nor preventing the induction of Fra2 has an effect on the NE induction of Aanat expression. These results are consistent with a lack of effect on the urinary output of 6-sulfatoxymelatonin, a major metabolite of melatonin, and an increase in the nocturnal expression of Dio2 in transgenic rats with knockdown of Fra-2 (25). However, the mechanism that accounts for the specificity of the Fra-2 on the expression of selective genes in the rat pineal gland remains unclear. Whether this is related to a difference in sensitivity between the AP-1 sites on the Aanat and Dio2 promoters toward the repressive effects of different Fra-2 complexes awaits further investigation. In this regard, it should be noted that JunB, which can form a repressive complex interacting with the AP-1 site, is also induced at night (41, 42, 43, 44), and it will be worthwhile to examine its contribution to the overall effect of the AP-1 complex on Dio2 expression.

In summary, using adenoviral transfection to modulate the expression of Fra2, we found that transcriptional repression by Fra-2 probably plays an important role in determining the time profile of the adrenergic induction of Dio2 expression in the rat pineal gland. During induction, Dio2 expression is up-regulated through an adrenergic-cAMP mechanism. To shape the time profile, cAMP regulates Dio2 expression indirectly by inducing Fra2 and the Fra-2/AP-1 complex functions as a repressor for Dio2 transcription. In this regard, Fra-2, but not ICER, appears to be important in shaping the Dio2 response. Because of the physiological importance of Dio2 and its role in regulating intracellular T3 level in various tissues (1, 2, 45, 46, 47, 48), it will be important to determine whether the expression of Dio2 is similarly regulated by Fra-2 and ICER in other tissues.


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

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 19, 2007

Abbreviations: AA-NAT, Arylalkylamine-N-acetyltransferase; AP-1, activator protein-1; CRE, cAMP response element; CREB, cAMP response element binding protein; Dio2, type II iodothyronine deiodinase; Fra-2, Fos-related antigen 2; Fra2-fl, adenovirus overexpressing the full-length Fra2 transcript; Fra2-si, adenovirus expressing siRNA complementary to the Fra2 transcript; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; ICER, inducible cAMP early repressor; Icer-fl, adenovirus overexpressing the full-length Icer transcript; Icer-si, adenovirus expressing siRNA complementary to the Icer transcript; NE, norepinephrine; siRNA, short interfering RNA.

Received February 5, 2007.

Accepted for publication April 11, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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